The location and identification of the enzyme system responsible for the fermentation of isomaltose in
Candida utilis
by John Edward Robbins
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by John Edward Robbins (1963)
Abstract:
In this investigation isomaltase was isolated and identified as the enzyme which was responsible for the
fermentation of isomaltose in the yeast strain, Candida utilis. The enzyme was located primarily in the
cell membranes and small amounts were found in the interior of the cell. The enzyme activity of the
membranes was far greater than that of the intracellular sap. Approximately eighty per cent of the
enzyme activity was extracted from the membranes by pH adjustment indicating that the enzyme was
held by virtue of electrical charge. In addition, evidence is presented that there are two molecular forms
of isomaltase present. There is also evidence that the yeast cells could utilize panose as well as
isomaltose and in fact they were able to grow well even when isomaltose and panose were the sole
sources of carbohydrate. However, there was no evidence under the conditions used where the yeast
cells could utilize isomaltotriose and higher homologues. THE LOCATION AND IDENTIFICATION OF THE ENZYME SYSTEM RESPONSIBLE
FOR THE FERMENTATION OF ISOMALTOSE.IN CANDIDA U T IL IS
by
JOHN EDWARD ROBBINS
A t h e s i s s u b m it t e d t o t h e G ra d u a te F a c u l t y in p a r t i a l
f u l f i l l m e n t o f t h e r e q u ir e m e n t s f o r t h e d e g re e
of
DOCTOR OF PHILOSOPHY
in
C h e m is try
Approved
Headr, M a jo r D ep artm en t
s
Dean,
G ra d u a te D i v i s i o n
MONTANA STATE COLLEGE
Bozeman, Montana
A u g u s t , -1 9 6 3
ACKNOWLEDGEMENT
I w is h t o e x p ress my s i n c e r e th an ks
D r,
K, J . G o e r in g ,
I w ould l i k e
t o my r e s e a r c h d i r e c t o r ,
f o r h is g u id a n c e th ro u g h o u t t h i s w o r k .
t o e x p re s s th an ks f o r t h e f i n a n c i a l
a s s is ta n c e
g iv e n by t h e Montana S t a t e C o l l e g e C h e m is try D e p a rtm e n t,
t h e C h e m is try
S t a t i o n , and t h e Research and Endowment Fund.
! w is h t o e x p re s s th a n k s f o r h e l p f u l
g iv e n by D r s .
B. N e ls o n , R. McBee, R. J» O 'C o n n o r, and
To my w i f e ,
I
I . K„ M i l l s .
P a t r i c i a , who has endured much, g iv e n encouragement
and h e lp th ro u g h o u t t h e e n t i r e
is e x t e n d e d .
s u g g e s tio n s and c r i t i c i s m
t i m e , a v e ry h ig h n o te o f a p p r e c i a t i o n
TABLE OF CONTENTS
PAGE
L IS T OF TABLES .................................. , ....................................................................................
L IS T OF FIGURES.
.......................................................................................................... ....
ABSTRACT .............................................................................
.
v
vi
..................................................... vi i I
I.
INTRODUCTION................................... .... .......................................................................
I
II.
MATERIALS AND METHODS U S E D ....................................... ......................................
3
P r e p a r a t i o n o f Is o m a lt o s e
............................. .......................................
A c id H y d r o l y s i s o f D e x t r a n ........................................................................
C o n v e rs io n o f M a lt o s e t o Is o m a lt o s e and Panose . . . . . .
S e p a r a t i o n o f Is o m a lt o s e . . . . . . . . . . . . . . . . .
Sugar A n a ly z e s .................................. . . . . . . . . . . . . . .
Chromatogram S o lv e n t System . . y ........................ . . . . . . .
C a r b o h y d r a t e C o lo r D e v e lo p e r ,
. . . . . . . . . . . . .
Yeast N u trie n t . . . . . . .
............................. . . . . . . . .
I d e n t i f i c a t i o n o f Is o m a lto s e . ................................................ ....
3
3
4
4
8
9
9
10
10
III.
EXPERIMENTS, RESULTS AND DISCUSSION...........................................%
- ’ ,.■■ ■
“
.i
. .
The A b i I i t y o f C andida u t i I i s t o U t i l i z e V a rio u s
C arb o h yd rates. . . V
L o c a t io n o f t h e Enzyme . , ....................................... ....
S o l u b i l i t y o f t h e Enzyme . . . . . . . . . . . . . . . . .
D . E . A . E . C e l l u l o s e Chromatography o f t h e Membrane
E x tra c t. . . . . .
.......................................................... . . . . . .
Optimum T e m p e ra tu re and Optimum pH ......................................................
Re v e r s i
b
i
I
i
t
y
I n h i b i t o r s o f C a rb o h y d ra s e Enzymes . . . . . .
.................... .
S p e c i f i c i t y o f t h e Enzyme System . . . . . . . . . . . . .
P u r i t y o f t h e Enzymes E l u t e d from t h e D . E . A . E .
C e I l u l o s e CoIumn
BV.
V.
SUMMARY.
.
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.
.
SUGGESTIONS FOR FUTURE RESEARCH
LITERATURE CITED
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.
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12
12
18
28
28
35
38
40
42
43
46
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48
50
LIST OF TABLES
TABLE
I.
II.
III.
IV .
Rgj
PAGE
V alu es o f M a lt o s e and Is o m a lt o s e
R a t i o o f G lu co se
In h ib ito rs
. . . . . . . . . . . .
: M a lt o s e w i t h T i m e .................................. ....
Used w i t h
the
Is o m a lt a s e S y s t e m ......................................
S p e c i f i c i t y o f Enzyme S ystem s.
. . . . . . . . . . . . . . .
11
13
41
41
LIST OF FIGURES
FIGURE
1.
PAGE
Chromatogram o f 1000 m l . F r a c t i o n s O b ta in e d from A c id
H y d r o l y s i s o f D e x t r a n .........................................................
6
2.
Chromatogram o f t h e F r a c t i o n s from t h e M a lt o s e C o n v e rs io n
w i t h C l a r a s e ......................................................................................................................... 7
3.
Wet C e l l
4.
U tiliz a tio n
5.
A Comparison Between C u l t u r e w i t h M a lt o s e as a S t a r t e r and
One w i t h o u t a S t a r t e r ............................. .... ..........................................
W eig h t vs T i m e ............................
of
15
I s o m a l t o s e , P a n o s e and D e x tra n
..................................
16
17
6.
Growth o f Candi da u t i l is as a F u n c t io n o f T im e ...........................
7.
Sugar U t i l i z a t i o n
8.
Comparison o f G lu co se vs M a lt o s e as S t a r t e r S u g a rs .. . . . .
21
9.
Enzyme A c t i v i t y o f t h e C e l l - F r e e M e d i u m ............................... .......
23
by C andida u t i l is
19
...........................................
20
10 .
C e l l - F r e e Medium A c t i v i t y
a t T h r e e pH V a l u e s .................................
11 .
L o c a t io n , o f Enzyme System .
12 .
The A c t i v i t y o f t h e Membrane E x t r a c t s ........................ .............................
27
13 .
