AN ABSTRACT OF THE THESIS OF
Tor Soren Nordmark
for the degree of Master of Science
in
Food Science and Technology presented on November 24. 1993 .
Title:
A Filter Paper Assay for Low Cellulase Activities and the
Cultivation of Trichoderma reesei on Acid Whev and Sweet Whev
Permeate
Abstract approved:
Michael H. Penner
The traditional filter paper assay for saccharifying cellulase
originally described by M. Mandels et al (1976) has been modified to
make
possible
low
activity
determinations
of
Trichoderma
cellulases. The enzymatic activity appears to decline during a
prolonged incubation period if no precautions have been taken. By
means of adding bovine serum albumin and potassium chloride as
protein stabilizers and sodium azide as an antimicrobial agent filter
paper activities in the range from 0.02 to 0.37 (IUPAC assay, 1987)
can be estimated by extending the incubation time
up to 20
hours.
Filter paper activity values obtained by this
method
may be
compared to those obtained by the IUPAC assay by using a conversion
factor from 1.4 to 1.7.
Acid whey and sweet whey permeate have been investigated as
media for growth and metabolite production by Trichoderma reesei
QM 9414 using shake flask cultures and spore inocula. In the case of
acid whey the mycelial growth after 2 weeks is 13 mg dry weight
/ml substrate. The specific growth rate is 0.29 / day.
The fungus
appears to metabolize the whey protein the first 2 weeks. The
alkalinity of acid whey rises continuously over a three week period
up to a pH of 8.5. In the case of whey permeate the maximal mycelial
weight gain is 4.4 mg/ml which appears after 8 days. A rise in net
soluble protein level comes after 3-5 days and reaches a maximum
value of 0.23 mg/ml after 2 weeks. The pH of whey permeate rises
continuously to 7.5 after 3 days and then slowly declines. The net
production of cellulases is low on both media. Dilution 1:6 of the
acid
whey,
supplementation
with
ammonium
adjustments did not enhance the production
sulfate
and
of cellulases.
pHAcid
whey supports a significant growth and sweet whey permeate shows
potential for extracellular protein production.
A literature review surveys the composition and uses of acid
whey,
environmental
aspects
of
whey
wastes,
the
fungus
Trichoderma reesei, the mode of action of the Trichoderma reesei
cellulase system
and
the structure of cellulose in cotton and wood.
A Filter Paper Assay for Low Cellulase Activities
and the Cultivation of Trichoderma reesei on Acid
Whey and Sweet Whey Permeate
by
Tor Soren Nordmark
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed November 24, 1993
Commencement June 1994
APPROVED:
Professor of Food Science and Technology in charge of major
y"
1
r
\
>lead of the Department of Food Science and Technology
,ry in v — | i »ii \y t . i
Dean of GrackJaie Schoo} \
Date thesis is presented
Typed by
November 24. 1993
Tor Soren Nordmark
Acknowledgement
I wish to thank Dr. Michael Penner for encouragement and
supportive ideas throughout the experimental work as well as
assistance during the preparation of this thesis. His ability to give
me freedom in determining research approaches and topics also
stimulated the progress. Dr. Alan Bakalinsky offered me laboratory
space and added valuable views to my microbiological perspectives.
This work has been supported by funds from the Western Dairy
Foods Research Center and the Oregon State University Agricultural
Experiment Station to which I wish to express my gratitude.
Finally I would like to express my gratefulness to Ean-Tun Liaw
and other colleagues in the department who generously made
arrangements in their own work in order to accommodate for my
experiments and with whom I had many helpful discussions.
I I
Table of Contents
1.
General Introduction and Literature Review
1.1
Project goals
1.2
Background
1.3
2.
1
1.2.1
Environmental aspects on whey wastes
2
1.2.2
Whey and whey permeate
4
1.2.3
Industrial processing of whey media
9
1.2.4
The microfungus Trichoderma reesei
11
1.2.5
The Trichoderma reesei cellulase system
14
1.2.6
Cellulose structure and cellulase action
16
1.2.7
The measurement of cellulolytic enzyme
activity
22
Research outline
24
The Application of the Filter Paper Assay for
Cellulase at Low Activity Levels
2.1
Introduction
2.2
Materials and methods
26
2.2.1
Materials
30
2.2.2
Enzyme preparation
30
11 I
2.2.3
2.3
2.4
3.
31
Results and discussion
2.3.1
Principles of an improved assay
33
2.3.2
Activity development studies
38
2.3.3
Stabilization studies
44
Conclusion
50
Cultivation of Trichoderma reesei on Acid Whey
and Sweet Whey Permeate
3.1
Introduction
3.2
Materials and methods
3.3
3.4
4.
Enzymatic hydrolysis
51
3.2.1
Substrates
54
3.2.2
Microbe
55
3.2.3
Incubation experiments
55
3.2.4
Reagents and analytical procedures
56
Results and discussion
3.3.1
The development of mycelial biomass
58
3.3.2
Metabolite production and effects on
substrate composition.
61
Final comments
Bibliography
80
82
Appendices
A.
A protocol for the maintenance and handling of
Trichoderma reesei cultures on potato dextrose
agar
97
B.
Spore counting on PDA plates by using Rose Bengal
102
C.
A protocol for the modified IUPAC filter paper
assay
103
D.
An experiment demonstrating the modified IUPAC
filter paper assay
109
E
The pigment production by Trichoderma reesei
on liquid media
113
F.
A protocol for preincubation and postincubation
processing when recovering proteins on whey
media
114
List of Figures
Fig. 1.
The taxonomy of T. longibrachiatum.
13
Fig. 2.
The Trichoderma reesei cellulase system.
15
Fig. 3.
Cellulose structure.
17
Fig. 4.
Attachment of cellulases to the cellulose.
19
Fig. 5.
Modes of cellulase action.
21
Fig. 6.
The relation between the amount of glucose
released in a filter paper assay and the logarithm
of (A) the enzyme concentration and (B) the
incubation time.
36
Fig. 7.
The level of reducing sugar as glucose produced by
Trichoderma reesei QM 9414 (A) and viride (B)
cellulase during a time extended filter paper assay
in the absence of stabilizers plotted against the
product of enzyme concentration (E as dilution
level) and time (t in hours).
40
Fig. 8.
The amount of reducing sugar as glucose released
by Trichoderma viride cellulase at different
concentrations of non-macerated Whatman #1
filter paper and cellulase activities.
41
Fig. 9.
Incubation of Trichoderma reesei QM 9414 cellulase
when the substrate (Whatman #1 filter paper) is
either (A) always present or (B) present only during
an assay hour ending by the times shown. In case A
four groups of different dilutions each cover the
points where E x t is equal to the original value,
whereas in case B all enzyme is undiluted.
43
V I
Fig. 10.
The level of reducing sugar as glucose produced by
Trichoderma reesei (A) and viride (B) cellulase
during time extended filter paper assays when
stabilizers are used plotted against the product of
enzyme concentration (E as dilution factor) and
time (t in hours). All standard deviation bars are
covered by the symbols.
47
Fig. 11.
The result of filter paper assays (triplicate
samples) using stabilized (S) and non-stabilized
(NS) Trichoderma reesei (A) and viride (B)
cellulase. The conversion factors (f) between the
apparent activities are 1.4 and 1.7 respectively.
48
Fig. 12.
The growth of T. reesei on filter-sterilized acid
whey.
59
Fig. 13.
The growth of T. reesei on filter-sterilized whey
permeate.
62
Fig. 14.
The development of pH when T. reesei is cultivated
on thermally sterilized acid whey.
63
Fig. 15.
The development of soluble protein as BSA when T.
reesei is grown on thermally sterilized acid whey.
65
Fig. 16.
The development of soluble protein as BSA when T.
reesei is grown on filter-sterilized acid whey.
66
Fig. 17.
The amount of glucose released in a filter paper
assay (IUPAC) when T. reesei is cultivated on
filter-sterilized acid whey.
68
Fig. 18.
The time course of pH when T. reesei is grown on
filter-sterilized acid whey that has been diluted
1:6.
69
VI I
Fig. 19.
The development of pH when T. reesei is grown on
filter-sterilized sweet whey permeate.
71
Fig. 20.
The development of soluble protein as BSA when T.
reesei is grown on filter-sterilized sweet whey
permeate.
72
Fig. 21.
The amount of glucose released in a filter paper
assay (IUPAC) when T. reesei is cultivated on
filter-sterilized sweet whey permeate.
74
Fig. 22.
The effect of 0.1 % (w/v) Solkafloc addition on the
level of soluble protein as BSA when T. reesei is
grown on filter-sterilized sweet whey permeate
76
Fig. 23.
The change in lactose level with and without added
0.1 % (w/v) Solkafloc when T. reesei is grown on
filter-sterilized sweet whey permeate.
78
Fig. 24.
If 0.001 % Rose Bengal is added to the PDA then the
7". reesei colonies will appear more distinct and
limited as can be seen from the photos above.
Higher concentrations can kill the fungus.
102
Fig. 25.
The amount of reducing sugar as glucose released
in a modified filter paper assay for different
incubation times and for 2 dilutions. T. viride
cellulase is used.
111
VIII
List of Tables
Table 1.
The chemical composition of acid whey.
Table 2.
Relative filter paper activities obtained
from modified assay data for different
dilutions compared with expected values
based on modified assay data for a stock
solution of T.viride cellulase.
112
Table 3.
The pigment production by Trichoderma
reesei on liquid media.
113
7
A FILTER PAPER ASSAY FOR LOW CELLULASE ACTIVITIES AND THE
CULTIVATION OF TRICHODERMA REESEION ACID WHEY AND SWEET
WHEY PERMEATE
1 . GENERAL INTRODUCTION AND LITERATURE REVIEW
1.1 PROJECT GOALS
The present study was undertaken as a part of the general
efforts to find useful applications for acid whey and other whey
products. One of the foci was to explore the properties of acid whey
as a substrate for the cultivation of the microfungus Trichoderma
reesei.
The growth of the fungus as well as its excretion of
metabolic
products
into
the
whey
were
of
interest.
Little
information in this matter is available and thus few processes or
projects have been started where acid whey is employed for the
production of useful commodities.
At the same time there was a need to develop and refine
analytical tools that could be utilized for whey products. Among the
metabolites produced by Trichoderma reesei is the cellulase system
- a group of enzymes that degrade the cellulosic part of such
materials as vegetable and fruit peel, cotton and wood. Thus the
applications of an assay designed for the
often
occurring
low
cellulolytic activities could be widespread. Obviously, access to
such
an assay would be of value when
investigating cellulase
production by Trichoderma reesei growing on acid whey.
Finally spin-off effects of this study would include extended
know-how about the
handling and processing
of whey
related
entities under cultivation conditions.
1.2
1.2.1
BACKGROUND
Environmental aspects on whey wastes
For every kg of milk used in cheese manufacture about 90 %
ends up as fluid whey. The U. S. dairy plants produce 20 billion kg of
whey annually (Abril and Stull 1989, Sell 1992) including 10 % acid
whey (Hui 1992). One third of this amount is directly utilized in
human foods or animal feeds. The remaining two thirds of the whey
is, for instance,
disposed
of
as
landfill
(irrigation)
by
pipeline
or truck transfer (Sell 1992). In areas with abundant rainfall and
vegetation some fertilization benefits can be obtained.
Even more
purposefully research has demonstrated that acid whey can be used
effectively
in reclaiming
sodic
soil
(Jones
et
al
1993).
However
the high levels of nitrogen, phosphorus and minerals in whey can kill
vegetation and the spread has thus to be managed carefully (Zall
1979).
The disposal of whey remains the greatest waste problem for
the dairy
industry
because
of
the
great
volume,
the
high
biological oxygen demand (BOD) and the often acidic properties.
Nearly 90 % of U. S.
(Sell 1992)
dairy plants are connected to
sewer
systems
and most of the whey waste is still transported to them
or directly into streams. A dairy plant may discharge 10-100,000 kg
whey daily. The BOD for whey is 30-60,000 mg/liter (the lower
range applies to acid whey). The lactose accounts for 90 % of this
value. Every 1000 kg whey requires 4.5 million liters of fresh
aerated water for its biological oxidation. This corresponds to the
daily BOD load from 470 people. Obviously concern is raised about
the damage to aquatic life support systems and the overload on
municipal sewage treatment
facilities.
