A cellobiose-phosphorylase from Clostridium thermocellum
by Charles J Sih
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Bacteriology
Montana State University
© Copyright by Charles J Sih (1955)
Abstract:
A cellobiose-phosphorylase was shown to be present in the cells of Clostridium thermocellum using
cell-free enzyme preparations.
This enzyme caused the phosphorolysis of cellobiose to glucose-1-phosphate and glucose. Its activity
was measured by following the decrease in inorganic phosphate or the increase in glucose-1-phosphate.
Phosphoglucomutase which destroys the glucose-1-phosphate was inactivated by freezing.
The phosphorylase was stable, being active over a wide range of temperature and pH. It was also
specific in its action, resembling other disaccharide phosphorylases in its specificity and inhibition by
the products of its action.
\ The finding of this enzyme gives a partial answer to the question of how Clostridium thermocellum
can use celldbiose as an energy source without being able to use exogenous glucose. A CELLOBIOSE -PH0SPH0RYLASE FROM CLOSTRIDIUM TRERMOn1RTTlUM
"by
CHARLES J . SIH
A THESIS
S u b m itted t o th e G rad u ate F a c u lty
in
p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts
f o r th e d eg ree o f
M aster o f S c ie n c e i n B a c te rio lo g y
at
Montana S t a t e C o lleg e
Approved:
C hairm an, Examining Committee
Bozeman, Montana
Ju n e , 1955
- 2 TABLE OF CONTENTS
3
ABSTRACT
I).
INTRODUCTION AND HISTORICAL REVIEW
5
<0 OO CD CO
ACKNOWLEDGMENTS
MATERIALS AND METHODS
C u ltu re
C u ltu re medium
P r e p a r a tio n o f la r g e c u ltu r e s
P r e p a r a tio n o f th e enzyme
Phosphorus d e te r m in a tio n
Sugar d e te r m in a tio n
P aper chrom atography
10
10
11
11
EXPERIMENTAL RESULTS
Growth e x p erim en t
C e l l - f r e e enzyme ex p erim en ts
Is o to p e ex p erim en ts
C h a r a c t e r i s t i c s o f th e c e llo b io s e p h o sp h o ry la se
E f f e c t o f te m p e ra tu re
E f f e c t o f pH
S p e c i f i c i t y o f th e p h o sp h o ry la se
I n h i b i t i o n b y g lu c o se
12
12
13
21
26
26
28
29
30
DISCUSSION
31
SUMMARY
35
REFERENCES
36
1 VTf i H
ACiOTOWLEDGMEM1S
The a u th o r ta k e s t h i s o p p o rtu n ity t o th a n k D r. R ic h a rd
H. McBee f o r h is g u id an ce th ro u g h o u t t h i s w ork, and Mr. W illis
J o h n sto n f o r h i s 'com petent s u p e r v is io n o f th e r a d io a c ti v e is o to p e
e x p e rim e n ts .
- U ABSTRACT
A c e lld b io s e -p h o s p h o ry la s e was shown t o b e p r e s e n t in th e c e l l s
o f C lo s trid iu m therm ocellum u s in g c e l l - f r e e enzyme p r e p a r a t io n s .
T h is enzyme cau sed th e p h o sp h o ro ly s i s o f c e lld b io s e t o
g lu c o s e - I -p h o sp h ate and g lu c o s e .
I t s a c t i v i t y was m easured b y fo llo w in g
th e d e c re a s e i n in o rg a n ic p h o sp h ate o r th e in c re a s e i n g lu c o s e - I -p h o sp h a te .
Phosphoglucom ntase w hich d e s tr o y s th e g lu c o s e - I -p h o sp h ate was in a c t iv a te d
by fre e z in g .
The p h o sp h o ry la se was s t a b l e , b e in g a c tiv e o v er a w ide ran g e
o f te m p e ra tu re and pH.
I t was a ls o s p e c i f i c i n i t s a c t i o n , re sem b lin g
o th e r d is a c c h a r id e p h o sp h o ry la se s i n i t s s p e c i f i c i t y and i n h i b i t i o n b y
th e p ro d u c ts o f i t s a c t i o n .
\
The f in d in g o f t h i s enzyme g iv e s a p a r t i a l answ er t o th e
q u e s tio n o f how C lo s trid iu m therm o cellu m can use c e lld b io s e a s an
e n e rg y so u rc e w ith o u t b e in g a b le t o u se exogenous g lu c o s e .
- 5 INTRODUCTION AND HISTORICAL REVIEW
Phosphoro ly s i s i s any p ro c e s s w hereby in o rg a n ic phosphorus
becomes e s t e r i f i e d .
I n one ty p e o f p h o sp h o ro ly s i s , p h o sp h o ric a c id is
s u b s t i t u t e d f o r w a te r i n a r e a c t i o n s im ila r t o h y d r o ly s is .
T h is r e s u l t s
i n th e fo rm a tio n o f a p h o sp h ate e s t e r in s te a d o f th e u s u a l h y d r o ly tic
p r o d u c t.
L iv in g system s w hich o f te n r e q u ir e th e e x p e n d itu re o f la rg e
amounts o f en erg y t o form su g ar p h o sp h ate s from m onosaccharides may be
a b le t o o b ta in th e s e same compounds from d is a c c h a r id e s and p o ly s a c ­
c h a rid e s b y phosphor o ly s i s w ith a s m a lle r o u tla y o f e n e rg y .
The p h o s­
p hor o l y t i c r e a c tio n s a re c a ta ly z e d b y a v a r i e t y o f enzymes c a ll e d
p h o sp h o ry la se s w hich a re w id e ly d i s t r i b u t e d i n an im al and p l a n t t i s s u e s
where th e y p la y im p o rta n t r o l e s in the. s y n th e s is and breakdow n o f many
su g a rs and p o ly s a c c h a r id e s .
The f i r s t r e c o g n itio n o f p h o s p h o ro ly s is o c c u rre d when C o ri and
C o ri ( 1936) u sed d ia ly z e d e x t r a c t s o f r a b b i t b r a i n , h e a r t , and l i v e r t o
d e m o n strate t h a t th e g ly c o ly tic r e a c tio n s w ere i n i t i a t e d b y th e fo rm a tio n
o f g lu c o s e - I -p h o sp h ate from g ly co g en and in o rg a n ic p h o s p h a te .
Hanes
( 1940a and b ) d is c o v e re d a s im ila r enzyme i n e x tr a c ts from p e as and
p o ta to e s w hich would c a ta ly z e th e phosphor o l y t i c breakdown o f s ta r c h and
g ly c o g e n .
The p h o s p h o ro ly s is o f d is a c c h a r id e s b y b a c t e r i a was f i r s t d is c o v e re d b y Kagan, L a tk e r , and Zfasman ( 194-2 ) and Doudoro f f , K aplan,
and H a ssid ( 1943) •
The form er group fo u n d a su c ro se p h o sp h o ry la se in
- 6 th e c e l l s o f L euconostoc m e se n te ro ld e s and th e l a t t e r re c o g n iz e d a s im ila r
enzyme i n th e c e l l s o f Pseudomonas s a c c h a r o p h ila .
The a c ti o n o f t h i s
enzyme may be r e p r e s e n te d as
S u cro se + H^PO^ —S u cro se Phosphor y la s e ^ G lu c o se -!-p h o sp h a te + F r u c to s e .
I n 1952, F i t t i n g and S cherp n o ted t h a t m a lto se was b ro k en down b y p h o s­
p h o r o ly s i s w ith an enzyme p r e p a r a tio n from th e c e l l s o f N e is s e r ia
m e n in g itid is :
M altose + HgPO^ —M altose Phosphoryla.se ^ G lu c o se -1 -p h o sp h ate + G lu co se.
The g lu c o s e - I -p h o sp h ate form ed b y th e s e r e a c tio n s i s r e a d i l y degraded b y
one o f s e v e r a l e n erg y y ie ld in g p r o c e s s e s .
The f a t e o f th e monosac­
c h a rid e w hich is a ls o form ed b y p h o s p h o ro ly s is i s n o t a s w e ll u n d e rsto o d .
Some b a c t e r i a such a s L euconostoc d ex tran icu m p o ly m erize th e su g ar t o form
a d e x tra n th u s g e t t i n g r i d o f a u s e le s s p r o d u c t.