G r a d i e n t E l u t i o n o f t h e Crude E x t r a c t
29
14.
The A c t i v i t y E x h i b i t e d by Two F r a c t i o n s E lu t e d from
D . E . A . E . C e l l u l o s e Column ....................................................
.
.
.
23
. _ ............................................
........................
26
. . . . . .
31
•:
15 .
T h r e e P r o t e i n s S e p a r a t e d from t h e Crude E x t r a c t
16.
The Change I n E l u t i o n
17.
R e d u c tio n
18.
The E f f e c t o f T e m p e ra tu re on A c t i v i t y o f t h e Is o m a lt a s e
Enzymes . . . . . . . . . . . . . . .
..................................
19.
.............................
P a t t e r n w i t h a New Column .
. .V
. .
in P r o t e i n a f t e r S u c c e s s iv e E l u t i o n s . . . . . . . .
.
32
33
34
. . .
36
The E f f e c t o f pH on t h e A c t i v i t y o f t h e Is o m a lt a s e Enzyme
Systems . . . . .
........................................................................ . . . . .
37
vi i
20.
R e v e r s i b i l i t y o f t h e E n z y m a tic R e a c t i o n ............................. ....
39
21.
E le c t r o p h o n e s is S e p a r a t i o n o f t h e Crude E x t r a c t
45
. . . . . . .
ABSTRACT
In t h i s i n v e s t i g a t i o n i soma.I t a s e was i s o l a t e d and i d e n t i f i e d as
t h e enzyme w h ich was r e s p o n s i b l e f o r t h e f e r m e n t a t i o n o f is o m a lt o s e in
t h e y e a s t s t r a i n , C andida u t i l i s .
The enzyme was lo c a t e d p r i m a r i l y in
t h e c e l l membranes and s m all amounts w e re found in t h e i n t e r i o r o f th e
c e ll.
The enzyme a c t i v i t y o f t h e membranes was f a r g r e a t e r th an t h a t
o f th e i n t r a c e l l u l a r sap.
A p p r o x im a t e ly e i g h t y p er c e n t o f t h e enzyme
a c t i v i t y was e x t r a c t e d from t h e membranes by pH a d ju s t m e n t i n d i c a t i n g
t h a t t h e enzyme was h e ld by v i r t u e o f e l e c t r i c a l c h a r g e .
In a d d i t i o n ^
e v id e n c e is p r e s e n t e d t h a t t h e r e a r e two m o l e c u l a r forms o f i soma I t a s e
p resen t.
T h e r e is a l s o e v id e n c e t h a t t h e y e a s t c e l l s c o u ld u t i l i z e
panose as w e l l as is o m a lt o s e and in f a c t t h e y w ere a b l e t o grow w e ll
even when is o m a lt o s e and panose w ere t h e s o l e sources o f c a r b o h y d r a t e .
However, t h e r e was no e v id e n c e under t h e c o n d i t i o n s used w here t h e
y e a s t c e l l s c o u ld u t i l i z e i soma I t o t r lo s e and h ig h e r homologues.
INTRODUCTION
Is o m a lt o s e has been c o n s id e r e d a n o n f e r m e n t a b le su g ar f o r many
years, y e t,
in t h e p a s t te n y e a r s s e v e r a l
2<&) have found t h a t
n u trie n t.
is o m a lt o s e d i s a p p e a r s
None o f th e s e
re s p o n s ib le fo r
s e rv e d t h i s
in v e s tig a to rs
th is
in v e s tig a to rs
u tiliz a tio n .
phenomenon s e v e r a l
17,
t h e enzyme
G o e rin g and M. J . H o u le ob­
tim e s w h i l e c o n d u c tin g r e s e a r c h on
f e r m e n t a t i o n s by Candida u t i l i s , N . R . R . L .
a l s o o b served t h a t
15,
from w o r t used as y e a s t
have i s o l a t e d
K, J .
(9»
Y 9 0 0 . lf
K. J .
G o erin g ( 2 4 )
f e r m e n t a t i o n stopped f o r a p e r io d o f t im e and then
c o n t in u e d once a g a in
in a c u l t u r e o f Saccharomyces c e r e v i s a e
in which
g lu c o s e , m a lt o s e and
is o m a lt o s e w ere used as t h e s o u rc e o f c a r b o h y d r a t e .
The m a lt o s e and g lu c o s e w ere f e rm e n te d f i r s t and is o m a lt o s e l a s t .
s u g g ested t h a t
p e rio d .
the fe rm e n ta tio n o f
T h is
is o m a lt o s e r e q u i r e d an a d a p t a t i o n
T h is was a l s o t h e c o n c lu s io n o f Okada ( 9 ) , who found s i m i l a r
e v id e n c e o f
is o m a lt o s e f e r m e n t a t i o n by Shizosaccharom yces pombe and,
a lt h o u g h he d id n o t
is o la te
t h e enzyme,
he su g g ested t h e f e r m e n t a t i o n
was t h ro u g h g lu c o s e produced by an e x t r a c e l l u l a r
p r o d u c t io n was
induced by c o n t a c t w i t h
The o b j e c t i v e o f t h i s
w h ich s u b c e l l u l a r
re s p o n s ib le fo r
fra c tio n
t h e enzyme e x i s t e d )
g a to rs b e lie v e t h a t c e r t a i n c e l l s
sugars and o t h e r n u t r i e n t s
*N ,R .R ..L . -
is o m a lt o s e .
i n v e s t i g a t i o n was t o
is o m a lt o s e f e r m e n t a t i o n
(5 ,
N o r t h e r n R e g io n a l
P e o ria , I l l i n o i s
i s o m a l t a s e , whose
lo c a te
and t o
(i.e .
to fin d
in
i s o l a t e t h e system
in Candida u t i l i s .
Some i n v e s t i ­
r e q u i r e permeases f o r t h e u p ta k e o f some
2 1 ).
O th e r
in v e s tig a to rs
feel
t h a t perme-
Research L a b o r a t o r i e s , Y e a s t s t r a i n
900,
ases a r e n o t r e q u i r e d a t a l l
and t h a t t h e u p ta k e o f n u t r i e n t s
a p a s s iv e phenomenon th an an a c t i v e one ( 1 6 ,
t im e was s p e n t . i n
the e a r l y
e x p e r im e n t s and t r y i n g
h ig h e r homologues
a p erm ease.
p art o f th is
in v e s tig a tio n
to p re p a re u n ifo rm ly
in an e f f o r t t o
18, 2 1 ) „
la b e le d
is more
A g r e a t deal o f
in d e s ig n in g
is o m a lt o s e and
i s o l a t e and f o l l o w t h e a c t i v i t y o f
MATERIALS AND METHODS USED
P re p a ra tio n o f
Is o m a lt o s e
Of t h e s e v e r a l methods f o r t h e p r e p a r a t i o n o f
in t h e l i t e r a t u r e ,
a l.
t h e method o f H u l t i n and Nordstrom ( 1 1 ) and Jeanes e t
( 1 3 ) was chosen because
Even a f t e r s e v e r a l
n o t be o b t a i n e d .
is o m a lt o s e d e s c r ib e d
tria ls
it
re s u lte d
in h ig h y i e l d s o f
u s in g t h i s m ethod,
is o m a lt o s e .
t h e r e p o r t e d y i e l d s co u ld
O th e r methods g i v i n g y i e l d s o f t e n p e r c e n t ( 4 ,
19)
w e re used because t h e p r e v io u s method was to o t im e consuming and th e
y i e l d s o b t a i n e d w e re n o t a p p r e c i a b l y b e t t e r th a n t e n p e r c e n t .