Thus
the load from one
dairy plant is equal to that from a medium size town (Zall 1979).
Acid whey has currently very limited
relatively more of such whey is being
applications
disposed
of.
and thus
Its acidic
character
causes
corrosion to metal tanks and pipelines thereby
increasing the cost of its handling. The use of ultrafiltration through
2
nm
membranes
for
the
purposes
of
protein
removal
and
concentration has in recent years emerged as a promising whey
treatment alternative. Lactose can then be removed by means of
reverse osmosis and the product has a BOD of only 3 % of its original
value (Sell 1992).
Agricultural waste, domestic refuse and industrial waste are
the three major waste sources in the human society. As indicated
above some management alternatives are to make use of the socalled waste (minimization [Snider 1992]) and to perform waste
treatment before disposal (Kharbanda and Stallworthy 1990). As
with all changes,
values
and
costs
are
involved
which may slow
down the progress towards a more harmonic relation to natural
resources. This thesis project should be seen as a contribution in
that direction.
1.2.2
Whev and whev permeate
Whey is the liquid drained from the curd when making cheese or
manufacturing casein. If the goal is to produce swiss or cheddar type
cheeses,
pasteurized
milk
is first
inoculated with
lactic acid
culture and then the enzyme rennet is added to coagulate the
caseins. The coagulum is cut into small blocks and heat treated to
expel whey, whereafter the whey is drained out. The cheese is then
generally left for ripening. The whey obtained from this procedure is
sweet whey that has a maximum titratable acidity of 0.16 % as
lactic acid (Code of Federal Regulations, April 1993) and usually a
pH within the range 5.8-6.6 (Zadow 1986). When cottage cheese or
quarg type cheeses are produced, skim milk is the common raw
material. It is pasteurized at 72
Short Time)
and a
0
C for 15 sec. (High Temperature
high amount of starter culture -
usually
Lactococcus lactis and cremoris together with a flavor giving strain
such as Leuconostoc
diacetylactis
citrovorum (Scott 1981) or Lactococcus
- are then added. Nowadays a small amount of rennet
is also often supplied to improve the firmness of the coagulum. Part
or
all of the lactic
culture
may
be replaced by
naturally
formed
lactic acid or by hydrochloric acid. The cottage cheese curd is heat
treated at 50-55 0C for 1-2 hours (Fox 1987) and the whey is then
removed. The quarg curd is traditionally not heat treated and the
final cheese product has a smoother consistency. Acid whey has a pH
in the range 4.0-5.0 and a titratable acidity of 0.4-0.6 % as lactic
acid (Zall 1979). If the acid present is due to the action of the
starter culture, a significant amount of lactose - approximately 0.7
% - is used up. Other cheesemaking procedures may result in
titratable acidity and pH values between those for sweet and acid
whey (Zadow 1986).
The whey composition varies with farm, breed, season, type of
cheese and aging of the whey. About half of the milk solids are
present in whey. Average data for acid whey are listed in Table 1.
Minerals are listed in order of descending concentration. From an
operational standpoint, however, proteins that are present in the pH
4.6 supernatant of milk are referred to as whey proteins. That
includes the
proteose peptone
fraction which is essentially a set of
heat stable fragments from betacasein (Hui 1993). Whey products
have GRAS status (Code of Federal Regulations, April 1993) and are
considered of good nutritional value for human use (Zadow 1986).
The whey proteins have a balanced amino acid composition and a high
digestibility (Hui 1992).
Whey and in particular acid whey is rich in
Table 1.
The chemical composition of acid whey.
Lactose
4.3 %
Lactic acid
0.7 %
Protein
0.7 %
Betalactoglobulins
Alfalactalbumins
Bovine serum albumin
Immunoglobulins (IgG)
Proteose peptones,
residual casein and
enzymes
Fat
(Sat'd : Monounsat'd
= 20 : 10 : 1)
50%
15%
5%
10%
20%
Polyunsat'd =
Ash
Major aminoacids
Glutamic acid
Aspartic acid
Leucine
Lysine
0.04 % (skim milk)
0.3-0.9 % (whole milk)
0.5 % (0.4 - 0.8 %)
Potassium
Calcium
P, Cl, Na, S, Mg
Zn, Fe, Cu, Mn, Se
1.4 g/kg
1.0 g/kg
Vitamins
B2
Pantothenic acid
B12
Water
1.2 ppm
3.3 ppm
1.5 ppb
93.7 %
8
minerals such
as calcium and
phosphorus.
The
contents
of water
soluble vitamins such as riboflavin are also of importance. The
lactose has low sweetness and that is considered an advantage in
some food applications. Whey cheeses - gjeitost or mysost type are popular in some countries. They are produced by cooking the
whey until the desired concentration of solids is achieved. People
who
do
not
have
sufficient
betagalactosidase
activity
in
their
digestive tract will have problems with these products due to
lactose
intolerance.
Available
indicate that all of the
composition
nutrients generally
data
for whey
also
required for fungal
growth are present. Unpasteurized whey has a high count of molds
and yeasts.
Ultrafiltration of whey results in whey permeate that can be
sweet or acid depending
on the raw material.
Common
membranes
have cut-off values around 15-20 kD and thus most proteinaceous
material is removed. The whey may first have been clarified from
phospholipoproteins by an initial heat treatment in the presence of
calcium and a following microfiltration using ceramic membranes
(Daufin et al 1992).
1.2.3 Industrial processing of whey media
Sweet whey aimed for use in foods must be processed within
hours of its removal from the curd.
Acid whey at a pH of 4.7 or
lower is more stable because of the inhibited bacterial growth. The
whey is first pasteurized and concentrated by means of vacuum
evaporation or reverse osmosis in the plant and then traditionally
spray-dried to yield whey powder. Caking and lumping in whey based
products may be due to lactose hygroscopicity. In order to avoid this
phenomenon, or to get delactosed whey powder, crystallization
should be performed before the spray-drying step (Hui 1992, Zadow
1986). Demineralized whey can be obtained by using electrodialysis.
Acid whey from a cottage cheese plant has reportedly been clarified
and treated with cationic and anionic columns to remove most of the
salt and lactose before it is used in vegetable oil based dips and
other products (Schexnayder 1991). Efforts to use acid whey in
fermentation processes have been made (Stewart and Gilliland 1982)
as well as to use it in sweet syrups after ultrafiltration and direct
enzymatic
ultrafiltrated
treatment
whey
(Abril
leaves
and
a
Stull
1989).
retentate,
i. e.
Clarified
whey
and
protein
10
concentrate (WPC). This product can be utilized in baking to improve
foaming properties. Upon heating dispersions of WPC can act as
gelling agents
mainly due to the content of betalactoglobulin. It may
be further processed to yield individual whey proteins or hydrolyzed
to yield peptides for specific dietetic purposes. A main application
of WPC's is in the production of baby formulas (Hui 1992).
Ultrafiltration of unpretreated whey and particularly acid whey
causes extensive membrane fouling and flux reduction. This varies
with membrane type but is generally due to the phospholipoproteins
and is markedly increased by the mineral contents, i. e. calcium.
Cottage cheese whey is rich in colloidal calcium phosphate (Heng and
Glatz 1991, Morr 1989, Pierre et al 1992). Sugars as lactose tend to
precipitate in the membrane pores (Kuo and Cheryan 1983). Removal
or modification of the phospholipoproteins or chelation
of the
calcium will improve the flux rate. It has also been observed that
increased transmembrane pressure results in increased
retention of
calcium and lactose when cottage cheese whey is ultrafiltrated
(Roehl 1990).
11
1.2.4
The microfungus Trichoderma reesei
Trichoderma sp. constitute up to 3 % of the total fungal
population in
character.
many forest soils especially those with an acidic
They
are
likely
to
be
found
in
abundance
in
the
rhizosphere in coniferous forests as well as in the litter of humid
mixed hardwood forests (Eveleigh 1985, Kendrick and Parkinson
1990). Pasture soils and a wide range of other habitats also contain
Trichoderma sp. As saprobes they are important in the natural
recycling of nutrients. They are metabolically versatile and degrade
a variety of carbonic substances such as cellulose, starch, pectin,
xylan,
laminaran and chitin.
Nitrogen sources like proteins and
ammonium compounds are all readily utilized.
Potassium
is a major
mineral requirement and it is also a major wood ash and whey
component. Trichoderma sp. can degrade hydrocarbons and are thus
found
in
oil-polluted
soils
and
even
in
jet
fuel,
rubber
and
polyethylenes (Eveleigh 1985). They are aerobic and have a septate
mycelium
chitin.
with
The
the
wild
longibrachiatum
cell
type
wall made up of layers of betaglucans and
ancestor
to
(Montenecourt 1983),
T.
reesei QM 9414 is
which
is a mesophilic
T.
soft-
12
rot fungus. Its general placement among living organisms can be
seen in Figure 1. There are still considerable problems with the
taxonomic
classification
of
species
(Herrera-Estrella
1993),
however, and the morphological differences between Trichoderma sp.
is a task for expert microbiologists (Eveleigh 1985). The colonies of
T. reesei QM 9414 often appear yellow due to pigment production.
Under restrained conditions and provided some short exposure to
blue light has occurred (see section 1.2.5), green rough-walled
conidia (asexual spores) are produced from tip cells in the branched
mycelium. Hyphal tips are also the centra for protein synthesis. The
presence of sterile mycelium makes colonies appear white.
The T. reesei QM 9414 genome has a size of
at
least
33
million basepairs that are located on seven different chromosomes
(Maentylae et al 1992). Cellulase genes are found on two of them.
Thus both exoglucanases (CBH) and one endoglucanase (EG) gene are
located on one single chromosome whereas another endoglucanase
gene is found on a different chromosome. These genes have been
expressed in hosts to obtain more pure cellulases for the study of
cellulase action.
The QM 9414
strain produces relatively small
13
Kingdom FUNGI
Class ASCOMYCETES
Subclass EUASCOMYCOTIDAE
Order HYPOCREALES
Family HYPOCREACEAE
Teleomorph:
Anamorph:
Genus HYPOCREA
TRICHODERMA
Species RUFA
LONGIBRACHIATUM
Strain:
First called T. viride
Renamed T. reeseiQM 6A
Fig. 1. The taxonomy of T. longibrachiatum.
14
amounts of betaglucosidase (BG). The cellulases display high
intrinsic
stability
at
room
temperature,
but
their
activity
is
affected by periods of drying or freezing and more so in the first
case than in the second (Sinsabaugh and Linkins 1989).
Although
not generally considered as an
invasive human
pathogen, T.viride has been able to cause fungal peritonitis in a
weakened individual (Loeppky et al 1983). Another patient was
proven to have a pulmonary fungus ball consisting solely of T. viride
mycelia (Escudero et al 1976).
People
with
specific
sensitivity or
who are already afflicted by a fungal skin disease should protect
themselves from direct contact with the mycelium.
1.2.5 The Trichoderma reesei cellulase system
Although an inducer of cellulase in Trichoderma
reesei is still
to be found, much information has been collected regarding the
regulatory mechanisms in this fungus (see Fig. 2).
Cellulose,
sophorose, maltose, lactose, glucose and many others are all able to
provoke synthesis of cellulases. A conidial cellulase
system
is
known to exist with a composition that differs from the mycelial
cellulase system.
