REGULATION OF CELLULASE ACTIVITY AND SYNTHESIS
IN CLOSTRIDIUM THEIRMOCELLUM
by
ERIC ARTHUR JOHNSON
University of California, Davis
(1976)
University of California, Davis
(1978)
B.S.,
M.S.,
Submitted to the Department of
Nutrition and Food Science
in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
November, 1983
c
Eric Arthur Johnson
1983
The author hereby grants to M.I.T. permission to reproduce and
to distribute copies of this thesis document in whole or in part.
Signature of Author:
Department of Nutrition and Food Science,
November 21, 1983
Certified by:
Thesis Supervisor
Accepted by:
Cha"i'rman, Committee on Graduate Students,
Department of Nutrition and Food Science
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This doctoral thesis has been examined by a Committee of
the Department of Nutrition and Food Science as follows:
Professor D.
I. C.
Wang
Chairman
Professor A.
L.
Demain
Thesis Advisor
Professor B. Magasanik
Professor A. J. Sinskey
_
U
-2-
REGULATION OF CELLULASE ACTIVITY AND SYNTHESIS
IN CLOSTRIDIUM THERMOCELLUM
by
ERIC ARTHUR JOHNSON
Submitted to the Department of Nutrition and Food Science
on November 21, 1983 in partial fulfillment of the
requirements for the Degree of Doctor of Philosophy in
Applied Microbiology
ABSTRACT
True cellulase activity was demonstrated in cell-free broths
from C. thermocellum. Enzyme preparations were highly active
on complex and crystalline cellulosic substrates, provided
they were supplemented with a sulfhydryl reducing agent and
calcium. Under these conditions, low concentrations (0.6 mg/
ml) of cotton, Avicel and filter paper were all extensively
solubilized at rates comparable with the cellulase from Trichoderma reesei but with fifty times less protein in the incubation.
Cellobiose was the predominant saccharification product
from Avicel.
Cellulase activity
was found to be inhibited by cellobiose and
inactivated by sulfhydryl reagents and iron chelators.
Cellobiose strongly inhibited the C. thermocellum cellulase when
the substrate was Avicel but was only mildly inhibitory to the
digestion of amorphous cellulose. Cellobiose inhibition was
relieved by the addition of 3-glucosidase. Analogues of cellobiose including salicin, lactose, and arbutin mildly inhibited
cellulase activity.
The crude dialyzed cellulase was inactivated by incubation in
a low concentration (0.2-0.4 mM) of dithiothreitol (DTT).
This
was caused by oxidation of the low DTT concentration in air to
form H 2 0 2 , which in turn oxidized cellulase
sulfhydryl groups.
Activity loss was prevented by exclusion of air, or by the
addition of catalase, EDTA, or an increased concentration (10
mM) of DTT. Crude cellulase from C. thermocellum was strongly
inhibited by sulfhydryl reagents including o-iodosobenzoate
(IB), N-ethylmaleimide (NEM), 5,5'-dithiobis (2-nitrobenzoic
acid) (DTNB),
p-chloromercuribenzoic acid (pCMB) and copper (Cu).
These inhibitions were prevented by 10 mM dithiothreitol.
Even
in the protective environment of a high DTT concentration, cellulase was inactivated by certain apolar chelating agents including o-phenanthroline and bipyridyl, such inactivation being preventable by the prior incubation of the chelator with a mixture
-3-
of Fe ++ and Fe +++
These data suggest that the thermophilic
clostridia-l cellulase, unlike the enzyme from aerobic fungi,
contains essential sulfhydryl groups and is stimulated by iron.
The component of the cellulase susceptible to sulfhydryl inactivation appears to be an enzyme participating in the breakdown
of crystalline cellulose (which I assume to be exo-3 (1+4)-glucanase) since the enzyme hydrolyzing amorphous cellulose (which
I assume to be endo-3-(1-4)-glucanase) was unaffected by oxidation or thiol reagents.
A minimal chemically defined medium was developed for C. thermocellum. The growth factors required are biotin, pyridoxamine,
vitamin B 1 2 , and p-aminobenzoic acid. This medium was used to
study the regulation of cellulase formation.
It was found that
the synthesis of extracellular cellulase is carefully regulated
in C. thermocellum. The specific titer (units cellulase activity per g cell) varied more than 100 fold depending on the carbon substrate present in the medium. Cellulase was produced in
highest specific titers on cellulose.
It was also formed on the
cellulose derivatives cellobiose and glucose, and on the sugars
fructose and sorbitol, which are not derived from cellulose, indicating that the cellulase system in C. thermocellum is prcduced
constitutively. The specific titer of cellulase was increased
when the cells were slowed in their formation of cellular energy.
This condition was imposed during growth on insoluble
cellulose, resulting in limitation of cellobiose, or during
adaptation to fructose or sorbitol, which were slowly assimilated. Starvation for carbon did not promote cellulase synthesis. Although very high specific titers were detected
during the growth lag on fructose and sorbitol, these values
declined sharply as the culture gradually adapted, and eventually reached a specific titer lower than observed on cellobiose. Cells growing on fructose underwent a shift in their
pyruvate metabolism and lactic acid production declined to low
levels.
This resulted in increased oxidative decarboxylation
of pyruvate accompanied by an increased ATP yield, and a decline in cellulase synthesis.
Cellulase formation was reestablished by inhibitors that shifted pyruvate metabolism
towards lactate production or by uncouplers that dissipated
the pH gradient across the cell membrane, thereby lowering the
energy level in the cells.
These results support the conclusion that extracellular cellulase formation is regulated by
catabolite repressionin C. thermocellum.
Thesis Supervisor:
Dr. Arnold L. Demain
Title:
Professor of Industrial Microbiology
-4-
ACKNOWLEDGMENTS
I would like to thank the many individuals who have generously contributed to this study.
I owe a special thanks to
Arnold Demain for his cooperation and support, to Daniel Wang
for his leadership, and to Charles Cooney and Anthony Sinskey
for their encouragement and guidance.
I am grateful to Boris
Magasanik for his interest, suggestions and enthusiasm during
this work.
I thank Geoff Halliwell and Irwin Hollander for their
collaboration, and Arthur Smith, Frederique Bouchot, Mary
Whitmer, Cristan Orrego, Cindy Allen, Mitsuji Sakajoh, Cindy
Tolman, Sue Groh, Herve Cellard and Beatriz Mendez for participation in various experiments.
I am grateful to W. H. Orme-Johnson, Edmund Lin, and
Elwyn Reese for their valuable suggestions.
I express thanks to Catherine Duong, Gerald Sanchez,
Mary-Louise Piret, Dan Gold, Jon Dordick and Nadine Solomon
for their special contributions, and Ruth Ayers for her expert
assistance.
I acknowledge National Distillers Co.,
Co.,
NSF, Eastman Kodak
Department of Energy and Archer Daniels Midland for fin-
ancial support.
Finally, I warmly thank Mary Whitmer for her valuable
help and friendship, and my parents for their everlasting encouragement.
-5-
TABLE OF CONTENTS
Page
Title Page
...............................................
1
Abstract .................................................
2
Acknowledgments ..........................................
4
Table of Contents ........................................
5
List of Figures ..........................................
7
List of Tables ...........................................
10
1.
Introduction .........................................
12
A.
General
..........................................
12
B.
C.
Historical .......................................
Physiological Properties of C. thermocellum ......
15
21
1.
2.
3.
General Properties ..............................
Carbon Substrate Utilization and Metabolism ..
Energy Metabolism and Endproduct Formation in
C. thermocellum ............................
21
22
Properties of C. thermocellum Cellulase ..........
...
Regulation of Cellulase Synthesis in Fungi and in
C. thermocellum .................................
Regulation of Cellulase Synthesis in C. thermocellum
..........................................
29
D.
E.
F.
G.
38
39
Selection of Mutants Affected in Extracellular En-
zymes ........................................
2.
26
Experimental Procedures
..............................
40
43
G.
Bacteria .........................................
Cultivation of Bacteria
..........................
Determination of C. thermocellum Nutritional Requirements ...................................
Source and Preparation of Cellulase ................
Measurement of Cellulase Activity ..................
CM-Cellulase Activity (endo-1,4- -D-glucanase, E.
C . 3.2.1.4) ..................................
Units of True Cellulase Activity ---.................
48
48
H.
Analysis of Cellulolytic End Products ..............
50
I.
J.
Analysis of Fermentation End Products .............. 50
Determination of Phosphate Uptake by Cells .......
.51
K.
Assay of ATP Concentration in Cells
A.
B.
C.
D.
E.
F.
--..............
43
43
44
45
47
51
-6-
Page
L.
M.
N.
0.
3.
Assay of Hydrogenase Activity ....................
Gel Electrophoresis ..............................
Purification of Cellulase ........................
Chemicals ........................................
Results
..............................................
F.
G.
Ca + and Sulfhydryl Reducing Compounds as Requirements of the Cellulase System of C. thermocellum ..........................................
Oxidative Inactivation of C. thermocellum Cellulase:
Evidence for Essential Sulfhydryls ....
Effect of Chelating Agents on Cellulase Activity:
Evidence that the C. thermocellum Cellulase
Requires Iron for Activity ...................
End Products of Avicel Saccharification ..........
Inhibition of Cellulase Activity by End Products
of Cellulolysis ..............................
Partial Purification of Cellulase ................
Construction of a Defined Medium for C. thermocel-
H.
Control of Cellulase Synthesis in C. thermocellum
A.
B.
C.
D.
E.
lum ..........................................
52
52
53
54
55
55
66
76
79
83
86
102
103
4.
Discussion ...........................................
135
5.
Recommendations for Future Research ..................
145a
6.
References
...........................................
146
-7-
LIST OF FIGURES
Figure
No
1
2
3
Title
Page
Biochemical Pathways of Sugar Catabolism in C.
thermocellum ..................................
23
Model for Cellulase Digestion of Insoluble Cellulose ........................................
32
A.
First Order Rates of Avicel Solubilization
(Measured at O.D. 660 nm) by Varying ConcentraRate of
B.
tions of Extracellular Protein.
Avicel Solubilization as a Function of Protein
Concentration
4
.................................
49
Clear Zones Produced by C. thermocellum After 8
Days Growth on Compression Milled Corn Stover
...
56
Influence of DTT Concentration on Avicel Hydrolysis by Clostridium thermocellum Cellulase
58
Influence of Ca 2 + on Avicel Hydrolysis by Clostridium thermocellum Cellulase ................
59
(A),
5
6
7
Avicel
(B),
or Amorphous Cellulose
Inhibition of C. thermocellum Cellulase by EDTA
...................
60
Solubilization of Native and Derived Celluloses
by Cellulase of Trichoderma reesei QM 9414 and
Clostridium thermocellum ......................
62
Hydrolysis of Cotton and Avicel under Optimal
Conditions by the Cellulases of Trichoderma
reesei RUT C-30 (Tr) and Clostridium thermocel................ ....
....
.
lum (Ct.) .......
64
Comparison of C. thermocellum and Trichoderma
reesei QM 9414 Cellulase Activities on Phosphoric Acid-Swollen Avicel and Microcrystalline
-........................................
Avicel.
65
Influence of Dithiothreitol Concentration on the
Activity of C. thermocellum Cellulase .........
67
and Its Reversal by Calcium
8
9
10
11
12
(C)
Influence of Anaerobic and Aerobic Atmospheres
on Inhibition of Cellulase by 0.4 mM DTT or 0.04
--..........................
mM Hydrogen Peroxide
69
-8-
Figure
No
13
Title
Page
Inhibition of Cellulase by Hydrogen Peroxide or
Low DTT in Air ................................
14
The Effect of Catalase and Superoxide Dismutase
(SOD) on the Inhibition of Cellulase by Low DTT
and Air .......................................
15
70
71
Solubilization of 3 g/l Avicel by 7 ig/ml Dialyzed Extracellular Protein in an Aerobic or
Anaerobic
16
17
18
19
20
21
22
Product
(90% N 2:5% CO 2:5% H2)
Formation
(Cellobiose,0
Atmosphere
;
....
81
Glucose,O )
During Saccharification of Avicel by C. thermocellum Dialyzed Culture Broth .....................
82
Inhibition of Cellulase Activity in Untreated
Culture Broths of C. thermocellum and T. reesei
by Cellobiose (A) or Glucose (B) .................
84
Inhibition of C. thermocellum Cellulase by Cellobiose (o) or Glucose (o) on Phosphoric AcidSwollen Avicel ................................
85
Relief of Cellobiose Inhibition of Cellulase
Activity by 3-Glucosidase from Aspergillus
phoenicis .....................................
88
Visible and Ultraviolet Adsorption Spectrum of
Dialyzed Cellulase in 20 mrM Tris, pH 8.8 ......
91
DEAE-Sepharose Chromatography of C. thermocellum
Extracellular Protein --.........................
92
Gel Filtration of Dialyzed, Total Extracellular
Protein on Ultrogel ACA 22 ....................
23
SDS-Polyacrylamide Gel Electrophoretic
PAGE) Patterns
94
(SDS-
(7% Acrylamide Gel) of C.
thermo-
cellum Extracellular Proteins Purified by DEAESepharose and Ultrogel ACA 22 Chromatography
24
25
26
..
SDS-PAGE of Purified Cellulase Proteins ........
95
96
Ion-Exchange HPLC Chromatography of Dialyzed,
Extracellular Proteins ........................
98
Ion-Exchange HPLC Chromatography of Dialyzed,
Extracellular Proteins ........................
99
-9-
Figure
Title
No
Page
27
SDS-PAGE of Crude Cellulase from C. thermocellum Grown in Different Carbon Sources and
Partially Purified by HPLC ...................... 100
28
SDS-PAGE of Fractions Obtained from Preparative
HPLC
(Lanes A-E)
..............................
101
29
Growth and Cellulase Formation in Cellobiose or
Fructose Defined Medium ......................... 109
30
Cellulase Production (units) as a Function of
Dry Cell Weight (mg) During the Lag in Fructose
31
32
or During Exponential Growth in Cellobiose ....
111
Effect of the Addition of Cellobiose on Cellulase Formation During the Growth Lag on Fructose ..........................................
112
Growth of C. thermocellum on Cellobiose or Avicel ...........................................
117
33
Transport of Inorganic Phosphate and Formation
of ApH by C. thermocellum ....................... 120
34
Effect of Serial Transfer on the Specific Titer
of Cellulase by C. thermocellum Culture Broths
35
123
Cellulase Synthesis by Cells Adapted to Fructose
(Figure _A),
Isolated on MJ-Avicel Agar,
Picked to Cellobiose Broth for One Transfer,
and Reinoculated to Fructose ................... 125
-10-
LIST OF TABLES
Table
No.
1
2
3
4
5
6
7
8
Title
Page
Isolation of Cellulose Decomposing Microorganisms .........................................
17
Effect of Hydroxyl Radical Scavengers on Oxidation of Cellulase by H 2 0 2 or Low DTT .......
72
Inhibition of Cellulase by Sulfhydryl Reagents
and Copper and Prevention by 10 mM DTT .......
74
CM-Cellulase Activity is Not Inhibited by Oxidation or Sulfhydryl Reagents ...................
75
Effect of Chelating Agents on Cellulase Activity Under Anaerobic, Reduced Conditions ......
77
Reversal of o-Phenanthroline (OP) Inhibition
of Cellulase by Prior Chelation of OP with
Metals .......................................
78
Influence of -Glucosidase on Cellulase Hydrolysis by C. thermocellum Cellulase ............
87
Inhibition of C. thermocellum Cellulase by Various Carbohydrates ...........................
89
Vitamin Requirements of C. thermocellum ATCC
27405 ........................................
104
10
Composition of CM3, GS and MJ Media ............
105
11
Formation of Ethanol, Acetic Acid, Lactic Acid,
and Avicel Hydrolyzing Activity in MJ and GS-2
Media ........................................
106
9
12
Cellulase Synthesis by Cells Previously Grown
in Cellobiose when Transferred as A Small Inoculum
13
14
(1% v/v) to Different Carbon Sources
..
108
Cellulose Synthesis by C. thermocellum Grown
on Cellobiose, and Inoculated (1% v/v) to Cellobiose or Avicel Media. Cultures were Grown
for 60 h .....................................
114
Growth and Cellulase Formation by Cells Adapted to Different Carbon Sources ..................
115
-11-
Table
No.
15
16
17
18
19
20
21
Title
Page
Formation of ATP, End Products and Cellulase
During Lags on Fructose and Glucose ...........
118
Difference in Product Formation by Cellobioseor Fructose-Adapted Cells in Cellobiose and
Fructose, Respectively ...........................
127
ATP Levels in C. thermocellum Grown on Soluble
Carbon Sources ................................
129
Influence of Gas Atmosphere on Growth of Fructose-Adapted Cells ...............................
130
Derepression of Cellulase Synthesis in FructoseAdapted Cells by Inhibitors of Pyruvate Decarboxylation ....................................
131
Changes in Cellulase Synthesis from Treatments
which Affect ATP Accumulation and Formation of
ApH ...........................................
133
Effect of Cyclic Guanosine Nucleotides on Cellulase Synthesis in Cellobiose Medium .........--
134
-12-
1.
INTRODUCTION
A.
General
The thermophilic anaerobe Clostridium thermocellum is
readily isolated from decaying cellulose in a wide variety of
habitats including soils, manures, composts,
intestinal con-
tents of animals, and marine and fresh water muds
152, 249).
Waksman and Skinner
(6, 50,
(240) suggested that the ther-
mophilic cellulolytic anaerobes, typified by C. thermocellum,
"stand in a group by themselves",
and are characterized by
their vigorous and rapid fermentation of cellulose.
The sig-
nificance of C. thermocellum in the global degradation of cellulose is not known, but it almost certainly plays a minor role
in the recycling of this abundant resource.
The primary degra-
dation of cellulose and other plant tissues occurs aerobically
through the action of fungi, and is especially prominent in
several ascomycetes and imperfect fungi including the genera
Aspergillus, Chaetomium, Fusarium and Trichoderma.
The initial
aerobic attack rapidly utilizes the amorphous forms of cellulose and leaves as residual material the highly crystalline and
lignified forms for digestion by other microorganisms including
C.
thermocellum.
A true cellulase
to saccharify
system is characterized
native cellulose
(e.g.
by its
cotton fibers)
over
ability
long
-13-
periods of time (84).
Cellulase is a complex enzyme system,
composed of endo- and exo-3-(1-4)-glucanase enzymes, which act
synergistically to degrade insoluble cellulose to cellobiose.
The nature of the processes involved in the anaerobic
decomposition of cellulose differs greatly from that found in
the presence of oxygen (232, 233, 237, 254).
A diversity of
microorganisms including protozoa, fungi, actinomycetes, myxobacters and aerobic unicellular bacteria thrive on cellulose
under aerobic conditions.
In the absence of oxygen, cellulose
decomposition occurs solely through the activities of bacteria,
especially by members of the genus Clostridium.
The aerobes
are generally versatile, capable of growing on a large number
of organic substrates.
On the other hand, the clostridia and
soil cytophagas are highly specialized organisms capable of
utilizing few if any carbon sources other than cellulose or its
sugar products
(50,
213).
The anaerobic fermentation of cellulose is believed (51,
254,
258) to be a cooperative process, carried out by interact-
ing populations of bacteria.
The primary hydrolytic decomposers
characteristically produce hydrogen gas and acidic end-products
which inhibit further growth unless the end-products are removed
by methanogens and sulfate reducers.
The reduced yields of cel-
lular energy in the absence of oxygen requires that cellulose be
efficiently
digested to support the microbial populations, and
provides a selective pressure for the evolution of an active
cellulase system.
-14-
Although the anaerobic bacteria grow very rapidly on
crystalline cellulose
(136, 242),
true cellulase activity
(i.e.,
the ability to completely saccharify native cellulose such as
cotton;
82-84),
has only been detected in the broths of ascomy-
cetous fungi, especially species of Trichoderma
The anaerobic bacteria are thought to
cellulase.
Eriksson has proposed
(55,
(83,
187,
144).
secrete little true
56)
that oxygen is in-
volved directly in the depolymerization of lignin and cellulose,
and in the coupling of their degradations.
rot fungi are known
(61, 72,
Furthermore, many
127) to produce hydrogen peroxide
as they enter the stationary phase of growth.