A c id H y d r o l y s i s o f D e x tra n
A b a c te ria l
d e x t r a n produced by L eu co n o sto c m e s e n t e r o id e s , N . R . R . L .
8 5 1 2 f r om s u c ro s e was o b t a i n e d by g ro w in g t h e o rg an ism
in t h e f o l l o w i n g
Medi urn:
S ucrose
10 grams /
100 ml o f H2 O
K2 HPOif
0 . 0 5 gram /
100 ml o f H2 O
Yeast E x tra c t
0. 25 gram /
100 ml o f H2O
MgSOii • 7H2 0
0 . 0 2 gram /
100 ml o f H2 O
NaCl
0 . 1 0 gram /
100 ml o f H2 O
The p r o d u c t io n o f t h e d e x t r a n may be r e p r e s e n t e d s im p le by t h e f o l ­
lo w in g e q u a t i o n :
n S u cro se + D e x t r a n s u c r a s e --------- n F r u c to s e 4- G lu co sen .
T h is d e x t r a n r e p o r t e d l y has 95%
^ N o r t h e r n R e g io n a l
(1 ,6 )
R esearch L a b o r a t o r i e s ,
l i n k a g e s w i t h s m all
B a c te ria 512,
amounts o f
P e o ria ,
Illin o is
<5^ ( 1 , 4 ) and < $ < 0 >3)«
F i f t y - t h r e e gram i o f d e x t r a n w e re d i s s o l v e d
by h e a t i n g .
The s o l u t i o n was s t i r r e d
c o n s ta n tly
o f w ater
o f 3N s u l f u r i c
f o r seven hours a t
c o o l e d , and t h e pH a d j u s t e d t o 6 . 0 by s lo w ly a d d in g 3N sodium
h y d r o x id w i t h a g i t a t i o n ,
fifte e n
lite rs
T h is s o l u t i o n was c o o le d and 540 m i l l i l i t e r s
a c i d was a d d e d .
90° C ,,
in 4 , 4 6
is o m a lto s e was produced
in a p p r o x im a t e ly
per cen t y i e l d .
C o n v e rs io n o f M a lt o s e t o
is o m a lt o s e and P an o se,
Is o m a lt o s e was a l s o s u c c e s s f u l l y p re p a r e d by t h e method o f Pazur
w h ich c o n v e r t s m a lt o s e t o
(1 9 ),
is o m a lt o s e and panose by means o f a fu n g a l
tra n s g lu c o s y la s e .
Four hundred grams o f B- m a lt o s e d i s s o l v e d
w e re added t o one l i t e r
grams o f th e c l a r a s e .
in 2 , 0
lite rs
o f w ater
o f a Takam ine c l a r a s e s o l u t i o n c o n t a i n i n g
T h is s o l u t i o n was
in c u b a te d a t 3 0 ° C,
ten
f o r 72 hours
and th e n a n a ly z e d f o r t h e su g ars p r e s e n t by paper c h ro m a to g ra p h y .
a n a ly s is
T h is
i n d i c a t e d a minimum c o n c e n t r a t i o n o f m a lt o s e and a h ig h concen­
t r a t i o n o f g lu c o s e ,
m in u te s t o
i s o m a lt o s e , and p an o se.
in a c tiv a te
The s o l u t i o n was b o i l e d f i v e
t h e enzyme f o l l o w e d by t h e a d d i t i o n o f f i f t y
o f b a k e r s ' y e a s t suspended
in one hundred m i l l i l i t e r s
o f w a te r.
hours t h e g lu c o s e and m a lt o s e w e re removed by f e r m e n t a t i o n
grams
A f t e r 24
l e a v i n g th e
is o m a lt o s e and paose in t h e r e a c t i o n m i x t u r e .
S e p a ra tio n o f
Is o m a lt o s e
A lt h o u g h c o m p le t e ly p u re
to g rap h y,
is o m a lt o s e was n o t o b t a i n e d ,
u s in g a c a r b o n - c e l i t e
column chroma­
column c o n t a i n i n g eq u al amounts o f each
- 5 "
by w e i g h t ,
p roved more s a t i s f a c t o r y
is o m a lt o s e from su g ar m i x t u r e s .
th an S ep h ad ex-G -25 f o r s e p a r a t i n g
A column o f D a rc o -G -6 0 and C e l i t e - 5 3 5
( 7 . 5 x 95 cm .) was p re p a re d by m ix in g t h e a b s o rb e n ts
th is
s lu rry
tra te d
in w a t e r and p o u rin g
i n t o t h e column, wh ich was p lu g g ed w i t h g la s s w o o l.
h y d r o c h l o r i c a c i d was a llo w e d
t o d r i p s lo w ly th ro u g h t h e column
f o r d e a c t i v a t i o n o f t h e carb o n and t o remove t r a c e s o f
a l k a l i n e ash from t h e c e l i t e .
u n til
Concen­
i r o n and th e
W ater was th e n passed t h r o u g h , t h e column
t h e e f f l u e n t was n e u t r a l .
The su g ars w ere p u t on t h e column in aqueous s o l u t i o n s
in c o n c e n t r a ­
t h a t w e re n o t le s s th a n 0, 25 p e r c e n t nor g r e a t e r th a n f i v e
tio n s
cen t.
For h ig h e f f i c i e n c y
n o t more th a n one gram o f su g ar m i x t u r e was
added f o r each 150 c . c . o f c a r b o n - c e l i t e .
w ith fo u r
lite rs
lite rs
o f w a te r,
of fifte e n
c o lle c te d .
F ig u re I ,
per
fiv e
lite rs
p er c e n t e t h a n o l .
S u c c e s s iv e e l u t i o n s w ere made
o f fiv e
F i f t e e n one l i t e r
A chromatogram o f th e s e v a r io u s
The s e p a r a t i o n o f
p er c e n t e t h a n o l
fra c tio n s
and s i x
f r a c t i o n s were
is d e p i c t e d
is o m a lt o s e and panose is d e p i c t e d
in
in F ig u r e 2
(2 3 ).
Pure is o m a lt o s e was o b t a i n e d
p ap er c h ro m a to g ra p h y .
a s o lu tio n o f
in amounts o f 2 0 - 3 0 m i l l i g r a m s
Whatman 3 mm p ap er
made u n t i l
(2 2 x 35 cm .) was p a i n t e d w i t h
is o m a lt o s e and h ig h e r homologues w i t h a a sm all
on a l i n e 6 cm. from t h e to p o f t h e p a p e r .
p a i n t brush
Repeated a p p l i c a t i o n s were
t h e r e s i d u e on t h e paper lo o ked a lm o s t c r y s t a l l i n e .
o f p ap er was th e n p la c e d
u sin g
in a c h r o m a t o c a b in e t f o r 24 h o u r s .