Access to some light is of importance in the
15
CONIDIA:
Constitutive cellulases:
cell wall bound:
50 % CBH II
25 % CBH I
EG III
plasma membrane bound:
betaglucosldase (alkaline)
inhibits CBH & EG
synthesis & activity
exo-exo synergistic ^^^
attack on crystalline ""™
cellulose
(all complete cellulases
known contain CBHs)
cellobiose (major product)
cello-oligosaccharides
d-cellobiono-1,5-lactone '
(from oxidized cellulose)
MYCELIUM:
Sporulation
within 24 hours
betalinked disaccharide
permease
4
CBH II expression
enhanced
responding nucleotide
palindrome in the CBH II
promotor
transglycosylation
via intracellular betaglucosldase
(provokable by sophorose)
or EG I
t
other intermediate
provoker(s)
cyclic AMP elevated
\
or
extracellular
betaglucosldase
(pH>S)
glucose
' repression of
betaglucosldase
synthesis
sophorose (pH 3 optimum)
/
"true" inducer
adenyl cyclase elevated
Light at 3B5 nm and 445 nm
peaks starts induction
gene transcription within 20 min. and with
constant ratio between CBH I, CBH II and EG I
translation on ribosomes/ER
(more ER present in hypersecreting mutants)
Light also stimulates:
* hyphal branching (on solid media)
* the TCA cycle whereby the ATP
level rises
vacuoles (with EG I) & Golgi bodies;
enzymatic regulation of cellulases
plasma membrane
Attack on cellulose:
-^
CBHs and most EGs contain cellulose
binding domains; cell wall heteroglycans
may have affinity for cellulose.
EGs rapidly hydrolyse amorphous
cellulose.
Synergism exists between CBH I, CBH II
and EGs.
Fig. 2.
Release ot induced cellulases:
up to 70% of soluble protein is cellulases,
all of which are N-glycosylated for heal
and proteolysis stability; CBHs and EGs
may be O-glycosylated to ease secretion.
Different enzyme systems are exhibited
on different substrates. One example:
60% CBH I
20% CBH II
10% EG I
EG III and betaolucosidase
The Trichoderma reesei cellulase system.
16
lifecycle of Trichoderma reesei primarily because it is required for
sporulation in this asexual fungus. Constitutive cellulases on the
spore surface start the breakdown of cellulose to smaller molecules
that can be recognized by the fungus as the sugars just mentioned
are recognized. Transport mechanisms carry the information to the
genetic material where transcription
occurs.
Following
translation
enzymatic modification of the cellulases takes place, whereafter
they are released from the cell surface (Farkas et al 1990, Kubicek
1987, Kubicek et al 1990, Kubicek et al 1993, Messner et al 1991,
Messner and Kubicek 1990 and Szakmary et al 1991).
1.2.6 Cellulose structure and cellulase action
Cellulose as a macromolecular substance with many origins has
been a hard object to get grasp of in terms of its many variants and
how the functional properties are related to the structure. Recent
work has cast new light on this issue (see Fig. 3-4). It is apparent
that efforts to quantify the interaction between cellulose and its
environment must be a complicated task.
Although
its smallest
repeating unit is cellobiose that in turn consists of 2 glucose
(glucopyranosyl) units,
there are numerous options for the design of
17
The structures considered are primarily mature native cotton and wood.
The chemical reactions only take place at the submicrofibhl level or lower.
Cellulose fiber
Macrofibril
Macrofibril (parallel) or
microfibril (parallel or
randomly woven) layers
wound around a central
axis; reversal of direction
occurs.
Up to a few hundreds
of microfibrils spaced
at 20 nm (but depends
on the degree of swelling).
Diameter less than 15
microns. Cavities and
pores may be present.
Submicrofibril
Microfibril
Consists of stacked unit crystal
cells of parallel cellulose chains
lined up in distinct planar layers.
Diameter 2-6 nm.The cellulose
chains - as well as the submicrofibrils themselves - may be
twisted together in some species.
9-12 submicrofibrils (elementary
fibrils) together with amorphous
regions every 100 nm of length.
Diameter 3-10 nm, length 0.11000 microns. Small 60 nm
long crystallites (micelles) of
stacked glucan sheets connected by
weak Van der Waal's forces can be
found.
Unit crystal cell
Cellulose molecule
Ribbon-like approx. 2-fold
helix of 2 cellulose chains
(1 staggered central chain
and 4 quarter chains) in all
polymorphs. The native
monoclinic unit crystal cell
dimensions are 0.8x0.8x1.0
nm depending on cellulose
source and polymorph.
Repeated 1.0 nm long
beta-1,4-linked anhydrocellobiose units. The D.P.
is 3,500-36,000 glucopyranosyl units, that are
in chair conformation and
alternatingly rotated half
a turn along the chain
length. Non-reducing ends
appear to be stereochemically identical.
Fig. 3.
Cellulose structure.
18
The six known polymorphs
result from a set of binding
options; they thereby differ
in density and surface area.
Most native cellulose is of
type I where k1, l-beta and
parallell arrangement
dominate.
Secondary structure
of intrachain H-bonds:
k1 = C6-0 and ring-0 bind to
C3-0 on the adjacent
glucopyranosyl residue
and k2 type bonding from
these 2 residues to the
connecting residues on
other cellobiose units.
K2= Ring-0 binds to C3-0 on
the adjacent residue.
Fig. 3 cont'd.
Cellulose structure.
Tertiary
structure:
A) Different lattice parameters.
B) Different interchain hydrogen
bonding pattern (types l-alpha
and the more stable l-beta).
C) Parallel or antiparallel (that
provides for intersheet H-bonds)
chain polarity.
19
Accessibility
Binding to
crystalline
surface
Binding to
amorphous
region
Fig. 4.
Dimensions and shape vary
with cellulose morphology,
type of enzyme and stage of
hydrolysis. The CBH's are
5x20 nm in size. Competitive
adsorption may occur.
Tightness of
binding
Increased crystallinity results
in increased affinity for both
CBH's and EG's. The glycosylation in their binding
domains may provide for
hydrophobic interactions that
are enhanced by decreased pH.
Synergism requires good
binding.
The surface area is
4 times larger than
in crystalline regions.
The binding domain in
the cellulase is of
less importance.
Water can be H-bonded
monomolecularly to the
hydroxylgroups (and on
crystalline surfaces); it
can also be loosely adsorbed
polymolecularly. Swelling
will reduce the crystallinity
left and increase accessibility
while water breaks H-bonds
and rearranges the higher
order structure of cellulose.
Attachment of cellulases to the cellulose.
20
the
whole
cellulosic
polymerization
(D.
complemented
with
properties
other
of
material.
P.)
and
many
Characteristics
crystallinity
other
substances
index
as
(C.
degree
of
must
be
I.)
data
such
as
the
adsorption
like
cellulases
to
accessible
cellulose surfaces over time. Some cellulosic materials appear more
inert than others. Thus filter paper and microcrystalline cellulose
(Avicel)
are
relatively
inert
substrates,
whereas
mechanically
treated, amorphous cellulose is much easier to degrade when using
cellulolytic enzymes. (Atalla and Vanderhart 1989, Coughlan 1992,
Dinwoodie
1989,
Kirk-Othmer 1979,
Nevell
and Zeronian
1985,
Seymour and Carraher 1988, Sarko 1985, Turbak and Sakthivel 1990,
Weimer et al 1991).
The
mechanisms
by which
cellulase
systems
degrade cellulose are slowly being revealed. The access to more pure
crystalline
cellulose
as
well
as
the
possibility
of
expressing
cellulase genes in hosts to obtain more pure or modified enzymes
have contributed considerably to this progress. The cellulase system
of T. reesei is the best studied and the features of its degradation of
cellulose are outlined in Figure 5. Endoglucanases (EG) make cuts in
the crystalline cellulose surface thereby leaving fragments that can
21
Synergism occurs primarily in the degradation of crystalline regions but not lor all types of substrate
and the extent varies considerably with the concentrations and conditions involved. The hydrolytic
mechanisms employed by the cellulase active sites have been described as lysozyme like and beta-amylase
like. The numbers 1-8 below refer to a time sequence of events. The T. reesei system is featured.
(1)
Diffusion by CBH's and EG's break the
weak H-bonds between the (nicrofibrils.
Microfibril
(2a) Endoglucanases (EG III)
EG's help prepare crystalline
regions for attack by CBH's but
easily degrade amorphous
regions, easier if the cellulose
is in a staggered array. They
attach readily along the
microfibril surface and
cleave internal glycosidic
bonds in the amorphous
cellulose, whereby free
chain ends become exposed
for action by CBH's.
(3a) CBH I
Binds along crystal edges and
cuts reducing ends. It cooperates synergisticaliy with EG's
in detachment and digestion
of surface submicrofibrils. It
facilitates EG's disruptive
work upon binding and cuts
the outermost chains in
the submicrofibril whiskers.
The CBH activity creates
clusters and plaques of
digestion products.
(3b) CBH II
This enzyme is more substrate
specific than CBH I and binds
preferentially to crystal tips.
Cooperates synergisticaliy with
EG's in amorphous parts and with
CBH I in crystalline parts when
cutting liberated submicrofibril
chains, since CBH I prepares new
sites for attack by CBH II.
CBH II removes cellobiose
units from the non-reducing end
It degrades amorphous
cellulose less efficiently, although
synergism between CBH I and
CBH II has been reported on
amorphous substrate. Complex
formation between CBH's occur.
Fig. 5.
Modes of cellulase action.
(4)
Possible water uptake after addition
of CBH's results in increased accessible surface area.
(2b) Endoglucanases (EG III)
Apart from amorphous regions, EG's
may make superficial cuts in the
crystalline surface. This activity is
facilitated if the area is weakened
by crystal faults, skewing of adjacent
sheets or binding of other enzymes,
intersheet hydrogen bonds would
have an opposite effect. Whiskerlike
submicrofibrils, that remain attached
to the lattice, are splayed off from
the surface. The EG binding domain
may help unzip the microfibril by
y disrupting the (H-) bonding pattern
V in the crystal structure. When acting
I (at the common pi) in a complex with
/ CBH, maximal synergism is obtained.
Only crystal faces with accessible
2,3- and 2,3,6- hydroxyl groups are
attacked.
EG I has minimal binding to
crystalline cellulose and appears not
to act synergisticaliy with CBH's.
Formation of Clusters (S) and
Plaques (6), composed of swollen
but water insoluble cellulose intermediates together with crystal and
chain fragments in various proportions.
Crossfracture (7) caused by the
action of an EG-CBH complex.
Beta-Glucosidase
Cleaves cellobiose to glucose units,
whereby product inhibition of EG and
CBH activities is decreased. Weak
inhibition by glucose of the synergism
between EG and CBH has been reported.
Some EG's may convert cellobiose
to glucose by transglycosylation
if cellulose is present.
22
be further degraded by the exoglucanases (CBH). The CBH's are not
required for the degradation of the amorphous regions of the
cellulose. Cellobiases (betaglucosidases) will cleave cellobiose to
glucose. (Beguin and Aubert 1992, Hoshino et al 1992, Klyosov 1990,
Sprey and Bochem 1991, 1992 and 1993, Tomme et al 1990, Walker
and Wilson 1991, White 1982).
1.2.7 The measurement of cellulolvtic enzyme activity
There is widespread interest in the application of cellulase
enzymes
to
utilization,
technologies
textile
associated
processing,
food
with
forest
processing
products
and
biomass
conversion.
These technologies are dependent on inexpensive
sources
cellulase
of
Consequently,
enzymes
considerable
with
research
high
specific
activity
is
activities.
directed
at
identifying novel cellulase enzyme systems with properties which
may be exploited for industrial purposes.
for
any
of these
studies
measurement of cellulase
degrade
native
cellulose
is
an
activity.
secrete
An obvious prerequisite
appropriate
method for
Microorganisms
a
which
the
readily
family of cellulolytic enzymes
which act in concert to solubilize/saccharify
crystalline
cellulose.
23
In many cases it is of primary interest to measure the specific
activity of the complete enzyme
microbe.
mixture
produced
by a given
This is in contrast to assays designed to measure the
activity of a specific component of the complete enzyme system.
The need for an assay of complete cellulase activity is particularly
true for the cellulases due to the well documented
synergism
between
that
individual
cellulase
enzymes.