However, an u n ex p lain ed
s i t u a t i o n i s e n c o u n te re d when th e l i v i n g c e l l s o f th e s e organism s w i l l use
d is a c c h a r id e s r a p i d l y b u t n o t th e m o n o sacch arid es, which a re b e lie v e d t o
be p ro d u c ts o f p h o s p h o ro ly s is .
therm ocellum
Such a s i t u a t i o n e x i s t s when C lo strid iu m
i s grown on c e ll o b io s e .
b u t n o t g lu c o s e .
T h is s p e c ie s w i l l u se c e I lo b lo se
G lucose i s n o t found fo llo w in g grow th on c e llo b io s e ,
e i t h e r as th e f r e e s u g a r o r a s a p o ly m e r.
B e fo re th e r e c o g n itio n o f p h o s p h o ry la s e s , th e grow th p e c u l i a r i ­
t i e s o f some o f th e c e llu lo se -d e c o m p o sin g b a c t e r i a o f th e genus Cytophaga
- 7 w ere e x p la in e d Tay H u tchinson and C la y to n (1919) and Im esenecki and
S o ln ts e v a in 1936 a s i n h i b i t i o n o f grow th and c e ll u lo s e d e co m p o sitio n b y
t r a c e s o f g lu c o se and o th e r re d u c in g s u g a r s .
They p o s tu la te d t h a t th e s e
w ere o b lig a te c e llu lo se -d e c o m p o sin g o rg a n ism s, cap ab le o f u t i l i z i n g
c e ll u lo s e d i r e c t l y w ith o u t i t s f i r s t b e in g h y d ro ly z e d t o s u g a r s .
S ta in e r
(19^2) found th e same phenomenon i f th e g lu c o se had b e en s t e r i l i z e d b y
h e a t.
I f , how ever, th e g lu c o se had b e e n s t e r i l i z e d b y f i l t r a t i o n , th e
Cytophaga u t i l i z e d i t even more r a p i d l y t h a n . c e l l u l o s e .
T h e re fo re , he
concluded t h a t h e a t s t e r i l i z a t i o n decomposed g lu c o se t o form to x ic
p ro d u c ts w hich i n h i b i t e d th e grow th o f some c e llu lo se -d e c o m p o sin g
o rg a n ism s.
On th e o th e r h a n d , many w orkers have fo u n d th e r a p i d u t i l i z a ­
t i o n o f d is a c c h a r id e s and p r a c t i c a l l y n o n - u t i l i z a t i o n o f m o n o sac c h arid es.
D o u d o ro ff, K aplan, and H a ssid ( 19^-3 ) fo u n d t h a t su c ro se was r a p i d l y
u t i l i z e d b y th e i n t a c t c e l l s o f Pseudomonas s a c c h a ro p h ila w hereas g l u ­
cose was o x id iz e d v e r y s lo w ly and f r u c to s e was n o t a tta c k e d .
Yet f r u c ­
to s e was th e p ro d u c t b o th o f th e p h o sp h o ro ly s i s and h y d r o ly s is o f su c ro se
b y b a c t e r i a l p r e p a r a t io n s .
D oud o ro ff, e t a; a l , , ( I9U9) a ls o fo u n d a s im ila r
anomalous s i t u a t i o n i n th e c e l l s o f a m utant s t r a i n o f E s c h e r ic h ia c o l l .
The l i v i n g c e l l s would m e ta b o liz e m a lto se r e a d i l y b u t n o t g lu c o s e .
How­
e v e r , when b a c t e r i a l - f r e e enzyme p r e p a r a tio n s were u s e d , g lu c o se was
form ed in th e breakdown o f m a lto s e .
C lo s trid iu m therm pcellum i s an a n a e ro b ic , th e r m o p h ilic , c e l l u ­
l o l y t i c o rg an ism .
I t was f i r s t o b ta in e d in p u re c u ltu r e b y McBee ( 19^8 )
- 8 ■who r e p o r te d t h a t t h i s organism a tta c k e d c e l l u l o s e and c e llo h io s e h u t n o t
g lu c o s e , r e g a r d le s s o f w h e th er th e g lu c o se was s t e r i l i z e d h y h e a t o r
filtra tio n .
The c e l l u l o s e was f i r s t b ro k e n down t o c e llo h io s e h y th e
enzyme c e l l u l a s e and th e l a t t e r was b e lie v e d t o h e f u r t h e r b ro k e n down
h y th e enzyme c e ll o b ia s e t o form two m o lecu les o f g lu c o s e .
S in c e t h i s
organism d id n o t u t i l i z e g lu c o se i n th e c u ltu r e medium, th e h y p o th e sis
t h a t c e llo h io s e was h y d ro ly z e d t o g lu c o se does n o t seem t o h e l o g i c a l .
The p r e s e n t s tu d y was th e r e f o r e u n d e rta k e n t o d eterm in e w h eth er
u n re c o g n ize d pathw ays o f c e llo h io s e u t i l i z a t i o n m ight o c c u r i n C lo s trid iu m
th erm o cellu m .
MATERIAIS AED METHODS
C u ltu re
C lo s trid iu m therm ocellu m , s t r a i n 157, was o b ta in e d from D r, R.
H. McBee who i s o l a t e d i t from s o i l i n 1946. The c h a r a c t e r i s t i c s o f t h i s
'
organism w ere g iv e n h y McBee ( 1954) . S to c k c u ltu r e s w ere m a in ta in e d in
r o l l e d tu b e s o f c e ll u lo s e a g a r u s in g H u n g aters (1950) a n a e ro b ic te c h n iq u e .
C u ltu re Medium
The b a s ic c u ltu r e medium u se d th ro u g h o u t t h i s work was composed
o f O.O75 p e r c e n t E aC l; 0 .0 3 p e r c e n t (HH^)2SOip* 0 .0 3 p e r c e n t KH2POip*
0*05 p e r c e n t K2HPO^;
0 .0 1 p e r c e n t % sb% 'f'0 .0 1 p e r c e n t CaCl2 ,* 0 .5 p e r
c e n t h a i t t m i l l e d c e l l u l o s e o r o th e r C arh o h y flrate; 0 .1 p e r c e n t y e a s t
e x t r a c t ; 0 .0 2 p e r c e n t sodium t h i o g l y c o l l a t e ; 0 .5 p e r c e n t HaHCOg and a .
s m a ll amount o f r e s a z u r i n a s an o x id a tio n - r e d u c tio n i n d i c a t o r .
B ecause
- 9 HaHCOg i s r e a d i l y decomposed b y h e a t , i t ^ srk fcq riliatecfc byf i l t r a t i o n o f a 10 p e r c e n t s o lu tio n and added t o th e medium a t a
te m p e ra tu re below 60 C.
w ith 2 p e r c e n t a g a r .
F o r th e r o l l e d tu b e s th e medium was s o l i d i f i e d
The c e ll u lo s e was p re p a re d i n a 5 p e r c e n t aqueous
s u sp e n sio n a c c o rd in g t o th e d ir e c tio n s o f Hungate ( 1950)•
P r e p a r a tio n o f Large C u ltu r e s .
Ten ml o f a l i q u i d c e ll u lo s e
medium was in o c u la te d w ith a co lo n y p ic k e d from a r o l l e d tu b e o f c e ll u lo s e
ag ar.
A f te r two o r t h r e e days' in c u b a tio n a t 55 C t h i s c u ltu r e was u sed
t o in o c u la te 100 ml o f medium.
A fte r th r e e days a t 55 C, t h i s 100 ml
s e rv e d a s an inoculum f o r two l i t e r s o f medium w hich was i n t u r n in c u b a te d .
f o r th r e e days and u sed a s th e inoculum f o r a f i v e g a llo n c a rb o y o f th e
medium.
T h is was in c u b a te d a t 55 C i n a la r g e w a te r b a t h .
S t e r i l e medium
i n th e carb o y was k e p t re d u c e d b y b u b b lin g carb o n d io x id e th ro u g h i t p r i o r
t o in o c u la tio n , s in c e i t had b e ep fo u n d t h a t u n le s s th e r e s a z u r i n in th e
medium was re d u c e d t o th e c o lo r le s s s t a t e , good grow th c o u ld n o t be
e x p e c te d .