H 2 0 2 may in turn
react with iron to form oxygen radicals active in the depolymerization of lignin
(61, 72)
and cellulose
(56,
127).
Aerobic
organisms have developed mechanisms to protect themselves from
the reactivity of 02'
idizing ability.
and have evolved to exploit its high ox-
Anaerobes do not have the benefit of being
able to use 02 for enzymatic purposes or as an electron acceptor during growth.
Thus, they must use a non-oxidative mechan-
ism for the degradation of plant tissues, e.g. lignin and cellulose, and their inefficient energy metabolism requires that
they have efficient degradative enzymes to provide themselves
with ample sugar for growth.
The ability of C. thermocellum to grow rapidly on cellulose, and its role in decaying partially attacked cellulose,
suggest that it synthesizes an extracellular cellulase system
that can efficiently saccharify crystalline cellulose.
This
-14a-
study was designed to elucidate the biochemical requirements
for its activity, and the physiological factors controlling its
synthesis.
-15-
B.
Historical
An understanding of the microbial decomposition of cellulose has developed from investigations on the associative and
antagonistic interactions of soil microorganisms, and the biological transformations that they carry out in their natural environment.
(232, 236,
Winogradsky
240)
(251, 235),
Omeliansky
(173), Waksman
and others recognized that the dead residues of
plants are particularly favorable habitats for many microbes.
These soil residues are gradually decomposed by successions of
microorganisms (88,
238) first being attacked by sugar fungi
(e.g. zygomycetes),
then by ascomycetes and imperfect fungi and
finally by bacteria and actinomycetes, which complete the transformation into humus
(234), which is itself very important for
the renewed growth of plants.
The connection between the activities of microorganisms
and the decay of cellulose was first established by Omeliansky,
the student of Winogradsky.
In 1895, he
(173) used an enrich-
ment medium with cellulose as the only source of carbon to isolate a culture of mesophilic anaerobic bacteria that fermented
cellulose.
Omeliansky found that the mixture of gases produced
during fermentation consisted of hydrogen and methane, the two
gases being produced by different microorganisms.
Heating the
dung or mud inoculum destroyed the capacity to produce methane,
and the hydrogen-producing bacterium could be obtained free from
-16-
the methanogen.
It was found to produce terminal endospores,
and to cling to cellulose fibers, but he could not maintain it
in pure culture.
Omeliansky also studied the rotting of cellulose in
manures and composts, which reach temperatures greater than
60*C during decay due to the metabolic activities of microbes
(231).
He noticed that the gas composition changed with depth,
consisting of methane and hydrogen and completely lacking oxygen at the lower depths of the pile.
Blaxall
In 1899, MacFayden and
(140) isolated thermophilic bacteria from manure which
vigorously fermented cellulose at 65*C.
Following these initial observations, many microbiologists isolated different groups of fungi and bacteria that had
the capacity to digest cellulose
(Table 1).
The existence of
various aerobic bacteria capable of degrading was demonstrated
in 1904 by Van Iterson and substantiated by Kellerman and his
associates
(120-122), who with relative ease isolated single
colonies that cleared cellulose suspended in agar.
The main or-
ganism responsible for the decomposition had a gliding motility,
and was later identified as Spirochaeta cytophaga
Sporocytophaga) by Hutchinson and Clayton
(110).
(now known as
The ease in
purifying the aerobic decomposers, as opposed to the extreme
difficulty in isolating the anaerobic cellulose digesters, led
Kellerman to challenge the findings of Omeliansky with respect
to the involvement of anaerobic bacteria in cellulose decompo-
Table
1
Isolation of Cellulose Decomposing Microorganisms
Group
An:erobic
Bacteria
Gliding Bacteria
(aerobic)
Generic Name
(G+C, Molar %)
Clostridium
(23-43)
Cellulolytic Species
Morphology and Physiology
Ecology
Rods, usually motile. SporeMesophilic to therformer.
mophilic.
Soil,mud,
C.
gut,ma-
C.
thermocellum
cellobioparum
Comments
Vigorous cellulose fermenters.
nure,
composts
albus
H.
flavefaciens
rumen
B.
G- rods, motile by gliding.
Strict aerobe, respiratory.
Only uses cellulose, cellobiose, glucose as C and
energy sources. Forms
resting stage (microcyst).
Soil.
Acetate,
Rods, aerobic.
prdpionate and succinate
are end products.
Soil.
G+ cocci.
Cellobiose preferred source, CU 2 and H 2
produced. Major non-gaseous products are lower
fatty acids.
rumen
Bacteroides
(40-55)
G- rods. Product mixtures
of acids including succinic,
formic, lactic, propionic.
Non-spore forming.
Require p-aminobenzoic and
biotin.
Sporocytophaga
k Spirochaeta
cytophaga)
(36)
Cytophaga
(33-42)
Omelianski, 1895-1897; cited
In Waksman and Skinner,
1926.
Viljoen et al., 1926.
Hungate,
R.
Ruminococcus
(39. 8-41.4)
References
1944.
Cellulolytic ruminococci
very limited in tneir
sugar fermenting
abilities.
Sijpesteijn,
al., 1958.
succinogenes
Cultures
Hungate, 1950. Bryant and
Doetsch, 1954.
S.
myxococcoides
Vigorous aerobic celluLong
lose fermenter.
adaptation to glucose.
Hutchinson
C.
hutchinsonii
show CO 2 uptake in fermentation
of cellulose or cellobiose.
1949.
Bryant et
flungate, 1963.
and Clayton, 1919.
Dubos, 1928.
Stanier, 1940.
Winogradsky, 1929.
and Norman, 1943.
Fuller
continued...
Table 1
(continued)
Group
Generic Name
(G+C, Molar %)
Morphology and Physiology
Ecology
Cellulolytic Species
Comments
References
Pseudomonads
Pseudomonas
(58-70)
(Cellfailicula)
(Cellvibrio)
Short rods; strict aerobes;
Gram-, mesophiles.
Soil,
freshwater,
marine
P. fluorescens
var. cellulosa
Cellvibrio fulvus
Probably not strong
fermenters of cellulose.
Winogradsky,
Actinomrycetes
and related
organisms
Streyto.ices
Aerial mycelium; highly ox-idative: acid generally not
produced from glucose.
Soil
S.
Many actinomyc2tes have
a limited capacity to
attack cellulose.
Waksman, 1919.
et al., 1913.
Irregular rods; respiratory;
acid produced from glucose. Temp. opt. is -3300
C. Coryneform bacteria.
Soil.
C. flavigena
U. uda
Relationship to 02 is
not clear, most strains
give significant yet reduced growth on glucose in absence of
oxygen.
Winogradsky, 1929.
Kellerman et al., 1913.
Clark, 1951
Facultative aerobe; opt.
growth 4t-550 C
Fresh
manure,
straw,
T. curvata
Henssen, 1957
N.
Metcalf and Brown, 1957
(69-75)
Cellulomonas
(72-73)
Thermomonospora
thermoviolaceus
1929.
McBeth
manure
compost
Nocardia
(2)
Aerial mycelium; aerobe
Soil
and
cellulans
manure.
continued...
Table 1
(continued)
Group
Fungi-Ascomycetes
Generic Name
(G+C. Molar %)
Fusarium
Trjcioderma
C'haetomium
Penicillium
Verticillium
Cephalosporium
Morphology and Physiology
Oxidative metabolism.
trating hyphae.
Pene-
Ecology
Soil.
Cellulolytic Species
F. solani
T. viride
Comments
Although some lower
fungi weakly degrade
cellulose (e.g. Saprolegnia) most of the
Fungi which secrete
potent cellulases are
Ascomycetes and ImImportant
perfects.
In initial, aerobic attack of cellulose. The
thermophilic molds
show greater cellulolytic ability than
thermophilic actinomycetes.
References
De Bary, 1886. Appel,
Waksman, 1916.
1907.
Scales, F.M., 1915.
Waksman and Skinner,
Fergus, 1969.
1926.
-20-
sition.
Omeliansky was also challenged by Winogradsky, his pro-
fessor, who believed cellulose decomposition to be an aerobic
process and to occur by an oxidative mechanism
the formation of uronic acids.
(236),
e.g. by
The anaerobic, mixed-culture
fermentation of cellulose was extremely vigorous, however, and
Pringsheim
(186),
Kroulik
(129),
Khouvine (125),
vincingly demonstrated its occurrence.
and others con-
They too, however,
failed to purify the bacterium responsible for the degradation.
Khouvine
(125) isolated an anaerobe, Bacillus cellulosae dis-
solvens, from human feces, but could not obtain single colonies
on solid medium.
Viljoen et al.
(229)
in 1926 isolated a ther-
mophilic cellulose digester from manure, which they named Clostridium thermocellum, but this organism lost its cellulolytic
capacity when subcultured in glucose medium.
The same trend
continued until the late 1930's, when Pochon (184) suggested
that the organisms were pure but were undergoing morphological
and physiological changes in a type of life cycle, and Enebo
(51, 53)
suggested that life of the anaerobic thermophilic cel-
lulose digesters was dependent on an obligate symbiosis.
last, however, Hungate
(102, 104)
At
successfully developed a
method for the isolation of pure cultures by meticulously diluting mesophilic populations through a series of tubes coated
inside with cellulose mineral salts agar.
abled McBee
This technique en-
(151, 152) in 1948 to obtain pure cultures of ther-
mophilic cellulolytic anaerobes, and facilitated the study of
their characteristics and physiology in pure culture.
-21-
C.
Physiological Properties of C. thermocellum
1.
General Properties
(11, 50,
153, 156, 167, 185,
248)
C. thermocellum occurs morphologically as a Gramnegative rod-shaped bacterium (0.5 x 2.5-5.0 pm),
erally motile by lateral flagella (153).
which is gen-
Cultures appear in
liquid media as single cells, in short or long chains, and on
solid media as single cells often with swollen terminal spores.
On agar medium, single cells grow into yellow or white lensshaped colonies and, when growing on insoluble cellulose, produce well-defined clear zones.
In the strains examined, the DNA base composition
is approximately 39% G+C
(167),
low for the clostridia.
temperature optimum for growth is 60-65*C.
The
C. thermocellum does
not produce hydrogen sulfide, and nitrate is not reduced to nitrite.
It is not proteolytic.
Fermentation products are H 2 '
CO 2 , acetic and lactic acids, and ethyl alcohol.
ties of succinic acid may be formed.
are not well defined;
Minor quanti-
Its nutrient requirements
it appears to require a number of amino
acids and vitamins as growth factors
(60).
C. thermocellum is
probably most closely related to the spore forming, fermentative
bacilli
(62),
digesters
and its physiology resembles ruminant cellulose
(12, 23-27, 94, 104-107, 208-210) and the fermentative
sarcinae (29, 130).
-22-
2.
Carbon Substrate Utilization and Metabolism
C. thermocellum ferments very few carbon substrates;
it trefers cellobiose and cellulose, and it will adapt for
growth on glucose (50, 6S
tol
(92).
, 73, 177),
fructose (170),
or sorbi-
It also grows very slowly on salicin but most strains
will not ferment other hexoses, hexitols, organic acids, amino
acids, pentoses or polysaccharides
(50, 63 , 132).
Alexander
(178) and McBee (153) reported that C. thermocellum grew on mannitol and xylose, respectively, but these results have not been
confirmed.
The biochemical pathways of sugar catabolism and
energy generation are summarized in Figure 1.
The fermentation
of fructose and sorbitol is initiated by phosphoenolpyruvatedependent phosphorylation and transport (173),
and probably
occurs by the bacterial sugar phosphotransferase system (PTS;
196).
The PTS is very common in saccharolytic anaerobes
195).
It may be inducible in C. thermocellum since Patni and
Alexander
(100,
(173) reported that it was formed only when the cells
were grown on fructose or mannitol.
In addition, the phospho-
fructokinase responsible for conversion of fructose-l-phosphate
to the common intermediate, fructose-1,6-diphosphate, is inducible in the clostridia (101).
Although PEP was shown to be the phosphoryl donor
in C. thermocellum cell extracts, the direct demonstration of a
-23-
Figure
1
Biochemical Pathways of Sugar Catabolism in C. thermocellum
cel lobiose
fructose
glucose
sorbitol
~2
I
I
'I
I~
ATP-
PEP-
dpendent
phospho-
t ransf erase
F-i-P
F-6-PF
F-1 6-diP
t
ATP
NADH
Lactate
-
-
--
PYRUVATE
acetyl-CoA
ferredoxin
K
2 Hi1
AT P
NAD
*7/
I
i
I
V
H2
/
Acetate
I
Ethandl
-24-
PTS by dissection of the system into its protein components, and
reconstitution with purified constituents from C. thermocellum
or a related organism (e.g. Staphylococcus aureus),
has not been
carried out.
Cellobiose is the principal product and glucose a
Both of
minor product of C. thermocellum cellulase activity.
these substrates are dependent on glycolytically-generated ATP
as the source of energy for their transport
(90, 170).
Cello-
biose and glucose uptake is inhibited by 1% oxygen and by sodium
arsenate, and partially by DCCD and sodium fluoride
supporting an energy requirement for ATP.
(170),
Uncouplers
(e.g. CCCP
and DNP) do not severly inhibit cellobiose and glucose uptake
(91); it is therefore unlikely that a proton motive force
is the direct drive in their transport.
that ATP may participate directly
(pmf)
The experiments suggest
(15, 197) in the uptake; the
available data does not rule out the direct involvement of
acetyl phosphate, the high-energy precursor of ATP formed after
the pyruvate branchpoint in energy metabolism (see below) and
known to be the direct energy donor in certain other "ATP"dependent transport systems
(97).
After its energy-dependent uptake, cellobiose is
metabolized by both a cellobiose phosphorylase
and a
-glucosidase
(2).
(4, 5, 7 , 207)
The phosphorylase is highly active in
C. thermocellum cell extracts
(5, 75),
approximately 10 times less than the
has a Km for cellobiose
-glucosidase, and is prob-
-25-
ably the main enzyme involved in cellobiose cleavage.
Cello-
biokinase activity has also been detected in C. thermocellum
cell extracts
(166).
A cellodextrin phosphorylase is present
in C. thermocellum, which has been purified and characterized
as an enzyme distinct from the cellobiose phosphorylase
Glucose and glucose-l-phosphate
(G-1-P) are gen-
erated in the cells by phosphorolysis of cellobiose.
converted to fructose-1,6-biphosphate
(203).
G-1-P is
(FDP) by phosphoglucomu-
tase, phospho-glucose isomerase, and 6-phosphofructokinase.
Conflicting results have been reported on glucose metabolism.
First of all, several investigators
(50, 152) observed that C.
thermocellum did not grow on extracellular glucose.
Alexander
(177) and Garcia-Martinez et al.
(68)
found, however,
that growth did occur, but only after a very long lag
in the presence of a high concentration
Patni and
(> 100 h)
(0.5%) of yeast extract.
Since glucose is generated internally from cellobiose, the delay
in growth may be caused by the need for induction of an uptake
system
(74,
91).
This was supported by measurement of the
transport of labeled glucose, which was very low in cellobioseadapted culture of C. thermocellum
(74).
However, another lab-
oratory has observed uptake of glucose by cellobiose-grown cells
of the same C. thermocellum strain
personal communication).
Ruminococcus
(C. Tolman and Dr. M. Roberts,
Other cellulolytic bacteria, e.g.
(12) and Sporocytophaga (210,
extended lags on glucose.
213) also exhibit
In Sporocytophaga, growth occurred
-26-
more rapidly on low concentrations of glucose, suggesting that
metabolism of glucose may inhibit growth of the culture
(210).
Glucose toxicity was also observed in a mesophilic, cellulolytic Clostridium (72).
The growth inhibition was not affected
by the inoculum density and thus is probably not due to the
excretion of a toxic metabolite.
Intracellular glucose is probably phosphorylated
by a glucokinase, using ATP as the phosphoryl donor.
Gluco-
kinase activity was present in cell extracts of C. thermocellum grown in cellobiose or glucose (177, 91),
fructose-grown cell extracts
(177).
but was low in
(177)
Patni and Alexander
suggested that cell extracts contained an inhibitor of glucokinase.
3.
Energy Metabolism and Endproduct Formation in
C. thermocellum
In anaerobes such as C. thermocellum, large quantities of sugar must be glycolytically catabolized to provide
energy for growth
(77, 107).
ATP for cell growth is obtained
by substrate level phosphorylations via 3-phosphoglycerate kinase, pyruvate kinase and acetate kinase.
The initial transport
and catabolism of sugars by C. thermocellum (see above;
Fig. 1)
produces the common intermediate, fructose-1,6-diphosphate (Fdi-P) which is glycolytically fermented to pyruvate where a
branch occurs in the catabolic pathway:
(1) reduction to lac-
-27-
tic acid by a FDP-activated, NAD-dependent lactate dehydrogenase
(132);
(2) oxidative decarboxylation to acetyl-CoA, CO
21
and reduced ferredoxin by pyruvate:ferredoxin oxidoreductase
(124).
The branch in pyruvate metabolism provides the
cells with a mechanism for the regulation of ATP formation and
NADH oxidation.
In many anaerobes and lactic acid bacteria a
restriction in the energy supply
(e.g. by carbon source limi-
tation in a chemostat) causes drastic shifts in the proportions
of end products formed
(28, 45,
70, 78,
95,
220, 221).
182,
A
shift toward lactate is often caused by an increased cellular
concentration of F-di-P, signalling energy excess
and activating L(+)-lactic dehydrogenase
(70).
(47, 70),
A switch from
acid to solvent production in Clostridium acetobutylicum is
thought to be associated with decreased rates of growth and
energy metabolism (70a).
Such a slowdown can be brought about
by several conditions, including treatment with inhibitors of
energy production or lowering the pH, and appears to be mechanistically
In
related to the onset of butnol production.
the saccharolytic clostridia such as C. thermocellum ATP,
yield is determined by regulating pyruvate decarboxylation,
which occurs by the phosphoroclastic reaction
(128, 253).
Acetyl phosphate, carbon dioxide and hydrogen are the products
of this reaction.
In Clostridium pasteurianum, phosphoroclas-
tic activity is strongly inhibited by acetyl phosphate
(17).
-28-
The oxidant acting as cofactor in the decarboxylation of pyruvate is the small iron sulfur protein, ferredoxin
(175).
Ferredoxin has a central role in the energy metabolism
of C. thermocellum.
It functions as a low potential electron
carrier from pyruvate or NADH oxidation (110, 175); it serves
to release reducing equivalents as hydrogen gas and as a
source of biosynthetic reducing power via NADP + :ferredoxin
oxidoreductase (118).
The oxidation of pyruvate by ferredoxin
(and ATP formation) is tightly linked to the reduction of hydrogen ions
(71,
77, 220).
Tewes and Thauer
(219) have pro-
posed that energy gain in saccharolytic clostridia depends on
the availability of oxidized ferredoxin in the cell.
Wolfe
and O'Kane (253) showed that artificial electron acceptors
can substitute for ferredoxin in the phosphoroclastic reaction, including neotetrazolium, and flavins in the presence of
oxygen, but not pyridine nucleotides.
Artificial oxidants pre-
vent the evolution of hydrogen, but they often stimulate the
activity, suggesting that the availability of oxidized ferredoxin may sometimes limit growth in clostridial cells.
The
biosynthesis of ferredoxin and other iron-sulfur proteins is
dependent on an adequate supply of iron in the medium.
Re-
striction of iron has dramatic effects on the fermentation of
carbohydrates in the clostridia.
In an iron deficient medium,
saccharolytic members of the genus Clostridium produce chiefly
lactic acid instead of the usual mixture of hydrogen, CO 2 '
-29-
solvents, and acetic, lactic and butyric acids
(71,
123, 176).
This shift in metabolism also occurs in the presence of relatively high concentrations of cyanide
with the iron sulfur proteins.
(123),
which combines
Pappenheimer and Shaskan
(176)
showed that the formation of extracellular lecithinase is
highly dependent on the concentration of iron in the medium.
The ferrous iron requirement in the phosphoroclastic reaction
is similar to that required by the aldolase of Clostridium perfringens
(14).