Substrates
are
particularly good for measuring complete cellulase systems are
those which contain an appreciable fraction of crystalline cellulose,
such as filter paper, Avicel and cotton. Thus the water soluble
carboxymethyl cellulose (CMC) is no longer used for the estimation
of total cellulase activity. Viscosimetric methods were designed for
evaluation of the degree of hydrolysis in the produced slurry. At the
present time the most widely used assay for total cellulase activity
is the IUPAC filter paper assay (Ghose 1987), the origin of which can
be traced back 25 years (Mandels and Weber 1969). The experimental
work described
in section 2 considerably extends the
range
of
detectable activities while building on the principles of the IUPAC
assay.
24
1.3 RESEARCH OUTLINE
Trichoderma reesei QM
9414 was
used to evaluate the
properties of acid whey as a growth substrate for Trichoderma
microfungi.
Acid whey batches were
obtained
from two different
regional dairy plants in order to work with commercial type
substrates. Ultrafiltrated whey was used as a complementary whey
based substrate. It is easier to process whey permeate than acid
whey. The employment of whey permeate also offered an opportunity
to determine the effects of the whey proteins on the outcome of the
experiments. Efforts were made to keep the whey media as close to
unprocessed characteristics as possible despite the necessity of
sterilization
and
pretreatment.
Laboratory
protocols
for
the
processing of the culture samples and in some cases improved assay
methods had to be developed before comparative studies could be
carried out. In most cases, the parameters monitored included fungal
growth, protein production,
pH of the medium and production of
cellulase enzyme activity. Experiments were designed to evaluate
the major period of fungal growth,
i. e.
prior to
extensive
mycelial
25
deterioration. Efforts were made to boost the yield of cellulase
enzymes in some cultures.
A major part of this thesis involved the development of a
cellulase
enzyme assay appropriate
for
enzyme
solutions
of
relatively low activity. The first part of this phase of the research
was to evaluate the fate of cellulase enzymes during prolonged
incubation under IUPAC filter paper assay conditions. The IUPAC
assay conditions were then modified to accommodate enzyme
preparations
identification
of
low
activity.
of appropriate
This
modification
enzyme stabilizers.
involved
the
To ensure a broad
applicability, a commercial cellulase preparation from Trichoderma
viride was tested in addition to Trichoderma reesei QM 9414.
26
2. THE APPLICATION OF THE FILTER PAPER ASSAY FOR
CELLULASE AT LOW ACTIVITY LEVELS
Key words: Cellulase activity, cellulase stability, filter paper
assay, incubation time, Trichoderma.
2.1 INTRODUCTION
Cellulases are produced by a large number of aerobic and
anaerobic fungi and bacteria. Trichoderma sp. are known for their
potent
ability
to
enzymatically
degrade
Cellulolytic enzymes have found
cellulosic
materials.
applications in the fields
of
forestry, biomass conversion, food processing and cellulosic waste
treatment.
The
amount,
type
and
cooperative
pattern
of the
molecules active in any given cellulase system depend on the source
of the enzyme, the substrate and the environmental conditions
(Cochet
1991).
Three
classes
of
cellulase
enzymes
cellobiohydrolases (CBH), endoglucanases (EG) and betaglucosidases
(BG) - are now well recognized. Only a few organisms produce
complete cellulase systems, i.e.
they possess a pronounced ability
(Stahlberg et al 1993) to break down crystalline cellulose.
27
In actual biomass material cellulose
in
other structures containing
is generally embedded
hemicelluloses,
lignin,
proteins,
lipids etc. It is difficult to assess cellulase enzyme preparations of
lignocellulosic
raw
materials.
Several
cellulase
assays
exist;
however, it has been difficult to correlate the different assays due
to the complexity of the substrates and the synergism between the
enzymes.
of
Currently,
filter paper
are
cellulase assays
widely
based
accepted. The
on
the
early
degradation
filter
paper
assay from 1969 (Mandels and Weber 1969) was modified a few
years
later
in
order to
of activities
and to
amorphous
and
et al
obtain
be
glucose
The
linearity
better account for a
crystalline substrates
(1976) suggested
should
good
that
calculated
released
assay
from
recommended
combined
the
50
by
mg
the
activity
(Griffin 1973).
international
on
a
over a wide range
piece
IUPAC
Mandels
enzyme
basis
of
of
filter
on
units
2
mg
paper.
Commission
on
Biotechnology is the filter paper assay described by Ghose (Ghose
1987). If appropriate dilutions have been made,
how
the
amount
a graph displaying
of reducing sugar as glucose varies with the
28
logarithm
of
concentration
the
enzyme
corresponding to
concentration
will
provide
exactly 2 mg glucose.
the
International
Units (IU) are defined as the number of (imoles/min of substrate
converted.
If each complete hydrolytic event is considered to
produce 1 molecule of reducing sugar (glucose), then 0.37 lU/ml only
need to be divided by the concentration (i.e. the inverse of the
dilution factor) in order to get the filter paper activity (FPA). Low
cellulase activities as defined by this assay are encountered in many
applications. A low level can be due to type of strain, growth
medium,
time
of
harvest
and
application
conditions.
The
measurement of low activities is difficult since the traditional plot
of reducing sugar versus the logarithm of the enzyme concentration
becomes less steep and is non-linear. Thus extrapolation of low
activity values is not appropriate. To assay low activity solutions
one
must
concentrate
samples
by
laborious
ultrafiltration
or
freezedrying, with a risk of some loss of activity (Roseiro et al
1993). No other method is currently available for the determination
of lUPAC-FPA's for low activity samples.
29
Forintek Canada Corp., Ottawa has described a filter paper assay
resembling lUPAC's except that lower amounts of glucose/ml
are
targeted,
the
the
activity
values
thereby
being
larger
than
lUPAC-FPA's (Chan et al 1989). Another approach for low activity
samples involves the addition of a small level of BG to cellulases
(Breuil et al 1992) of low BG activity as is the case for Trichoderma
reesei. Apart from BG addition the ratio between FPA and cellobiase
(BG) activity has been suggested as a measure of the hydrolytic
potential of cellulase enzyme preparations (Cochet 1991). The
significance of any of these filter paper based assays obviously
depends on how the enzyme is used and the substrate employed.
The method described in the following permits the estimation of
lUPAC-FPA's at levels up to 20 times lower than those
previously
assessable. No additional sample processing and no expensive
reagents are required.
30
2.2 MATERIALS AND METHODS
2.2.1
Materials
Reagent components were dinitrosalicylic acid (Sigma Chemical
Co., MO)
and
phenol and
from
sodium
sodium
Mallinckrodt
cellulase
hydroxide,
metabisulfite
Specialty
stabilizing
agents
potassium
all
of
Chemicals
tested
were
sodium tartrate,
which
were
obtained
Co.,
KY.
Potential
sorbitol,
D-mannitol,
inositol, glycerol, L-ascorbic acid, bovine serum albumin (fraction
V), heavy mineral oil, potassium chloride, calcium chloride and
magnesium sulfate. The antibacterial agent sodium azide was added
at 0.02 M concentration to the citrate buffer.
2.2.2
Enzvme preparation
Trichoderma reesei QM 9414, from which a complete enzyme
system is obtained, was grown in our laboratory using shake-flask
cultures
for
7
days
at
27 - 28.5
0
C
in
dim
light.
The
hammer-milled spruce cellulose, Solkafloc SW 40 (James River Inc.,
Berlin,
NH), was used at the concentration level of 8 g/l for
cellulase
ammonium
production
sulfate,
together
2 g/l
with
Mandel's
medium
monobasic potassium phosphate,
[ 1.5 g/l
0.32 g/l
31
calcium chloride, 0.32 g/l
magnesium sulfate heptahydrate, 1
ml/I
trace metal solution (containing ferrous and manganese sulfate, zinc
chloride and cobalt chloride), 0.31 g/l urea, 0.8 g/l proteose peptone
(DIFCO Laboratories, Ml), 1 m/l Tween 80].
pH-adjusted
to 4.8,
filters and
subsequently
Minitan
ultrafiltration
filtered through
1.2 fim
concentrated
unit
(Millipore
The flask contents were
glass
20-fold
Corp.,
microfiber
by
MA).
using
The
a
enzyme
fraction was first precipitated by addition of cold acetone (70 % of
the total volume). Following centrifugation, the enzyme precipitate
was washed three times with 100 % acetone, then vacuum dried at +
2 "C
using a Rotovapor equipment (Buchi Co.). The result was a
crude enzyme powder, which was stored at + 4 0C. The commercial
preparation
Trichoderma
Cellulysin
viride
(Calbiochem
cellulase
was
Corp.,
employed
CA)
containing
without
further
processing.
2.2.3
Enzymatic hydrolysis
IUPAC filter paper assay conditions were used if not otherwise
stated. All filter papers (Whatman #1) were weighed to
1
accuracy. Enzyme stock solutions were made up by
crude
dissolving
mg
32
cellulase
powder
in
double distilled water immediately before use.
The reducing properties of the hydrolysates were evaluated by the
dinitrosalicylic acid assay in all cases (Breuil and Saddler 1985,
Miller 1959).
33
2.3 RESULTS AND DISCUSSION
2.3.1
Principles of an improved assay
The classical Michaelis-Menten theory based on the initial action
of a single enzyme species (E) on a well defined substrate (S) states
that when So » Eo the initial velocity is:
Vo = (Vmax x So)/(KM + So) = (kcat X EQ X SO)/(KM + So)
In case So » KM then VQ is proportional to Eo and the rate of product
(P) formed is:
dP/dt = - dS/dt = VQ = kca, x Eo
Integration of the Michaelis-Menten equation in the same case shows
that P is proportional to time (t) (Kuchel and Ralston 1988).
However, for several reasons these relations cannot be taken for
granted under the condition used in the filter paper assay (Schwald
et al 1988, Mandels et al 1976, Mandels et al 1978). The cellulase
system
consists
of
multiple
enzyme
species
acting
34
synergistically, activators and inhibitors might be present and the
substrate is not of a single type. Steady-state conditions are mostly
lacking
(Tomme et al 1990). In particular, an extension of the
incubation time could cause a decline in enzymatic activity due to
denaturation processes.
Laboratories utilizing
IUPAC filter paper
assay conditions have presented data for 0.5 - 2 hour incubations
employing cellulase activities in the range 0.2 - 0.7 FPU which
suggests that a change in
hydrolysis time may substitute for an
equal change in enzyme activity (concentration) provided that the
same level of substrate conversion is achieved (Ghose 1987, Mandels
et al 1976). This means that E and t may appear
Ext
in
an
symmetrically
as
integrated rate equation, it does not mean that P is
proportional to E and t or that the enzymatic hydrolysis follows
Michaelis-Menten kinetics in the filter paper assay. It is recognized
on empirical basis that, at enzyme concentrations releasing close to
2 mg reducing sugar as glucose in the IUPAC one hour assay,
glucose
level
is
related
to
the
logarithm
of
the
the
enzyme
concentration by a proportionality factor (k), that represents type of
substrate, type of cellulase and other assay conditions:
35
A P = k x A log E
Thus a plot of P vs. log E for different dilutions of an enzyme sample
is a straight line (Reese and Mandels 1971). The same plot for the
same dilutions of an enzyme sample of another activity is also a
straight line parallel to the former line since the k-value is the
same. If Eactive is independent of time, then time can substitute for
enzyme concentration (see below) and a plot of P vs. log t for
different incubation times is also a straight line with the same
k-value as above. An enzyme acting at a m-fold lower concentration
but over a m-fold longer incubation time would result in the same
line as the original enzyme solution would give.
Figure 6 is a
theoretical plot of P against log E and log t for conditions when E
does not change during the incubation period.
The reaction rate in general in the assay is a
function
of
So
and the time dependent variables E, S, A (activator concentration)
and I (inhibitor concentration). If variations in So, A and I between
assays can be considered insignificant and assuming that the effect
of
substrate
recalcitration
etc.
is
time
independent
if
the
final
36
mg glucose
X-cellulase: sample
# 1 and dilution 1:1
(A)
X-cellulase: sample
# 2 and dilution 1:1
(alt sample # 1 and
dilution 1:2)
log E
mg glucose
X-cellulase: sample
# 1 and incubation
time 1 hour
(B)
X-cellulase: sample # 2
and incubation time 1
hour (alt. sample # 1
and incubation time 0.5
hour)
log t
Fig. 6. The relation between the amount of glucose released
in a filter paper assay and the logarithm of (A) the
enzyme concentration and (B) the incubation time.