T his f lu s h in g w ith carb o n d io x id e was n e c e s s a ry f o r th e f i r s t
f i f t e e n ho u rs o f in c u b a tio n ; a f t e r w hich tim e th e organism s produced s u f ­
f i c i e n t OOg and Hg t o keep th e medium i n a re d u c ed s t a t e .
A m ech an ical
s t i r r e r was th e n u sed t o d is p e r s e th e re m a in in g c e ll u lo s e th ro u g h o u t th e
medium, s in c e i t was found t h a t fe rm e n ta tio n p ro ceed ed more r a p i d l y when
th e c e l l u l o s e was k e p t suspended.
When a l l th e c e ll u lo s e had d is a p p e a re d
(a b o u t 48 h o u rs ) th e c u ltu r e was n e u tr a liz e d w ith a p p ro x im a te ly 50 ml o f
g l a c i a l a c e t i c a c id t o p re v e n t a n a l k a l i n e c o n d itio n due t o th e lo s s o f
- 10
COg.
The c e l l s were th e n h a rv e s te d from th e c u ltu r e w ith a cream
s e p a r a to r and u sed a t once f o r making c e l l - f r e e enzyme p r e p a r a t io n s .
The
c e l l y i e l d was u s u a ll y a b o u t 20 g w et w eig h t from e ac h f i v e g a llo n c u l t u r e .
P r e p a r a tio n o f th e Enzyme
The c e l l s w ere b ro k e n em ploying M cIlw ain's ( 19^8 ) method as
recommended b y H a y a ish i and S ta n ie r ( l 95l ) .
G rin d in g , o f th e c e l l s was
done i n a m o rta r a t 0 t o 5 C f o r t e n m in u tes w ith tw ic e t h e i r w eig h t o f
alum ina (A lcoa C hem icals alum ina A-30l ) ,
About 40 ml o f .M/35 sodium
a c e t a t e - b a r b i t a l b u f f e r a t pH 7 . 0 was added d u rin g th e g rin d in g t o e x ­
t r a c t th e s o lu b le enzym es.
by c e n trifu g a tio n .
o f th e e x p e rim e n ts .
cru d e enzyme.
The alum ina and in s o lu b le d e b r is were removed
The sp eed o f c e n tr if u g a tio n was v a r ie d w ith th e c o u rse
The e x t r a c t s o b ta in e d w ere u sed a s th e so u rc e o f
The tr e a tm e n t o f th e e x t r a c t a ls o v a r ie d a c c o rd in g t o th e
ex perim ent i n w hich i t was u s e d .
Phosphorus D e te rm in a tio n
The F is k e and Subbarow (1925) method o f in o rg a n ic p h o sp h ate
d e te r m in a tio n was u sed fo llo w in g p r e c i p i t a t i o n o f p r o te in s w ith 100 p e r
c e n t t r i c h l o r o a c e t i c a c id .
H e a t- la b il e o rg a n ic p h o sp h ates w ere d eterm in ed
b y th e same method fo llo w in g h y d ro ly s is w ith I H h y d ro c h lo ric , a c id f o r
sev en m inutes in a b o i l i n g w a te r b a t h . . In o rg a n ic p h o sp h ate i s d e sig n a te d
a s P i , th e l a b i l e o rg a n ic p h o sp h ate as P ^ y .
- 11
S ugar D e te rm in a tio n
R educing su g a rs w ere d e term in e d "by th e Folin-M alm ros (1929)
m ethod.
P ap er Chrom atography
A scending p a p e r chrom atography was u sed t o s e p a r a te p h o sp h ate
d e r iv a tiv e s o f s u g a r s .
The method o f Hanes and Isherw ood ( 1949) was
m o d ifie d , u sin g t e r t ia r y - b u ta n o l- p i c r i c a c id - w a te r (80 m l, 4 g , 20 ml)
and n - h u ta n o l- a c e tic a c id - w a te r ( j k m l, 19 m l, $0 ml) (Benson e t a d .,
1950) a s s o lv e n t s .
S o lu tio n s c o n ta in in g unknown m ix tu re s o f su g ar p h o s ­
p h a te s were s p o tte d a t 2 .5 cm i n t e r v a l s alo n g th e s t a r t i n g l i n e o f a
s h e e t o f f i l t e r p a p e r (Whatman
p i p e t t e and a llo w ed t o a i r d r y .
Ho. I and 4 ) b y means o f a c a p i l l a r y
The p a p e r was th e n hung i n a b a t t e r y
j a r c o n ta in in g th e s o lv e n t m ix tu re so t h a t th e edge o f th e p a p e r n e ar th e
unknown s p o ts d ip p ed in to th e s o lv e n t .
The j a r was th e n c o v ered w ith a
g la s s p l a t e and s e a le d w ith s c o tc h ta p e t o p re v e n t e v a p o r a tio n o f th e
s o lv e n t .
A fte r a p e r io d o f a t l e a s t 15 h o u rs , th e f i l t e r p a p e r was
removed from th e j a r , d r ie d i n an oven a t 55 C, and d ev elo p ed t o d e te c t
p h o sp h ate compounds.
The p a p e rs w ere sp ra y e d w ith a s o l u t i o n c o n ta in in g
5 ml o f 50 p e r c e n t p e r c h lo r ic a c id , 10 ml o f I N HCl and 25 ml o f 4 p e r
c e n t ammonium m olybdate p e r IpO m l.
The sp ra y e d p a p e rs w ere d r ie d a g a in
a t 55 C f o r a few m inutes t o remove e x c e ss w a te r , th e n t r a n s f e r r e d t o an
85 C oven, i n w hich th e y w ere h e a te d f o r sev en m in u te s .
T h is tre a tm e n t
even c au ses enough h y d ro ly s is o f su ch r e s i s t a n t e s t e r s as.
- 12
3 -phospho- g ly c e r ic a c id and g lu c o s e - 6 -p h o sp h ate t o re n d e r them d e te c ta b le
in m inute amounts (Hanes and Isherw ood 1949) .
The p o s i t i o n o f th e
p h o sp h o ric e s t e r s was v i s i b l e a t t h i s s ta g e , b u t t o d e v elo p th e c o lo r
f u l l y , th e p a p e rs were a llo w ed t o r e g a in m o is tu re from th e a i r and th e n
p la c e d in a j a r c o n ta in in g H2S f o r f i v e m in u te s .
The p h o s p h o r ic .e s te r s
th e n a p p ea re d as i n te n s e ly b lu e s p o ts a g a in s t a b u f f b ack g ro u n d .
The
ty p e o f compound c o u ld b e d e term in e d b y i t s p o s i t i o n on t h e .chrom atogram .
EXPERIMENTAL RESULTS
Growth E xperim ent
A p r e lim in a r y e x am in atio n o f C lo s trid iu m th erm o cellu m f o r th e
p re s e n c e o f a c e llo b io s e p h o sp h o ry la se was made b y a sim p le grow th e x p e r i­
m ent.
A l i t e r c u ltu r e o f th e organism was grown in th e b a s a l medium
c o n ta in in g c e ll o b io s e .
A f te r th r e e days o f in c u b a tio n , n o t o n ly was
v ig o ro u s grow th a p p a r e n t, b u t a ls o a d e c re a s e i n in o rg a n ic p h o sp h ate was
n o te d .
However, when 0 .5 p e r c e n t g lu c o se was s u b s t i t u t e d f o r c e llo b io s e
i n th e above medium, no grow th was o b ta in e d and no d e c re a se in in o rg a n ic
p h o sp h ate was n o te d (T ab le I ) .
T h is co n firm ed th e e a r l i e r o b s e rv a tio n
t h a t g lu c o se was n o t u t i l i z e d b y t h i s o rg an ism , and s u p p o rte d ,the th e o ry
t h a t a p h o sp h o ry la se m ight b e p r e s e n t i n t h i s organism .
P h o sp h ate d e te rm i
n a tio n s a f t e r 30 days in c u b a tio n showed a n even l a r g e r u p ta k e o f p h o sp h ate
i n th e c e llo b io s e c u ltu r e w ith no change i n th e g lu c o se c u l t u r e .
- 13 -
TABLE I .
Changes i n in o rg a n ic p h o sp h ate o f th e c u ltu r e medium
d u rin g grow th o f C lo s trid iu m th erm o cellu m .