Mueller
(160, 161) found that Clostridium
tetani lost the ability to ferment glucose in an iron deficient
medium, and that this deficiency was reversed by adding small
quantities of glutamine
(162).
These authors also observed
that a factor in casein digest, subsequently identified as glutamine, increased the ethanolic
tion by C. tetani
(162).
fermentation and toxin produc-
In addition, they showed that the
gaseous fermentation products had a detrimental effect on toxin
production (160).
It is clear there is a relationship between
fermentation product patterns, iron availability, and toxin
production in the saccharolytic clostridia.
D.
Properties of C. thermocellum Cellulase
115, 116, 169)
(3, 50, 7E,
A true cellulase is an enzyme complex composed of at
least two enzymatic activities
(cellobiohydrolase and endo-3-
-30-
glucanase) that is capable of completely saccharifying complex
cellulosic substrates such as cotton
(83).
Endo-3-glucanase
is commonly assayed using CMC as the substrate, but there is
not a simple assay for cellobiohydrolase in an enzyme mixture.
Many microorganisms produce extracellular fluids which degrade
amorphous or derived forms of cellulose such as phosphoric
acid-swollen cellulose and carboxymethylcellulose, but only
the culture broths of certain fungi (e.g. Trichoderma spp.,
Fusarium spp. and Chaetomium spp.)
have been demonstrated to
completely saccharify cotton or filter
paper
(83,
144 ).
Al-
though Clostridium thermocellum and other bacteria grow on
these complex substrates, a potent cell-free cellulase has not
yet been isolated from a prokaryote.
The first report of enzymic decomposition of cellulose
was in 1912, when Pringsheim (186) demonstrated the presence
of a cellulose-destroying enzyme in a culture anaerobically
fermenting cellulose at 551C.
He stopped growth by the addi-
tion of iodoform and demonstrated that cellobiose and glucose
accumulate as products.
When the temperature was raised to
67*C, cellobiose was the only hydrolytic product, and when
lowered to 20'C, glucose was the predominant product.
From
these results, he postulated the presence of two hydrolytic
enzymes, one which converted cellulose to cellobiose and another which hydrolyzed cellobiose to glucose.
A classical
study by Reese and Levinson (191) provided further evidence
-31-
that
cellulase
is
a multienzyme
system. They proposed the "C"
concept, C 1 being an enzyme that modifies the crystalline cellulose and renders it susceptible to attack by ordinary hydrolytic enzymes such as C :
Crystalline
Cellulose
C1
Reactive
> Cellulose
C
Cellobiose
+ Glucose
-
Cellobiose
Glucose
Non-T c, e Cellulolytic Organisms
True Cellulolytic Organisms
This theory generated a great deal of research and controversy
into the mechanism of cellulose degradation.
Fungal cellulose is not a single enzyme;
it is a sys-
tem generally composed of three major enzymic components
187,
246):
1,4- -D-glucan cellobiohydrolase
endo--l,4--D-glucanase
(E.C. 3.2.1.4) and
(84,
(E.C. 3.2.1.91),
-glucosidase
(E.C. 3.2.1.21), which acts synergistically to entirely decompose cellulose to glucose.
(Fig. 2),
In the currently accepted scheme
endo-glucanase randomly cleaves internal glucosidic
bonds within an unbroken glucan chain, creating non-reducing
chain ends which then become the substrates for cellobiohydrolase.
Cellobiohydrolase erodes away the cellulose crystal by
splitting out cellobiose units;
glucose by
-glucosidase.
these are then hydrolyzed to
-
-
mu--om-N.,
-
-32-
Figure 2
Model for Cellulase Digestion of Insoluble Cellulose
(Brown, 1982)
A
B
(EG
UEIG
calobwe
1
non-reducn end
C
D
ca
- EG
(
-
2-Glucose
E
C
EG
A schematic representation of cellulase action. (A) The Trichoderma cellulase enzyme
system consists of three major enzymes: endoglucanase (EG). cellobiohydrolase (CBH), and 6glucosidase (O-G). EG binds randomly to the surface of the cellulose microfibril. The catalytic action
of EG breaks a glucosyl bond within a glucan chain. (B) EG leaves the microfibril surface. The nick
in the glucan chain exposes a reducing and a nonreducing end. (C) CBH can act only on the free
nonreducing end of a glucan chain. The catalytic action of CBH cleaves a cellobiose unit from the
nonreducing chain end. (D) Cellobiose is released into solution, where it is split into glucose
monomers by P-G. CBH moves to the newly created free nonreducing end and continues to cleave
cellobiose units from the glucan chain. (E) EG nicks continuous glucan chains, but releases little
soluble reducing sugar. CBH is catalytically active only at glucan chain ends. Thus, EG creates sites
at which CBH may act. The result is synergistic degradation of cellulose.
-33-
The cellulase from Trichoderma has the highly desirable property of being able to act on thick slurries of celIn 1968, Katz and Reese
lulose.
of 30%
(119) reported the production
glucose from a 50% slurry of heated ball-milled cellu-
lose in 15 days.
The reaction mixture contained a high cellu-
lase concentration, and was supplemented with Aspergillus
luchiensis cellobiase to alleviate product inhibition.
This
saccharifying ability has yet to be duplicated by another cellulase system.
It should be emphasized, however, that the
Trichoderma cellulase has a very low specific activity on ins.oluble cellulose, which further decreases with highly crystalline celluloses such as cotton, Avicel, or biomass wastes.
This limitation has hindered its commercial utilization.
rent Trichoderma mutants
(RUT-NG-14 and RUT-C30) produce 20
g/l extracellular protein which is predominantly
lulase.
0.6 -
Cur-
(78.5%) cel-
The specific activity of the cellulase is only about
0.7 filter paper units/mg protein.
To achieve a 40-50%
conversion of a 10-30% slurry of cellulose in 24-48 hours requires about 10 filter paper units/g cellulose for a susceptible substrate, and about 20 units/g for a more resistant
substrate
(7, 146).
Thus the requirement is 15-30 g of enzyme
protein per kg of substrate, a remarkably high figure.
In
general, extracellular polysaccharases active on insoluble substrates have a low molecular activity of 102 to 104 bonds
cleaved/min/enzyme molecule, compared to 10
to 108 bonds/min/
-34-
enzyme molecule for enzymes acting on small soluble substrates.
A low molecular activity requires that the producing organism
make a large quantity of the polysaccharase in order to grow at
its maximum rate, and thus the release of fermentable sugar
usually limits the growth rate on an insoluble substrate.
By far the majority of studies on cellulases have been
done on the aerobic, saprophytic fungi, and it is likely that
different biochemical processes are responsible for cellulose
degradation by other organisms.
Many of the wood-rotting fungi
(e.g. various basidiomycetes) possibly employ an oxidative
mechanism for cellulose and lignin decomposition
127).
(56, 61,
73-,
The biochemical properties of cellulases of anaerobes
are not yet known.
A serious problem plaguing the study of bacterial cellulases has been the low cellulase activity detectable in culture filtrates.
Cultures of C. thermocellum grow faster than
T. reesei on native cellulose, yet the assay of cellulase activity in the bacterial broth is generally about 100 times less
per unit volume of broth
(75, 169).
It has been suggested that
contact between cells and cellulose is necessary in some microorganisms for effective depolymerization, but this does not
seem to be the case with C. thermocellum, since large and distinct clearing zones are formed when C. thermocellum is grown
on agar media containing cellulose as the insoluble carbon
source
(116).
-35-
C. thermocellum has received some attention for its
ability to carry out a limited attack on derived forms of
cellulose
(3, 169).
The presence of CMCase and weak cellu-
lase activity has been demonstrated by Ng and Zeikus
(167).
The activity was reported to be "oxygen stable", low in "exoglucanase",
to 70 0 C.
and resistant to inactivation by temperatures up
They later found (169) that the cellulase activity
was inhibited by Hg +,
and that this inhibition was partially
relieved by excess dithiothreitol.
The cellulase had a pH op-
timum of about 5.4, much lower than the optimum pH of the organism for growth, which is 7.0-7.5.
Enebo
(51-53) showed that the cellulase of C. thermo-
cellulaseum is quite sensitive to inhibition by certain metal
ios
nluig
ions
Ca 2+,
g+
including
Ag
or Mg 2+.
Cu+
,
Cu
2+
,
Hg
3+
,
Fe
3+
and Cr
2+
,
but not to Mn
,
This inhibit-ion could be reversed at low ion
concentrations by adding peptone, which probably sequestered
the inhibitory metal ions.
Ng and Zeikus
thermocellum cellulase is inhibited by Hg
Mn +,
Ca2+ and Mg2+ had no effect.
(169) found that C.
2+ , Cu
12+ and Zn2+
2+
The basis for inhibition
of bacterial cellulase by metal ions is not known.
Enebo
(53) also demonstrated that thermophilic cellu-
lase activity from C. thermocellulaseum was inhibited by cellobiose, and less strongly by lactose.
(76,
More recent studies
205) have suggested that C. thermocellum cellulase is re-
sistant to end-product inhibition, but this discrepancy remains
to be resolved.
-36-
The composition of the thermophilic cellulase has not
yet been satisfactorily determined.
(186),
and later in 1954
(53),
It was suggested in 1912
that the cellulase from C.
thermocellum and related thermophilic clostridia consisted of
two enzymic components, since different products accumulated
in the presence of inhibitors, such as iodoform.
The purifi-
cation of C. thermocellum cellulase has been hindered by low
amounts of extracellular protein, binding of enzymes to cellulose, tendency of the proteins to form aggregates, and low recovery of the components, especially exo- -glucanase, during
purification.
Ait et al.
(3) partially purified a protein by
preparative polyacrylamide electrophoresis which produced reducing equivalents after 1 h from fibrous cellulose.
covered 10%
of the activity applied to the gel.
They re-
Petre et al.
(181) were able to separate the cellulase complex into distinct
protein fractions by gel chromatography in the presence of urea.
One of these was further purified and characterized as an endo-1,4-glucanase.
The final purification was about 100 fold and
the recovery exceeded 100%; the most highly purified fraction
contained 60 percent of the initial CMCase activity.
This pro-
tein was probably active as a monomer, had a molecular weight
of about 56,000 daltons, a pI of 6.2, and an optimum pH of 6.0.
The endo- -glucanase was not affected by EDTA or several monovalent and divalent ions, was not oxygen sensitive, and was unaffected by several sulfhydryl reagents.
Ng and Zeikus
(168)
-37-
also purified 22-fold an endo- -glucanase from broths of C.
thermocellum grown on glucose.
From 6.7 g of broth protein,
they obtained 21 mg of enzyme, and they attributed this low
yield to major losses during ion exchange chromatography.
They
suggested that this enzyme is a prominent component of the cellulase complex, accounting for over 25%
endo- -glucanase activity.
of the extracellular
The enzyme appears distinct from
the endoglucanase purified by Petre et al.
(181); it has a
molecular weight of 88,000 daltons, contains about 11% carbohydrate, has a pI of 6.7, an optimum pH of 5.2, and is most
active at 62*C.
The purified protein had a low methionine con-
tent and completely lacked cysteine.
An endoglucanase from a
new thermophilic clostridium has recently been purified
(41).
The enzyme is large, having a molecular weight of 91,000 to
99,000.
Recently, workers at the Pasteur Institute
(39)
have
managed to clone into Escherichia coli two endoglucanases from
C. thermocellum.
The genes for the two enzymes were examined
by nucleic acid hydridization, and were found not to be homologous, and to be separated on the C. thermocellum chromosome.
Other workers have also cloned and expressed endoglucanases
from cellulolytic organisms including Cellulomonas fimi
and Thermomonospora sp.
(37).
(248)
These genetic studies will
facilitate detailed characterization of endoglucanase activities but no one has yet been able to clone cellobiohydrolase
-38-
or an enzyme responsible for true cellulolytic activity in
these bacteria.
E.
Regulation of Cellulase Synthesis in Fungi and in C.
thermocellum (50, 67, 68,144, 187)
The nutritional and environmental factors influencing
cellulase formation have been little studied in bacteria, but
have been investigated in the fungi, especially in Trichoderma
(144).
Trichoderma is a saprophytic, aerobic fungal genus,
generally imperfect, but of ascomycetous origin.
Members of
this genus secrete a true cellulase.
The rapid metabolism of cellobiose, glucose or other
soluble carbon sources drastically decreases cellulase synthesis in cellulolytic fungi and bacteria
(171).
The decrease in
cellulase synthesis during rapid growth on the products of
cellulolysis indicates this regulation is due to carbon catabolite repression (144, 171, 187).
The cellulase in T. reesei is reported to be an inducible enzyme system, which is formed in highest quantities when
the fungus is grown on cellulose.
Since cellulose is insoluble,
limiting quantities of solubilized products must trigger induction.
Cellulase in Trichoderma and many other cellulolytic
organisms
(144,
187) has been reported to be induced by a prod-
uct derived from cellulose, originally thought to be cellobiose
-39-
(148).
Mandels et al.
(147) later showed that sophorose
-D-glucopyranosyl-D-glucose),
as a transglycosylation product
inducer.
In 1979
(214),
(2-0-
probably formed from cellobiose
(138),
is a much more potent
it was confirmed that sophorose stim-
ulates CMCase synthesis in T. reesei.
Synthesis of CMCase
stopped when the mycelium was removed from the inducer.
The slow metabolism of sophorose may be responsible
for its effectiveness as an inducer of CMCase.
sophorose by mycelia was quite slow
other sugars)
CO 2 and H 2 0
The uptake of
(1/5 to 1/10 the rate of
(214) and the disaccharide was catabolized to
(138).
Induction was maximal at the slowest re-
spiration rates and was influenced by varying the pH or carbon
source.
These data suggest that it may be the slow metabolism
of sophorose, and not its combination with a repressor protein, which is responsible for its stimulation of cellulase
synthesis.
Cellulase synthesis in Myrothecium verrucaria is
reported to be regulated solely by carbon catabolite repression
(84).
F.
Regulation of Cellulase Synthesis in C. thermocellum
(50, 68
An understanding of the control of cellulase synthesis
in bacteria has been hindered by the difficulty in obtaining
true cellulase activity from the culture broths.
In C. ther-
-40-
mocellulaseum, very closely related to C. thermocellum
152),
(53,
broths with high activity on derived cellulose were ob-
tained when insoluble cellulose was the carbon source;
the
addition of cellobiose or glucose to C. thermocellulaseum
cellulose fermentations markedly lowered the amount of cellulose fermented (53).
Cellobiose was the more inhibitory of
the two sugars, presumably because of its 3-fold higher rate
of metabolism.
This suggested that cellulase synthesis by
C. thermocellulaseum is sensitive to catabolite repression.
In contrast to these earlier findings with C. thermocellulaseum, which apparently differs from C. thermocellum
only in its ability to ferment maltose and xylose, catabolite
repression has not been reported to influence cellulase synthesis in C. thermocellum.
stitutively
(3, 68:,
76,
CMCase appears to be produced con-
87, 152),
and nutritional and envir-
onmental conditions that improve cell growth
(e.g. prevention
of a pH drop) generally also result in improved CMCase volumetric activity.
G.
Selection of Mutants Affected in Extracellular Enzymes
An initial objective of this study was to develop selection methods for the isolation of C. thermocellum mutants
affected in extracellular enzyme formation or activity.
There
have been many techniques and strategies developed for the se-
-41-
lection of microbial mutants that produce increased quantities
of an intracellular enzyme or metabolite
(46).
Environmental
conditions can be devised so that an individual
(B) producing
more of an intracellular enzyme or product than the parent
(A)
will be better fit to multiply in the environment and will increase at a growth rate p A(1+S) = yB where P is the specific
growth rate and S is the selection coefficient.
The selection
coefficient is therefore defined as S =
A.
greater than 0, the fitness
(yB~4A
When S is
(f) of B is greater than A, and
positive selection occurs for B, i.e.,
pB >A*
When S is 0,
the environment is neutral with respect to the trait under consideration and no selection occurs, and when S is negative, selection occurs against the B strain.
Several techniques are
known for increasing the fitness of a strain over-producing an
For example, Horiuchi et al.
intracellular enzyme.
able to select for hyperproducers of
(100) were
-galactosidase by limit-
ing the supply of lactose in continuous culture.
The develop-
ment of selective conditions for extracellular enzymes is not
as straightforward.
In a mass culture, it would not be advan-
tageous for an individual cell to overproduce an extracellular
enzyme, which could be exploited by other members of the population.
Furthermore, such a trait would be unstable in a
homogeneous environment such as a fermentor because the occurrence of a non-producing mutant would result in faster growth
than the overproducer.
The process of extracellular enzyme
-42-
evolution is interesting because it requires that selection in
a population operates on a trait which is potentially harmful
to an individual possessing the trait.
It is a difficult prob-
lem to devise environmental conditions that would enable extracellular enzyme producers to outcompete non-producers and increase in proportion in a mass population.
A direct approach
is to study the regulation of the enzyme activity and synthesis
in the producing organism.
An understanding of its control and
functions in the individual cells might explain its capacity to
evolve in a microbial population
(228).
-43-
2.
EXPERIMENTAL PROCEDURES
A.
Bacteria
The ATCC 27405 strain of Clostridium thermocellum was
used throughout this study.
Coy anaerobic
It was periodically plated in a
(90% N 2 :5% CO2:5% H 2 ) glove box on defined med-
ium containing Avicel as the carbon source.
A cellulolytic
colony was recovered with a toothpick, grown in cellobiose
broth and reisolated on an Avicel plate.
This was grown in
cellobiose minimal broth and stored at 4*C.
The clostridial
cultures could be maintained by lyophilization or at -80*C in
50% V/V glycerol.
B.
Cultivation of Bacteria
The development of a minimal, defined medium (MJ) is
described in the Results section of this thesis, and was used
for growth experiments unless otherwise noted.
pared by mixing the basal salts
It was pre-
(1.5 g KH 2 PO 4 , 2.9 g K 2 HPO 4 ,
2.1 g urea, 1.0 g cysteine hydrochloride, 10.0 g morpholinopropane sulfonic acid, 1.0 mg resazurin and 3.0 g of sodium
citrate in 850 ml H 2 0).
This was then boiled to remove 02 and
transferred to an anaerobic chamber with an atmosphere of 90%
N 2,
5% CO2 and 5% H 2 .
The medium was dispensed and capped in
-44-
Hungate pressure tubes
(Bellco) inside the chamber, and ster-
ilized for 15 min at 121 0 C.
A 1OX
trace salts solution (10.0
g/l MgCl 2 .6H 2 0, 1.5 g/l CaCl 2 .2H 2 0 and 12.5 mg/l FeSO 4 .7H 20)
was autoclaved separately and combined with a filter-sterilized 1000 X vitamin solution (20 mg/l biotin, 200 mg/l pyridoxamine hydrochloride, 40 mg/l p-aminobenzoic acid and 20 mg/
1 vitamin B 1 2 ), which was then added by syringe.
The carbon
sources were then added at 5 g/l, except where noted.
All
cultivations were done in Hungate tubes at 60*C and growth was
measured by optical density in a Turner model 330 spectrophoOne mg dry cell weight per ml corresponds to 1.8 op-
tometer.
tical density units at 660 nm.
Sampling of tubes during ex-
periments was usually done in the anaerobic glove box to prevent leakage of air and changes in the physiology of C. thermocellum.
Growth on cellulose
(Avicel PH105) was determined
by microscopic counts in a Petroff-Hausser chamber.
Viable
counts were obtained by plating on GS-2 medium (114)
in the
anaerobic chamber.
C.
Determination of C. thermocellum Nutritional Requirements
To determine amino acid and vitamin requirements, an
initial inoculum of C. thermocellum ATCC 27405 was grown in
GS-2 medium for 24 h at 60 0 C.
It was then added at 2 ml/liter
to GS-2 medium lacking yeast extract.
Cells were cultured for
-45-
24 h, the carryover of growth factors allowing growth to occur
in this first transfer.
The value of such a starvation step
in developing inocula is well documented (42).
These cells
were used to inoculate 10 ml of GS-2 medium lacking yeast extract, but containing various combinations of growth factors
(second transfer).
The third transfer was made to identical
media, and growth (absorbance at 660 nm) was measured after
40 h of incubation.
D.