The s h e e t
S t r i p s w ere
c u t from t h e edges o f t h e chromatogram and d ip p e d i n t o C - D - I ,
( s e e page 9)
and th e n h e a te d t o
The s t r i p
l o c a t e t h e bands of. t h e d i f f e r e n t s u g a r s .
- 6 -
Sfd
12
3
4
S
F r a c t i o n Number
6 -7 8 tI IO 11 12
H i gher
Homologues
15"
= Uiiu
6 0
Is o m a lt o t r i o s e
0
oOOO
Is o m a lto s e
M a l to s e
0
Glucose
OOOOo
F ig u r e
I.
0 0 0 0
D ire c tio n
of
Sol vent
Chromatogram o f 1000 m l. F r a c t i o n s O b ta in e d
from A c id H y d r o l y s i s o f D e x tra n
- 7 -
F r a c t i o n Number
o
I soma I t o s e
M a lto s e
0
Glucose
0
F ig u r e 2 .
q
6-
0
0
0
0
7.
a
O
U
CF
Panose
I
Q
2.
O
I.
O
Std
G/.M
Chromatogram o f t h e F r a c t i o n s from t h e
M a lt o s e C o n v e rs io n w i t h C la r a s e
D ire c tio n
of
Sol ven t
=* 8 w
c o n ta in in g
is o m a lt o s e was c u t o u t and t h e
is o m a lt o s e e l u t e d w i t h w a t e r .
T h is s o l u t i o n was c o n c e n t r a t e d by h e a t i n g a t 8 0 ° C. under vacuum.
Sugar A n a ly z e s
Sugar m i x t u r e s ,
such as t h e p ro d u c ts
from t h e a c i d h y d r o l y s i s o f
t h e d e x t r a n , w e re a n a ly z e d by q u a n t i t a t i v e paper chrom atography ( 4 ) .
The su g ars w e re l o c a t e d on t h e chromatogram by d e v e lo p in g t h e spots o f
t h e s e p a r a t e d unknown and a m i x t u r e o f known sugars w i t h C - D - I .
sp o ts on t h e s t r i p
c o n ta in in g
t h e unknown w e re then used t o
s p o ts on an u n developed s t r i p o f t h e unknown.
w ith w a te r,
d ilu te d
to f i f t y
m illilite rs ,
s u l f u r i c a c i d method o f Dubois and G i l l e s
h y d ro ly s is o f d e x tra n ,
cent
i s o m a lt o s e ,
a fte r
These sp o ts w ere e l u t e d
and a n a ly z e d by t h e p h e n o l(6 ).
The m i x t u r e from t h e a c i d
i soma I t o t r lo s e and f o r t y
is o m a lt o s e a c c o u n ted f o r $8 p er c e n t ,
19 p er c e n t , and t h e h ig h e r homologues 23 p er c e n t .
s o l u t i o n was used as t h e s u b s t r a t e s o l u t i o n
On t h e b a s is
i soma I t o t r i o s e
U n less o t h e r w i s e s t a t e d
in t h i s
in v e s tig a tio n .
Is o m a lt o s e a c co u n ted f o r s e v e n ty p er c e n t o f t h e m icro m o les o f g lu c o s e
t h e p a n o s e - is o m a lt o s e m i x t u r e .
per
p e r c e n t h ig h e r
homologues c a l c u l a t e d on t h e b a s is o f m icro m o les o f g lu c o s e .
th is
l o c a t e th e
t h e g lu c o s e was removed, c o n t a in e d f o r t y
tw e n ty p er c e n t
o f re d u c in g s u g a r ,
The
An exam ple o f th e s e c a l c u l a t i o n s
P h e n o l-s u lfu r ic a c id a n a ly s is g a v e :
ls o m a lt o s e
-
20 / j g
o f g lu co se
ls o m a lto trio s e
-
10^ g
o f g lu co se
H ig h e r homo logues
-
20 >ig
o f g lu c o s e
in
fo llo w s .
as
Is o m a lt o s e
20
m
-
o f g lu c o s e
—
20 x 100 /
0 .1 1
p moles o f g lu c o s e
20 pg o f
is o m a lt o s e
10 jjg o f
i soma I t o t r i o s e
20 )jg o f
i soma I t p p e n t a o s e ~ 0 .0 2 2 jj moles o f
T o ta l
=
50 = 40%
0 . 0 5 5 M moles o f
J=
0 . 0 1 8 p moles o f
moles o f r e d u c in g su g ar
p e r c e n t re d u c in g su g ar
=
|som al t o s e
i soma I t o t r lo se
i soma I t o p e n ta o s e
= 0 .0 9 5 M moles
55 x 100 /
95 = 58 p e r c e n t .
A m o d if i c a t i o n o f t h e method o f Sumner ( 2 0 ) was used t o a n a ly z e f o r
re d u c in g s u g a r s .
S in c e t h e r e a g e n t w i l l
d e te r io r a te w ith
t im e
n e c e s s a ry t o r u n a s t a n d a r d w i t h each t e s t as a p r e c a u t i o n .
i t was
The s ta n d a rd
c u rv e was based on m a l t o s e .
Chromatogram S o lv e n t System
The s o l v e n t system used t o d e v e lo p a l l
t h e chromatograms
i n v e s t i g a t i o n was a m i x t u r e o f b u t a n o l , p y r i d i n e ,
t o volume r a t i o o f 6 : 4 : 3 r e s p e c t i v e l y
number one f i l t e r
and w a t e r
in t h i s
in a volume
u n le s s d e s c r ib e d o t h e r w i s e .
p ap er was used f o r a l l
chrom atogram s.
Whatman
P r o b a b ly due t o
more u n if o r m t e m p e r a t u r e and atm o sp h ere s a t u r a t i o n a g r e a t
improvement in
t h e s e p a r a t i o n s o f t h e su g ars o c c u r r e d when a new Research S p e c i a l t i e s .
c h r o m a t o c a b in e t was used*
C a r b o h y d r a t e C o lo r D e v e lo p e r
The c a r b o h y d r a t e c o l o r d e v e lo p e r used was t h a t o f Gorden e t a l » ( 8 ) .
The chromatograms w ere d ip p ed
in C - D - I , d r i e d ,
and th en h e a te d w i t h a
M a s t e r h e a t b lo w e r t o d e v e lo p t h e c o l o r o f t h e s p o t s .
The c o l o r o f t h e
- 10 s p o ts
in c o n j u n c t i o n w i t h t h e Rg j *
v a lu e s w e re used f o r
id e n tific a tio n
o f t h e v a r io u s s u g a r s .
Yeast N u trie n t
The n u t r i e n t
fo r the y e a s t c u ltu r e s
s t a n c e s p er gram o f sugar
c o n t a in e d t h e f o l l o w i n g sub­
in one hundred m i l l i l i t e r s
Urea
0 . 1 2 gram
Ca(I-^POi t) 2
0 . 0 3 gram
KCl
0 .0 1 gram
MgSOit
•7H20
o f n u trie n t:
0 .0 1 gram.