S u b s tr a te
G lucose
C e llo b io se
P i - mg
In itia l
2 .8 1
2 .8 6
A f te r th r e e days
2 .8 l
2 .6 4
A f te r one month
2 .8 1
2 .6 l
Ce 11-F ree Enzyme Experim ents
I n o r d e r t o s tu d y more c o m p le te ly th e p h o sp h o ry la se which had .
Been d em o n strated i n o n ly a p resu m p tiv e manner w ith a l i v i n g c u l t u r e , i t
was n e c e s s a ry t o use enzyme p r e p a r a tio n s f r e e o f l i v in g c e l l s .
These
were p re p a re d b y g rin d in g th e c e l l s o b ta in e d from f i v e g a llo n c u ltu r e s
in th e manner a lr e a d y o u tlin e d .
E xperim ent I .
An enzyme p r e p a r a t io n o b ta in e d from th e g rin d in g
o f 20 g o f c e l l p a s te w ith Uo g o f alum ina was e x tr a c te d w ith 40 ml o f
a n M/3 5 s o d iu m -a c e ta te - b a r b i t a l b u f f e r , pH 7 . 0 .
The alum ina and la r g e r
c e l l p a r t i c l e s were removed b y c e n tr if u g in g f o r 20 m inutes a t a r e l a t i v e
c e n t r i f u g a l f o r c e o f a b o u t 2000 G.
w ith a p i p e t t e .
The s u p e rn a ta n t f l u i d was drawn o f f
Such p r e p a r a tio n s y ie ld e d 25 t o 30 ml o f a n o p a le s c e n t
crude enzyme m ix tu re .
A 6 .1 5 #
p o r ti o n o f t h i s f l u i d was p la c e d i n each
o f s i x 20 ml t e s t tu b e s , two o f w hich were th e n p la c e d i n a b o i l i n g w ater
b a th f o r 15-20 m inutes t o i n a c t i v a t e th e enzym es.
S u f f i c i e n t 0 .0 1 M
p h o sp h ate s o l u tio n was added t o g iv e a n in o rg a n ic p h o sp h ate c o n c e n tra tio n
o f 0 .1 6 m g/ml.
Magnesium s u l f a t e was added a s an enzyme a c t i v a t o r in a
f i n a l c o n c e n tr a tio n o f I . 63 mg/ml, and 0 .0 3 4 mg/ml o f sodium f l u o r i d e was
u sed t o i n h i b i t p h o sp h a ta se s w hich m ight i n t e r f e r e w ith th e accu m u latio n
o f o r g a n i c p h o s p h a te s .
To one b o ile d c o n tr o l tu b e and t o two o f th e o th e r
tu b e s was added 1 . 63 mg/ml o f g lu c o s e .
was added t o th e o th e r th r e e t u b e s .
A s i m i l a r q u a n tity o f c e llo b io s e
In o rg a n ic p h o sp h ate s and h e a t - l a b i l e
p h o sp h a te s w ere d e term in e d im m ed iately a f t e r th e a d d itio n o f th e su g ars
and a l s o fo llo w in g a tw o-hour in c u b a tio n a t 37 C.
T here was no s i g n i f i c a n t p h o sp h ate change i n th e tu b e s t o
w hich g lu c o se had b e e n added (T ab le I I ) .
The a d d itio n o f c e ll o b io s e ,
how ever, r e s u l t e d in a d e c re a s e o f th e in o rg a n ic p h o s p h a te , in d ic a tin g
t h a t p hosphoro ly s i s o f th e c e llo b io s e had ta k e n p la c e .
B ut th e r e was
no e v id en c e t h a t g lu c o s e - I -p hosph ate was a p ro d u c t o f t h i s r e a c t i o n .
- 15 TABLE I I
Changes i n p h o sp h ate when a c e l l - f r e e enzyme p r e p a r a tio n a c ts on
g lu c o se and c e ll d b io s e . R e s u lts a re th o s e o f d u p lic a te e x p e rim e n ts .
_______________________ S u b s tr a te ________________________
G lucose
C o n tro l
C e lld b io se
C o n tro l
P i - mg
In itia l
0 .8 1
0 .8 l
0 .8 0
0 .8 0
0 .7 9
0 .8 1
F in a l
0 .? 2
0 .9 5
0 .8 3
0 .6 4
0 .^ 8
0 .8 2
+ 0.11
+0.1k*
+0.03
-0 .1 5
-0 .2 1
+0.01
In itia l
0 .0 0
0 .0 0
0 .0 1
0 .0 4
0 .0 2
0 .0 1
F in a l
0 .0 0
0 .0 1
0 .0 5
0 .0 1
0 .0 6
0 .0 1
0 .0 0
+ 0.01
+0 .0 4
-0 .0 3
+0 .0 4
0 .0 0
In itia l
0 .8 1
0 .8 1
0 .8 1
0 .8 4
0 .8 1
0.83
F in a l
0 .9 2
0 .9 6
0 .8 8
0 .6 6
0 .6 4
0 .8 3
+ 0.11
+0.15
+0.07
- 0 .1 8
-0 .1 7
0 .0 0
S x T - inE
P t o t a l - mg
*The in c re a s e i n in o rg a n ic phosp h ate w ith g lu c o se is p ro b a b ly due to
th e a c ti o n o f n o n - s p e c if ic p h o sp h ata ses on o rg a n ic p h o sp h ate s from
th e ground c e l l s .
- 16 E xperim ent 2 .
A r e p e t i t i o n o f E xperim ent I , u s in g an enzyme .
p r e p a r a tio n w hich had b e e n fr o z e n f o r s e v e r a l days gave s i m i l a r r e s u l t s
w ith r e s p e c t t o in o rg a n ic p h o sp h ate (T ab le I I I ) , and in a d d itio n showed
an in c re a s e i n th e h e a t - l a b i l e p h o sp h a te .
The d e c re a se i n th e in o rg a n ic phosphorus f r a c t i o n was.
p r o p o r tio n a l t o th e in c re a s e in th e 7 m inute h e a t - l a b i l e o rg a n ic p h o s­
p h a te f r a c t i o n w hich c o n ta i n s ' th o s e e s t e r s i n w hich th e p h o sp h ate r a d i c a l
i s a tta c h e d a t th e number I p o s i t i o n on th e s u g a r, such a s g lu c o s e -1 p h o s p h a te .
These a r e h e a t l a b i l e in d i l u t e a c id s o lu tio n s a s compared
t o th e h e a t s t a b l e g lu c o s e - 6 -p h o sp h a te .
T h e re fo re , th e 7 -m inute h e a t-
l a b i l e o rg a n ic p h o sp h ate f r a c t i o n may b e d eterm in ed b y s u b tr a c ti n g th e
c o n c e n tra tio n o f th e in o rg a n ic p h o sp h ate f r a c t i o n p r i o r t o h y d ro ly s is
from t h a t fo llo w in g h e a tin g f o r 7 m inutes a t 100 C i n I N HGI .
T his
,accu m u la tio n o f h e a t - l a b i l e p h o sp h ate was e n co u n te re d o n ly when th e
enzyme p r e p a r a tio n had b een fr o z e n f o r a few d a y s.
O th erw ise th e r e was
a d e c re a se i n in o rg a n ic phosphorus b u t no c o rre sp o n d in g in c re a s e in th e
7-minut.e phosphorus f r a c t i o n .
T his was a t t r i b u t e d t o th e p re s e n c e o f
phosphogTucom utase, a n enzyme w hich c o n v e rts g lu c o s e - I -p h o sp h ate (h e a t
l a b i l e ) t o g lu c o s e - 6 -p h o sp h ate (h e a t s t a b l e ) .
phosphogIu c omuta s e was in a c t iv a te d b y f r e e z i n g .
I t a p p ea re d t h a t th e
- 17 TABLE I I I
Changes in p h o sp h ate when a fro z e n enzyme p r e p a r a tio n a c te d on g lu c o se
and c e I l o h io s e . R e s u lts a re th o s e o f d u p lic a te e x p e rim e n ts .
________________________S u b s tr a te _______________________
G lucose
C o n tro l
C e llo h io se
C o n tro l
P i - mg
1 .0 7
1 .0 6
0 .9 9
1 .0 7
1 .0 7
1.05
F in a l
1 .0 7
1 .0 ?