Source and Preparation of Cellulase
Initially, a crude cellulase preparation was obtained
from B. Faison who used C. thermocellum ATCC 27405 grown in a
12-liter Microferm fermentor
on CM-4
cellobiose medium
(New Brunswick Scientific Co.)
( 92 ) at 60*C and 60 rpm for 68 h.
In her work, the broth was chilled to 4*C and then centrifuged
at 18,000 X g for 20 min to remove cells.
fluid was treated with solid
stored overnight at 4*C.
The supernatant
(NH 4 ) 2 so 4 at 80%
saturation and
The precipitate was harvested by
centrifugation, dissolved in 50 mM sodium citrate buffer (pH
5.7),
reprecipitated by the addition of 4 volumes of saturated
(NH4 ) 2 so 4 , and again stored overnight before dissolution in citrate buffer.
Biogel P-2
This preparation was desalted by passage through
(Bio-Rad Laboratories, Richmond, CA),
mg of protein per ml, and stored frozen
sis, M.I.T.,
Cambridge, MA, 1981).
diluted to 1
(B. Faison, S.M. The-
-46-
A second batch of enzyme was prepared similarly but
with C. thermocellum grown in a 100-liter fermentor containing 0.5% Solka-Floc SW-40
source.
(Brown Co.,
Berlin, NH) as carbon
The fermentor was stirred slowly at 50 rpm and gassed
with N 2 for the first 19 h of the 60 h fermentation.
At 60 h,
cysteine hydrochloride was added to a final concentration of
0.1% and the cells were removed in a Sharples centrifuge
(type M47-16Y, Sharples Corp., Philadelphia, PA).
This extra-
cellular preparation has retained its cellulolytic activity
for at least a month at 4*C, and for at least 2 years at -20*C.
[- 200 pg protein ml
The broth
by the Coomassie blue method
(19)] was freeze-dried for 48-72 h in shallow enamel pans in a
Virtis freeze drier, reconstituted in water and dialyzed at
room temperature against four changes of 20 mM Na succinate
buffer
(pH 5.8) for 6 h.
obtained
(-
The clear yellow-green preparation
312 pg protein ml~
) was stored at -20*C,
and
when required was thawed and used without further purification.
Dried powders from T. reesei were prepared at the Natick Laboratories
(T. reesei QM 9414 cellulase, 0.61 mg of
protein per ml, 0.32 filter paper units
reesei RUT C-30, 0.53 FPU/mg).
[FPU]
per mg. and T.
These were prepared in solu-
tion at 5 FPU/ml and 9 FPU/ml, respectively, which is equivalent to their broth concentrations.
-47-
Measurement of Cellulase Activity
E.
A simple procedure was developed in this study for
Activity was de-
routine measurement of cellulase activity.
termined by decrease in turbidity (660 nm) of an Avicel sus(Type PH 105, 20 jiM particles, FMC Corp., Marcus Hook,
pension
PA)
(see Results).
Three mg samples of Avicel were suspended
in Hungate tubes in 3 ml of 0.1 M sodium succinate buffer
5.8),
(pH
0.5 ml of 0.1 M DTT, 0.5 ml of 0.1 M CaCl 2 .2H 2 0 plus
various volumes of enzyme and water to 5 ml.
tion of cellulose
This concentra-
(0.6 mg/ml) was used so that cellulolysis
would proceed to completion
(81-84) within a relatively short
time with minimum interference from products.
In collaboration with M. Sakajoh and G. Halliwell,
cellulase activity was also -measured on Avicel and nonpowdered
celluloses by loss in dry weight.
paper
cotten
Three milligrams of filter
(Whatman No. 1 filter paper circles) or nonabsorbent
(SP cotton coil, C-9355-3, American Scientific Products,
Bedford, MA)
were incubated with the Clostridium cellulase
Residual cellulose
under the same conditions as with Avicel.
was determined colorimetrically with K2
20 7
(116).
When inhibitors or stimulants were added to the cellulase reaction mixture, they were first dissolved in water or
ethanol.
Stock metal ion solutions were prepared at 1 M in
dilute HCl.
The chelating agents o-phenanthroline and 2,2'-
dipyridyl were dissolved at 1 M in ethanol.
-48-
F.
CM-Cellulase Activity (endo-1,4-3-D-glucanase, E.C.
3.2.1.4)
CM-cellulase activity was determined by reduction in
viscosity of a 0.25%
(w/v) carboxymethyl cellulose
(CMC) (Type
7H4, Hercules cellulose gum, lot 48594) solution in 25 mM Na
succinate
(pH 5.8).
cal of time
The activity is expressed as the recipro-
(min) required for 5 ml to flow from an Ostwald
viscometer at 60*C, and is corrected for the flow of buffer.
The activity was linear with protein concentration up to at
least 15 pg protein per 5 ml assay.
tivity are given as
[l/min (assay) -
Units of CM-cellulase acYmin
(H2 0)]
X 10.
The
standard deviation for assays done on different days was 0.8.
G.
Units of True Cellulase Activity
The C. thermocellum cellulase activity can be measured
by loss in weight of non-absorbent cotton, micro-crystalline
Avicel, or filter paper
(see Results and (116)).
The loss in
dry weight correlates with the decrease in optical density at
660 nm of an Avicel suspension.
formed is cellobiose
The principal sugar product
(see Results).
Avicel concentration (0.6 g/l),
Under conditions of a low
the decrease in optical density
is first order and correlates with protein concentration
(Fig-
ure 3) up to approximately 5 pg/ml of extracellular protein.
In a Turner model 330 spectrophotometer, the degradation by
-49-
Figure 3
A.
First Order Rates of Avicel Solubilization (Measured
at O.D. 660 nm) by Varying Concentrations of Extracellular Protein.
B.
Rate of Avicel Solubilization as a Function of Protein Concentration.
4r
L
p
-
0 Oug
1.6
N
3.1
-N
3
62
-8
1UNIT
x
2
125
-
0o
-4
go
18,1
I I
0
a
40
hours
I
80
0
20
ug protein
40
-50-
this concentration of cellulase
(5 iig/ml) corresponds to an
O.D. decrease of 0.25 in 24 h at 601C.
For the purpose of this
thesis, one cellulase unit is defined as the amount of cellulase which gives a first order rate that completely degrades 1
mg of Avicel in 24 h.
This degradation corresponds to a first
order rate content of 0.02 h 1.
This assay avoids the trouble-
some measurement of reducing sugars in the presence of DTT, and
measures the complete saccharification of microcrystalline cellulose.
H.
Analysis of Cellulolytic End Products
Products of saccharification were determined by HPLC
on a column of HP X-87-H
of 0.5 ml/min.
(Bio-Rad) at 60*C with a flow rate
The solvent was H2SO4 in glass distilled H 20,
pH 2.1-2.2.
I.
Analysis of Fermentation End Products
Ethanol and acetic acid were measured by gas-liquid
chromatography on Chromosorb 101 columns with samples acidified with 1.5 M HCl and n-propanol as an internal standard.
Lactic acid was measured enzymatically
Bulletin No. 826 UV).
(Sigma Chemical Co.,
-51-
J.
Determination of Phosphate Uptake by Cells
Cells were grown in CM-4 medium, centrifuged at 44C,
and washed and resuspended in NMR buffer
Na
2
HPO 4 ,
100 mM PIPES,
50 mM MOPS
(5 mM KH 2 PO 4 , 5 mM
and 85 mM NaCl).
Cells were
kept cold and anaerobic in 3 ml aliquots in NMR tubes.
When
assayed for phosphate uptake and membrane energization, 0.1
ml of 20%
(w/v) carbon source was added to cells previously
warmed to 60*C.
magnet, and
They were immediately inserted into the Bruker
3 1 P-NMR
was employed to monitor intracellular and
extracellular inorganic phosphate.
K.
Assay of ATP Concentration in Cells
The procedures of Cole et al.
(35) were used for sam-
ple preparation and Holm-Hansen and Karl
(96)
for the lucifer-
Two milliliter samples of growing cells were re-
ase assay.
moved with syringe, and rapidly added into 0.5 ml of ice-cold
perchloric acid.
After 10 min on ice, the extract was vortexed
and neutralized with 1 M KOH; the precipitate was removed by
centrifugation and the extract kept at -20*C until the ATP determination.
This was done exactly by the published procedure
(96) and the error between identical standard samples was less
than 3%.
A Packard scintillation counter with one channel at
1385 V and the coincidence control switched out was used for
-52-
the measurement.
Counting was done for 0.1 min, and 2-20 mM
ATP standards were run at the same time.
L.
Assay of Hydrogenase Activity
Hydrogenase was assayed in centrifuged broths at 50*
C in a glove box atmosphere
control.
(88% N 2 :5%
CO 2 :7%
H 2 ) or in a N 2
To 3 ml of 50 mM Tris-HCl, pH 8.5 containing 1.5 mM
methyl viologen was added 0.1 ml to 0.5 ml broth, and the increase in O.D. at 578 nm was recorded.
M.
Gel Electrophoresis
Polyacrylamide gel electrophoresis
(SDS-PAGE; 131) was
used to qualitatively characterize C. thermocellum's extracellular proteins.
Slab-gels containing 3 percent
(stacking gel)
or 5 percent acrylamide were prepared from a stock solution of
30 percent by weight of acrylamide and 0.8 percent by weight
of N,N'-bis-methylene acrylamide, according to the following
recipe
(per gel):
Running
Gel
H 20
1.5 M Tris-HCl, pH 8.8
0.5 M Tris-HCl, pH 6.8
10% SDS
Bis-Acrylamide Mixture
10% Ammonium Persulfate
(5%)
17.1 ml
7.5 "
0.3 "
5.0 "
0.1 "
Stacking
Gel
6.3 ml
2.5 "
0.3 "
1.0
0.1
-53-
This solution was degassed, mixed with 10-20 il of
TEMED, and immediately poured using a syringe.
The running gel
was overlayed with isobutanol, and blotted dry with a Kimwipe
before pouring the stack.
The electrode buffer
(pH 8.3) con-
tained 0.025 M Tris, 0.192 M glycine and 0.1 percent SDS.
Samples
(80
il containing 5-50 jg protein) were mixed with 20
pl of 5-fold sample buffer
SDS
(10 ml); glycerol
water
(4 ml)
5 min.
),
(1.25 M Tris, pH 6.8
(10 ml);
(25 ml); 0.75% bromophenol blue
25%
(1 ml);
and immersed in a boiling water bath for
High molecular weight protein standards
(30,000 -
200,000; Sigma MW-SDS-200) were always electrophoresed as
markers.
The standards were carbonic anhydrase, 29,000 MW;
egg albumin, 45,000 MW; bovine albumin, 66,000 MW; phosphorylase B, 97,400 MW; E. coli
and myosin, 205,000 MW.
-galactosidase, 116,000 MW;
Electrophoresis was done at 20 mA/
gel loading, and 40 mA/gel running, until the bromophenol dye
reached the bottom of the gel
(about 3-5 h).
were stained for 1-2 h in Coomassie blue
The proteins
(filtered solution of
1.25 g Coomassie blue Brilliant R, 250 ml methanol, 250 ml
water and 45 ml glacial acetic acid) and destained in 10%
methanol/7% acetic acid.
N.
Purification of Cellulase
The extracellular cellulase from C. thermocellum was
partially purified by ion exchange chromatography and gel fil-
-54-
Ion exchange chromatography was performed with a
tration.
DEAE-sepharose open column
(-
2.5 x
12 cm) or by HPLC
(cour-
tesy of Cindy Allen at Waters Associates) using a vinyl ionexchange resin.
The cellulase and ion-exchange resins were
equilibrated with 20 mM Tris-HCl, pH 8.8, and after adsorption, the cellulase was eluted in a KCl gradient to 0.5 M salt
(see Results).
22 column
(-
Gel filtration was done with an Ultrogel ACA
1.8 x 85 cm),
equilibrated with 50 mM Tris-HCl,
pH 7.1 containing 100 mM NaCl or with a Fractogel Column
equilibrated with the same buffer containing 300 mrM NaCl.
Standards
(Sigma Chemical Co.)
for molecular weight calibra-
tion in gel filtration were hemocyanin, 3.1 x 106 MW;
globulin, 669,000 MW;
thyro-
ferritin, 440,000 MW; catalase, 232,000
MW; and aldolase, 156,000.
0.
Chemicals
Bovine blood superoxide dismutase, sulfhydryl compounds, reducing agents, cellobiose and metal chelators were
from Sigma.
Hydrogen peroxide was from Mallinckrodt.
gillus niger catalase was from U.S. Biochemical Corp.
AsperGlucose
was from Anachemia and fructose and sodium citrate were from
Baker.
Cellodextrins were provided by Herve Cellard.
triose was a gift from M. Ladisch.
of analytical quality.
Cello-
All other chemicals were
-55-
3.
RESULTS
A.
Ca 2 + and Sulfhydryl Reducing Compounds as Requirements
of the Cellulase System of C. thermocellum
A major impediment to the use of bacterial cellulases
has been their reportedly weak activity compared to the fungal
In this first section of my thesis, I describe ex-
enzymes.
periments which resulted in improvement of C.
thermocellum
cellulase activity.
C. thermocellum plated on agar medium containing insoluble cellulose gave rise to colonies surrounded by clear zones
in 5 to 7 days, suggesting the presence of an extracellular
cellulase
(Fig. 4).
However, extracellular broth from the or-
ganism, prepared on cellobiose medium, showed only weak cellulolytic activity
(0.2 to 0.4 mg reducing sugars ml
in the filter paper assay (.145),
(76, 169).
broth h- )
confirming earlier findings
Since C. thermocellum is an anaerobe, it seemed
plausible that reducing conditions may be necessary for cellulase activity.
However, reducing agents interfere drastically
with assays measuring liberation of reducing
sugars, and
it was therefore necessary to use a method that did not depend
on the detection of reducing equivalents.
I decided to develop
a turbidimetric assay with powdered Avicel as the substrate.
slight but significant decrease in turbidity was observed when
succinate replaced citrate as the buffer and a dramatic in-
A
-56-
Figure 4
Clear Zones Produced by C. thermocellum After 8 Days Growth
on Compression Milled Corn Stover (A), Avicel (B), or Amorphous Cellulose (C)
0
0
0
0
0~
0
*0A
-57-
crease in activity occurred when a sulfhydryl reducing compound
was added
(Figure 5).
DTT had little effect on the initial rate of Avicel hydrolysis but greatly increased the ultimate extent of breakdown
In the presence of 5 mM DTT, Avicel was completely
(Fig. 5).
solubilized;
ter 24 h.
in the absence of DTT, solubilization stopped af-
Other effective reducing agents included cysteine,
sodium dithionite, glutathione and mercaPtoethanol.
DTT
(5 mM)
had no effect on the enzymatic solubilization of phosphoric
acid-swollen Avicel or trinitrophenylcarboxymethyl-cellulose
(205).
The cellulase activity
of the C.
preparation was stimulated by Ca2+
thermocellum enzyme
(Fig. 6).
EDTA (10 mM) in-
hibited completely the cellulolysis of Avicel by the Biogeltreated preparation even in the presence of DTT
Addition of MgCl 2
(Figure 6).
(7 mM) improved cellulolysis slightly whereas
7 mM CaCl 2 was far more effective and enabled the enzyme preparation to achieve complete solubilization of the substrate. A
dialyzed preparation of C. thermocellum culture broth was
strongly inhibited by EDTA (Figure 7);
this inhibition was re-
versed by calcium confirming the necessity of this ion.
In the presence of calcium and DTT, the first order rate
of Avicel solubilization is proportional to broth protein added
(Fig. 3, Materials and Methods),
tions less than - 6 vig/ml.
but only at protein concentra-
This proportionality can be extended
-58-
Figure 5
Influence of DTT Concentration on Avicel Hydrolysis by ClostridFifty Micrograms of Biogel-Treated
ium thermocellum Cellulase.
Extracellular Protein were Incubated with 3 mg Avicel and The
Absorbance (660 nm) of the Suspension was Determined with Time.
Succinate Buffer with No Ca 2 + was Used.
0.4
No DTT
-
E
0.3-
C
S
0.2
2mM DTT
S0.1.-
5mM DTT
0
40
80
120
Hours of Hydrolysis
160
-59-
Figure 6
Influence of Ca 2 + on Avicel Hydrolysis by Clostridium
thermocellum Cellulase. Reaction Conditions were the
Same as in Fig. 2, but the Incubation Mixtures Contained 5 mM DTT and Either 20 mM EDTA, 7 mM CaCl 2 ,
or 7 mM MgCl 2.
EDTA
0.3
E
o
0.2
0
00
CaCC2
0
20
40
60
so
100
Hours of Hydrolysis
-60-
Figure 7
Inhibition of C. thermocellum Cellulase by EDTA and
Its Reversal by Calcium. Fifty Lil of Dialyzed
Enzyme were Used.
+2,5 or 10 mM
0.4
EDTA
E
C
0
(0 0.3
C
0
+10
mM EDTA
(n
(n.
Z-2H 2 0
0.2
=3
No EDTA, No Ca
+5 mM EDTA
0.1
10 mM Ca
2+
+2 mM EDTA
10 mM Ca 2 +
0
+10 mM Ca
I
I
0
40
20
Hours
60
2 +
2
+
-61-
to higher protein concentrations by increasing the substrate
conc.
Therefore, the rate of hydrolysis of insoluble Avicel
appears to be limited by the availability of susceptible sites
for attack.
The following experiments were done
(in collaboration
with Drs. M. Sakajoh and G. Halliwell) to determine whether
C. thermocellum cellulase in the presence of Ca2+ and DTT is
capable of extensively solubilizing complex celluloses including cotton and filter paper and whether it could do it as fast
as the cellulase from Trichoderma reesei.
Equal broth volumes
(1 ml) of the C. thermocellum (0.2 mg protein ml 1) and T.
reesei QM 9414
(9.5 mg protein ml~
) cellulase were examined
quantitatively for their ability to solubilize cotton, filter
paper, and Avicel as measured by loss in weight.
The crude
Trichoderma cellulase acted rapidly on filter paper, less so
on Avicel and slowest on cotton
(Figure 8),
whereas the Clos-
tridium enzyme showed the reverse pattern, cotton being saccharified most rapidly and faster than the Trichoderma cellulase.
Filter paper, however, showed greater resistance to hydrolysis
by the clostridial cellulase, particularly in the early stages.
Of note was the Clostridium enzyme's ability to achieve essentially complete hydrolysis of all three forms of cellulose in
the manner expected of a true cellulase.
The previous experiment comparing T. reesei and C. thermocellum cellulases
(Figure 8) employed a temperature of 37*C
-62-
Figure 8
Solubilization of Native and Derived Celluloses by Cellulase of Trichoderma reesei QM 9414 and Clostridium thermocellum. One Milliliter of Culture Broth (Fresh or Reconstituted) was Incubated at 37 0 C and pH 4.8 Acetate
Buffer (Trichoderma) or 60*C and pH 5.5 Succinate Buffer
(Clostridium) with Cotton, Filter Paper or Avicel. The
Clostridial Enzyme Incubation Mixture Contained 7 mM
CaCl 2 and 10 mM DTT.
100
80.
~
reesei
\T.
------------------C. thermocallum
4
A
\
o
FILTER PAPER
AVICEL
\COTTON
60-
#0
0
4 0-
20Q.)
0
21
42
63
Hours of Hydrolysis
84
96
-63-
for Trichoderma, this temperature being optimal for the cellobiohydrolase
(85).
(C1 ) component of T. koningii over long periods
However, in the short-term (1 h) assay employed by Man-
dels et al.
(145),
50*C is used, being optimal for the CM-
cellulase component in the culture filtrate.
Therefore, it was
of interest to compare the cellulase of T. reesei at 50*C with
the clostridial cellulase
(at 601C).
For this experiment, 1
ml of the best available T. reesei preparation
Mandels
[RUT C-30;
(144)] was used at a culture broth strength
The results measured turbidimetrically on Avicel
as loss in weight of Avicel and cotton
found in Figure 8.
(9 FPU ml
).
(Figure 9a) and
(Figure 9b) confirm those
In addition, the Trichoderma enzyme showed
slightly improved activity on both substrates at 50*C compared
with 37*C, rates that were equalled or exceeded by the Clostridium cellulase.