The n u t r i e n t was s t e r i l i z e d
by a u t o c l a v i n g
fo r
f i f t e e n m in u te s a t
th e n c o o le d t o room t e m p e r a t u r e and t h e pH a d j u s t e d t o 4 . 5 w i t h
The t y p e o f s u g ar was v a r i e d
n u t r i e n t was p re p a r e d
a e ro b ic a lly
c e lls
The c u l t u r e s w e re in c u b a te d
In o r d e r t o remove t h e
in v e s ti­
t h e c u l t u r e s w e re c e n t r i f u g e d a t 4 , 0 0 0 x g .
Is o m a lt o s e
Is o m a lt o s e was
id e n tifie d
by p ap er chrom atography and o p t i c a l
A v a lu e f o r th e m ig r a tio n
g iv e n by W h i s t l e r
p y rid in e ,
*R g]
in t h e above m a n n e r.
in a th e rm o s t a t e d sh a k e r a t 2 0 ° C .
Id e n tific a tio n o f
tio n ,
I NHCl.
in d i f f e r e n t e x p e rim e n ts b u t o t h e r w i s e a l l
from t h e media f o r t h e v a r i o u s e x p e r im e n t s th ro u g h o u t t h i s
g a tio n ,
15 p s i ,
(4 )
and w a t e r
as 0 . 4 3
ra tio o f
ro ta ­
is o m a lto s e t o g lu c o s e ,
in a s o l v e n t m i x t u r e o f e t h y l
in a volume r a t i o o f 8 : 2 : 1
re s p e c tiv e ly .
, was
ac e ta te ,
The sugar
is t h e r a t i o o f m i g r a t i o n d i s t a n c e o f a sugar as compared t o t h e
m i g r a t i o n d i s t a n c e o f g lu c o s e .
t h a t was t e n t a t i v e l y
i d e n t i f i e d as
is o m a lt o s e gave an i d e n t i c a l
v a lu e when t h e same s o l v e n t system was u s e d .
The Rgj
s o l v e n t systems f o r m a lt o s e and is o m a lt o s e a r e g iv e n
v a lu e s
Rg]
in two
in T a b l e
I.
The
d evelo p m en t t im e was Zk hours and t h e t e m p e r a t u r e was 3 0 ° C.
TABLE I
Rgl
V a lu e s o f M a lt o s e and
8 E th yl A c e ta te
2 P y rid in e
I W ater
L i t . V a lu e
Obs. V a lu e
1 .0 0
1 .0 0
0 .6 6
0 .6 5
0 .4 3
0 .4 3
Sol ven ts
Glucose
M a ltose
Is o m a lt o s e
W. Pigman ( 3 )
i s o m a lt o s e .
i d e n t i f i e d as
Is o m a lto s e
gave a s p e c i f i c r o t a t i o n
A s p e c ific
6 Butanol
4 P y rid in e
3 W ater
1 .0 0
0 .6 8
0 .4 7
v a lu e o f / 1 2 0 ° f o r
r o t a t i o n o f / 1 2 0 . 5 ° was o b t a i n e d f o r t h e sugar
is o m a lt o s e w hich
In c o n j u n c t i o n w i t h t h e c h ro m a to g ra p h ic
i d e n t i f i c a t i o n was c o n s id e r e d ample p r o o f o f
id e n tity ,
A r ig o r o u s
f i c a t i o n o f panose was c o n s id e r e d un n ecessary s in c e Pazur ( 1 9 )
id e n tifie d
is o m a lt o s e and panose as b e in g t h e o n ly p ro d u c ts o f t h e r e a c t i o n .
p o s itio n s o f the
id e n ti­
The
is o m a lt o s e and panose sp o ts on t h e chromatograms c o r r e s ­
ponded v e ry w e l l w i t h t h e s e shown by P a z u r .
EXPERIMENTS, RESULTS, AND DISCUSSION
The A b i l i t y o f Candjd a u t i l is t o U t i l i z e
A com parison o f t h e u t i l i z a t i o n
V a r io u s C a rb o h y d ra te s
o f g lu c o s e and m a lt o s e was made
o v e r t h e ran g e o f z e r o t o one hundred p e r c e n t o f g lu c o s e .
w e re w ith d ra w n h o u r ly
from t h e c u l t u r e s
f o r f o u r hours and t h e sugar
c o n c e n t r a t i o n s w ere d e t e rm in e d by paper chrom atography
a r e g iv e n
in T a b l e
(4 ).
The r e s u l t s
11.
The d a t a o b t a i n e d d e f i n i t e l y
more r e a d i l y
Samples
than m a lto s e ,
i t was
m a lt o s e c o n c e n t r a t i o n was d e c i d e l y
m a lt o s e was u t i l i z e d ,
showed t h a t t h e y e a s t used g lu co se
i n t e r e s t i n g t o n o t e t h a t when t h e
l a r g e r th a n t h a t o f g l u c o s e ,
th e
o r d is a p p e a r e d , more r a p i d l y ; when t h e r a t i o o f
g lu c o s e t o m a lt o s e was one o r
la rg e r,
g lu c o s e was u t i l i z e d more r a p i d l y .
In t h e case o f t h e one hundred p e r c e n t g lu c o s e c u l t u r e , m a lt o s e was
p ro d u c e d .
tu re .
G lu co se was produced in t h e one hundred p e r c e n t m a lt o s e c u l ­
T h is
i n d i c a t e d an e q u i l i b r i u m w h ich m ig h t be caused by t h e p resen ce
o f an e x t r a c e l l u l a r m a l t a s e .
T h re e s e p a r a t e c u l t u r e s w ere p re p a re d t o compare t h e a b i l i t y
y e a s t to u t i l i z e
i somal t o s e and panose w i t h
: '
see i f
'
o f the
r e s p e c t t o m a l t o s e , and t o
.
'
i t w ould be p o s s i b l e f o r t h e y e a s t t o u t i l i z e
V
d extran .
S in c e an
.
a d a p t a t i o n p e r i o d was a n t i c i p a t e d ,
the
0 . 5 p e r c e n t m a lt o s e was
is o m a lt o s e - p a n o s e and one p er c e n t m a lt o s e
The t o t a l
w e ig h t.
in c lu d e d
in
in t h e d e x t r a n c u l t u r e .
c a r b o h y d r a t e c o n c e n t r a t i o n was a p p r o x im a t e ly two p e r c e n t by
A c u l t u r e c o n t a i n i n g one p er c e n t m a lt o s e was
The w e t c e l l
in c lu d e d .
w e ig h t o f th e s e c u l t u r e s was d e te rm in e d a t t h e end o f
- 1 3 “
TABLE I I
R a t i o o f G lucose
0ZcG •- 0ZoM1
1 st
hour r a t i o
: M a lt o s e wi th Time
2nd
hour r a t i o
3rd
hour r a t i o
4 th
hour r a t i o
10 - 90
1 .7
: 8 .3
I .2
: 8 .8
I .0
20 - 80
3.1
: 6 ,9
1 .9
: 8 .1
2 .2 : 7 .8
30 - 70
4 .3
: 5 .7
1 .9
: 8 .1
I .0
: 1 .0
C. U.
40 -
60
5 .2
: 4 .8
3 .4
: 6 .6
4 .0
: 6 .0
C. U.
50 - 50
5 .5
: 4 .5
4 .4
: 5 .6
3 .5
: 6 .5
C. U.
60 - 40
4 .2
: 5 .8
4 .5
: 5 .5
3 .3
: 6 .7
C. U.
70 “ 30
2 .5
: 7 .5
5 .7
: 4 .3
3 .8
: 6 .2
C. U.
80 - 20
7 .2
: 2 .8
4 .9
: 5 .1
6 .4
: 3 .5
C, U,
90 -
10
8 .3
: 1 .7
6 .7
: 3 .3
4 .6
: 5 .4
C. U.
100 -
0
9 .2
: 0 .8
6 .7
: 3 .3
5 .1
: 4 .9
c.
u.