37
degree of substrate conversion is the same in the assays, then
integration of the rate equation yields the amount of product formed:
t
P=
J f [E(t)] dt
0
If as suggested above P is a function of Eo x t only and thus no
change in cellulolytic activity takes place over time, then a m-fold
decrease in the original enzyme concentration together with a
m-fold increase in the incubation time will always result in the
same P value.
Thus if P is plotted against Eo x t for a set of
different enzyme concentrations, then this set of curves will cover
each other. If on the other hand E varies with time, then the plots for
different enzyme concentrations will appear separated from each
other since, for instance, E may be lowered over a prolonged time
owing to denaturation .
Stabilization of the enzymatic activity could thus allow the
application of an extended time assay when only small amounts of
cellulase are available. Assuming the validity of the model the FPA
of the original sample in the low activity range could
be
calculated
38
from
FPA = [f x 0.37]/[conc. x #hours]
(filter paper units)
where f is a factor that accounts for any difference in hydrolysis
rate due to any necessary assay modification. The experiments were
designed
to
demonstrate
the
validity
of
this
hypothesis
by
investigating the development of cellulase activity over several
hours and trying to find means for maintaining the original activity.
2.3.2 Activity development studies
Any diagram of reducing sugar vs. the product of enzyme
concentration and incubation time for a set of different dilutions
would reveal if there is any time dependent decline in enzymatic
activity (Selwyn 1965). Four different dilutions (1:1, 1:3, 1:9 and
1:27) in duplicates of Trichoderma
reesei
incubated with filter paper during periods
cellulase
of four
were
each
different lengths
(1, 3, 9 and 27 hours). The subsequent DNS assay resulted in the data
plot in Figure 7:A. Since different dilutions appear to give rise to
different graphs the enzyme activity seems to
vary
with
time,
i.e.
39
consistent with
a
loss
of enzyme
activity.
Multistep
thermal
denaturation (Dominguez et al 1992, Lencki et al 1992, Baker et al
1992),
proteolytic
activity
(Haab
inhibition (Huang and Penner 1991,
et al 1981)
et
al
1990)
and
substrate
Liaw and Penner 1990,
Mandels
may be causative agents separately or in combination.
Trichoderma viride cellulase was employed in a study (Fig. 7:B)
that was otherwise identical to the previous experiment. The same
conclusions as before can be drawn in this case and thus an activity
decline is expected to occur in case of other Trichoderma cellulase
preparations as well.
The possibility exists that substrate inhibition takes place with
filter
paper as a substrate under the assay conditions. Therefore
duplicates of 3 different concentrations of
cellulase
were
each
concentrations of
filter
inhibition can
incubated
paper.
No
together
Trichoderma
with
significance
4
for
viride
different
substrate
be extracted from the result shown in Figure 8.
It
may be noted that the glucose level is still relatively dependent on
the substrate concentration at the 50 mg level. Since the Whatman
#1 filter papers have a weight variation
range of ±10 %
(Whatman
40
mg glucose
(A)
Dilution 1:27
-o— Dilution 1:9
Ext
-o— Dilution 1:3
-A
Dilution 1:1
mg glucose
(B)
Ext
Fig. 7. The level of reducing sugar as glucose produced by
Trichoderma reesei QM 9414 (A) and viride (B) cellulase
during a time extended filter paper assay in the absence
of stabilizers plotted against the product of enzyme
concentration (E as dilution level) and time (t in hours).
41
...••■•0
3 -
mg glucose
2-
0
0
*
*»
a
6
1 -
^••-•o
0- —u—T
50
0
1
100
0
1
150
1
200
250
mg filterpaper
—□— 0.005 FPU
—O— 0.02 FPU
—■ o—•
0.08 FPU
Fig. 8. The amount of reducing sugar as glucose released by
Trichoderma viride cellulase at different concentrations
of non-macerated Whatman # 1 filter paper and cellulase
activities.
Co. 1993), weighing is advisable when the filter paper assay is
carried out.
The next experiments (Fig. 9) were conducted in order to further
explore the role of the substrate in the hydrolytic process when the
amount of product formed is kept close to the required 2 mg in the
42
filter paper assay. The reducing sugar time course for Trichoderma
reesei
cellulase
was
followed
when
substrate
was
either
continuously present or present only during an assay hour ending by
the times indicated and the enzyme thus
first
preincubated.
In
the
first case duplicates of 4 different concentrations were incubated
for periods of different lengths so that the product E x t equal to the
original value should be contained in each group of dilutions. As the
figure shows dilution and time are not interchangeable with regard
to the amount of glucose obtained. Rather there is a continuous
decline in activity, thus prohibiting the direct employment of a time
extended filter paper activity concept.
This
is
apparent
also
from
the trend in the second case with an undiluted sample that always
has the original E x t value. These data points lie closely to the data
points from the diluted samples with
the
same
E x t value.
Thus
substrate inhibition is not likely to be a major factor whereas
protease activity, thermal denaturation of grace period or biphasic
type (Lencki et al 1992) and lack of steady-state conditions (Selwyn
1965) remain as possible reasons for the observed time courses.
Measures must be taken in order to prevent an activity change.
43
mg glucose
(A)
i—i—i—i—i—r
9
10
11
12
13
14
15
hours
mg glucose
(B)
i—i—i—i—r
10
11
12
13
14
15
hours
Fig. 9.
Incubation of Trichoderma reesei QM 9414 cellulase
when the substrate (Whatman # 1 filter paper) is
either (A) always present or (B) present only during
an assay hour ending by the times shown. In case A
four groups of different dilutions each cover the
points where E x t is equal to the original value,
whereas in case B all enzyme is undiluted.
44
2.3.3 Stabilization studies
Several conditions can be expected to influence the determined
cellulase
activity.
Commonly
used
protein
stabilizers
include
various sugars, polyhydroxyalcohols, aminoacids and kosmotropic
salts (Kristjansson and Kinsella 1991, Timasheff 1992, Timasheff
and
Arakawa 1989).
Both cellulose and cellulase show some
susceptibility to oxygen attack (Rodriguez et al 1991, Scott 1965).
Metal ions can alter the rate of the reactions (Forouhi and Gunn
1983). Fibers and other macromolecules have a general promoting
and stabilizing effect on
the
cellulase
(Volkin and Klibanov 1989).
Proteins such as BSA can serve as artificial
substrates
for
protein
degrading agents and thus act to preserve cellulase molecules
(Reese and Mandels 1980, Whitaker 1972). Shaking can cause
significant denaturation with loss of apparent activity due mainly to
surface tension forces but also to shear forces
(Tjerneld et al
1991). I have continued the work on stabilization of cellulases that
was conducted by Reese and Mandels in 1980. My pilot screening
studies (data not shown) revealed that sorbitol and L-ascorbic acid
increased the detected level of reducing sugar in the DNS assay.
45
The L-ascorbic acid also appeared to cause a continuous decline in
absorbance readings when the DNS assay was carried out.
The use of
degassed buffer and pure heavy mineral oil covering the incubated
volume did not result in any significant increase in filter paper
activity. Bovine serum albumin appeared to have a good stabilizing
effect and even to increase the apparent FPA. Potassium chloride
also showed good stabilizing properties (Manonmani and Joseph
1993)
but
decreased
lowered
the
the
apparent
activities
FPA.
Glycerol
and
considerably.
Calcium
chloride
magnesium sulfate both caused formation of a precipitate
mannitol
when
DNS reagent was added. Sodium azide at the concentration
did
not
significantly
Mandels
1980).
significantly
influence
Shaking
lowered
the
of
the
the
activity
activity
enzyme
values.
values
until
The
and
the
employed
(Reese
and
foam
formation
most
promising
stabilizers were bovine serum albumin and potassium chloride in
combination.
Accordingly
we
then
employed
potassium
chloride
(1 M)
together with bovine serum albumin (1 mg/ml) as stabilizers
in the
next study where
Trichoderma viride cellulase
Trichoderma
were
utilized.
reesei
The
cellulase
and
experiments were
46
conducted in the same way as the first experiments in order to see
if
a
time
independent enzymatic activity could be achieved. Figure
10 shows that the graphs for different dilutions cover each other
within a 5 % margin. That is not more than the variation in FPA that
can be found when a crude sample is analyzed for cellulolytic
activity. Thus the original stabilized cellulolytic activity appears to
have
been
maintained.
The
results
from
these
two
enzyme
preparations suggest that enzyme concentration and incubation time
can be considered as interchangeable entities when the stabilizers
are used.
protein
I hypothesize that the presence of a large amount of added
and
the
enhanced water structure due to potassium
chloride can be expected to slow down the cellulolytic activity.
Triplicates of cellulase from Trichoderma
viride
were
incubated with
reesei and Trichoderma
and without added
stabilizers
at
dilutions suitable for obtaining a filter paper activity in each case
(i. e. production of 2 mg glucose in a one hour assay).
shown in
Figure 11
where the
lines
The result is
are parallel as expected and
from which the conversion factors (f), i.e. the ratio between the
dilution factors required to reach the 2 mg glucose level,
were
47
mg glucose
(A)
Dilution 1:27
■o
Dilution 1:9
-o—
Dilution 1:3
Ext
Dilution 1:1
mg glucose
(B)
Ext
Fig. 10. The level of reducing sugar as glucose produced by
Trichoderma reesei (A) and viride (B) cellulase during
time extended filter paper assays when stabilizers are
used plotted against the product of enzyme concentration
(E as dilution factor) and time (t in hours). All standard
deviation bars are covered by the symbols.
48
(A)
mg glucose
dilution (log scale)
mg glucose
(B)
0.10
1.00
dilution (log scale)
Fig. 11. The result of filter paper assays (triplicate samples)
using stabilized (S) and non-stabilized (NS) Trichoderma
reesei (A) and viride (B) cellulase. The conversion
factors (f) between the apparent activities are 1.4 and
1.7 respectively.
49
determined
to
variation
this factor should be
in
1.4
and
1.7
Trichoderma cellulase preparations.
respectively. Thus some minor
expected
between different
50
2.4
CONCLUSION
Low filter paper activities from various Trichoderma sp. can be
determined from a modified filter paper assay,
where bovine
serum
albumin, potassium chloride and sodium azide are added and the
incubation time is prolonged up to 20 hours. The slower reaction
rate necessitates the employment of a conversion factor between
the unit defined by this modified assay and the regular FPU. The
equation presented here provides the number of filter paper units
(IUPAC) within a minor error margin.
51
3. CULTIVATION OF TRICHODERMA REESEI QM 9414 ON
ACID WHEY AND SWEET WHEY PERMEATE
Key
words: Acid whey, growth,
whey permeate.
soluble protein,
Trichoderma,
3.1 INTRODUCTION
Acid whey as a fairly rich medium could be expected to support
considerable fungal growth (Smith and Berry 1975). T. reesei grows
on Solkafloc and glucose media despite pH values as low as 3 (Brown
et al 1990) and has thus proven to be acid tolerant enough for use on
acid whey that has a normal pH range from 4 to 5. Acid whey
permeate has proven to yield high ethanol concentrations when
fermented by Saccharomyces
fragilis (Chen 1991). Sweet whey
permeate is used as a growth substrate for the production of
ethanol, lactic acid, citric acid, single cell protein, bakers' yeast
and antibiotics (Hui 1992 and Champagne et al 1990) by other
microbes such as Lactococcus lactis (Christopherson and Zottola
1989),
Lactobacillus
bulgaricus, Kluyveromyces
fragilis,
Kluyveromyces bulgaricus (Zadow 1986) and Aspergillus
(Maddox 1983).
niger
Earlier studies have shown that deproteinated whey
is more promising than sweet whey as a medium for
cellulase
52
production by
1982).