The activities of the Clostridium and Trichoderma cellulases were also compared on a specific protein basis
10).
(Figure
Interestingly, the Clostridium cellulase showed a higher
specific
activity
on Avicel,
but a much lower activity
phoric acid-swollen cellulose.
on phos-
The solubilization of Avicel re-
quires exo- and endo-glucanase activities; digestion of swollen
cellulose requires mainly endo-glucanase.
These data together
with the preference of the bacterial cellulase for highly crystalline cotton suggests the presence of a prominent exo-glucanase component in the C. thermocellum complex.
-64-
Figure 9
Hydrolysis of Cotton and Avicel under Optimal Conditions by the
Cellulases of Trichoderma reesei RUT C-30 (Tr) and Clostridium
thermocellum (Ct).
The Trichoderma Enzyme Incubation was Done
in pH 4.8 Acetate Buffer and the Clostridium Enzyme Incubation
in pH 5.8 Acetate Buffer.
(A) Turbidimetric Measurement of
Avicel Hydrolysis.
(B) Colorimetric Determination of Residual Cellulose using Avicel or Cotton. For each Organism, 1 ml
of Culture Broth (Fresh or Reconstituted)was Used.
-100
B
100
A
Cotton
Avicel
SO
0
0
0
0
0
W
60 -
Tr 37C
0
%4-
0
(-
Tr 37*C
U)
\
.0.,
s0*C
\
40
Ct 604C
Tr
20
U)
Tr
3rC
AVICEL
2
;20
\Ct
60*C
Tr
Ct
r6
0
609C
21
42
0
21
Hours of Hydrolysis
42
-65-
Figure 10
Comparison of C. thermocellum and Trichoderma reesei QM 9414
Cellulase Activities on Phosphoric Acid-Swollen Avicel and
Microcrystalline Avicel.
Fifty *ig of Crude Broth Protein
were Used in the Incubations.
'~
E
*4
Avicel
Swollen Avicel
c
C
I
.3
.3
CT
C)
.2
,21
.1
II
C
CT
0
TA
80
40
Hours
120
0
L-
I
I
2
I
I
4
I
-
6
Hours
Id
-J.
i
22
-66-
B.
Oxidative Inactivation of C. thermocellum Cellulase:
Evidence for Essential Sulfhydryls
The stimulatory effect of DTT suggested that sulfhydryl
reduction may be important for Clostridium cellulase activity.
When dialyzed culture broth was treated with various concentrations of DTT, it was observed that stimulation occurred at
high DTT
(10 mM) and surprisingly that inhibition occurred at
a lower concentration
(0.1 -
0.5 mM)
in low DTT was completely prevented or reversed
addition of 10 mM DTT.
Activity loss
(Figure 11).
Some other enzymes
(within 24 h) by
(e.g. rhodanese and
glyceraldehyde-3-p-dehydrogenase) show similar behavior with
DTT or other thiols
(40, 226),
and it has been postulated that
inhibition at a low concentration of thiol is due to the formation of hydrogen peroxide, which inactivates the enzyme:
H2C
H 2C
SH
H-C-OH
H-C-OH
+
HO-C-H
H 2C
S
SH
02
2
+HO
metal ions
HO-C-H
H2C
S
The hydrogen peroxide could oxidize cellulase directly
or could be converted to hydroxyl radical, which is a very potent oxidant:
-67-
Figure 11
Influence of Dithiothreitol Concentration on the
Activity
Activity of C. thermocellum Cellulase.
is Expressed as the Change in O.D.(660 nm) of an
Avicel Suspension after 24 Hours.
I
I
I
.3
--. 2
,2
.2 .4 r1
5
10
15
dithiothreitol c onCc.(mm)
20
-68-
Fe 2+
+
H 2 02
OH a
--
+
OH
+
Fe
3
+
destructive
To test this, I treated C. thermocellum cellulase with 0.4 mM
DTT under aerobic and anaerobic conditions
(Figure 12).
Under
anaerobic conditions, H 2 0 2 should not be formed, and inactivation should not occur.
There was no inhibition of cellulase
activity by 0.4 mM DTT in the absence of air
(Figure 12);
the presence of air, cellulase was strongly inhibited.
in
Fur-
ther, cellulase was inhibited by 0.04 mM H 2 0 2 under aerobic or
anaerobic conditions
(Figure 12).
The inhibition by H 2 0 2 was
prevented when 10 mM DTT was added to the reaction mixture
(Figure 13).
Inactivation of cellulase by low DTT was partially
prevented by adding catalase
(10 U/ml) from Aspergillus niger
and removing the azide from the incubation, which is inhibitory
to catalase
(Figure 14).
Cellulase was not protected by bovine
superoxide dismutase (10 U/ml),
by copper EDTA which is known
to dismutate superoxide ion, or by hydroxyl radical scavengers
including mannitol or sodium formate at 20 mM (Table 2).
By
far the best protectant from oxidation was millimolar quantities of DTT.
The stimulatory effect of DTT on cellulase activity and
the reversible inhibition by H 2 0 2 suggests that there is essential thiols necessary for activity.
Dialyzed cellulase was
treated with various sulfhydryl reagents which differ in their
-69-
Figure 12
Influence of Anaerobic and Aerobic Atmospheres on Inhibition
of Cellulase by 0.4 mM OTT or 0.04 m.M Hydrogen Peroxide. Also Shown is-the Effect of 10 mM OTT. Anaerobic Atmosphere
was Obtained by Sealing the Hungate Tubes in a Coy Anaerobic
Chamber (90% N 2 :5% CO 2 :5% H 2 ).
C
Anaerobic
Aerobic
0.4
E
C
0
0.04 mM H2 0 2
C
0
0.3-
0.04 mM H2 0 2
0.4 mM DT T
.)
C
(n
co
0.2-
C!)
0.1-
0.4 or 10 mM DTT
10 mM DT T
0
40
80
120
0
Hours
40
80
120
-70-
Figure 13
inhibition of Cellulase by Hydrogen Peroxide or Low DTT in Air.
Open Symbols Represent the Addition of 10 mM DTT after 1 h.
A
A
0.4
0.4 mM H 202
E
c
0
(0
0.04 mM
0.3
HT
(n
C
0.2
0.0
0
4 mM H2 0 2 ,.
0.4 nM H20
2
10 mM DTT t
d
{or 0 .4 mM OTT
0.1
OC
0
20
Time
40
(Hours)
60
-71-
Figure 14
The Effect of Catalase and Superoxide Dismutase (SOD)
Inhibition of Cellulase by Low DTT and Air.
on the
0.4-
E
C
0
0
(D
0.3
0
()
C- 0.2.
0.4 mM DTT
C-
0.4 mM DTT
+SOD
a>
0.1
0.4 mM DTT
+ Catalase
d
10 mM DTT
0
10
20
30
Time
40
50
(hours)
60
70
-72-
Table 2
Effect of Hydroxyl Radical Scavengers on Oxidation
of Cellulase by H 2 0 2 or Low DTT.
%activity
aerobic
anaerobic
none
100
104
0
none
65
100
0. 1
0
sodium formate, 20mM
78
100
4
0.1
0
mannitol,
20mM
45
100
5
0. 1
0
ethanol,
300mM
46
101
6
0
0.05
none
59
47
7
0
0.05
sodium formate, 20mM
46
63
8
0
0.05
mannitol,
20mM
56
56
9
0
0.05
ethanol , 300mM
50
47
10
0.1
0.05
63
99
11
0.1
0.05
sodium formate,
70
99
12
0.1
0.05
mannitol,
20mM
89
98
13
0.1
0.05
ethanol,
300mM
54
94
14
0.1
0
100
104
15
0
0.05
DTT,
10mM
96
102
16
0.1
0.05
DTT,
10mM
97
102
DTT(mM)
H 2 0 2 (mM)
1
10
0
2
0.1
3
Scavenger
none
DTT, 10mM
20mM
100% activity corresponded to the complete degradation of 3mg Avicel by 15.6ug
extracellular protein in 65h.
-73-
mode of action including N-ethylmaleimide
double bond),
(NEM; addition to
5,5'-dithiobis-(nitrobenzoic acid)
dation); o-iodosobenzoate (IB; oxidation);
zoic acid
(p-CMB; mercaptide formation),
alkylation).
(DTNB; oxi-
p-chloromercuriben-
and iodoacetate
(IA;
Inhibition occurred in the presence of all these
(Table 3) and protection or reversal by DTT varied
reagents
with the reagents.
Complete protection by DTT was observed in
the case of DTNB or TB and only partial restoration of activity was observed with NEM, pCMB or IA.
The C. thermocellum
cellulase was strongly inhibited by 5 iM copper, known to
catalyze air oxidation of sulfhydryls.
Low concentrations of
metal chelators protected the cellulase from air oxidation
(data not shown).
The cellulase of T. reesei is known to be comprised of
endo-3-1,4-glucanase and exo--1,4-glucanase components.
It
was of interest to determine if the endo-glucanase activity in
the C. thermocellum cellulase is susceptible to oxidation.
Endo- -glucanase can be measured by depolymerization of CMC,
but there is no direct way of measuring exo-3-1,4-glucanase activity in a crude extracellular preparation.
I observed that
endo-S-glucanase activity in C. thermocellum cellulase was not
inhibited by low concentrations of DTT or by H 2 0 2
(Table 4).
The CMCase activity was also not affected by p-CMB, DTNB, or
NEM, although these are strong inhibitors of true cellulase
activity.
These results suggest that the exo-f-1,4-glucanase
activity is the component sensitive to oxidation.
74-
Table 3
Inhibition of Cellulase 1 by Sulfhydryl Reagents
and Copper and Prevention by 10 mM DTT
Reagent Added
Addition of
%
10 mM DTT
Activity 2
+
71
100
+
29
77
+
5
78
+
15
92
+
35
62
None
1 mM H 2 0 2
2 mM o-iodosobenzoate
0.2 mM 5,5'-dithiobis-2-nitrobenzoic
acid (DTNB)
1 mM N-ethylmaleimide
(NEM)
50
1 mM iodoacetic acid (IA)
0.02 mM p-chloromercuribenzoic acid
(~pCMB)
+
70
+
2
27
+
11
98
5 i'M CuSOg'5H 2 O
1 15.6 Lg of dialyzed, extracellular protein was
Incubations were aerobic + 10 mM DTT.
2 Decrease in turbidity after
48 h.
100%
used.
activity corresponded to
the complete depolymerization of 3 mg Avicel in 60 h.
-75-
Table 4
CM-Cellulase Activity is Not Inhibited
by Oxidation or Sulfhydryl Reagents
Treatment
Activity(min'
10
None
DTT,
12
10 mM
H2 02 ,
0.5 mM
11
DTNB,
0.2 mM
10
p-CMB,
)i
20
M
7.5 pg of dialyzed enzyme protein was used.
observed in the absence of enzyme.
10
No activity was
-76-
C.
EviEffect of Chelating Agents on Cellulase Activity:
dence that the C. thermocellum Cellulase Requires Iron
for Activity
The experiments described above indicate that inactivation of cellulase in low DTT takes place via the formation of
hydrogen peroxide, which then oxidizes essential thiols.
Un-
expectedly, the apolar chelators o-phenanthroline and 2,2'dipyridyl
(but not the polar chelators EDTA or 8-hydroxyquino-
line) were found to strongly inhibit DTT-protected cellulase in
an anaerobic atmosphere
(Table 5).
o-Phenanthroline inhibition
did not occur if the chelator was first combined with a mixture
of ferrous and ferric iron
(Table 6),
and was partially preven-
ted if the chelator was mixed with zinc and ferric ions.
How-
ever, inhibition still occurred if the chelator was first combined with ferrous iron alone.
o-Phenanthroline binds zinc and
ferrous ions quite strongly, but ferric ions only weakly
1974).
(Bragg,
It is likely that zinc or ferrous iron tied-up the in-
hibitory chelator, and enabled ferric ion to stimulate the
cellulase activity.
It is also possible that the combination
of ferric and ferrous ions provided an oxidation potential in
which the enzyme was active.
Characterization of the role of
iron in purified cellulase and redox titrations should distinguish between these hypotheses.
The possibility of iron being a component of cellulase
was investigated in collaboration with Drs.
Sue Groh and W. H.
-77-
Table 5
Effect of Chelating Agents on Cellulase
Activity Under Anaerobic , Reduced Conditions
Chelator
None
o-phenanthroline
Conc.
(mM)
-
1
4
10
20
% Activity 2
100
77
71
45
21
8-hydroxyquinoline
1
2.5
100
99
EDTA
1
100
5
10
20
83
78
54
2,2'-dipyridvl
i Experimental conditions identical to Table 3 except that
air atmosphere was replaced by 90% N2:5% CO2:5% H2.
DTT
concentration was 10 mM.
2
100% activity corresponded to the complete depolymerization of 3 mg Avicel in 65 h.
-78-
Table 6
Reversal of o-Phenanthroline (OP) Inhibition of
Cellulase by Prior Chelation1 of OP with Metals
Addition
% Activity
100
None
29
20 mM OP
+ 6 mM Fe2+
34
+ 6 mM Zn2+
34
+
0.5 mM Fe 3
29
+ 6 mM Fe 2 + + 0.5 mM Fe 3 +
100
+ 6 mM Zn2+ + 0.5 mM Fe3 +
69
Metals (as dissolved as chloride salts in HCl) were combined with OP before addition to enzyme incubation. Experimental conditions identical to Table 5. Inactive
were manganese, cobalt, copper, molybdenum, magnesium and
nickel.
-79-
Orme-Johnson of MIT's Chemistry Department.
of dialyzed, crude cellulase
presence of 0.12 mM iron.
Chemical analysis
(312 pg protein per ml) showed the
Electron paramagnetic spectroscopy
(epr) revealed that there was a high concentration of high spin
ferric iron associated with the proteins.
The iron signal in-
creased in intensity in a preparation of cellulase partially
purified by ion-exchange chromatography.
The epr analysis
showed that the iron, however, is bound non-specifically, i.e.,
it is not present in heme or an iron-sulfur cluster, and therefore the only definitive way to show its involvement in cellulolytic activity is by complete purification of the cellulase
and detection of iron.
The presence of ferric iron in extra-
cellular hydrolytic enzymes is very unusual, having only been
reported in the periplasmic acid phosphatase from the yeast,
Saccharomyces rouxii
(10).
D. End Products of Avicel Saccharification
In the previous results of this study, I showed that the
cellulase
from C. thermocellum has a specific activity on crys-
talline cellulose 50 to 70 times higher than reconstituted
Trichoderma reesei
(146).
This high activity is observed when
a low concentration of purified, crystalline cellulose
(e.g.
cotton or Avicel) is used as substrate and aerobic incubation
is carried out in the presence of a sulfhydryl reducing agent
and calcium.
However, when the Avicel concentration was in-
-80-
creased to 3 g/l '(5-fold increase),
cellulolysis in air stopped
after one-quarter of the cellulose was digested
probably due to oxidation of the cellulase.
of 90% N 2 : 5% H 2 : 5% CO 2
(Figure 15),
In an atmosphere
(anaerobic) the reaction went to com-
pletion.
Achieving complete saccharification anaerobically of a
reasonable cellulose concentration (3 g/1) provided the means
to determine sugar products formed during the digestion of microcrystalline Avicel.
Previously, Gordon
(75) showed that
cellobiose was the principal product from the digestion of
phosphoric acid-swollen cellulose, which is non-crystalline.
Small quantities of cellotriose and glucose were also formed.
Similarly, I found cellobiose to be the major product of Avicel
saccharification at all stages of digestion
(Figure 16).
Cel-
lobiose was assayed by HPLC; this major peak cochromatographed
with a cellobiose standard, and was converted entirely to glucose when treated with
(data not shown).
-glucosidase from Aspergillus phoenicii
I also detected smaller concentrations of
glucose, which increased in the later stages of saccharification, especially at the higher substrate level.
was detected.
No cellotriose
-81-
Figure 15
Solubilization of 3 g/1 Avicel by 7 ig/ml Dialyzed
Extracellular Protein in an Aerobic or Anaerobic
(90% N2:5% C02:5% H2) Atmosphere.
DTT and CaCl 2 were 10 mM.
E
C
0
1.2Aerobic
C
0
c.0.8
-
0.4
0
Anaerobic
41
0
80
40
Hours
-82-
Figure 16
Product Formation
(Cellobiose, O ; Glucose, 0 ) During Saccharif-
ication of Avicel by C. thermocellum Dialyzed Culture Broth.
The Reactions on 3 g/l or 0.6 g/l Avicel were Conducted in HunSaccharification was
gate Tubes in an Anaerobic Atmosphere.
Followed by Turbidity (o) and the Products were Determined by
DTT and CaCl 2 were 10
HPLC on a Column of HPX-87-H (Bio-Rad).
MM.
3 g/l
Avical
A
1.2
3
1'0
E
Cellobios e42
0.8
0
(D) 0.6
0
0
Glucose
0.4
0
C:
0.2
0
0
Q.)
0
0
0.4w
20
40
60
0.6 g/l Avical
0
a
--
0.3
Cellobiose
0.6
<0
00
0.1
0.2
Glucose
0
20
30
Hours of Hydrolysis
0
-83-
Inhibition of Cellulase Activity by End Products of
Cellulolysis
E.
The next series of experiments describes the inhibition
of C. thermocellum cellulase by the end-products, cellobiose and
glucose.
When microcrystalline cellulose
(Avicel) and untreated
culture broth were incubated in the standard assay (10 mM DTT
and Ca 2+,
aerobic, see Materials and Methods) it was found that
C. thermocellum cellulase is strongly inhibited by cellobiose
and is much less susceptible to inhibition by glucose
17).
Approximately 50%
(Figure
inhibition occurred at 2.5 mg/ml cello-
biose and the cellulase was completely inhibited at 20 mg/ml. A
maximum of 20%
inhibition was observed with 60 mg/ml glucose.
The C. thermocellum cellulase was not inhibited strongly when
the substrate was non-crystalline;
a maximum of 50%
inhibition
by cellobiose was observed on phosphoric acid-swollen cellulose
(Figure 18) and no inhibition occurred when TNP-CMC was the substrate
(data not shown).
Inhibition of the Clostridium and Trichoderma cellulases
(Figure 17).
were compared
The fungal cellulase was less in-
hibited by cellobiose than the bacterial cellulase at equal
broth volumes.
The T. reesei cellulase was much more strongly
inhibited by glucose; this may be due to the presence of
-glu-
cosidase in the fungal broth, which is sensitive to inhibition
by glucose.
a-glucosidase has not been found to any significant
level in the Clostridium culture broths.
-84-
Figure 17
Inhibition of Cellulase Activity in Untreated Culture Broths
of C. thermocellum and T. reesei by Cellobiose (A) or Glucose
(B). Various Volumes of Culture Broth were Used. Avicel was
the Substrate and the Duration of the Experiment was 20 h.
Maximal (100%) Activity for C. thermocellum Corresponds to the
Degradation of 0.73 mg (0.1 ml Containing 20 ,.g Protein), 0.93
mg (0.2 ml; 40 ig protein) and 1.7 mg (0.5 ml, 100 ,ig Protein).
For T. reesei, 100% Corresponds to 1.7 mg (0.5 ml Containing 6
mg Protein).
A. Cellobiose
100
80
Tr (0.5ml)
60
Cl (0.SmI)
40
Ct (0.I2mI
2d~
C
(0.1"t)
4-rn
0
A
I
I
B. Glucose
C
Ct (0.5m)
0
0~
Ct
(0.(m)
60
40
T r (o0
SmO)
20I
oJ
f
0
S
10
15
20
Sugar (mq/ml)
40
60
-85-
Figure 18
Inhibition of C. thermocellum Cellulase by Cellobiose (e) or
Activity was
Glucose (o) on Phosphoric Acid-Swollen Avicel.
Measured by Decrease in Turbidity of a Swollen Cellulose SusFifty 4g of Crude Depension after 3 h Incubation at 601C.
salted Enzyme was used in this Experiment.
Avicel
Swollen
100
Avicel
100
o
80
Glucose
80
60-
60
Cellob iose
40
40
20.