: 10
1 .5
: 8 .5
3 .2
: 6 .8
c.
u.
0 -
100
.0
% G - % M --
2
C. U. —
: 1 .0
% G lu co se t o % M a lt o s e
t h e su g ars w ere c o m p le t e ly u t i l i z e d
C.
U .2
C. U.
- 14 24 hours and e v e r y s i x t h
m illilite rs
hour t h e r e a f t e r
o f the c u lt u r e ,
c e n t r i f u g i n g o u t t h e c e l l s , w e ig h in g th e
y e a s t pack and th e n c a l c u l a t i n g
t h e p e r c e n t w e ig h t o f w et c e l l s on th e
b a s is o f t e n grams f o r t h e t o t a l
F ig u r e 3 .
The u t i l i z a t i o n
f o r t h r e e days by removing te n
a liq u o t.
These r e s u l t s a r e g iv e n
o f sugar was f o l l o w e d w i t h p ap er chrom atography
by s p o t t i n g t h e n u t r i e n t on a chromatogram each t im e a c e l l
d e t e r m in e d .
These r e s u l t s a r e g iv e n
The pH a t
m a lt o s e c u l t u r e .
frozen
fo r
6 .3
w e ig h t was
in F i g u r e 4 .
t h e end o f f i v e days was o b s e rv e d t o be 7 . 0
is o m a lt o s e - p a n o s e c u l t u r e ,
in
fo r
f o r t h e d e x t r a n c u l t u r e and 5 . 2
th e
fo r the
The y e a s t c e l l s w ere removed by c e n t r i f u g i n g and then
f u t u r e e x p e rim e n ts .
The is o m a lt o s e - p a n o s e c u l t u r e grew v e ry w e l l
panose w e re u t i l i z e d
and b o th
as e v id e n c e d by t h e chrom atogram s.
y e a s t was u n a b le t o u t i l i z e
the d e x tr a n .
In a d d i t i o n a l
w h ich y e a s t was used t h a t had a d a p te d t o
to ad ap t to u t i l i z a t i o n
is o m a lt o s e ,
is o m a lto s e and
A p p a re n tly the
e x p e rim e n ts
in
t h e y e a s t was u n a b le
o f d e x tra n .
A c u l t u r e was p re p a r e d c o n t a i n i n g two p er c e n t
is o m a lt o s e and panose
w i t h no m a lt o s e as a s t a r t e r sugar and i n o c u l a t e d w i t h y e a s t t h a t had n o t
been p r e v i o u s l y exposed t o
is o m a lt o s e o r p an o se.
c u l t u r e was compared t o t h a t o f t h e
is o m a lt o s e - p a n o s e c u l t u r e
I t was a p p a r e n t from t h e two g row th r a t e s
re q u ire d .
The g ro w th r a t e o f t h i s
in F ig u r e 5 .
t h a t an a d a p t a t i o n p e r io d was
The c e l l s w ere removed from t h e medium and f r o z e n .
Two c u l t u r e s w ere p re p a r e d c o n t a i n i n g
1 . 5 per c e n t
is o m a lt o s e ,
i somaI t o t r i o s e and h ig h e r homologues and 0 , 5 p er c e n t g lu c o s e
and 0 . 5 p e r c e n t m a lt o s e
in t h e o t h e r .
in one
T h is was done in o r d e r t o a s c e r t a i n
- 15 -
D e x tra n
M a l t o s e C o n tro l
Panose & Is o m a lto s e
70
t im e
F i g u r e 3.
80
in hours
Wet C e ll
W eight vs Time
G/M
I
2
e
—
© —
KEY
H ig h ers
Panose
(8> ®
x
I soma Ito s
M a l to se
G l ucose
_____________ LJ_______________ ________________
24 hour sample
42 hour sample
48 hour sample
54 hour sample
66 hour sample
g lu c o s e / m a lto s e
I soma I t o s e ,
Panose / m a lt o s e
D e x tra n / m a lto s e
— —
F ig u r e 4 .
-
M a lt o s e c o n t r o l
U tiliz a tio n
of
I soma I t o s e ,
Panose, and D extran
17 -
- M a l t o s e , Is o m a lto s e & Panose
- Is o m a lto s e / Panose
% w et c e l I w e ig h t
0
^
20
90
30
tim e
F ig u r e 5 .
100
in hours —
A Comparison Between C u l t u r e w i t h M a lt o s e
as a S t a r t e r and One w i t h o u t a S t a r t e r
-1 8 w h e th e r t h e s t a r t e r
and, a t
su g ar would
t h e same t i m e ,
in flu e n c e the u t i l i z a t i o n
t o check th e a b i l i t y
w ere w ith d ra w n a f t e r 24 hours and e v e r y s i x t h
7,
and 8 show t h e c e l l
o f the n u t r i e n t
w e ig h ts ,
th ro u g h o u t t h e
is o m a lto s e
o f y e ast to u t i l i z e
h ig h e r m o l e c u l a r w e ig h t su g ars c o n t a i n i n g the«=< ( 1 , 6 )
6,
of
any o f t h e
lin k a g e s .
hour t h e r e a f t e r .
Samples
F ig u re s
re d u c in g sugar v a lu e s and chromatograms
i n c u b a t io n p e r i o d .
A f t e r 72 hours t h e y w ere
removed from t h e medium and b oth t h e c e l I - f r e e medium and t h e c e l l s w ere
saved f o r
f u t u r e e x p e r im e n t s .
T h e re was a p p a r e n t l y no d i f f e r e n c e
in t h e g row th o f t h e y e a s t w h e th e r
g lu c o s e o r m a lt o s e was u s e d ; however, e x a m in a t io n o f t h e chromatograms
in d ic a te d
t h a t the
is o m a lt o s e was u t i l i z e d
at a s lig h tly
t h e c u l t u r e w i t h g lu c o s e as a s t a r t e r s u g a r .
s u p p o rte d t h i s o b s e r v a t i o n .
fa s te r
in
The re d u c in g sugar v a lu e s
The chromatograms a l s o
in d ic a te d
m a l t o t r i o s e and t h e h ig h e r homologues w ere w ere n o t u t i l i z e d
I t was i n t e r e s t i n g
ra te
t o n o te t h e d e c re a s e in c e l l
th at
is o -
by th e y e a s t .
w e ig h t a t a p p r o x im a t e ly
t h e same t im e t h e su g ar d is a p p e a r e d from t h e n u t r i e n t and th e n an in c r e a s e
in t h e c e l l
w e ig h t a few hours
on t h e c e l l
membrane f o r h y d r o l y s i s by is o m a lt a s e a t a r a t e
the a c tu a l
u tiliz a tio n .
la te r.