Trichoderma
reesei MCG 80 (Allen and Andreotti
Some drawbacks are that whey is deficient in inorganic
nitrogen and has a high content of mineral salts that may interfere
with growth (Marth 1970) and processing (Moore-Landecker 1990,
Zadow 1986). Certain agents in whey media as well as autoclaving of
these
media have been found to
inhibit the growth
of or the
metabolite production in some bacteria and viruses (Marth 1970,
Anderson et al 1986). Environmental pollution by whey wastes has
long been a problem for the food industry (Sell 1992). By using acid
whey or whey permeate as a growth medium for fungi, a twofold
goal
could be accomplished:
reduction of whey wastes and
a
facilitated production of a wide range of utilizable products. Since a
significant decrease in the cost of cellulase production could be
expected if the cultivation of
Trichoderma reesei on whey media
results in a fair yield of cellulase, an objective of the study was to
explore the feasibility of such a technique. Other applications of
Trichoderma sp. are the production of mycelial protein for feedstock
purposes
and
materials
as
their ability
well
as
to
transform
xenobiotics
many
(Eveleigh
complex
1985).
natural
They
are
53
considered to possess disease biocontrol potential (Jackson et al
1991). Trichoderma species are known to produce mycotoxins such
as trichotoxin A (a cyclic polypeptide with some antimicrobial
activity) (Taber and Taber 1978), trichodermin (a trichothecene) and
trichoviridin (an isocyanide) (Eveleigh 1985). The small amounts of
these
compounds
have
motivated
the
Food
Additives
and
Contaminants Committee of the Ministry of Agriculture, Fisheries
and Food in the United Kingdom to classify Trichoderma reesei in
group B, i. e. substances that on the available evidence may be
regarded provisionally acceptable for use in food but about which
further information should be gathered (Chaplin and Bucke 1990). In
the U. S. Trichoderma metabolites do not yet appear in the GRAS list
(Code of Federal Regulations april 1993). Trichoderma
strains also
produce yellow pigments such as anthroquinones (Eveleigh 1985).
However,
no data have been presented in the literature on the
growth of T. reesei on acid whey. The purpose of the present study
was to fill this information gap.
54
3.2
3.2.1
MATERIALS AND METHODS
Substrates
Fresh acid whey (pH 4.6 and 4.4 respectively) was obtained from
Sunny Brooks Dairy, Corvallis, OR and from Echo Springs Dairy,
Eugene, OR. Whey permeate (pH 6.3) was provided by the Department
of Food Science at Utah State University, Logan, UT. These media
were vacuum-filtered in several steps using Celite powder (D3877
followed by D 5384, both from Sigma Chemical Co., MO) and 1.2 jim
glass
fiber
filters.
Sterilization
was
normally
carried
out
by
filtration through 0.2 jim Nalgene cellulose nitrate membranes (VWR
Scientific). In the initial study on acid whey (Fig. 14 and 15) thermal
sterilization
was
conducted
at
121
0
C
for
15
min.
The
hammer-milled spruce cellulose, Solkafloc SW 40 (James River Inc.,
Berlin, NH), at 8 g/l was normally used as the control together with
Mandels'
medium
[1.5 g/l
ammonium
sulfate,
2
g/l
monobasic
potassium phosphate, 0.32 g/l calcium chloride, 0.32 g/l
magnesium
sulfate
(containing
heptahydrate,
1
ml/I
trace
metal
solution
ferrous and manganese sulfate, zinc chloride and cobalt chloride),
0.31
g/l
urea,
0.8 g/l
proteose peptone (DIFCO Laboratories,
Ml),
55
1 ml/l Tween 80]. This control was always adjusted to pH 6.5 and
thermally sterilized at 121
whey permeate (Fig.
0
C for 15 min. In the growth study on
13) a control consisting of 8 g/l
maltose
monohydrate together with Mandels' medium was employed and
filter-sterilization was conducted as described above.
3.2.2
Microbe
Trichoderma reesei QM 9414 was obtained as a freeze-dried
preparation from ATCC and grown on potato dextrose agar (DIFCO
0
Laboratories, Detroit, Ml) slants or plates at 30
C for 1-2 weeks in
the laboratory (Smith and Onions 1983). An aqueous spore suspension
(3-125 million CFU/ml) was used as inoculum (0.1-0.4 ml/100 ml
medium).
3.2.3
Incubation experiments
The inoculated media were contained in
Erlenmeyer flasks
(duplicates or triplicates) with glasswool plugs and aluminum foil
caps. The maximum liquid to volume ratio was 100 ml in a 250 mL
flask.
The
incubations were
performed
at
28
0
C
in
an
Orbit
Environshaker (Lab.-Line Instruments, Inc., Melrose Park, IL) with a
speed setting of 100 r. p. m.
56
3.2.4
Reagents and analytical procedures
Spore counting was carried out on potato dextrose agar (PDA) and
on PDA with added Rose Bengal at 0.001 %. Soluble protein was
determined according to the Bradford
method
(reagent provided
by
Bio-Rad Laboratories, Richmond, CA) with bovine serum albumin as
standard. Samples aimed for determination of cellulolytic activity
were centrifuged at 5000 g for 1 hour using Centricon polysulfone
ultrafiltration membranes with 10 kD M. W. cut-off (Amicon, Inc.,
Beverly, MA),
then subjected to size exclusion chromatography by
using Econopac 10 DG desalting columns with 6 kD
polyacrylamide gel
subsequently
(Bio-Rad
freeze-dried
Laboratories,
and diluted
M. W. cut-off
Richmond,
as appropriate
CA)
and
for the
following assay. Glucose levels were assayed in accordance with the
IUPAC method (Ghose 1987) for filter paper activity determination
employing the dinitrosalicylic acid method. Cellulolytic activity was
determined by a low-activity variant of the IUPAC method developed
in our laboratory and using potassium chloride (1 M) and bovine
serum albumin (1 mg/ml) as protein stabilizers and sodium azide
(0.02 M)
as an antimicrobial.
Lactose was assayed by using
a
57
kit from
employing
Boehringer
lactase,
Mannheim
Biochemicals,
hexokinase
and
Indianapolis,
IN,
glucose-6-phosphate
dehydrogenase. All reagent chemicals were of analytical grade.
Mycelial growth was assayed by vacuum filtering the sample and
washing it with at least 3 x 10 ml cold filtered tap water on a 1.2
|im glass fiber disk until no foam appeared, drying the retentate at
102 0C for 16 hours, cooling it in a desiccator and weighing.
58
3.3
3.3.1
RESULTS
AND
DISCUSSION
The development of mvcelial biomass
When active growth starts it involves only apical and subapical
hyphal cells (Nielsen 1993) and then the degree of branching and the
pellet size will determine
much of the
growth
(Garraway and Evans 1984). T. reesei QM 9414
characteristics
grows well on acid
whey as can be seen in Figure 12. The lag phase may parallel the
finding that the lag on cellulosic media using a spore inoculum is
long and unpredictable (Sternberg 1976b). The doubling time was
found to be 2.4 days which
corresponds
rate of 0.29 (= In 2 / 2.4 ) per day.
to
a
specific growth
After 2 weeks and a mycelial dry
weight gain of 13 mg/ml the growth has not reached the stationary
phase. As a comparison T. viride QM 9123 growing on glucose (23
mg/ml) supplemented with Mandels' medium reached
a maximum of
11 mg/ml (Brown and Halsted 1975). The mold Aspergillus niger has
been shown to have a specific growth rate of 0.62/day on grape
waste, which was considered a good growth (Kuzmanova et al 1991).
Microfungi in general contain 19-47 % protein (Smith and Berry
1975).
Protein data are sometimes based on
Kjeldahl
nitrogen
59
100
mg mycelial
dry weight/ml
(log scale)
10
days
□
Acid whey # 1
O
Acid whey # 2
O
Dil. acid whey # 1
A
Dil. acid whey # 2
S
Dil. acid whey (adjusted) # 1
♦
Dil. acid whey (adjusted) # 2
Fig. 12. The growth of 7. reeseion
filter-sterilized acid whey.
60
values
multiplied by 6.25. However, the high amount of non-protein
nitrogen in microfungi means that the corresponding amount of real
protein is lower.
Figure 12 also shows the effect of dilution 1:6 of
acid whey. The low level of nutrients causes the time scale to shrink
(Megee et al 1970) and the maximum mycelial mass to decrease. The
maximum mycelial mass in the case of undiluted acid whey is
predicted to be approximately 22 mg/ml
factor above. A duplicate set of
were
and
the
based
diluted
on the dilution
acid
whey
flasks
supplemented with ammonium sulfate to 14 mM (Garraway
Evans
1984).
influenced growth,
This
adjustment
indicating
that
does
not
ammonium
appear
nitrogen
to have
is
not
growth limiting. The diluted acid whey graphs in Figure 12 are
consistent with cell death occurring after 7 days. The death phase
may involve cell autolysis or turnover (Sikyta
Sternberg
and
Dorval
1979),
1983,
Freitag
1989,
in which case a declining growth
curve appears. No stationary phase was observed in any of the
experiments reported in Figure 12. The absence of a true stationary
phase often occurs among filamentous fungi (Garraway and Evans
1984, Schaffner and Toledo 1991, Wilke et al 1983).
61
On sweet whey permeate (Fig. 13) the maximum mycelial yield
is 4.4 mg/ml. The 0.8 % maltose control reached 3.3 mg/ml. This can
be compared with a 0.5 % maltose culture which yielded a maximum
2.2 mg/ml mycelial weight using T. viride (Mandels and Reese 1957).
The growth curves on diluted acid whey and whey permeate are
similar. This suggests that neither lactose nor mineral constituents
are growth limiting, since the peptide content is about the same in
both media. The higher final biomass yield in the
case
of
undiluted
acid whey is thus likely to result from its higher protein content
relative to the diluted and permeate media. Since the carbon and
mineral contents vary considerably between the
media
reasonable
growth
to
assume
that
peptide
nitrogen
is
it
is
limiting.
The findings above support that acid whey is superior to sweet whey
permeate as a growth medium.
3.3.2
Metabolite production and effects on substrate composition
Figure 14 shows how pH changes when thermally sterilized, pH-
adjusted and non-supplemented acid wheys are used as substrates.
The first pH-changes are likely to be a result of the thermal
treatment since no such variations occur if the acid whey is
filter-
62
mg mycelial
dry weight/ml
□
Whey permeate # 1
O
Whey permeate # 2
O
0.8 % Maltose # 1
A
0.8 % Maltose # 2
Fig. 13. The growth of T. reeseion
filter-sterilized whey permeate.
63
PH
days
—o
Acid whey pH 4.4
• o
Acid whey pH 5.0
—-o—-
Acid whey pH 5.5
—A—
Acid whey pH 6.0
---ffl---
Acid whey pH 6.5
--♦--
Solkafloc pH 6.5
Fig. 14. The change in pH when T. reesei is
cultivated on thermally sterilized
acid whey.
64
sterilized (data not shown). The following one unit rise in pH
parallels a decline in soluble protein level (Fig. 15). Also in the case
of filter-sterilized acid whey there is a considerable decline in
soluble protein (Fig. 16). These two findings appear to support the
hypothesis that whey proteins are digested and ammonia released
into the medium.
At any particular time point metabolite production
is generally inversely related to growth (Megee et al 1970). There is
a
50-60 %
sterilized
difference in the initial protein values for the thermally
(Fig. 15)
and
the
filter-sterilized
(Fig. 16)
acid
whey
preparations. This difference is similar to the percentage of whey
proteins that can be heat coagulated (Hui 1992).
It is
also
apparent
from the figures that the net specific protein production rate is
similar on acid whey and Solkafloc medium (Fig. 15). The maximal
net
soluble
protein
production
is
0.17
mg/ml
for
thermally
sterilized and 0.19 mg/ml for filter-sterilized acid whey. These
values are likely to reflect actual metabolite production since there
was no indication of cell lysis and the rise comes immediately after
the digestion phase. As mentioned below the development of
enzymatic activity parallels the rise in soluble protein
level
in
the
65
Incubation
Cold
storage
mg
protein/ml
Acid whey pH 4.4
-O
Acid whey pH 5.5
-O-—
Acid whey pH 6.5
-A—
Solkafloc pH 6.5
Fig. 15. Change in soluble protein as BSA when
T. reesei is grown on thermally sterilized
acid whey.