20
Cel lobiose
0
0
10
20
Sug ar
it
I
I
30
40
( m g/ m I)
50
60
0
0
10
20
30
40
Sugar (mg/ml)
50
-86-
As shown above, cellobiose but not glucose is an inhibitor of C. thermocellum cellulase activity.
To determine whe-
-glucosidase would benefit the activity of clostridial
ther
cellulase by destroying cellobiose, a
(10 units/ml) with the bacterial cellulase at 45*C
was incubated
(Table 7).
-glucosidase preparation
The addition of
S-glucosidase promoted a 16%
in-
crease in cellulose hydrolysis in the absence of added cellobiose and increased the activity five-fold in the presence of
cellobiose.
Similar results were obtained with a heat-stable
a-glucosidase from Aspergillus phoenicis
(Figure 19).
Glucose
did not affect activity in the presence or absence of added Bglucosidase.
A number of sugars other than cellobiose and glucose
were tested to see whether they inhibited C. thermocellum celluCertain glucosides and galactosides, e.g. sali-
lase
(Table 8).
cin,
lactose and arbutin were fairly inhibitory, but none was
as inhibitory as cellobiose.
F.
Partial Purification of Cellulase
Partial purification of cellulase was done to determine
the proteins involved in cellulolysis and to provide a means by
which changes in activity observed during growth on various carbon sources
(see later) could be attributed to activation of
the cellulase or to changes in the biosynthesis of cellulase
proteins in the broth.
Table 7
Influence of O-Glucosidase on Cellulase
Hydrolysis by C. thermocellum Cellulasel
Additions
Variation
0-glucosidase
(10 units/ml)
Cellobiose
(10 mg/ml)
1
+
2
Glucose
(10 mg/ml)
Cellulase
Activity (mg Avicel
hydrolyzed in 24 h)
-
+
1.16
-
+
0.24
-
+
1.35
3
+
4
+
+
-
+
1.29
5
+
+
+
+
1.29
6
+
+
4
1.29
+
+
7
8
+
~
1.16
0
1
Reactions were done at 4500 in succinate buffer with Avicel as substrate as described in Methods.
I
00
-88-
Figure 19
Relief of Cellobiose Inhibition of Cellulase Activity by SFifty uil of DialGlucosidase from Aspergillus phoenicis.
yzed Culture Broth were Used.
0.4<
-..+ Cellobiose (10 mg/ml)
C
0
0
0.3
+ Cellobiose +,-Glucosidase +
Glucose (10 mg/ml)
0
C
>
0.2+Cellobiose +jS-Glucosidase
(10 Units/mI)
U)
0.1
No
+,-Glucosidase or
+-Glucosidase +Glucose
oAdto
0
20
60
Hours
100
-89-
Table 8
INHIBITION OF C. THERMOCELLUM CELLULASE
BY VARIOUS CARBOHYDRATES'
Concentration
Inhibitor
none
glucose
2-deoxyglucose
(mg/ml)
Activity
(%)
100
40
76
67
3
100
40
33
gentiobiose
40
42
maltose
40
62
trehalose
40
83
sucrose
40
100
cellobiose
10
20
40
10
0
0
lactose
10
20
40
60
67
50
17
12
arbutin
10
20
40
60
salicin
10
20
40
67
51
18
12
45
36
17
glucose- 1-phosphate
methyl-
xylan
laminarin
-D - glucoside
40
3
3
100
100
1
Maximum (100%)
Assays were done with Avicel as substrate.
h.
in
24
solubilization
responds to 1.21 mg Avicel
activity cor-
-90-
The source of cellulase was the cell-free fluid from a
(see Mate-
culture grown in a 100 liter fermentor on Solka-floc
rials and Methods).
It has retained approximately 80% of its
activity during 2 years of storage at -200C.
The proteins were
concentrated by freeze-drying, reconstituted in water, and dialyzed for 4 h at room temperature against 4-5 changes of 20 mM
Tris-HCl, pH 8.8.
It was found that large losses of activity
(> 80%) occurred during ammonium sulfate precipitation, and
this method was therefore not used for concentration.
The dialyzed, crude extracellular preparation had a
greenish-brown color, and had a slight absorbance in the visible
range (Figure 20).
In addition to the usual protein absorbance
at 280 nm, the dialyzed-concentrate had a very high absorbance
at 260 nm.
It is not known whether this is due to binding of
nucleotides or other UV-absorbing materials by the extracellular proteins.
The cellulase was initially purified by column chromatography on DEAE-sepharose
(Figure 21);
tightly to the ion-exchanger, and most
the cellulase bound
of the contaminating
proteins were eluted before the cellulase in a KCl gradient.
The cellulase eluted with part of the main protein peak;
fore it was probably contaminated with much protein;
tion, large losses of activity
ion exchange chromatography.
there-
in addi-
(- 80-95%) took place during
The active
concentrated and applied to a gel filtration
fractions
column
were
then
(Ultrogel
-91-
Figure 20
Visible and Ultraviolet Adsorption Spectrum of Dialyzed Cellulase in 20 mM Tris, pH 8.8
2
-
7-
-V:
f-
--
HTET~z.z~z
-
-
I
-
-
17,~
4t
C0
}-
-
~
S-
,
0
PQ
-
-
~
--
-
4----
-
-
w
0n
02
0
WAVELENGTH
-92-
Figure 21
DEAE-Sepharose Chromatography of C. thermocellum Extracellular
Protein. The Protein was Dialyzed Against 20 mM Tris-HCl, pH
8.8, and Applied to the Column Equilibrated with the Same Buffer. A KCl Gradient (0-0.5 M) was Run from Fractions 8 to 40.
Five ml Fractions were Collected and Analyzed for Protein and
Cellulase Activity.
180-
Cellulase
-3
0.5M KCI
Protein
A
100 -
/
2
xE
A-1=
100
-,
20- 20
0
a0.
10
20
fraction
30
40
-93-
The cellulase chromatographed as a large complex, elu-
ACA 22).
ting in the void volume
fractions
(MW
>
1.5 x 10 6)
(Figure 22).
Active
(18-22) were examined by SDS-polyacrylamide gel elec(SDS-PAGE) using a 7% acrylamide gel
trophoresis
(Figure 23);
(MW
three major bands of large molecular weight were observed
In addition, several minor bands were
218 K, 112 K and 85 K).
A major contaminating protein in the DEAE-treated
observed.
protein was removed by gel filtration
(lane C).
A second preparation was purified as described above.
Again, a large loss
(92%) of activity occurred during chroma-
tography on DEAE-sepharose, whereas the recovery from gel filtration exceeded 100%.
phoresed by SDS-PAGE
The purified preparation was electro-
(using a 5% acrylamide gel) and compared
it appears that cellulase comprises 2
(Figure 24);
to the crude
major and 1 minor bands of
tively.
It
is
difficult
- 100 K,
to test
78 K, and 220 K, respec-
whether these proteins are com-
ponents of a pure enzyme, since it forms a large complex that
barely migrates into a native polyacrylamide gel
(3% -
12%
gradient concentration of acrylamide) and chromatographs in the
void volume of Ultrogel ACA 22, whose exclusion limit is
6
x 106.
>
1.5
Thus, it is necessary to find a gel filtration medium
with a large exclusion, e.g. 2-5 x 10 6,
gation to test
for purity.
It
or to use ultracentrifu-
is interesting
that
other anaero-
bic bacteria are also known to produce extremely large cellulolytic complexes
(183, 25 ).
This characteristic is different
300
,ftI
a AI
IuI
lase
c
0
c
0
T
200
10
0
Ui)
L
Protein
c
4i)
0
7
\00
-5
C,)
4-
C
0
9
w
10
I
20
L
30
Fraction
Figure
40
22
Gel Filtration of Dialyzed, Total Extracellular Protein on Ultrogel
The Protein (2 ml, - 15 I,/ml, - 0.5 mg protein/ml) was ApACA 22.
plied to Ultrogel Previously Equilibrated with 50 mM Tris-HCl, pH
7.1, 100 mM NaCl, and 2.7 nil Fractions were Collected.
-L
50
-95-
Figure 23
SDS-Polyacrylamide Gel Electrophoretic (SDS-PAGE) Patterns (7%
Acrylamide Gel) of C. thermocellum Extracellular Proteins Purified by DEAE-Sepharose and Ultrogel ACA 22 Chromatography.
Lane A, Molecular Weight Standards (Sigma Kit MW-SDS-200): My-Galactosidase, 116; Phosphorylase B,
osin, 205,000 Daltons;
97,400 D; Bovine Albumin, 66,000 D; Egg Albumin, 45,000 D;
Proteins were ElectrophoCarbonic Anhydrase,29,000 Daltons.
resed at 20( mA to Load a 3% Stacking Gel, then at 40 mA per
Lane B,
Running Gel, Removed and Stained in Coomassie Blue.
Cellulase Activity Fraction (80 pl) after DEAE-Sepharose and
Ultrogel Treatment; Lane C, Non-Cellulolytic Fraction (80 'l)
from Same Treatment Recovered from Ultrogel; Lane D, Same as B
except 20 pl were Applied.
- 95a-
a
b
C
d
-218
K
205Km~
L
116Kbmo
- M-112 K
97 K-
8 85K
66 K-
ie.
~
45Kp.
I-
A
q.s.ummd
-96Figure 24
SDS-PAGE of Purified Cellulase Proteins.
(see Legend to Fig.
23);
Lane B,
Crude,
Lane A, Standards
Dialyzed Extracellu-
Conditions were
lar Proteins; Lane C, Purified Cellulase.
the Same as in Fig. 23 except a 5% Acrylamide Gel was Used.
a
4f .
.IIL
b
c
-97-
from the cellulases of aerobic fungi, which readily separate in
solution into exo- and endo-glucanases.
The large losses of activity
(~
90%) that occur during
ion-exchange chromatography were eliminated by using HPLC with
an ion-exchange vinyl resin column.
Using HPLC, 70-80% of the
cellulase activity was routinely recovered.
very rapid
(30-60 min),
This procedure was
and resolved the extracellular protein
mixture into many more peaks than had been obtained on an open
column
(Figure 25, compare
to Figure 21).
The versatility of
the machine allowed the programming of non-linear gradients to
achieve optimal separation
were compared.
(Figure 26).
Two separations by HPLC
In the first, 3 ml of crude cellulase was
applied, and a linear gradient was run to 0.5 M, during which
most of the contaminating proteins were separated.
Then, the
salt concentration was stepped to 1 M, eluting the remainder of
the cellulase.
However, the cellulase preparation obtained was
contaminated with many non-cellulolytic proteins
lane a).
We decided
(seeFigure 26)
(Figure 27,
to run a non-linear gra-
dient to 0.3 M salt to get rid of contaminating proteins, then
to step to 0.7 M KCl to elute the cellulase, and finally to step
to 1 M to eliminate further contaminants.
applied on a preparative scale:
This technique was
40 ml of crude
(-
20 mg pro-
tein, 600 units cellulase) were applied, and chromatographed as
described above.
The cellulase recovery was 75%, and the prep-
aration obtained had 3 major bands
(Figure 28).
It is likely
-98-
Figure 25
Ion-Exchange HPLC Chromatography of Dialyzed, Extracellular
Proteins. Three ml of Dialyzed Broth was Applied to the IonExchange Column, the Column was Washed with 20 m.M Tris-HCl,
pH 8.8, a Linear KC1 Gradient to 0.5 M KC1 was Applied, and
Finally the Salt Concentration was Stepped to 1 M to Elute
the Remaining Proteins. The Recovery of Cellulase was 79%.
r
--
------
-
I
O.D. 280 nm
Cellulasel
4
04
2
K CI
f\
,<KCI
F-- ----
0
0
-
20
Time (min)
40
-l 0
-99-
Figure 26
Ion-Exchange HPLC Chromatography of Dialyzed, Extracellular
Proteins.
Same Procedure as Fig. 25 except 2 ml Crude Cellulase was Applied, and a Non-Linear Gradient was Applied
as Described in the Text. Recovery was 74%.
P
--
I
CIM
I
I
jI ri
60
0.D. 280
ci,
Co
h
nm....I~
II
401
I~~t
ii
liii II
0.5
CeIuIa%se
,4vAsj
I
ci)
()
20F-
I
\
//
I
-J
01-I
0
30
Time (min)
II
60
0
-100Figure 27
SDS-PAGE of Crude Cellulase from C. thermocellum Grown in Different Carbon Sources and Partially Purified by HPLC (see Fig.
25).
Lane A, Active Cellulose Fraction from HPLC; Lane B,
Broth from Cells Grown in MI-cb Medium; Lane C, Broth from MJAvicel Medium; Land D, Broth from MJ-Fructose. Eighty pl of
Sample were Electrophoresed.
For Examination of Broths, Three
ml Samples were Dialyzed, Lyophilized, and Redissolved in 0.5
ml 50 mM Tris-HCl, pH 7.1.
Arrows at Left of Figure Point to
Probable Cellulase Proteins Based on Previous Characterization.
Lane E Contains Molecular Weight Standards.
a
b
C
!PV
*
.
-
d
e
-101-
Figure 28
SDS-PAGE of Fractions Obtained from Preparative HPLC
Also Electrophoresed are Crude Broths;
Lane F,
Lane G, MJ-Avicel; Lane H, MJ-Cellobiose.
Molec-
(see Text).
E)
MJ-Fructose;
(Lanes A-
Arrows at Left Point
ular Weight Standards were Run in Lane I.
to Probable Cellulase Proteins.
a
b
C
d
e
f
g
h
r
218-205
-116
112--
-102-
that these are components of the C. thermocellum cellulase. The
final purification of cellulase from C. thermocellum depends on
the utilization of a technique after HPLC which can separate the
cellulase based on its size.
G.
Construction of a Defined Medium for C. thermocellum
(113)
To study the regulation of cellulase synthesis by C.
thermocellum it is desirable to use a minimal, chemically defined growth medium.
Unfortunately, only a complicated defined
medium has been described previously (60),
tains several unnecessary nutrients.
which probably con-
The next few experiments
describe the development of a minimal medium.
Early studies by Madia and Demain
(personal communica-
tion) noted requirements for biotin and vitamin B 6 ; the latter
was supplied by pyridoxine.
However, this medium failed to sus-
tain growth through repeated transfers.
Pyridoxal or pyridoxa-
mine was next shown to support higher growth rates than pyridoxine, but this was not sufficient for serial subculture.
Superiority of pyridoxamine and pyridoxal over pyridoxine has
been observed with other bacteria
(42).
It was next noted that
methionine addition to biotin and pyridoxamine showed a positive effect on growth, but again it was not sufficient for serial
subculture.
Since auxotrophic requirements for vitamin B 1 2 or
-103-
p-aminobenzoic acid are sometimes compensated for by methionine
(43),
these vitamins were added to biotin plus pyridoxamine.
Not only was good growth obtained (Table 9),
but also the cul-
ture remained vigorous through at least 10 subcultures.
The chemically defined medium
in Table 10.
(MJ medium) is described
It is identical to the complex medium GS-2, with
the exception that 6 g of yeast extract per liter is replaced
by 2 mg of pyridoxamine hydrochloride, 0.4 mg of p-aminobenzoic acid, 0.2 mg of biotin, and 0.2 mg of vitamin B 1 2 per liter.
This minimal medium supports good growth, product formation, and
cellulase synthesis by C. thermocellum (Table 11),
and also
supports the growth of other C. thermocellum strains including
LQ8
(170),
NCIB
(2),
and the C-9 ethanol-tolerant mutant devel-
oped by Herrero and Gomez
(92).
However, the ethanol-tolerant,
lactate non-producing strain S7-19, developed by Avgerinos
published),
(un-
was found to require supplementary addition of tryp-
tophan for growth (data not shown).
It can be seen from Table 10 that MJ medium contains
sodium citrate
(3 g/l)
to prevent precipitation of salts.
addition, however, may cause a decrease in cellulase titer
This
(K.
Shimada and A. L. Demain, personal communication).
H.
Control of Cellulase Synthesis in C. thermocellum
Previous studies
(3, 50, 68,
87) on the regulation of
synthesis of C. thermocellum cellulase have concluded that car-
Table 9
Vitamin Requirements of C. thermocellum ATCC 274051
Additives to GS-2 Medium Without Yeast Extract (mg/liter)
Biotin
Pyridoxamine
p-Aminobenzoic
B12
Pyridoxine
Pyridoxal
Growth at 40 h
(0.2)
(2)
Acid (0.4)
(0.1)
(2)
(2)
(absorbance at 660 nn)
+
+
+
+
-
-
0.70
+
-
+
+
-
+
0.75
-
+
+
+
-
-
0.10
+
-
+
+
-
-
0.01
+
+
-
+
-
-
0.02
+
+
+
-
-
-
0.02
+
-
+
+
+
-
0.05
1
GS-2 medium supported growth to an absorbance of 0.87.
H
Q
-105-
Table 10
Composition of CM3, GS and MJ Media
CM3
Ingredients
MJ
GS'
KH 2 PO 4
1.5
g
1.5
g
1.5
g
K 2 HPO 4
2.9
g
2.9
g
2.9
g
(NH4 ) 2 SO 4
1.3
g
-
-
Urea
1.0
MgCl 2 . 6H 2 0
CaC
2
150
.2H 2 0
g
mg
-
2.1
g
2.1
g
1.0
g
1.0
g
mg
150
mg
150
FeSO 4 . 6H 2 0
1.25 mg
1.25 mg
1.25 mg
Cysteine-hydrochloride
1.0
g
1.0
g
1.0
g
Resazurin
2.0
mg
2.0
mg
2.0
mg
g
10.0
g
10.0
g
10.0
g
10.0
g
6.0
g
-
Cellobiose
Morpholinopropane
fonic acid
10.0
sul-
Yeast extract
2.0
Pyridoxamine hydrochloride
-
Biotin
g
-
-
2.0
0.2
mgmg
p-Aminobenzoic acid
-
-
0.4
mg
Vitamin B 1 2
Sodium citrate.2H 2 0
-
-
0.2
mg
-
-
3.0
g
Final volume
pH
1 liter
7.0
1 liter
7.4
1 liter
7.4
GS-2 is identical to GS except it contains 3 g/l sodium
citrate.
-106-
Table 11
FORMATION OF ETHANOL, ACETIC ACID, LACTIC ACID, AND
AVICEL HYDROLYZING ACTIVITY IN MJ AND GS-2 MEDIA'
Medium
Dry
cell wt.
(g/liter)
Ethanol
(g/liter)
Acetic
Acid
(g/liter)
Lactic
Acid
(g/liter)
Blue
Avicelase
MJ
0.43
0.29
1.15
0.45
0.90
0.10
GS-2
0.47
0.42
1.20
0.52
0.73
0.25
2
1
(h
)
Cultures were grown for 24 h at 600C.
2
Specific growth rate.
3
Expressed as change in optical density (595 nm) after 24 h by 0. 1 ml culture broth. This degree of activity is equivalent to that shown by a lyophilized broth filtrate of Trichoderma reesei QM 9414 when used at 0.1 mg
of protein per ml of assay mixture.
-107-
boxymethylcellulase (CMCase) is a constitutive enzyme that is
produced in relatively constant specific titers irrespective
of the conditions of growth.
With the finding of true cellu-
lase activity in culture fluids
from C.
thermocellum and the
construction of a defined medium, it became possible to reinvestigate the regulation of cellulase.
It is shown in this
section of the progress report that true cellulase formation
is strongly influenced by the metabolism of the carbon substrate.
The ability to grow on cellulose is a stable character
in C. thermocellum populations.
When C. thermocellum was ser-
ially transferred to cellobiose medium, and then plated on
cellulose agar in a glove box under anaerobic conditions, the
entire population grew and formed clearing zones.
Single col-
onies were picked and grown in cellobiose broth as inoculum,
and then transferred to fructose, sorbitol or glucose
12).
(Table
C. thermocellum initially grew very poorly on these car-
bon sources from a cellobiose inoculum.
fructose and sorbitol
The poor growth in
(but not the poor growth in glucose)
was accompanied by a 9-fold increase in the specific titer of
cellulase.
When broths from cellobiose and fructose cultures
were mixed, no inhibition of cellulase activity was observed,
suggesting that the difference in activities was due to different amounts of cellulase proteins.
The kinetics of growth and cellulase formation were examined in fructose and cellobiose
(Figure 29).