1.0 0
0 .9 4
0 .9 0
1.03
0 .0 0
-0 .0 1
+ 0 .0 1
-0 .1 3
-0 .1 7
-0.02
In itia l
0 .0 0
0 .0 0
0 .0 2
0 .0 0
0 .0 0
0 .0 1
F in a l
0 .0 1
0 .0 1
0 .0 2
0 .1 1
0 .1 3
0 .0 0
+0.01
+ 0.01
0 .0 0
+ 0.11
+0.13
-0 .0 1
In itia l
1 .0 7
1 .0 6
1 .0 1
1 .07
1 .0 7
1.0 6
F in a l
1 .0 2
1 .0 6
1.0 2
1.05
1 .0 3
1.03
-0 .0 5
0 .0 0
+0.01
-0 .0 2
-0 .0 4
-0 .0 3
S
In itia l
P to ta l
- 18 E xperim ent 3 .
The e f f e c t . o f f r e e z in g o f th e enzyme shown in
E xperim ent 2 was confirm ed on two a d d i t i o n a l enzyme p r e p a r a t io n s .
The
changes i n p h o sp h ate s i n th e p re se n c e o f c e llo b io s e w ere d eterm in ed on a
p o r ti o n o f th e enzyme when i t was p re p a re d and a g a in a f t e r h av in g been
fr o z e n f o r one week (T ab le IV ).
I n b o th c a s e s , i t was fo u n d t h a t th e
phosphoglucorautase had b e e n in a c t iv a te d and t h a t g lu c o s e - I -phosphate
had a c c u m u la te d .
E xperim ent k .
To determ in e w h eth er th e e f f e c t o f f r e e z in g th e
enzyme p r e p a r a tio n was t r u l y due t o an i n a c t i v a t i o n o f phosphoglucorautase
o r p o s s ib ly t o some o th e r r e a c t i o n , an ex p erim en t u sin g g l u c o s e - 1 - p h o s "
'phate a s th e s u b s t r a t e was s e t up w ith f r e s h enzyme and w ith enzyme w hich
had b een fr o z e n f o r one w eek.
F r e s h ly p re p a re d enzyme c o n v e rte d n e a r ly
a l l o f th e g lu c o s e - I -p h o sp h ate t o a h e a t s t a b l e compound, presum ably
g lu c o s e - 6 -p h o sp h a te , w hereas th e fr o z e n enzyme had l i t t l e , i f an y , e f f e c t
on th e c o n c e n tra tio n o f h e a t - l a b i l e p h o s p h a te , in d ic a tin g t h a t th e
g lu c o s e - I -p h o sp h ate rem ained u n a lte r e d (T ab le V ).
-
- 19 TABLE IV
Changes in p h o sp h ate when f r e s h l y p re p a re d and fr o z e n
enzyme p r e p a r a tio n s were a llo w ed t o a c t on c e ll o h io s e .
R e s u lts a re th o se o f d u p lic a te e x p e rim e n ts .
____________________ Enzyme______________________
F r e s h ly p re p a re d __________ F ro zen f o r one week
P i - mg
In itia l
0 .7 3
0 .7 3
0 .7 7
0 .7 7
F in a l
0 .5 1
0 ,k 9
0 .5 9
0 .5 9
-0 .2 2
-0 .2 4
-0 .1 8
-0 .1 8
In itia l
0 .0 0
0 .0 0
0.00
0.00
F in a l
0 .0 0
0 .0 0
0.10
0.11
+ 0.10
+ 0.11
PA7 - ° e
0.00
0.00
P t o t a l - mg
In itia l
O.73
O.73
0 .7 7
0 .7 7
F in a l
0 .5 1
0 .4 9
O.69
0 .7 0
0.22
-0 .2 4
-0 .0 8
-0 .0 7
-
- 20
TABLE V
Changes in p h o sp h ate when f r e s h and fro z e n enzyme were
a llo w ed t o a c t on g lu c o s e - I -p h o s p h a te . R e s u lts a re
th o s e o f d u p lic a te e x p e rim e n ts .
Enzyme
F r e s h ly p re p a re d
F ro zen f o r one week
S u b s tr a te
G lucose - I -phosphate
G lucose - I -p h o sp h ate
P i - mg
In itia l
0 . 2b
0 .2 4
0.2 2
0 .2 0
F in a l
0 .2 2
0 .2 3
0 .1 8
0 .1 5
-0 .0 2
-0 .0 1
- 0.0 4
-0 .0 5
In itia l
0 .6 9
0 .7 4
1 .2 7
1 .2 7
F in a l
0 .1 7
0 .1 6
1.23
1 .3 1
-0 .5 2
-O.58
-0 .0 4
+0 .0 4
In itia l
0 .9 3
0 .9 8
1.49
1 .4 6
F in a l
0 .3 9
0 .3 9
1 .4l
1 .4 6
-0 .5 k
-0.5 9
-0 .0 8
0 .0 0
Pa 7 " nS
P t o t a l - mg
- 21 Is o to p e E xperim ents
The s tu d y o f th e changes i n in o rg a n ic and h e a t - l a b i l e p h o sp h ate
su p p o rte d th e " b e lie f t h a t C lo s trid iu m therm ocellum was c ap a b le o f
s p l i t t i n g c e llo b io s e b y p h o sp h o ro ly si s b u t f a i l e d t o d em o n strate con­
c lu s i v e ly t h a t in o rg a n ic p h o sp h ate was in c o rp o ra te d in to , th e p h o sp h ate
e s t e r s o f g lu c o se b y t h i s mechanism.
I t ap p eared t h a t t h i s problem
c o u ld b e s t be s o lv e d th ro u g h th e use o f in o rg a n ic p h o sp h ate e n ric h e d
w ith r a d io a c ti v e p h o sp h o ru s, p 3^ .
O rganic p h o sp h ates d e riv e d from th e
in o rg a n ic p h o sp h ate c o u ld th e n be a t l e a s t p a r t i a l l y s e p a ra te d b y
p a p e r chrom atography and th e r a d i o a c t i v i t y o f each group o f compounds
d e te rm in e d .
The
was o b ta in e d from O ak rid g e, T en n essee, a s an aqueous
s o lu tio n o f o r th o -E^PO^ w ith a s p e c i f i c a c t i v i t y o f ab o u t
0 .025 m g /m illic u r ie .
T h is s o lu tio n was d i l u t e d 100 tim e s and added t o
enzyme p r e p a r a tio n s i n q u a n t i t i e s s u f f i c i e n t t o g iv e a s a t i s f a c t o r y
c o u n t.
A c e l l - f r e e enzyme p r e p a r a tio n e n ric h e d w ith P^^ was s e t up
i n d u p lic a te w ith g lu c o se and w ith c e llo b io s e as s u b s t r a t e s .
enzyme c o n tr o ls were in c lu d e d f o r each s u g a r .
B o ile d
C hrom atographic a n a ly se s
were made im m ed iately a f t e r adding th e s u b s tr a te and a g a in fo llo w in g two
hours* in c u b a tio n a t 37 0 .
Each ch rom atographic a n a ly s is was perform ed
i n d u p lic a te , one b e in g developed in th e t e r t i a r y b u t a n o l - p i c r i c a c id w a te r s o lv e n t and th e o th e r w ith th e n - b u ta n o l- a c e tic a c id - w a te r s o lv e n t..
- 22 Development was f o r 15 h o u rs a t room te m p e ra tu re .
C o n tro ls o f known
p h o sp h o ric e s t e r s w ere in c lu d e d on each chromatogram.
The r a d i o a c t i v i t y o f th e v a rio u s p h o sp h ate e s t e r s form ed d u rin g
th e c o u rse o f th e ex p erim en t was d e term in e d w ith a g e ig e r c o u n te r.
The
s p o ts w ere sim p ly c u t from th e p a p e r a f t e r c o lo r developm ent and co u n ted
d i r e c t l y u s in g a g e ig e r tu b e w ith a t h i n m ica window.
P ap er chrom atography d id n o t s e p a r a te g lu c o s e - I -p h o sp h ate and
g lu c o s e - 6 -p h o sp h a te .
S in c e th e y were in te r c o n v e r tib le h y enzymes in th e
e x p e rim e n ta l sy stem , no a tte m p t was made t o s e p a ra te them b y th e use o f
o th e r d e v e lo p in g s o lv e n t s .