66
mg
protein/ml
days
Acid whey # 1
-o
Acid whey # 2
-o-—
Solkafloc # 1
-A—
Solkafloc # 2
Fig. 16. Change in soluble protein as BSA
when T. reesei is grown on
filter-sterilized acid whey.
67
experiment employing filter-sterilized whey permeate
and
also
in
the case of the filter-sterilized acid whey (Fig. 16 and 17).
I have found that acid whey which
has
been
diluted and supplemented with ammonium sulfate,
initial pH dip as the Solkafloc
medium
filter-sterilized,
shows
a
similar
(data not shown).
The
development of pH when Trichoderma reesei is cultivated using a
cellulosic carbon source such
as
Solkafloc
together
with
medium is well known. There is a rapid decrease initially to
Mandels'
pH 3
or
less (Garraway and Evans 1984) due to consumption by the mold of
ammonia from the ammonium sulfate (Sternberg 1976b). Such low pH
values can inactivate
produced
enzymes (Sternberg
1976a)
and
favor protease formation on peptone media. Peptones can also
substitute for carbohydrates as
a
carbon
source. When the
ammonium ions are used up the pH starts to rise due to release
of ammonia when peptones are digested (Moore-Landecker 1990).
Simultaneously the fungus starts production of digestive enzymes,
for instance cellulases if a suitable provoker (Kubicek et al 1993) is
present such as cellulose.
The filter-sterilized and diluted but
unsupplemented acid whey displays a strong rise in pH (Fig. 18).
The
68
1.5-
mg glucose
0.5-
days
Acid whey # 1
•o
Acid whey # 2
-o—
Solkafloc # 1
Solkafloc # 2
Fig. 17. The amount of glucose released in a filter
paper assay (IUPAC) when T. reesei is
cultivated on filter-sterilized acid whey.
69
PH
□
Dil. acid whey # 1
O
Dil. acid whey # 2
O
Solkafloc # 1
A
Solkafloc # 2
Fig. 18. The time course of pH when T. reesei
is grown on filter-sterilized acid
whey that has been diluted 1:6.
70
rise is the same as on undiluted acid whey (data not shown). The pH
profiles for growth on the diluted and undiluted acid wheys do not
correlate with those for growth on whey permeate. This observation
may
be
a
result
of
the
differences
in
protein
and
peptide
composition and/or mineral contents for the acid wheys and whey
permeate. The buffering capacities of the two media could also
differ.
The increased risk for irreversible denaturation
of proteins
and enzymes when pH rises above 8-9 (Townend 1978), especially at
elevated temperatures, could harm the yield of desired
metabolites.
When sweet whey permeate is used as a substrate there is also
a pH increase initially (Fig. 19).
The low amount of peptides in whey
permeate will limit the rise in pH and favor an earlier start of net
protein production (Fig. 20). Thus a marked increase in soluble
protein takes place after 3-5 days and a maximum value of 0.23 mg
protein/ml is attained after 2 weeks. The long term trends with
declining pH can be explained as an effect of organic acid production
under conditions of oxygen access and ongoing autolysis.
T. reesei produces pigment compounds that are released into
liquid and solid growth media. Visible spectra of
these
pigments
in
71
PH
days
Whey permeate # 1
Whey permeate # 2
-o-
Solkafloc # 1
Solkafloc # 2
Fig. 19. The development of pH when T. reesei
is grown on filter-sterilized sweet
whey permeate.
72
0.5
0.4-
mg
0.3
protein/ml
0.2
0.1-
days
Whey permeate # 1
■o- •■•••
Whey permeate # 2
■o—
Solkafloc # 1
-A—
Solkafloc # 2
Fig. 20. Change in soluble protein as BSA when
T. reesei is grown on filter-sterilized
sweet whey permeate.
73
cone, sulfuric acid reveals a marked increase in absorption of
wavelengths
shorter
than
470
nm.
The
apparent
pigment
concentration is correlated with the pH of the media. Visually, the
color of the growth media changes with incubation time. This color
change appears related to the pH of the culture broth. I have
compared the pigmentation during growth on acid whey,
Solkafloc media at initial pH values of 4.4 and 6.5.
Acid
lactose
and
whey gives
the strongest color development reaching an apparent maximum
after
1-2
weeks.
Lactose
pigmentation consistent with a
and
Solkafloc
smaller
final
media
mycelial
give
weight
less
and
lower pH values due to the lower peptide contents. See appendix E
for further information on culture pigmentation.
Figure 21 shows the cellulolytic filter paper activity measured
as glucose after incubation on whey permeate. The development is
consistent with the development of extracellular protein (Sternberg
1976b) (Fig. 20) and demonstrates that a small cellulase activity is
produced on unsupplemented whey permeate. In terms of IUPAC filter
paper activities the difference between the Solkafloc and the whey
permeate is much bigger due to the
nonlinear
relationship
between
74
mg glucose
days
Whey permeate # 1
•o-
Whey permeate # 2
-o-
Solkafloc # 1
Solkafloc # 2
Fig. 21. The amount of glucose released in a filter paper
assay (IUPAC) when T. reesei is cultivated on
filter-sterilized sweet whey permeate.
75
the amount of glucose released in the assay and the cellulase
concentration.
Supplementation of whey permeate with
0.1
%
Solkafloc appeared to increase the soluble protein level by 25 % (Fig.
22). ANOVA analysis (Fischer's protected LSD) shows a 90 %
significance for the differences on day 3. The result is consistent
with
a
source
substitution
in
the
by
case
Solkafloc
of
the
for
some
supplemented
lactose
as carbon
whey
permeate.
Addition of 0.2-0.5 % cellulose to 4-6 % lactose media has been
found to enhance the soluble protein level and the cellulase
using the strains T. reesei CL-847 (Warzywoda et al 1983)
activity
and
C-5
(Chaudhuri and Sahai 1993a). Figure 17 illustrates the development
of cellulase activity on acid whey. It is apparent that the net
cellulase production
is low.
This is probably
in
part due to
extracellular proteases emitted into the medium because of the
protein and peptide contents in acid whey and whey permeate (Haab
et al 1990). No protease inhibitors are known that could prevent
digestion of cellulases to more than 50 % (Haab et al 1990). Dilution
of the acid whey 1:6 resulted in even lower cellulase enzyme
activities (data not shown) and the IUPAC filter paper activity
after
76
mg
protein/ml
Whey permeate
Fig. 22.
•■••O
Whey permeate + Solkafloc
-O—-
Solkafloc
The effect of 0.1 % (w/v) Solkafloc addition
on the level of soluble protein as BSA when
T. reesei is grown on filter-sterilized whey
permeate.
77
7 days was determined to 0.02 FPU. This finding suggests that some
whey proteins present after the dilution are inhibitory to cellulase
production and/or that the medium was deficient in growth factors.
Cellulase is assumed to originate in the apical hyphal wall (Gow
et al 1993) where, for instance, lactose is known to provoke
cellulase synthesis (Ryu et al 1979). When cellulase is produced
industrially on lactose media the pH is generally kept close to 5.
Cultivation on different media results in different
pH
profiles
and
cellulase production is not tied to a- particular profile (Esterbauer et
al 1991). Consumption of lactose by the mold when growing on
whey
permeate is demonstrated in Figure 23. It is clear that after 10 days
growth there was only limited reduction in the lactose content of
the media. The high lactose content (4.5-5 %) in whey (Hui 1993),
appears to favor growth (Chaudhuri and Sahai 1993b) instead of
metabolite production (Mandels and Reese 1957, Farkas et al 1987).
High concentrations of rapidly metabolized carbon sources strongly
repress cellulase formation (Mandels and Weber 1969).
High peptone concentrations in cellulosic media are strongly
inhibitory to cellulase production
(Mandels and Weber 1969).
The
78
g lactose/I
E3 DayO
H Day 10
Whey
permeate
Fig. 23.
Whey
permeate +
Solkafloc
The change in lactose level with and without
added 0.1 % (w/v) Solkafloc when T. reesei is
grown on filter-sterilized sweet whey permeate.
79
experiments above have all resulted in low net yield of cellulase. It
thus seems unlikely that acid whey or whey permeate without
further processing could be used as media for cellulase production
by Trichoderma reesei QM 9414.
80
3.4 FINAL COMMENTS
It appears of interest to conduct further studies on acid whey
which showed potential for mycelial mass production. The focus
when molds are grown is perhaps more often on the secondary
metabolites,
i. e.
products that appear when
there
is a
nutrient
deficiency and which do not have any obvious function for the fungus.
Primary metabolites and digestive enzymes are also of interest in
many applications. As far as proteinaceous metabolites are of
concern sweet whey permeate appears to be a better alternative
than acid whey. If a precultured broth had been used instead of a
spore inoculum the lag period could have been shorter (Sternberg
1976b) than in the previous experiments. This can be of economical
importance in an industrial setting. Acid whey permeate could be
worth a separate study due to its low protein content and the
economical benefits of acid whey utilization. The productivity of
other
T.
reesei
enzymes
than
cellulase
such
as
glucanases,
chitinases (Peberdy and Ferenczy 1985), pectinases (Haltmeier et al
1983) and xylanases (Poutanen and Puls 1989) on whey media need
to be
addressed. T.
reesei
has
also
been
called
a
promising
81
alternative
host for protein production
(Penttilae
et al
1991).
Optimal conditions for the production of extracellular proteins will
be dependent on nutritional and temperature requirements of the
fungus,
handling
of
the
substrate,
ranges
for
denaturation,
precipitation and aggregation of the protein, ease of harvesting and
of the costs involved in each case.
82
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95
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APPENDICES
97
A.
A PROTOCOL FOR THE MAINTENANCE AND HANDLING OF
TRICHODERMA REESEI CULTURES ON POTATO DEXTROSE
AGAR
Media
1)
preparation
and
inoculum
Trichoderma reesei grows well on potato dextrose agar (PDA).
Since it is a rich medium it tends to give rise to excessive
mycelial growth. Weaker media, such as potato carrot agar
(PCA) and malt extract agar (MA), are also suitable growth
media (Smith and Onions 1983).
Addition of leaf litter or a
piece of filter paper can improve the growth. One third of the
volume of a screw capped test tube should be PDA, i. e. 6 ml
agar in a 17 ml test tube. Plates often offer better sporulation
conditions than tubes;
fill them with 16-20 ml PDA.
Media formulation
Potato dextrose agar (DIFCO)
39 mg/ ml distilled water
or
Diced potatoes
Glucose
Agar
Dist. water
300
20
15
1.0
g
g
g
L
98
Boil finely diced potatoes in 500 ml of water
until thoroughly cooked; filter through cheesecloth
and add water to filtrate to 1.0 L. The agar is
dissolved in the filtrate by heating, and the glucose
is added prior to sterilization (ATCC Catalogue of
Filamentous Fungi 1991).
2)
Use a revived freeze-dried fungal preparation from ATCC or a
contaminant free sample from an old plate or slant with good
sporulation as inoculum.
3)
Transfers should be made during night or morning hours that are
reasonably dust free.
4)
Minimize the necessary heat treatment of the PDA. Preheat the
PDA in a waterbath past 90 0C but do not boil the agar before
sterilization at 121
0
C for 15 min. This should give an agar with
a pH in the range from 5.5 to 5.7.
5)
The filling of the agar and the spore transfer should be done in a
sterile hood. Wash shelf and table areas with ethanol (>70 %)
before use. Wear gloves.
6)
The agar should be close to solidification (40-50 0C) when it is
poured into the tubes by using a disposable sterile pipette.
99
The tubes should then be tilted so that the
reaches the neck of the tube.
Allow
the
agar
water
barely
vapor
to
completely leave the plate or tube so that enough oxygen is
present for the growth and no condensation of water will occur.
Inoculation
7)
Inoculate the agar within hours after solidification. Surface
streaks are better than inoculation with a spore suspension.
Sterilize the inoculation needle in a flame and move it into the
hood.