Growth was rapid
-108-
Table 12
Cellulase Synthesis by Cells Previously Grown in
Cellobiose when Transferred as A Small Inoculum
(1% v/v) to Different Carbon Sources
Medium
lag (h)
axim um DCW1
(mg/ml)
Maximum Cellulase
(units/mg DCW)
(units/ml)
No C Source
(starved)
0
0.08
0.16
2.0
Cellobiose
0
0.48
3.6
7.5
Glucose
120
0.26
2.7
10.7
Fructose
110
0.13
5.9
45.4
Sorbitol
84
0.17
6.4
37.6
1
Cultures were harvested after reaching stationary phase.
-109-
Figure
29
Growth and Cellulase Formation in Cellobiose or Fructose DeThe Inoculum (1%
fined Medium. Carbon Sources were 10 g/l.
v/v) was a Culture Growing Exponentially in Cellobiose.
4
4
cellobiose
-2
2-
fructose
X
044
2
2-
0
40
hours
80
-110-
in cellobiose and cellulase volumetric titer paralleled growth.
Cells inoculated to fructose from a cellobiose inoculum had a
60-70 h growth lag, during which cellulase readily accumulated
in the medium.
The accumulation of cellulase as a function of
increase in dry cell weight was fifty times higher during adaptation to fructose than during growth on cellobiose
(Figure 30).
When the cells began to grow on fructose, the differential rate
of cellulase synthesis (159) dropped to 12, still 5-6 times
higher than on cellobiose
(Figure 30).
The addition of cellobiose to a culture in the lag phase
on fructose (Figure 31) caused a rapid burst of growth and cessation of cellulase synthesis.
The extent of growth and de-
crease in cellulase synthesis depended on the dose of cellobiose.
When the added cellobiose was exhausted, the growth lag
on fructose was reestablished and formation of cellulase again
occurred at its high rate.
Therefore, the addition of a desired
carbon source to cells slowly growing on fructose promoted rapid
growth and a sharp decline in cellulase synthesis.
The response to cellobiose/fructose is analogous to 3galactosidase synthesis during diauxic growth of methyl-3-Dthiogalactoside- induced Escherichia coli on glucose plus lactose
(54, 194).
rate and little
glucose,
The glucose is initially metabolized at a rapid
-galactosidase is made.
After exhaustion of
-galactosidase is made at a high differential rate
during the lag on lactose, which drops slightly when growth begins.
Under appropriate conditions of growth, not only glucose,
-111-
Figure 30
Cellulase Production (units) as a Function of Dry Cell Weight
(mg) During the Lag in Fructose or During Exponential Growth
in Cellobiose
6
I
41fru
C',
.4.'
0
G)
C',
C.,
2F-
40
cb
0
0.2
0.4
dcw
-112-
Figure 31
1Or
Effect of the Addition of Cellobiose on Cellulase
Formation During the Growth Lag on Fructose
C.29/1
40
no additions
6H
gi
19/1
CE
2 g/ I
2k
I
I
0
I
I
I
I
0.1
0.2
0.3
0.4
dcw(mg/ml)
-113-
but all readily utilizable carbon sources, act as repressors
of
3-galactosidase.
3-
In an E. coli strain constitutive for
galactosidase synthesis, the enzyme titer is inversely correlated with the doubling time of the culture
(149) and with
the quantity of "high energy" phosphate in the cells
142);
(165,
the poorer the carbon source, the higher is the enzyme
activity.
The derepression of cellulase formation during adaptation to fructose, suggests that rapid catabolism of cellobiose
(the principal product of cellulolysis and C. thermocellum's
preferred energy source) leads to catabolite repression of cellulase synthesis.
This concept was supported by limitation of
catabolism by growth on insoluble crystalline cellulose.
Under
this condition, growth is limited by the supply of soluble carbon source
(cellobiose) (136, 243).
I observed a major increase
in cellulase titer with cellobiose limitation
Fur-
(Table 13).
thermore, when cellobiose was limited by fed-batch addition,
the cellulase titer increased.
In this experiment
(Table 13),
surprisingly little variation in protein titer was observed,
suggesting the limiting cellulase activity may be a minor component of the extracellular proteins.
The derepression of cellulase formation in fructose was
not limited to prior growth in cellobiose;
cells previously
adapted to glucose and inoculated to fructose also substantially
increased their specific titer of cellulase
(Table 14).
This
high specific titer also dropped when growth was established,
Table 13
Cellulase Synthesis by C. thermocellum Grown on Cellobiose, and Inoculated
(1% v/v) to Cellobiose or Avicel Media. Cultures were Grown for 60 h.
Carbon Source
(final conc. 2 g/1)
Cellobiose
Avicel
(Av)
(cb)
Maximum
DCW
(mg/m]
Maximum
Cellulase
(units/ml)
(units/mg DCW)
Extracellular
Protein
(pg/m1)
0.33
1.4
4.3
62.5
0.22
6.4
29.2
65.0
1-h
H
cb + Av
0.24
5.6
23.2
70.0
cb1
0.19
2.9
15.3
38.0
Av1
0.14
5.1
36.4
65.0
1
Added in three doses of 0.5 g/l after limitation of growth at each stage.
Table 14
Growth and Cellulase Formation by Cells Adapted
to Different Carbon Sources
1
2
Medium
lag (h)
-1
p (h )
Sorbitol
Fructose
Cellobiose
Glucose
110
110
0
125
nd
nd
0.35
nd
no C source
0-
Sorbitol
Fructose
Cellobiose
Glucose
70
30
0
30
no C source
0
Fructose
Sorbitol
Fructose
Cellobiose
Glucose
5
7
3
12
Sorbitol
Sorbitol
Fructose
Cellobiose
Glucose
15
10
0
15
no C source
0
Adapted
Inoculum
maxium
DCW
(ng/hl)
Cellulase
(units/ml)
Cellulase
(units/mg DCW)
3
Cellobiose
Glucose
1
3
0.12
0.13
0.38
0.26
6.5
6.0
3.2
2.0
47
46
8.4
7.9
0.08
0.12
1.5
0.14
0.11
0.44
0.41
7.3
5.1
3.3
1.6
53
47
7.6
4.0
0.04
0
0
0.20
0.30
0.45
0.14
0.31
0.43
0.56
0.47
0.6
0.5
0.5
0.1
2.0
1.1
0.9
0.2
0.20
0.25
0.25
0.25
0.31
0.39
0.40
0.38
0.79
0.86
0.94
0.26
2.5
2.2
2.3
0.7
0.05
0
0
nd
nd
0.21
0.18
-
-
Inocula developed for at least 8 serial transfers.
Exponential growrth did not occur.
2
Carbon sources were 0.5% (w/v).
Hn
I
-116-
indicating that the catabolism of the
sugars was responsible
for
the repression.
It is clear that the formation of extracellular cellu-
lase in C. thermocellum is regulated by the carbon source and
growth rate of the cells.
sor;
Cellobiose is not a unique repres-
the rapid metabolism of other substrates also controls
cellulase synthesis.
Limiting the supply of cellobiose by growth
on Avicel substantially increases the
cellulase.
formation of extracellular
These data suggest that the rate of catabolism and
energy production by the cells controls the levels of cellulase.
To test this,
I measured ATP levels in cells grown in carbon-
source excess
(cellobiose) or under carbon-source limitation
(Avicel) (Figure 32).
It was observed in cellobiose that ATP
concentration reached a very high level during rapid growth, but
plummeted when the culture entered stationary phase.
In con-
trast, ATP levels remained low during growth on Avicel, and
declined slightly when growth stopped.
As previously observed,
the cellulase titer was higher in the Avicel medium.
Carbon substrate limitation also occurred during adaptations to fructose, sorbitol or glucose, from a cellobiose inoculum, but only in fructose and sorbitol was an increased specific titer of cellulase observed.
Adaptations to fructose and
glucose were studied in more detail
(Table 15).
During the lUO
h adaptation to fructose the culture was able to glycolytically
ferment the sugar to low quantities of end products and to form
relatively
low amounts of ATP.
A
high differential rate of
W
-117-
Figure 32
Growth of C. thermocellum on Cellobiose or Avicel
a
S
a
I
m
U
*1
~1
-J
'U
U
CELLOBIOSE
AVICEL
CELLS
CELLS
-ATP
CELLU LASE
OA
CELLULASE
ATP
O
40
80
0
HOURS
40
80
Table 15
Formation of ATP, End Products and Cellulase During
Lags on Fructose and Glucose
C Source
Tine
(h)
Cells
(mg/ml)
Cellulase
(U/ml)
(U/mg)
ATP
(nruoles/mg)
Acetate
(mg/ml)
Ethanol
(rng/ml)
Lactate
(mg/ml)
fructose
23
0.06
0.2
3.3
0.57
0.08
0.05
0.03
"
57
0.06
0.45
7.5
0.72
0.11
0.12
0.02
"
100
0.083
1.6
20.0
0.88
0.15
0.17
0.02
glucose
"
"
23
57
100
0.05
0.06
0.06
0.1
0.17
0.27
2.0
2.8
4.5
0.50
0.10
0.02
0.19
0.13
0.11
0.03
0.06
0.08
0.03
0.05
0.04
no C~source
100
0.05
0.0
0
0.10
0.0
0.0
0.0
cb control
60
0.38
1.1
2.9
n.d.
0.27
0.54
1.97
fru control
60
0.40
1.0
2.5
n.d.
0.32
0.96
0.28
2
1
Fructose inoculum; other inoculated from cellobiose inoculum.
2
Not determined in this experiment, see Table 17.
I
H
03
-119-
cellulase synthesis was detected during this slow glycolytic
metabolism.
In contrast, cells exposed to glucose declined
drastically in ATP levels, and did not accumulate fermentation
products, indicating they were blocked in glucose uptake or in
glycolysis.
The extremely low ATP concentrations and absence
of glycolytic metabolism in cells exposed to glucose or in the
absence of a carbon source precluded the synthesis of cellulase proteins.
Hernandez suggested
(91) that the extended lag (>
100 h)
on glucose from a cellobiose inoculum is caused by the need for
induction of a transport system.
However, the lack of uptake
is probably not solely responsible for the lag.
Experiments in
Dr. M. Roberts' laboratory (C. Tolman, personal communication)
have demonstrated by
1 3 C-NMR
that labeled glucose is transported
during short incubation periods (<
Similarly, I observed
1 h).
that cellobiose-grown cells, washed and resuspended in buffer,
are able to transport glucose,
as. indicated by their ability to
form a pH gradient across the membrane
(Figure 33).
The proton
gradient was rapidly formed in cellobiose or glucose, but not
in fructose or in the absence of a carbon source
shown).
The proton gradient was measured by the uptake of in-
organic phosphate (Figure 33),
transport (197),
(197).
(data not
which depends on ApH for its
formed by ATP hydrolysis by the membrane ATPase
Thus, cells exposed to glucose deplete their ATP levels
by the ATPase and possibly by glucokinase
(177),
and cannot re-
generate the energy due to a block in glycolysis, as reflected
-120-
Figure 33
Transport of Inorganic Phosphate and Formation of ApH by
C. thermocellum
The cells were grown in cellobiose medium and washed and
resuspended in NMR buffer (see Materials and Methods).
They were dosed with glucose, cellobiose or fructose and
the rate of Pi uptake determined by 3 1 P-NMR. The numbers
indicate minutes of incubation in the presence of sugar.
The tall peak is extracellular Pi; intracellular Pi deRefer to ApH
velops as peak immediately to the left.
rapidly formed in cellobiose.
-121-
glucose
cel lobiose
12
1
5
5
fructose
15
~vI~Nv~p
30
8
10
~AAPPA
(~PJ12
tJV\PV\45
20
Ar
60
-122-
by the absence of end product formation
(Table 15).
The
lucif-
erase assay confirmed that ATP concentration drops to a drastically low level in glucose (Table 15),
in the absence of a carbon substrate
much lower than detected
(Table 15).
These results
suggest that exposure to glucose causes a depletion of ATP and
a paralysis of metabolism.
The depletion of ATP might be caused
solely by the membrane ATPase, which in Clostridium pasteurianum
has the interesting property of being activated by phosphoenolpyruvate and fructose
1,6-bisphosphate
(Morris, personal commun-
ication).
Depletion might also occur by a kinase/phosphatase
cycle (86)
or by the formation of a reserve polysaccharide such
as glycogen
(106).
Although the specific titer of cellulase was high in
cells adapting to fructose or sorbitol
(Table 12),
continued
cycles of growth on these substrates led to a decrease in cellulase production, until specific titers were obtained which were
consistently less than cultures maintained in cellobiose or
glucose
(Figure 34).
adapted to fructose.
It was observed that cells only gradually
Initially the lag phase was
1OU h, decreas-
ing to 30 h on the second transfer, than to 14 hours, and finally
to 3 to 4 h; growth reached a constant final cell den-
sity of about 0.4 g dcw/l.
This adaptation and rapidity of
fructose utilization occurred concomitantly with the drop in
cellulase titer.
Fermentation broths were analyzed by SDS-polyacrylamide
gel electrophoresis to determine whether the quantities of cel-
-123-
40
fructose
E
sorbitOl
207S
cellobiose
glucose
01
6
24
serial transfer
Figure 34
Effect of Serial Transfer on the Specific Titer of Cellulase by
C. thermocellum Culture Broths.
Cultures were Grown in Cellobiose and then Inoculated to the Various Carbon Sources.
Each
Transfer Corresponds to Five Mass Doublings.
-124-
lulase proteins varied after growth in fructose, cellobiose or
Avicel
(Figure 27,
electrophoresed.
page 100).
Growth was
Identical volumes of broth were
approximately the same
on each of
the carbon sources and the cellulase activities were:
4.0 U/ml;
cellobiose, 1.2 U/ml and fructose, 1.0 U/ml.
Avicel,
The re-
sults show that growth in Avicel yields higher concentration of
cellulase components, and confirms that cellulase synthesis is
regulated in C.
thermocellum.
The drop in cellulase titer on serial transfer
in fruc-
tose was not caused by the selection of cellulase non-producing
mutants, as shown by plating experiments.
In the fructose adap-
ted culture, equal numbers of cells grew on cellulose or fructose agar and the cell recovery was more than 70%.
Furthermore,
when individual cells from the Avicel agar plates were isolated,
growth in cellobiose, and transferred back to fructose, they underwent another cycle of increased cellulase synthesis followed
in later transfers by a very low specific titer
(Figure 35).
These experiments demonstrate that a genetic change had not
occurred in the cells.
I considered the possibility that cycles of growth in
fructose or sorbitol depleted the cells of an inducer which was
made during growth on the cellulose derivatives
glucose) but not on fructose or sorbitol.
(cellobiose or
Cellobiose or cello-
dextrins would be plausible inducers of cellulase, especially
since they are initial products of cellulolysis
(75),
and are
known to be made in C. thermocellum extracts from glucose and
-125-
10 F.
iU
S
U)
C
U)
U
WE
U
5i.
20
0
I
I
20
.40
I
i
I
=A-
20
I
40
mass doublings
Figure 35
Cellulase Synthesis by Cells Adapted to Fructose (Figure 12),
Isolated on MJ-Avicel Agar, Picked to Cellobiose Broth for
Growth and CelOne Transfer, and Reinoculated to Fructose.
lulase was Assayed During Sequential Cycles of Growth in
Fructose Medium. The Different Symbols Represent Individual
Isolates.
-126-
glucose-l-phosphate
(204).
However, fructose or sorbitol adap-
ted cultures were not "induced" for cellulase synthesis by one
cycle of growth in cellobiose
lings
(Table 14);
after 15 mass doub-
(3 cycles) the cellulase titer gradually returned to the
level on serial transfer in cellobiose
(2.1 units/ml)
(data not
Fructose-adapted cultures were also not induced by
shown).
growth in a cell-free medium preconditioned with cellobioseadapted cells.
Although the fructose-adapted cells grew well
in the conditioned medium with fresh cellobiose added as the
carbon source, they did not rapidly increase their production
of cellulase.
rivatives
Finally, the addition
(0.1 g/l) of cellulose de-
(including glucose, glucose-l-phosphate, cellobiose,
cellotriose, cellotetraose, a mixture of soluble dextrins),
to
fructose or cellobiose medium failed to boost the production of
cellulase from a fructose inoculum in a single transfer
mass doublings).
(- 5
Cellobiose analogues including lactose, sali-
cin and thiocellobiose were also inactive.
Taken together,
the above experiments argue against the requirement for a cellulose-derived inducer in cellulase synthesis.
Analysis of soluble fermentation products from fructose
and cellobiose adapted cultures revealed significantly different patterns
(Table 16).
In fructose,
the formation of lactic
acid was almost completely turned off, possibly due to a decrease in fructose-1,6-diphosphate, which activates lactic de-
hydrogenase.
The diminished lactic acid formation leaves more
pyruvate available for oxidative decarboxylation, with possible
-127-
Table 16
Difference in Product Formation by Cellobioseor Fructose-Adapted Cells in Cellobiose and
Fructose, Respectively
Final
C Source
pH
Acetate
Lactate
Cells
EtOH
(g/l)
(MM)
(mM)
(MM)
cb
6.4
0.56
7.6
5.6
4.6
fru
6.9
0.34
4.7
3.3
0.3
-128-
increased ATP yield
(Figure 1 ).
This was shown directly by
measurement of the ATP levels in the cells with firefly luciferase;
ATP was elevated in the tructose-adapted cells
(Table 17).
The increased decarboxylation of pyruvate also requires that
NADH be reoxidized by a mechanism not involving lactic dehydrogenase
(LDH).
In the clostridia, this is mainly accomplished
by NADH:ferredoxin oxidoreductase
hydrogen.
NAD
(E0
(j9) and formation of molecular
The reduction of ferredoxin
= -320
(E0
= -398 mV) by reduced
mV) is thermodynamically unfavorable, and is in-
hibited by the accumulation of low partial pressures of hydrogen gas
(77,118Y.
Indeed, fructose-adapted cultures did not
grow in an atmosphere of hydrogen
(Table 18),
unless supplemen-
ted with cellobiose which probably allowed the formation of lactate.
These data suggest that the stronger repression of cellu-
lase synthesis in fructose than in cellobiose adapted cells is
related to the increased formation of ATP from decarboxylation
of pyruvate.
This was supported by the action of inhibitors
which block this pathway of pyruvate metabolism.
The addition
of potassium cyanide, known to shift an alcoholic fermentation
to a homolactic fermentation in saccharolytic clostridia (123),
caused increased formation ot lactate and significantly increased
cellulase production in the culture
(Table 19).
Similarly, the
addition of methyl viologen, which reacts with hydrogenase,
blocking the decarboxylation pathway, caused an increase in
cellulase formation
(Table 19).
-129-
Table 17
ATP Levels in C. thermocellum Grown on
Soluble Carbon Sources
1
nmoles ATP/
mg DCW
Growth Phase
+ 0.35
exponential
3.25 + 0.17
exponential
Inoculum
C Source
cb
cb
2.1
fru
fru
cb
none
1
Carbon sources were at 5 g/l.
1.30
-130-
Table 18
Influence of Gas Atmosphere on Growth
of Fructose-Adapted Cells
Atmosphere
N2
Carbon
Source
+0.5 mg/ml
Cellobiose
Final
pH
DCW
(g/l)
Fructose
no
6.7
0.42
yes
6.8
0.40
no
7.3
0.14
yes
6.8
0.40
no
6.7
0.38
yes
6.7
0.39
"
H2
Gas mix
1
Glove box atmosphere
(90% N 2:
5% H 2 : 5% CO 2 ).
-131-
Table 19
Derepression of Cellulase Synthesis in Fructose-Adapted
Cells by Inhibitors of Pyruvate Decarboxylation
Additioni
DCW
(mg/ml)
Cellulase
U/mg DCW
U/ml
Lactic
Acid (mrM)
None
0.44
0.7
1.6
0.2
10
mM KCN
0.36
2.6
7.2
2.7
5
mM KCN
0.33
2.7
8.2
nd
1
iM methyl viologen
0.31
2.9
9.4
nd
2.5
PM methyl viologen
0.30
3.4
11.3
nd
1
mM dinitrophenol
0.37
1.8
4.9
nd
2
Additions were made
The carbon source was fructose (5 g/1).
to freshly prepared media before inoculation with a log phase
culture in fructose medium.