P o s s ib ly t h e
is o m a lt o s e was abso rb ed
C o n s e q u e n tly t h e c e l l w e ig h t
sugar had been removed from t h e n u t r i e n t .
f a s t e r than
in c r e a s e d a f t e r
The momentary d e c re a s e
the
in c e l l
w e ig h t was p r o b a b ly due t o t h e d e la y e d e f f e c t o f a d a p t a t i o n .
L o c a t io n o f t h e Enzyme
Okada ( 9 )
th a t the y e a s t,
su g g ested t h a t
f e r m e n t a t i o n o c c u r r e d t h ro u g h g lu c o s e and
Shizosaccharom yces pombe, had produced an e x t r a c e l l u l a r
- 19 -
A
- M a lt o s e / Is o m a lto s e and
H ig h e r Homologues
Q - Glucose / Is o m a lto s e and
H ig h er Homologues
tim e
F ig u r e 6 .
in hours —
Growth o f Candida u t i I is
as a F u n c tio n o f Time
- 20
Q - M a lt o s e /
H+ hydro I y z e d d e x t r a n
A
H+T iy d ro ly z e d d e x t r a n
tim e
F ig u r e 7•
Sugar
- G lucose /
In hours
U tiliz a tio n
by C a n d ida u t i l is
I d e n t i f i c a t ion
o f sugars
G/M G
I ^
O
O
Q
f -f
9
O
O
O
Q
O
0 0 0
0
0
O
0
0
0 0 0
O
0
0
0
H igher homologues
o f is o m a lto s e
I soma I t o s e
O
M a lto se
G lucose
O
1
N>
I - 24 hours
2 — 30 hours
3 4 -
G - g lu c o s e
36 hours
42 hours
- m a lto s e
5 48 hours
6 54 hours
7
8
-
60 hours
66 hours
0.01 m l . o f n u t r i e n t s p o t t e d f o r each
F ig u r e 8 .
Comparison o f Glucose vs M a lto s e as S t a r t e r Sugars
22
i somal t a s e whose synthesis was
Because o f t h i s ,
f i v e days was checked f o r enzyme a c t i v i t y
in c u b a tin g a ten m i l l i l i t e r
i s o m a lt o s e ,
a t room t e m p e r a t u r e ,
in a c tiv a te
is o m a lt o s e .
t h e c e l l - f r e e medium o f a c u l t u r e t h a t had grown on
is o m a lt o s e and panose f o r
c o n ta in in g
induced by t h e presen ce o f
a l i q u o t w it h ten m i l l i l i t e r s
by
o f a s o lu tio n
i soma I t o t r i o s e and h ig h e r homologues
A p o r t i o n o f t h e m i x t u r e was b o i l e d
f o r Zk hours
im m e d ia te ly t o
t h e enzyme and s p o t t e d on a chromatogram s t r i p as a c o n t r o l ,
T h e r e was e v id e n c e o f h y d r o l y t i c a c t i v i t y
wh ich Was due t o t h e enzyme,
i soma I t a s e , as shown in F i g u r e 9 .
S in c e t h e f i n a l
t o s e was u t i l i z e d
g ro w th o f y e a s t ,
p re lim in a ry
pH was 7 . 0
in t h e p r e v io u s s tu d y
and because a pH o f 4 . 5
t h e pH v a lu e s o f 5 . 0 ,
s tu d y o f t h e optimum pH.
t h e use o f c i t r i c
in wh ich
is o m a l­
is recommended f o r optimum
6 ,0 ,
and 7 . 0 w ere chosen as a
These pH v a lu e s w e re o b t a in e d by
a c id - p h o s p h a t e b u f f e r
(2 ),
The t e s t s o l u t i o n s were
p re p a r e d by m ix in g equal amounts o f c e l l - f r e e medium, b u f f e r and th e
s u b s tra te ; a s o lu tio n o f
homologues.
i s o m a lt o s e ,
i soma I t o t r i o s e ,
and t h e h ig h e r
These samples w e re in c u b a te d f o r 48 h o u r s .
' v,
The r e s u l t s o f t h i s
--I1
'
e x p e r im e n t a r e shown in F ig u r e
10.
chromatogram t h e optimum pH was n o t e l u c i d a t e d by t h e f a c t
a c t i v i t y was e x h i b i t e d a t pH 6 . 0 .
From thd
t h a t th e l e a s t
The chromatogram d e f i n i t e l y
t h e c o n c lu s io n o f p r e v io u s e x p e r im e n t w h ic h
su p p o rted
i n d i c a t e d t h e p re s e n c e o f an
I soma I t a s e .
Several
subsequent a t t e m p t s
t o o b t a i n enzyme a c t i v i t y
f e e medium proved t o be u n s u c c e s s f u l .
a p p ro x im a te ly ten f o l d ,
a v e ry sm all
in t h e c e l l -
By c o n c e n t r a t i n g t h e c e l l - f r e e medium
amount o f g lu c o s e was produced from
- 23 -
C o n tro l
Sample a f t e r
24 hours
H ig h e rs
D ire c tio n
of
Sol vent
Is o m a lto s e
Ma I t o s e
G l ucose
F ig u r e 9 .
G/M
Enzyme A c t i v i t y o f t h e Cel I - F r e e Medium
C o n tro l
pH o f Enzyme S o l u t i o n
D ire c tio n
of
Sol ven t
H ig h e r
Homologues
Is o m a lto s e
M a l to s e
Glucose
F ig u r e
10 .
C e l l - F r e e Medium A c t i v i t y a t T h re e pH V alues
-24 is o m a l t o s e .
c e llu la r
u s u a lly
fo r
In v ie w o f t h i s ,
i t ap p e a re d t h a t t h e enzyme was i n t r a ­
r a t h e r th a n b e in g an exoenzyme.
in 72 hours and t h e above c u l t u r e had been in c u b a te d
reach ed
120 h o u rs ,
S in c e maximum g row th was
t h e r e was a p o s s i b i l i t y
th a t the c e lls
in t h e c u l t u r e
had a u t o l y z e d t o such an e x t e n t as t o r e l e a s e t h e enzyme.
A la rg e c u lt u r e o f y e a s t,
p re p a r e d w i t h g l u c o s e - 0 . 5 p er c e n t , and
is o m a lt o s e and t h e h ig h e r homol o g u e s - I .5 p e r c e n t , was
th re e days.