8)
Smear the tube area close to the screwcap with a ring of
petroleum jelly. Thereby the risk of infection by dust mites is
decreased (Dade 1960).
Incubation
9)
Incubate under dark conditions immediately after inoculation.
The best temperature for sporulation is 30
0
C. Keep the tube
caps unscrewed the first 24 hours and then tighten them but not
completely.
100
10) The agar should turn yellow after 2 days.
11) After 3-4 days expose the mycelium to daylight or blue light for
at least a few seconds.
12) Use the cultures after 7-10 days if the spores will be utilized
for inoculation of liquid media.
Culture
preservation
13) At least one tube should be stored at -20 0C for 24 hours before
placement in regular cold storage since
adults,
instars and eggs of mites will then be killed
nymphal
(Jong 1981).
14) Store well sporulated cultures in a dark area at 4 0C.
Mites are mostly immobile and the fungal growth,
variation and
metabolism is retarded at that temperature (Smith and Onions
1983).
15) The agar tends to dry out and curl after several months at 4 0C.
Transfers
frequent
can
be
transfers
contamination
with
species and others.
done
in
order
Rhizopus
after
to
4-6
months.
decrease
the
Avoid
risk
of
stolonifer, many Penicillium
101
16) Liquid
nitrogen storage and freeze-drying of cultures are
superior techniques for long-term preservation.
102
B.
SPORE COUNTING ON PDA PLATES BY USING ROSE BENGAL
PDA + 0.001 % Rose Bengal
Dilution 10-8
PDA
Dilution 10-8
PDA + 0.001 % Rose Bengal
Dilution 10-7
PDA
Dilution 10-7
Fig. 24.
If 0.001% Rose Bengal is added to the PDA then the
T. reesei colonies will appear more distinct and
limited as can be seen from the photos above. Higher
concentrations can kill the fungus.
103
C.
A PROTOCOL FOR THE MODIFIED lUPAC FILTER PAPER
ASSAY
Introduction
This assay permits the estimation of total cellulase activities
in accordance with the lUPAC assay (Ghose 1987) in the range from
0.37 FPU to 0.02 FPU by employing certain enzyme stabilizing agent
and extending the incubation time up to one day. The assay has the
advantages and disadvantages that accompany the regular lUPAC
assay. Cellulosic substrates other than filter paper may result in
different rankings of the cellulase activity.
If the intention is to get lUPAC filter paper activities and not
only relative activities, a new Trichoderma cellulase preparation
should
be
assayed
by this
method
for determination
of
the
conversion factor (f) between the regular lUPAC filter paper unit and
the unit defined by the modified assay. This is done by carrying out a
one hour assay under lUPAC conditions but with stabilizers added to
half the number of incubated enzyme tubes.
104
Ingredient
1)
preparation
Dinitrosalicylic acid (DNS) reagent:
Sodium hydroxide
Distilled water
19.8 g
1416 ml
Dissolve the above and add:
3,5-Dinitrosalicylic
When all
following:
the
acid
above
10.6 g
has
been
dissolved
Rochelle salt
(=Potassium sodium tartrate)
Phenol (melt in a 50 0C water bath)
Sodium metabisulfite
306 g
add
the
7.6 m I
8.3 g
Titrate 3 ml of the DNS reagent with phenolphtalein
with 0.1 N HCI. Some addition of NaOH to the reagent is
normally necessary to get the HCI volume into the
required range from 5 to 6 ml (1 ml 0.1 N HCI = 2 g NaOH).
The final pH is 13-14.
Store
this
solution at
room
temperature
0
shorter times and at + 4 C for longer periods.
2)
Substrate:
Whatman #1
filter paper (dry)
50 mg
for
105
This amount corresponds to approximately 50 x 12
mm. Trim it to ± 1 mg. Curl the papers (around a
thin stainless steel rod).
3)
Sodium citrate buffer:
Sodium hydroxide
Citric acid monohydrate
13.75 g
52.50 g
Dissolve the above in distilled water so that the
final volume is 250 ml. This stock solution should
have a pH close to 3.9 and can be stored at + 4 0C until
used.
Then take 100 ml solution and add distilled water
until the volume is 1950 ml. Add 5 N sodium
hydroxide solution until the pH is almost 4.80. Then
add distilled water until the total volume is 2000
ml. Use this solution fresh or store at + 1 0C for up
to a week.
4)
Glucose standards:
Glucose
(dry in vacuum or at 60 0C for 5 hours)
1.00 g
Add distilled water until the total volume is 100
ml and mix thoroughly. Dispense small quantities
into plastic vials and store them at - 20 0C until
used.
Then make 5 dilutions of the above 10 mg/ml stock
as follows:
106
Concentratioii
(mg/ml)
Stock
(ml)
Dist. water
(ml)
6.67
5.00
3.33
2.00
1.00
2
2
1
1
1
1
2
2
4
9
Mix each solution thoroughly and store at + 4 "C
until used the same day.
5)
Sodium citrate buffer with stabilizers:
Sodium citrate buffer
Bovine serum albumin
Potassium chloride
Sodium azide
100 ml
150 mg
11.18g
30 mg
Use this solution fresh.
Place the amounts under 6) to 8) below in screw-capped test tubes
of 25 ml volume or more. Store them at + 4 0C until used in the
assay.
6)
Substrate blank solutions:
Reagent blank solutions:
citrate buffer
7)
1.5 ml
Assay standards:
citrate buffer
glucose standard (dilution)
1.0 ml
0.5 ml
107
8)
Enzyme solutions:
Enzyme blank solutions:
citrate buffer
(w. stabilizers)
enzyme
1.0 ml
0.5 m I
Prepare for 3 data points in duplicates (see 14) below)
by taking 6 x 2 x 0.5 ml (enzyme solutions and enzyme
blank solutions) from the original enzyme preparation.
The incubation times must be chosen so that both data
points above and below 2 mg glucose are obtained in the
assay. Label the tubes according to the incubation times
which should be in a geometric series. Transfer the 0.5
ml solutions slowly and take care not to leave any
remnants of solution.
Performing
9)
the
assay
Preincubate the solutions under 6) to 8) for 10 min. at + 50
C. Then start the regular incubation by adding one filter paper
to each tube. Stab the papers gently with a glass rod so that
they stay under the liquid surface. Cover the tubes.
0
10)
After the desired incubation period stop the reaction by
adding 3 ml DNS. Boil the tubes for exactly 5 minutes in
a vigorously boiling water bath. Keep screw caps on.
Immediately cool in an ice bath.
11)
Add 20 ml distilled water to each test tube and invert the
tubes several times so that the pulp moves from end to end.
Let the tubes stand for 20 min.
12)
Calibrate the spectrophotometer by using the reagent
blanks. Remove samples from the top third of the test tube.
Read the absorbances at 540 nm. Make water dilutions as
108
required to get the absorbances between 0.1
that the pH is still highly alkaline.
and 0.7. Be sure
Calculations
13)
Get a regression line for the assay standards. Translate the
blank corrected absorbances to mg reducing sugar as glucose
released in the assay (i. e. in 0.5 ml).
14)
Plot grams of glucose vs. the logarithm of the number of hours
on a semilogarithmic paper. Find the number of hours that
corresponds to 2 mg glucose.
15)
Calculate the IUPAC filter paper activity from:
FPA = f x 0.37 / [(dilution)-i x # hours]
If only relative activities are of interest, the factor f above
can be excluded.
109
D.
AN EXPERIMENT DEMONSTRATING
FILTER PAPER ASSAY
THE
MODIFIED
IUPAC
Enzyme
A 6.18 mg/ml stock solution of a commercial T. viride cellulase
(Cellulysin) was made.
Materials
Bovine serum albumin (1 mg/ml) and potassium chloride (1 M)
were present in the incubated volume. Sodium azide was utilized as
an antimicrobial agent at 0.087 % and 0.013 % concentration in test
runs 1 and 2 respectively.
Test
runs
In both runs 1:1, 1:1.5 and 1:2 dilutions of the stock solution
were incubated in duplicates with stabilizers for one hour in order
to determine the apparent filter paper activities.
Duplicates of a
1:7 dilution were incubated for 5 and 10 hours in test run 1 and for
3.5,
5 and 7 hours in test run 2.
Duplicates of a
1:14 dilution were
110
incubated for 5, 10 and 20 hours in test run 1 and for 7, 10 and 14
hours in test run
2.
Result
Figure 25 shows how the amount of reducing sugar varies with
the incubation time for the two dilutions in the second test run. The
intersection with the 2 mg line gives the number of hours to be used
in the calculation of the filter paper activity. Table 2 shows relative
activity
data.
Apparently,
similar
activities
are
obtained
independent of the incubation times. This talks in favor of the
modified assay as a useful tool when testing samples of low
cellulase activity.
111
mg glucose
hours (log scale)
Fig. 25.
D
Dilution 1:7
O
Dilution 1:7
o
Dilution 1:14
A
Dilution 1:14
The amount of reducing sugar as glucose
released in a modified filter paper assay
for different incubation times and for 2
dilutions. T. viride cellulase is used.
112
Table 2.
Relative filter paper activities obtained
from modified assay data for different
dilutions compared with expected values
based on modified assay data for a stock
solution of 7". viride cellulase.
TEST RUN
Apparent FPA Average
Av./7
0.57
# 1, undiluted
0.55
# 2, undiluted
0.56
0.069
|# 1, dil. 1:7
0.087
#2, dil. 1:7
0.078
# 1, dil. 1:14
0.038
0.04
# 2, dil. 1;14
0.039
SE(Mean)
jAv./14
0.08!
0.04
0.01
:
0.009
0.001
1 13
E.
THE PIGMENT PRODUCTION BY TRICHODERMA
REESEI ON LIQUID MEDIA
Table 3.
DAYS
The pigment production by Trichoderma
reesei on liquid media.
ACID WHEY
pH4.4
0 whiteish
pH6.5
LACTOSE
pH 4.4
jpH 6.5
iSOLCAFUX
pH4.4
pH6.5
whiteish
colorless
jcolorless
i
whiteish
whiteish
3 milky yellow yellowish
6 yellowish
(pH=5.4)
9 egg yolk
yellow
(pH=5.8)
14 yellow
20 brownish
lyellowish
egg yolk
yellow
yellowish
brownish
yellow
lemon yellow ibrownish
(pH=4.4)
lyellow
(pH=6.9)
dark yellow
brownish
yellowish
white
ilemon yellow pale yellowish yellowish gray
!(pH=4.4)
(pH=3.2)
lemorM^ellow^ brownish
yellow
m=m
!(PH=6.1)
i
:
f
i
S
i
i
(PH-4.1)
grayish yellow
gray
1 14
F.
A PROTOCOL FOR PREINCUBATION AND POSTINCUBATION
PROCESSING WHEN RECOVERING PROTEINS ON WHEY
MEDIA
Acid whey or whey permeate
PREINCUBATION STEPS
FILTRATION
1.
1.2 [im glass fiber / vacuum
2. 1.2 urn glass fiber with a 0.5 " layer
of Celite D 3877 / vacuum
3.
1.2 |im glass fiber with a 0.25" layer
of Celite D 5384 / vacuum
4.
1.2 (im glass fiber / vacuum
FILTERSTERILIZATION
Nalgene 0.2 jim cellulose
nitrate filter cups / vacuum
115
POSTINCUBATION TREATMENT
FOR PROTEIN RECOVERY;
FILTRATION
1.2 |xm glass fiber / vacuum
ULTRAFILTRATION
Amicon's Centricon 10
YM membrane filters (10 kD cut-off)
Centrifuge at 5000 g/1 h 15 min/4 "C
Turn filter unit and centrifuge at 800 g/2 min/4 0C
Volume adjustment w. dist. water
SIZE EXCLUSION CHROMATOGRAPHY
BioRad's Econopac 10 DG columns with
polyacrylamide gel (6 kD cut-off)
Gravity flow / 4 0C
Preserve the gel after use in 0.02 % aq. NaNa
LYOPHILIZATION
-60 0C/7 urn Hg/60 h
I
Store in vacuum desiccator at 4 0C
(or store at -20 0C)