. T y p ic a l chromatograms .of known p h o sp h o ric e s t e r s and th o se
form ed b y p h o s p h o ro ly s is a re p re s e n te d d ia g ra m m a tic a lly in F ig u re I
( t e r t i a r y - b u t a n o l - p i c r i c a c id - w a te r) and F ig u re 2 ( n - b u ta n o l- a c e tic
a c id - w a te r ) .
R a d i o a c tiv ity was found o n ly in th e s p o t s .r e p r e s e n tin g th e
in o rg a n ic p h o s p h a te , g lu c o s e - I -p h o sp h ate and g lu c o s e - 6 -phophate m ix tu re , '
and th e hexose d ip h o s p h a te .
There was no s i g n i f i c a n t e s t e r i f i c a t i o n o f
r a d io a c tiv e 'p h o s p h a te i n an y o f th e c o n tr o l tu b e s ( b o ile d enzyme) o r
th o s e c o n ta in in g g lu c o se a s a s u b s tr a te (.Table V I) .
There w as, how ever,
th e in c o rp o ra tio n o f m easurable q u a n t i t i e s o f r a d io a c tiv e phosphorus
i n t o th e m ix tu re o f g lu c o s e - I -phosphate and g lu c o se -6 -p h o s p h a te .
I t is
b e lie v e d t h a t most o f t h i s was in th e form o f g lu c o se - I -p h o sp h ate s in c e
th e c o lo r o f t h i s s p o t developed v e r y r a p i d l y , in d ic a tin g th e p re se n c e
- 23 -
F ig u re I .
D iagram m atic r e p r e s e n ta tio n o f a chromatogram o f o rg a n ic
p h o sp h ate a f t e r developm ent w ith t e r t i a r y - b u t a n o l - p i c r i c
a c id - w a te r .
- 2k -
In o rg a n ic
o rth o p h o sp h a te
3 -p h o sp h o g ly c erate
Glu c o s e -6 -phosphate
F r u c to s e - 6 -phosphate
G lu c o se - I -phosphate
F r u c to s e - 1 , 6 -d ip h o sp h a te
The known p h o sp h o ric e s t e r s a re s p o tte d a lo n g t h i s line
F ig u re 2 .
The
unknown
D iagram m atic r e p r e s e n ta tio n o f a chromatogram o f o rg an ic
p h o sp h ate s a f t e r d ev elo p in g w ith n - b u ta n o l- a c e tic a c id w a te r .
- 25 TABLE VI
R a d i o a c tiv ity o f s p o ts c u t from chromatograms p re p a re d b e fo re and a f t e r
enzyme a c ti o n on c e ll o b io s e .
I n i t i a l - 0 h r.
S u b s tr a te
Pi
T e r t I a r y - b u ta n o l- p l c r Ic a c id -w a te r
F in a l - 2 h r s in c u b a tio n a t 3'
X
X
+
Hexose
+
Hexose
G lucose I & 6 DiPOh
Pi
G lucose I & 6 DiPOU
G lucose
570
B. G.
No sp o t
494
B . G.
No sp o t
G lucose
726
B . G.
No sp o t
466
6
No sp o t
C o n tro l
U-rJk
No sp o t
717
B . G.
No sp o t
C e llo b io s e
381
B . G.
No sp o t
536
30
No sp o t
C e llo b io se
659
B . G.
No sp o t
532
21
No sp o t
C o n tro l
!+69
B . G.
No sp o t
463
B . G.
No sp o t
I n it ia l - 0 h r.
8
N- b u ta n o l- a c e tic a c id -w a te r
F i n a l - 2 h r s in c u b a tio n a t 37 C
r
Pi
G lucose I Sc 6
Hexose
DiPOi4
G lucose
413
No sp o t
11
G lucose
520
No s p o t
B . G.
762
No sp o t
C o n tro l
387
No s p o t
9
512
No sp o t
C e llo b io s e
351
No s p o t
7
656
92
15
C e llo b io s e
654
No s p o t
B. G.
318
42
13
C o n tro l
620
No s p o t
B. G.
385
No s p o t
S u b s tr a te
Hexose
DiPOu
Pi
655
No sp o t
14
8
G lucose I Sc 6
B. G.
B . G.
P32 wag th e so u rc e o f in o rg a n ic phosphorus
Counts a re in term s o f c o r r e c te d counts p e r m inute above background
X - th e unknown p h o sp h o ric e s t e r
B . G. - background
o f th e more e a s i l y h y d ro ly z a b le compound.
These r e s u l t s co nfirm ed th e
p re se n c e o f a c e llo b io s e phosphory la s e w hich could, s p l i t c e llo b io s e w ith
th e fo rm a tio n o f g lu c o s e - I -p h o sp h a te .
The r e l a t i v e l y la r g e v a r i a t i o n s i n a c t i v i t y shown betw een
th e v a rio u s tu b e s in th e same exp erim en t a re due t o th e u se o f drops o f
l i q u i d o f u n e q u al s iz e i n th e p r e p a r a tio n o f th e chrom atogram s.
These
v a r i a t i o n s a re n o t d e s ir a b le b u t do n o t a f f e c t th e r e l a t i v e r e s u l t s .
The f a i l u r e t o f i n d a s p o t on th e chromatogram d ev elo p ed w ith
one s o lv e n t w hereas i t was v i s i b l e when a n o th e r s o lv e n t was u sed
p ro b a b ly in d ic a te s th e p re s e n c e o f u n id e n tif ie d p h o s p h a te . e s t e r s e x tr a c te d
from th e c e l l s along w ith th e enzym es.
These were a s s o c ia te d w ith th e
.
g lu c o s e - I -phosphate and g lu c o s e -6 -p h o sp h ate when th e t e r t i a r y - b u t a n o l —
p i c r i c a c id -w a te r s o lv e n t was u sed and w ith th e hexose d ip h o sp h ate when
th e n - b u ta n o l- a c e tic a c id -w a te r s o lv e n t was em ployed.
T h is w andering
f r a c t i o n c o n ta in e d no m easurable r a d i o a c t i v i t y and d id n o t in flu e n c e th e
r e s u l t s o f th e e x p e rim e n t.
■
C h a r a c te r is tic s o f th e C e llo b io s e -P h o sp h o ry lase
E f f e c t o f te m p e ra tu re .
The p h o sp h o ry la se was q u ite s t a b l e ,
showing v e r y l i t t l e change in a c t i v i t y fo llo w in g s to ra g e f o r s e v e r a l weeks
I t was a ls o a c ti v e over a r e l a t i v e l y wide ran g e o f te m p e r a tu r e s .
Maxima
and minima were n o t d e te rm in e d , b u t a c t i v i t y was q u ite h ig h oyer th e ran g e
o f 25 C and 55 C (T ab le V I I ) .
A d e c re a se was n o te d a t 65 C, b u t th e
enzyme was s t i l l a c ti v e a t 69 C, w hich i s above th e upper l i m i t o f
-
2?
-
TABLE V II
E f f e c t o f te m p e ra tu re upon a c t i v i t y o f c e lld b io s e p h o sp h o ry la se as
r e f l e c t e d b y changes in p h o s p h a te . R e s u lts a re th o se o f d u p lic a te
e x p e rim e n ts .
_____________________ Tem perature_______________________
________________ 25 C
~
37 C
55 C
65 C
P i - mg
In itia l
1 .1 0
1 .1 0
1 .0 8
1.0 9
1.05
1.0 5
1 .0 1
1.02
F in a l
0 .8 9
0 .9 0
0 . 9U
0 .9 1
0 .9 0
0 .9 0
0 .9 1
0.9 2
-0 .2 1
-0 .2 0
-O .llt
-0 .1 8
-0 .1 5
-0 .1 5
-0 .1 0
-0 .1 0
In itia l
0 .0 0
0 .0 0
0 .0 0
0 .0 0
0 .0 0
0 .0 0
0 .0 0
0 .0 0
F in a l
0 .1 9
0 .1 7
O.O7
0 .1 2
0 .0 9
0 .0 8
0 .0 8
0.0U
+0 .1 9
+0 .1 7
+0 .0 7
+0.12
+0.09
+0.08
+0.08
+0»0U
a7
mS
P t o t a l - mg
I n itia l
1 .1 0
1 .1 0
1 .0 8
1.0 9
1 .0 5
1.05
1 .0 1
1.02
F in a l
1 .0 8
1 .0 7
1 .0 0
1 .02
0 .9 9
0 .9 8
0 .9 9
0 .96
-0 .0 2
- 0 .0 3
-0 .0 8
-0 .0 7
-0 .0 6
-0 .0 7
-0 .0 2
-0 • 06
- 28 grow th o f C lo s trid iu m th erm o cellu m .