Not determined.
-132-
The results presented above indicate that cellulase is
constitutively produced on the different carbon substrates, but
that its differential rate of synthesis is controlled by catabolite repression (141).
This was further supported by the in-
creased cellulase synthesis observed when the cells were treated
with various energy metabolism inhibitors
For exam-
(Table 20).
ple, treatments which are known to dissipate pH gradients resulted in increased cellulase formation, due to the dissipation of
cellular energy levels
(Table 20).
For example, an uncoupler
(carbonylcyanide m-chlorophenylhydrazone, CCCP),
hibitor
(N,N'-dicyclohexylcarbodimide, DCCD),
all which dissipate the pH gradient
levels
(126),
an ATPase in-
and organic acids,
(193, 12b) and decrease ATP
stimulated cellulase accumulation.
On the other
hand, imposing a pH gradient by adding a strong acid
a membrane-permeable weak base
cellulase.
(Tris) reduced the production of
These treatments are tigntly linked to the phos-
phorylation potential,
crease
(HCl) or
and either increase
(e.g. HCl) or de-
(e.g. CCCP) the energy sufficiency of the cells.
In conclusion, cellulase in C. thermocellum is a constitutively produced enzyme system, whose rate of formation is
regulated by the rate of catabolism and energy sufficiency of
the cells.
known;
The molecular mechanism of this regulation is not
the addition of 2-10 mM cAMP or cGmP had no effect on
growth or cellulase formation in C. thermocellum (data not shown),
although the cAMP dibutyryl derivative did stimulate cellulase
formation in cellobiose medium
(Table 21).
-133-
Table 20
Changes in Cellulase Synthesis from Treatments which
Affect ATP Accumulation and Formation of ApH
C Source
Expt.
Addition
Final
pH
DCW
(mg/ml)
6.6
6.7
6.8
6.3
6.9
6.9
0.42
0.25
0.17
0.34
0.50
0.37
1.0
2.0
4.1
0.25
1.7
3.2
6.25
6.3
6.2
0.39
0.25
0.36
2.5
2.4
0.13
0.42
0.38
0.34
0.46
0.52
0.7
4.6
3.1
1.7
0.4
U/ml
U/mg
I:
fru
cb
None
10 PM
100 PM
10 mM
20 mM
5 pM
CCCP
CCCP
HCi
KOH
DCCD
None
10 PM CCCP
10 mM HCi
2.4
8.0
24
0.8
3.4
8.7
6.4
9.6
0.36
Expt. II:
fru
None
Sodium pyruvate,
2.5 mM
Sodium fumarate, 12.5 mM
Sodium acetate,
4
mM
Tris, 4 mM
1.6
12.1
9.1
3.7
0.8
1
Additions were made to freshly prepared media before inoculation with a log phase culture growing on the same carbon
source used in the experiments.
-134-
Table
Effect of Cyclic
21
Nucleotides on
Cellulase Synthesis in Cellobiose Medium
D.C.W.
(mg/ml)
Cellulase
(U/ml)
U/mg
1
None
0.53
0.80
1.50
2
0.025 mM dibutyryl cGMP
0.33
0.45
1.30
3
0.025 mM dibutyryl cAMP
0.50
1.50
3.00
4
0.1
0.50
1.70
3.40
mM
"
"
-135-
4.
DISCUSSION
The present study shows clearly the presence of true cellulase activity in a bacterial extracellular preparation.
C.
thermocellum produces an extracellular enzyme which, in the
presence of Ca2+ and DTT, has the ability to solubilize native
and derived forms of cellulose
(cotton, filter paper and Avicel)
at a rate and to an extent comparable with T. reesei cellulase.
The crude clostridial cellulase works effectively at protein
concentrations fifty times less than the Trichoderma enzyme,
and thus appears to have a much higher specific activity.
Fur-
thermore, the clostridial cellulase comprises only 30-35% of
the crude broth protein, as shown by partial purification, and
therefore probably has a specific activity at least 100-fold
improved over the Trichoderma cellulase.
The protein secreted
by T. reesei is known to be at least 85% cellulase
(7).
should be stressed, however, that low concentrations
It
(< 3 g/l)
of cellulose were used in the present study, and saccharification of concentrated slurries of cellulose
(e.g. 15%) have not
yet been accomplished with the bacterial enzyme.
The Clostridium cellulase displays an unusual preference for
highly crystalline substrates.
Filter paper was the preferred
substrate for the Trichoderma cellulase but was a poor substrate
for the bacterial cellulase in the initial stages of hydrolysis.
In contrast, cotton presented less of a problem to the Clostridium
-136-
enzyme then to the Trichoderma cellulase.
This high specific
activity on resistant substrates reflects C. thermocellum's
ability to proliferate under thermophilic, anaerobic conditions
on partially digested plant tissues.
As an anaerobe depending
solely on glycolysis for its cellular energy, C. thermocellum
cannot afford to produce high quantities of extracellular protein.
I observed two biochemical properties of the C. thermocellum
cellulase which probably contribute to its effective solubilization of cellulose.
The crude enzyme contains sulfhydryl groups
essential for activity and may also employ iron in cellulose
depolymerization.
The participation of sulfhydryl groups has
been demonstrated in hydrolytic enzymes including papain and
ficin
(224) in which the sulfhydryl acts directly as a nucleo-
philic catalyst.
Sulfhydryls also have a strong affinity for
metals, such as Ca 2+,
Cu2+ and Fe3+
(224, 250).
The lecithinase
from Bacillus cereus and the a-toxin from Clostridium welchii
are activated by Ca2+
groups from oxidation.
(32),
possibly due to protection of SH
The binding of ferric iron to protein
may provide a strong acid catalyst (250).
I found that the
cellulase from C. thermocellum was inhibited by o-phenanthroline,
and this inhibition was reversed by the addition of iron.
Fur-
thermore, clostridial cellulase purified by dialysis and column
chromatography contained a high concentration of ferric ion.
These results suggest iron as a component in the cellulase.
-137-
Iron is not commonly found in hydrolytic enzymes, although ferric iron was recently reported to stimulate the acid phosphatase
from Saccharomyces rouxii
(10).
It is interesting that the aldo-
lase isolated from clostridia is a thiol enzyme which requires
iron (14),
unlike the aldolase from enteric bacteria.
The sensitivity of the Clostridium cellulase to oxidation
clearly differentiates the bacterial enzyme complex from the
cellulases of aerobic fungi including Sporotrichum pulverulenturn, Polyporus adustus, Myrothecium verrucaria and Trichoderma
viride, whose cellulases work most effectively in the presence
of oxygen (56).
The cellulase from T. reesei is not reported
to have essential sulfhydryls and is inactivated by 3.2 mM DTT
(190).
This is probably due to the disruption of stabilizing
disulfide bonds.
It is not surprising that T. reesei does not
contain sulfhydryl groups in its extracellular cellulase, since
it operates in an aerobic environment.
sulfhydryls
The presence of free
(i.e., cysteine) in extracellular proteins of aero-
bic organisms is rare (57),
and nearly all the cysteine residues
are present as the disulfide cystine (222).
The presence of
cystine residues in aerobic extracellular proteins is generally
conservative, without much variation in positioning of the cystines or their frequency of appearance (222).
In contrast, free
cysteine residues appear sporadically, and are usually eliminated by natural selection, since their exposure in aerobic organisms leads to drastic oxidation and polymerization (222).
-138-
The occurrence of cysteine or cystine in extracellular proteins
in anaerobic, facultative and aerobic bacteria was considered
by Fahey et al.
(57) to be determined by the oxidation state of
the environment in which the protein functions.
For example,
clostripain from the anaerobe Clostridium histolyticum is a
sulfhydryl protein, serine protease from the aerobe Streptomyces griseus is a disulfide-containing protein, and most extracellular proteins from facultative bacteria contain neither
cysteine nor cystine
(57).
The cellulase from the rumen
anaerobe Ruminococcus albus is sensitive to oxidation
(211) but
it has not been reported whether this is caused by sulfhydryl
inactivation.
The reactivity of thiols
to combine with essential metals
(113) and their ability
(250) might provide anaerobic
bacteria with catalytic abilities not observed with aerobes.
It is likely that the component of C. thermocellum cellulase
which contains essential sulfhydryl is a cellobiohydrolase
-1,4-glucanase),
(exo-
since CMCase activity was found to be unaffec-
ted by oxidation or sulfhydryl reagents.
Two distinct endo-6-
(1,4)-glucanase (CMCases) have been purified from C. thermocellum
(168, 181) and neither of these enzymes are affected by sulfhydryl reagents.
The CMCase purified by Ng and Zeikus
pletely lacks cysteine.
(168) com-
The further purification and character-
ization of the C. thermocellum cellulase complex should help determine the role of sulfhydryls and metal ions in its catalytic
activity.
-139-
Despite earlier observations that hydrolysis of dyed-CMC
and dyed-Avicel by C. thermocellum cellulase was not inhibited
by cellobiose or glucose
(68),
I have found in the present
work that hydrolysis of crystalline cellulose is inhibited by
cellobiose.
While this thesis was in preparation, Petre et al.
(181) reported that purified endo- -glucanase of C. thermocellum
is relatively insensitive to cellobiose using TNP-CMC as substrate.
The effect of cellobiose is therefore dependent on the
nature of the substrate.
Reese et al,
(188) first showed that
cellobiose may have anomolous effects on cellulase activity depending on the substrate used and the incubation conditions;
inhibition or even stimulation may occur with CMC or derived
celluloses.
An important limitation in cellulose hydrolysis is enzyme
inhibition by cellobiose, especially when the substrate is
highly crystalline and the enzyme preparation is low in
glucosidase.
-
With respect to cellobiose inhibition, our stud-
ies point to a similarity between C. thermocellum and Trichoderma.
(84),
The inhibition of Trichoderma cellulase is competitive
and increases with resistance of the cellulose to break-
down (84).
Addition of
-glucosidase preparation of high spe-
cific activity to cellulose saccharification mixtures leads to
cellobiose hydrolysis and thus alleviates the inhibition of the
cellulase by its product (215).
The sensitivities of the fungal and bacterial cellulases to
glucose inhibition are strikingly different
(Figure 17);
this
-140-
may be the result
the broths.
of different
-glucosidase
concentrations
S-Glucosidase is known to greatly enhance cellu-
lose hydrolysis
(215)
T. reesei RUT-C30
and is present in low concentrations
(215).
in
On the other hand, C. thermocellum is
not known to produce an extracellular
possesses a periplasmic
-glucosidase
cellobiose phosphorylase
(4),
-glucosidase although it
(2) and a periplasmic
which together convert cellobiose
to glucose and glucose-l-phosphate.
The improvement in cellu-
lose saccharification observed in the presence of added
cosidase
in
-glu-
(Table 7) suggests that it would be useful to develop
strains which secrete cellobiase.
A potentially important finding in this study is that cellobiose analogs such as salicin, arbutin and lactose also inhibit
C.
thermocellum cellulase
(Table 8
).
It was previously repor-
ted that the cellulase of C. thermocellulaseum is inhibited by
cellobiose and lactose
(53).
Since salicin, arbutin and lactose
are not carbon sources for growth of C. thermocellum, it may be
possible to use these analogs as selective agents for isolation
of strains of C. thermocellum affected in cellulase synthesis or
activity.
In this study, I have shown that C. thermocellum requires
only four growth factors
(biotin, vitamin B 6,
p-aminobenzoic
acid and vitamin B 1 2) in a chemically defined medium.
Its vita-
min requirements are similar to the cellulose digesters in the
rumen of cattle and sheep (107).
Growth of these anaerobes is
-141-
frequently stimulated by vitamin B 1 2 , biotin and p-amonibenzoic
acid.
C. thermocellum has an unusual requirement for vitamin
B 6 ' which is generally not required by cellulolytic anaerobes
(24,
107) or soil bacteria (137).
There are stiking similarities in the nutrition and physiol-
ogy of rumen cellulose digesters
(Ruminococcus and Bacteroides),
free-living Sporocytophaga and C. thermocellum.
These organ-
isms rapidly use cellobiose, but only reluctantly use the hexoses fructose, glucose and mannose
sources fail to support growth.
(12, 68,
210).
Other energy
The organisms characteristically
produce a yellow-orange pigment when growing on cellulose.
The
ruminococci and Bacteroides appear to have an obligate requirement for CO 2 or bicarbonate, and characteristically produce high
concentrations of succinic acid.
In contrast, the cellulolytic
clostridia generally don't produce much succinate.
Of particu-
lar interest is the preference among these organisms for cellobiose, confirming this disaccharide as the main product of cellulose digestion.
cellobiose
The cells employ a phosphorolytic cleavage of
(4, 12, 107).
these cells.
This is an energy-saving mechanism in
C. thermocellum possesses more than one pathway
for the dissimilation of cellobiose.
biokinase
(166) and
-glucosidase
It reportedly has a cello-
(2) for the catabolism of
cellobiose, and also possesses a cellodextrin phosphorylase
(204) which may function in the cells to synthesize cellodex-
trins from cellobiose, possibly as a reserve polysaccharide
(105).
-142-
Although the regulation of cellodextrin formation has not been
studied in the clostridia, the formation of glycogen in bacteria
and its phosphorolytic cleavage is regulated by allosteric mechanisms,
(31),
by catabolite repression,
and by covalent modification
suggesting an important role for this process in microor-
ganisms.
As demonstrated in the present study, the degradation of
cellulose by C. thermocellum appears to be well-coordinated with
the growth and energy needs of the cell.
Formation of cellulase
occurs at the most rapid differential rate when the cells are
growing slowly on cellulose or on fructose.
Little cellulase is
made during rapid growth on cellobiose or when cellobiose-grown
cells
are exposed to glucose,
in which even slow growth does not
occur possibly due to severe decline in ATP levels.
sis of extracellular cellulase occurs
The synthe-
in highest quantity when
cells are presented with an environment in which they are capable
of growth, but are deprived of carbon substrate and energy.
Cellulase is repressed during growth on rapidly metabolized carbon sources.
The severity of repression appears to be related
to the degree of pyruvate decarboxylation and the level of ATP
available to the cells
(48, 172).
Oxidative decarboxylation of
pyruvate supplies more energy than does reduction to lactate,
and lowers the specific titer of cellulase.
Other studies have
established that production of xylanases and cellulases is constitutive in the rumen bacteria
(67,
183).
The rate of their
-143-
formation is inversely related to the growth rate of the cells
in carbon-limited continuous culture.
Very little work has been done on the control of catabolic
pathways in the clostridia, and my review of the literature found
only mechanisms for the inhibition or activation of key catabolic
enzymes.
In Clostridium tetanomorphum,
the first
enzyme of
threonine degradation, threonine deaminase, is activated by ADP,
GDP or IDP
(164, 223, 247)).
dryl inhibitors (164).
The enzyme is inhibited by sulfhy-
Purification and characterization of the
enzyme has shown that it has an allosteric site responsible for
activation (164).
In the same organism, glucose fermentation
was shown by Anthony and Guest (8) to be delayed in preference
for more favorable energy sources (probably amino acids).
They
concluded that glycolytic enzymes necessary for glucose fermentation are inhibited, but not repressed, by a catabolite of amino
acid metabolism.
Evidence is presented in this thesis that C. thermocellum
is able to control the synthesis of cellulase by catabolite repression.
Since C. thermocellum is only able to dissimilate
cellulose, cellobiose, glucose, fructose and sorbitol as carbon
sources, it seems unlikely that catabolite repression evolved
to enable the bacterium to select its most desired energy source.
Instead, the regulatory mechanism may provide protection from
excess energy metabolism (.1,
13,
63, 192).
In both gram-posi-
tive and gram-negative bacteria, excessive metabolism through
-144-
Embden-Meyerhof pathway, in which the terminal steps are overloaded due to deficiency of electron acceptors, results in the
formation of glycolyticbyproducts
which are toxic to the cells.
(e.g. methylglyoxal)
Formation of lethal
(38)
methylgly-
oxal has been demonstrated in Clostridium acetobutvlicum (182),
Clostridium pasteurianum (38),
and Streptococcus faecalis
(180),
especially at high cell densities and high temperatures.
From the results presented above, it appears that cellulose
degradation in C. thermocellum is controlled by cellulase inhibition and oxidative inactivation, and by catabolite repression.
The mechanism of catabolite repression was not elucida-
ted; it may involve phosphorylated glycolytic intermediates and
highly phosphorylated nucleotides, which are involved in catabolite repression in the spore-forming bacilli
(64, 139).
The
effector signalling energy deficiency is probably not cyclic
AMP(21,179) which has not been isolated from anaerobes
(21,
111,
179) or sporeformers (21).
In conclusion, this study shows that the thermophilic anaerobe, C. thermocellum, synthesizes a true cellulase of an anaerobic nature and high specific activity on crystalline cellulose.
C. thermocellum possesses controls to regulate both the activity
and formation of its efficient extracellular enzyme.
Judging
from the primitiveness of the saccharolytic clostridia, thought
(174, 241) to closely resemble the first organisms present on
earth some 3.5 billion years ago, further elucidation of the
-145-
control of metabolism in C. thermocellum might provide fundamental insights into the development of higher organisms.
-145a-
5.
RECOMMENDATIONS FOR FUTURE RESEARCH
A.
Activity of the Cellulase
Compared to the Trichoderma cellulase, the crude Clostridium cellulase appears to have novel biochemical requirements
and an exceptionally high specific activity on crystalline substrates.
The bacterial cellulase complex active on Avicel and
cotton should be purified and characterized for its specific
activity, molecular weight, peptide composition, sulfhydryl and
metal content, and substrate preferences.
In addition, the pos-
sible roles of sulfhydryls and iron in catalysis and aggregation
should be examined using the purified enzyme.
The individual
reactions involved in the degradation of insoluble cellulose
could be separated and characterized.
It would be useful to
carry out hydrolysis studies on thick slurries of cellulose,
and then on agricultural residues.
The sensitivity of the
Clostridium cellulase to oxygen inactivation suggests that a
novel, non-oxidative mechanism might be active in the depolymerization of lignin, which could be investigated in the extracellular protein preparation.
B.
Formation of the Cellulase
Evidence is presented in this thesis for the regulation
of cellulase synthesis by the energy requirements of the cells.
-145b-
The differential rates of cellulase synthesis
(units cellulase
synthesized per unit protein synthesis) on various carbon
sources should be determined using the uptake of a radioactive amino acid as an indicator of protein synthesis and
turnover.
The changes in the levels of the polypeptides in the
cellulase complex could be determined on the different carbon
sources using electrophoresis or antibodies with purified
cellulase as standard.
To understand the regulation of cellulase synthesis
in
Clostridium thermocellum, the relationship between energy production by glycolysis and cellulase synthesis should be studied.
Specifically, the control of the branch resulting in pyruvate
reduction or oxidative decarboxylation should be investigated.
The oxidative branch employs iron sulfur proteins
(ferredoxin
and hydrogenase) whose levels and activities might be influenced
by the availability of iron, hydrogen gas and electron acceptors
in the medium.
The simple, fermentative metabolism of C. ther-
mocellum suggests that it
may be possible to monitor energy
production and catabolite repression by a simple and nondestructive method, e.g. by the uptake of inorganic phosphate.
C. thermocellum experiences a sharp decline in ATP
levels during the stationary phase of growth and when adapting
to glucose.
Probably the extremely low levels of ATP prevent
the synthesis of cellulase under these conditions.
The role
of proton ATPase, energy-dissipating futile cycles
(e.g. glu-
cokinase/phosphatase),
and energy storage as polysaccharide
-145c-
should be investigated as causes for the lack of growth on glucose.
C.
Selection of Mutants Affected in Extracellular Cellulase Formation
An understanding of the regulation of cellulase activity and synthesis and its functions for C. thermocellum (e.g.
cellulose hydrolysis and metal scavenging) would suggest
methods for the enrichment of mutants changed in cellulase formation.
For example, limiting energy production by availability
of carbon source, by including a non-metabolizable inhibitor of
cellulase in the medium, or by limiting the iron supply would
provide a selective pressure for increased cellulase formation.
These mutants could be isolated and tested for cellulase
formation and changes in composition of the enzyme.
-146-
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