E f f e c t of pH.
A s e r i e s o f a c e ta te - b a r b i t a l "buffers o f pH
ra n g in g from U.5 to 8 .0 was used t o d eterm in e th e pH ran g e over which
th e p h o sp h o ry la se was a c t i v e .
A c tiv ity was m easured as th e amount o f
in o rg a n ic p h o sp h ate ta k e n u p .
The c e ll o h l o s e -p h o sp h o ry lase was a c tiv e
over th e e n t i r e ran g e employed h u t th e maximum a c t i v i t y was betw een
pH 6 .8 and 7 . 0 .
The pH changed d u rin g th e c o u rse o f th e e x p e rim e n t.
The
v a lu e s g iv e n (T able V I I I ) a re th o s e found a t th e end o f th e e x p erim en t,
when th e m ix tu re s were checked w ith in d ic a to r p a p e r .
TABLE V I I I .
E f f e c t o f pH on a c t i v i t y o f c e H o b lo s e p h o sp h o ry la se as r e f l e c t e d in
changes in p h o s p h a te .
pH
5 .6
5 .9
6 .0
6 .2
6 .6
6 .8
7 .0
7 .4
In itia l
1.28
1 .3 0
1 .2 9
1.2 9
1.2 9
1.2 8
1 .2 9
1.2 7
F in a l
1.22
1 .1 1
1 .0 7
1.05
1.0 5
1 .0 0
1 .0 1
1.0 1
-0 .0 6
-0 .1 9
-0 .2 2
-0.24
-0 .2 4
-0 .2 8
-0 .2 8
-0 .2 6
P i - mg
- 29 -
S p e c i f i c i t y o f th e phosphory l a s e .
Enzymes a re u s u a ll y q u ite
s p e c i f i c in t h a t th e y c a ta ly z e o n ly a c e r t a i n ty p e o f ch em ical r e a c tio n
o r a re a c ti v e on compounds having s im ila r s t r u c t u r e s .
S in ce th e p h o s-
p h o ry la s e was a c ti v e on c e I l o b lo s e , a £ -g lu e o s id e , i t was t e s t e d f o r
a c t i v i t y on th e jf - g l u c o s ld e s a l l c l n , and on th e f - g a l a c t o s i d e s m e lib io s e ,
r a f f in o s e , and l a c t o s e .
and t r e h a l o s e .
O ther su g a rs t e s t e d in c lu d e d m a lto s e , s u c r o s e ,
The f -g lu c o sid e g e n tio b lo s e was n o t a v a i l a b l e .
The c e llo b io s e p h o sp h o ry la se was n o t a c tiv e on any o f th e
su g a rs o r g lu c o s id e s t e s t e d o th e r th a n c e llo b io s e as in d ic a te d b y th e
changes in th e c o n c e n tra tio n o f in o rg a n ic p h o sp h ate (T ab le IX ).
This
h ig h deg ree o f s p e c i f i c i t y is com parable t o t h a t found in o th e r p h o sp h o r y la s e s .
TABLE H
A c tio n o f c e llo b io s e p h o sp h o ry la se on v a rio u s su g ars as r e f l e c t e d by
p h o sp h ate ch an g es. R e s u lts a re th o s e o f d u p lic a te e x p e rim e n ts.
________________________ S u b s tr a te _________________________
L a c to se
M altose
M elib io se
R a ffin o s e
P i - mg
O
O
-d " -d " O
OnOn O
O
CVJ 0 \ CO
ONCO O
d d o
In itia l
F in a l
S ucrose
In itia l
F in a l
0 .8 8
0 .8 8
0 .0 0
0 .9 0
0 .9 1
+ 0.01
0 .8 9
0 .9 1
+0.02
0 .9 1
0 .9 0
-0 .0 1
0 .9 1
0 .9 1
0 .0 0
0 .9 2
O.9U
+0.02
S u b s tr a te
T reh a lo se
S a lic in
0 .9 3
0.95
+0.02
0 .9 2
0 .9 1
-0 .0 1
0 .9 1
0 .9 6
+0.05
O.89
0 .8 9
0 .0 0
0 .9 0
0 .9 1
+ 0 .0 1
0 .9 1
0 . 9%
+0.03
C e llo b io se
O.89
0 .7 7
-0 .1 2
0.9 1
O.78
- 0.1 3
- 30 I n h i b i t i o n b y g lu c o s e *
I t was o b serv ed b y D oudoroff ( 19^ 3 )
t h a t su c ro se phosphory la s e was in h ib ite d b y th e a cc u m u latio n o f th e f r e e
m onosaccharide w hich i t p ro d u c e d .
S in ce th e p ro d u c ts o f a p h o sp h o ry la se
a c tin g on c e llo b io s e sh o u ld be g lu c o s e - I -p h o sp h ate and g lu c o s e , an
ex p erim en t was s e t up u sin g I . 63 mg/ml o f c e llo b io s e in one tu b e , and
I .6 3 mg/ml each o f c e llo b io s e and g lu c o se in th e o th e r t u b e .
The amount
o f in o rg a n ic p h o sp h ate ta k e n up i n th e tu b e c o n ta in in g c e llo b io s e was
more th a n tw ic e th e p h o sp h ate up tak e in th e tu b e t o which g lu c o se had
b e en added (T ab le X ).
The c e llo b io s e phosphory l a s e ap p ears t o b e s im ila r
t o su c ro se p h o sp h o ry la se w ith r e s p e c t t o p ro d u c t i n h i b i t i o n .
TABLE X
I n h i b i t i o n o f c e llo b io s e p h o sp h o ry la se a c t i v i t y b y g lu c o s e .
R e s u lts a re th o s e o f d u p lic a te e x p e rim e n ts .
____________________ S u b s tr a te ___________________
____________________ C e llo b io se _____________ C e llo b io se and G lucose
P i - mg
In itia l
O.89
0 .9 1
» . 9©
©*91
F in a l
0 .7 7
0 .7 8
0 . 8$
0 . 8$
-0 .1 3
-0.0 5
-0 .0 6
-
0 .1 2
- 31 DISCUSSION
The o b s e rv a tio n made b y McBee ( 19^8 ) t h a t C lo s trid in m
therm ocellum
c o u ld use c e l l u l o s e and c e llo b io s e a s e n e rg y so u rc e s b u t
c o u ld n o t u t i l i z e g lu c o se was n o t a d e q u a te ly e x p la in e d a t t h a t tim e .
The
f in d in g o f a c e llo b io s e p h o sp h o ry la se in th e c e l l s o f t h i s organism n o t
o n ly h e lp s t o c l a r i f y th e mechanism w hereby i t u ses c e llo b io s e w ith o u t
h y d r o ly s is t o g lu c o s e , b u t a ls o adds a p r e v io u s ly u n re p o rte d enzyme to
th e l i s t o f d is a c c h a r id e p h o s p h o ry la s e s .
I t a ls o le a d s ’ t o th e s p e c u la ­
t i o n t h a t d!s a c c h a r id e p h o sp h o ry la se s may b e q u ite common i n n a tu re b u t
have n o t b e e n found b ecau se i t was b e lie v e d t h a t th e s u c ro s e p h o sp h o ry ­
la s e r e p o r te d b y Kagan et_ a l . , ( 19^2 ) and b y D oudoroff e t a l . , ( 1943)
was a unique ty p e o f enzyme.
r e v e a l t h e i r p re s e n c e .
A s e a rc h f o r o th e r p h o sp h o ry la se s may
The im portance o f t h i s ty p e o f enzyme t o an
organism , is , a p p a re n t when e n e rg y y i e l d from a complex s u b s t r a t e is
c o n s id e re d .
The sim ple h y rd o ly s i s o f c e llo b io s e t o two m o lecu les o f g l u ­
cose may produce a su g a r fe rm e n ta b le b y many o rg an ism s, b u t a f a i r l y
la r g e e x p e n d itu re o f e n erg y i s r e q u ir e d t o change th e g lu c o se t o
g lu c o s e - 6 -p h o sp h a te , th e f i r s t s te p r e q u ir e d f o r th 6 u t i l i z a t i o n o f th e
su g ar.
I f th e d is a c c h a r id e i s s p l i t b y p h o sp h o ro l^ si s , how ever, th e
r e a c t i o n i s ex o th erm al and p roduces g lu c o se - I -p h o sp h a te , w hich may
r e a d i l y be c o n v e rte d t o g lu c6 s,e-6 -p h o sp h ate b y th e enzyme p h o sp h o g lu co - ■
r
m u ta se .
The organism is th e r e f o r e a b le t o form one m o lecu le o f
g lu c o s e - 6 -p h o sp h ate from each d is a c c h a r id e m olecule w ith o u t an en erg y
-
> ■
' h(
- 32 e x p e n d itu r e .
T h is ty p e o f p h o sp h o ro ly si s i s v e ry s im ila r t o h y d rd ly s i s }
th e o n ly d if f e r e n c e "being t h a t p h o sp h o ric a c id e n te r s in to , th e r e a c tio n
in ste a.d o f w a te r .
The c e lld b io s e p h o sp h o ry la se w hich can "be l i b e r a t e d from th e
c e l l s o f C lo s trid iu m therm ocellum b y g rin d in g w ith powdered alum ina has
c h a r a c t e r i s t i c s v e ry s im ila r t o th o s e o f th e su c ro se and m a lto se p h o sp h o ry la s e s w hich have b een s tu d ie d (Doudoro f f , 1943) ( F i t t i n g and Scherp
1952)-
I t i s a c ti v e ov er a wide pH ra n g e w ith an optimum a c t i v i t y n e ar
n e u tra lity .
I t i s h ig h ly s p e c i f i c , b e in g a c ti v e on c e lld b io s e b u t none
o f th e o th e r compounds t e s t e d .
I t sh o u ld b e p o s s ib le t o s y n th e s iz e
c e lld b io s e from g lu c o se and g lu c o s e - I -p h o sp h ate b y means o f t h i s enzyme.
I t i s a ls o p o s s ib le t h a t a s e r i e s o f d is a c c h a r id e s n o t found in n a tu re
c o u ld be s y n th e s iz e d i f o th e r m onosaccharides were s u b s t i t u t e d f o r th e
g lu c o se in th e above r e a c t i o n .
Such compounds have b een s y n th e s iz e d b y
th e use o f s u c ro se p h o sp h o ry la se (Doudoroff, H a ssid , and B a r k e r , 1944) .
The f a t e o f th e m olecule o f g lu c o se fo rm ed .b y p h o s p h o ro ly s is
o f c e lld b io s e h as n o t b e en d e te rm in e d .
S in c e g lu c o se does n o t accumu­
l a t e in c u ltu r e s o f C lo s trid iu m therm ocellum i t must be u sed b y th e
o rganism , even though g lu c o se added t o th e medium is n o t u t i l i z e d .
There a re s e v e r a l p o s s ib le e x p la n a tio n s f o r t h i s phenomenon.
c e l l w a ll may n o t be perm eable t o g lu c o s e .
The
The p h o sp h o ry la se may serv e
t o c a r r y c e lld b io s e th ro u g h th e c e l l w a ll in to an o r d e r ly sequence o f
enzymes where g lu c o se can be u t i l i z e d .
T h is i s s u p p o rte d b y th e ob sery a
- 33 t i o n t h a t a g lu c o k in a se i s p r e s e n t i n th e enzymes e x tr a c te d from th e .
c e lls .
The compound' decomposed "by th e p h o sp h o ry la se in th e c e l l may he
a d e r iv a tiv e o f c e llo h io s e in s te a d o f f r e e c e ll o h io s e .
T h is would
l i b e r a t e a g lu c o se d e r iv a tiv e in s te a d o f f r e e g lu c o s e .
The e v id en c e f o r
th e e x is te n c e o f a c e llo h io k in a s e w hich m ight make a c e llo h io s e p h o sp h ate
i s n e g lig ib le h u t i t i s s t i l l a p o s s i b i l i t y t h a t has n o t b een r u l e d o u t.
W hatever mechanism i s o p e ra tiv e b e s id e s th e p h o sp h o ry la se is
s t i l l n o t d e f i n i t e l y knotm and i s a m a tte r f o r f u r t h e r s tu d y .
The
p o s s ib le methods o f c e llo h io s e u t i l i z a t i o n a re in d ic a te d i n r e a c tio n s
• o u tlin e d (F ig u re 3 ) .
R e a c tio n I i s t h a t w hich is fo u n d in b a c t e r i a c o n ta in in g a
c e llo h ia s e and w hich a r e c a p a b le o f u t i l i z i n g g lu c o s e .
R e a c tio n 2 is
a summary o f r e a c tio n s t h a t have b een observed, i n enzyme p r e p a r a tio n s
from C lo s trid iu m th erm o cellu m .
1
The g lu c o k in a se has b e en w e ll e s ta b lis h e d
a lth o u g h i t i s n o t a p a r t o f t h i s t h e s i s .
R e a c tio n 3 r e q u ir e s th e
p re s e n c e o f a h y p o th e tic a l d is a c c h a r id e k in a s e , c e llo h io k in a s e , w hich is
p o s tu la te d b u t h as n o t b e en p ro v e n t o e x i s t .
The o b s e rv a tio n s
p r e s e n te d i n t h i s s tu d y le a d t o th e c o n c lu s io n t h a t r e a c t i o n 2 i s th e
one w hich i s m ost l i k e l y t o occur i n c e l l s o f C lo s trid iu m therm ocellum
grow ing on c e ll o h io s e .
C e llo h lo k ln a s e
C e llo h io se
CeI lo h lo s e -X -phosphate
P hosphorylase
■> G lucose -I-PO4 + G lucose X - PO,
C e llo -
Phosphoglucdmutase
G lucose
G lu c o se - 6 -POj4
G lucokinase
G lu c o se - 6 -PO,
G lu c o se -I-PO. + G lucose
Phosphog lu c o rautase
G lu c o se - 6 -POj4
G lu co k in ase
G lu co se-6 -PO,
F ig u re 3 » P o s s ib le methods o f c e llo b io s e u t i l i z a t i o n h y C lo strid iu m therm ocellum
SUMMARY
C e l l - f r e e e x t r a c t s o f C lo s trid iu m therm oce Hum w ere found to
c o n ta in ah enzyme, c e llo h io s e -p h o s p h o ry l a s e , which c a ta ly z e s th e
fo llo w in g r e a c t i o n :
G lu co se.
C e llo h io s e + HgPO^ __________^ G lu c o se - I -phosphate
T his r e a c t i o n was confirm ed h y th e use o f c h em ica l methods
t o fo llo w changes i n o rg a n ic and in o rg a n ic p h o sp h ate s and a ls o h y th e
use o f r a d io a c ti v e p 32 a s a t r a c e r .
I t was found t h a t i n th e p re sen c e
o f c e ll o h io s e , th e enzyme cau sed a d e c re a se i n th e in o rg a n ic p hosphate
and a c o rre sp o n d in g in c r e a s e i n g lu c o s e - I ^p h o sp h ate.
The fo rm a tio n o f
g Iu c ose - I -p h o sp h a te , how ever, c o u ld n o t h e d e te c te d u n le s s phosphoglucom utase was in a c t i v a t e d .
T h is c o u ld h e done h y s to r in g th e
enzyme p r e p a r a tio n in th e fr o z e n s t a t e f o r a few d a y s.
.
The p h o sp h o ry la se was a c tiv e o v er a wide ra n g e o f pH and
te m p e ra tu re .
I t was a ls o s p e c i f i c in i t s a c ti o n , as i t e x e r te d
a c t i v i t y o n ly oh c e ll o h io s e .
T h is phosphoro l y t i c r e a c t i o n , how ever, s t i l l does n o t e x p la in
th e n o n - u t i l i z a t i o n o f g lu c o se i n th e c u ltu r e medium, a phenomenon
w hich r e q u ir e s f u r t h e r in v e s t i g a t i o n .
- .30. "V j
LITERATURE CITED
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