The w et c e l l s
m a t e l y e i g h t y g ram s.
h a r v e s t e d from t h i s
in c u b a te d f o r
c u l t u r e weig h ed a p p r o x i ­
About h a l f o f t h e s e c e l l s was s t o r e d by f r e e z i n g
and t h e o t h e r h a l f was r u p t u r e d by g r i n d i n g w i t h sand in a m o r t a r .
membranes and c e l l
m illilite rs
c e ll
sap w ere removed from t h e sand by wash in g w i t h ten
o f d i s t i l l e d w a te r.
sap by c e n t r i f u g i n g a t
The membranes w ere removed from th e
1 0 ,0 0 0 x g f o r
tw e n ty m i n u t e s .
in t h e c e n t r i f u g e tubes w e re a b o u t f o u r c e n t i m e t e r s
to S utto n e t a l ,
The
(21)
t h e p e l l e t s h o u ld c o n t a i n
tom and t h e membranes o f t h e r u p t u r e d c e l l s
in h e i g h t .
in ta c t c e lls
on top..
t h e membrane f r a c t i o n was used in t h e e x p e r i m e n t ,
The p e l l e t s
A c c o rd in g
a t the b o t­
To i n s u r e t h a t o n ly
t h e to p o n e - f o u r t h and
bottom o n e - f o u r t h o f t h e p e l l e t w ere d i s c a r d e d .
The membranes w e re resuspended in c i t r i c
pH 5 . 0 and m ixed w i t h an equal
of
volume o f s u b s t r a t e s o l u t i o n ,
is o m a lt o s e and t h e h ig h e r homologues,
m ixed w i t h an equal
a t pH 5 . 0 .
a c id - p h o s p h a t e b u f f e r o f
a s o lu tio n
The s u p e r n a t a n t s o l u t i o n was
volume o f t h e s u b s t r a t e s o l u t i o n w h ich was b u f f e r e d
A p o r t i o n o f each o f t h e above t e s t s o l u t i o n s was b o i l e d
to
i n a c t i v a t e t h e enzyme and th e s e w ere s p o t t e d on chromatogram paper as
c o n tro ls .
The t e s t s o l u t i o n s w ere in c u b a te d a t room t e m p e r a t u r e f o r 24
- 25 "
hours and th en s p o t t e d on chromatogram p a p e r .
d e v e lo p e d f o r 24 h o u r s .
The chromatogram
a s s o c ia te d w ith
present
It
in t h e
F i g u r e 11 d e p i c t s
in d ic a te d
The chromatogram was
t h e r e s u l t s o f t h e e x p e r im e n t .
t h a t t h e m a jo r p o r t i o n o f t h e enzyme was
t h e membrane o f t h e c e l l
b u t t h e r e was d e f i n i t e l y
i n t r a c e l l u l a r sap.
has been known t h a t "bound” enzymes,
i.e .
enzymes h e ld by v i r t u e
o f c h a r g e , m ig h t be r e l e a s e d by m e r e ly a d j u s t i n g t h e pH.
and V. B ru ski
enzyme
(7 )
rep o rted th is
in work w i t h
K. J .
Goering
- am ylase from f u n g i .
They found t h a t t h e pH had t o be a d j u s t e d t o 8 . 0 b e f o r e t h e am ylase was
re le a s e d .
More r e c e n t l y ,
H. 0 .
on t h e p e c t i n e s t e r a s e s o f f r u i t
L e n c k e r and H. S w i f t
(10)
H u l t i n and A . S. L e v in e ( 1 2 )
p u l p , and R. L . H o l t z e r ,
in t h e i r work
J. L.
Van
in t h e i r work w i t h am ylase from zymogens and
m icrosom es, r e p o r t e d t h a t an a l k a l i n e
pH was r e q u i r e d
fo r re le a s e o f
th ese, enzymes.
W it h t h i s
in m in d ,
t h r e e samples o f y e a s t c e l l s o f t e n grams each
w e re e x t r a c t e d w i t h b u f f e r s o l u t i o n s o f pH 7 , 0 ,
8 . 0 and 9 . 0 ,
C itric
a c id - p h o s p h a t e b u f f e r was used t o o b t a i n pH 7 . 0 and c a r b o n a t e b u f f e r t o
o b t a i n pH 8 . 0 and 9 . 0 .
These e x t r a c t s w e re t e s t e d
by m ix in g them w i t h an equal
t h e pH t o 5 . 0 w i t h c i t r i c
checked f o r any r e s i d u a l
tio n e d .
It
f o r enzyme a c t i v i t y
volume o f s u b s t r a t e s o l u t i o n and a d j u s t i n g
a c id .
a c tiv ity
The e x t r a c t e d membranes w e re a l s o
in t h e same manner as p r e v i o u s l y men­
The r e s u l t s o f t h e s e t e s t s a r e d e p i c t e d
in F i g u r e
12 ,
seemed a p p a r e n t from t h e chromatogram th a t, t h e enzyme was e x ­
t r a c t e d most e f f i c i e n t l y
in a c t i v i t y ,
a t pH 8 . 0 .
A t pH 9 . 0 t h e enzyme s u f f e r e d a loss
p ro b a b ly due t o d e n a t u r a t i o n .
— 26 -
Cel I
Membranes
C e ll
C o n tro l
C o n tro l
H ig h e r
Ol Ig o s a c c h a r id e s
Is o m a lto s e
M a l to s e
GI u cose
C o n t r o l -M - C o n t r o l
C o n t r o l -S - C o n t r o l
F ig u re 11.
f o r t h e membranes
f o r t h e c e l l sap
L o c a t io n o f Enzyme System
- 27 -
G/M
H i gher
O l i g o s a c c h a r i d6
E x tra c t
7
CoR&rol E t i .
-----1®------- --- r5?---5,
s
§
Is o m a lto se
M a l to s e
O
O
0
0
8
E & t.
9
fa tt.
I
a
I I
0
E x t r a c t e d Membranes
Membrane
PH
PH
pH
Ta
CoR^r-oL-
I I
I
O
O
O
0
0
O
0 (3 0
O
0
(V
G l ucose
o
0
xv
0.01 m l . a p p l i e d
F ig u r e
12.
The A c t i v i t y
to each sp o t
o f th e Membrane E x t r a c t s
“ 28 -
S o lu b ility
o f t h e Enzyme
In an e f f o r t
s u lfa te
to
i s o l a t e t h e enzyme from t h e e x t r a c t s , an ammonium
f r a c t i o n a t i o n was p erfo rm ed
(2 ).
The o n ly f r a c t i o n
any p r e c i p i t a t e was t h e n i n e t y p e r c e n t s a t u r a t e d
o f p r e c i p i t a t e was e x t r e m e ly sm all
in t h i s
fra c tio n .
T h is
th a t y ie ld e d
fra c tio n .
The amount
and t h e r e was no m e a s u ra b le a c t i v i t y
i n d i c a t e d t h e enzyme was h i g h l y s o l u b l e .
D . E . A . E . C e l l u l o s e Chromatography o f t h e Membrane E x t r a c t
The c ru d e enzyme e x t r a c t was f r a c t i o n a t e d on a column o f D . E . A . E ,
c e llu lo s e * w ith g ra d ie n t c i t r i c
was p re p a r e d by a l l o w i n g
b u f f e r o f pH 5 . 0 ,
a c id - p h o s p h a t e b u f f e r .
t h e d ry m a t e r i a l
T h is was t r a n s f e r r e d
The a b s o rb e n t
to e q u i l i b r a t e over n ig h t w ith
t o a column 1 ,5 x 39 c e n t im e t e r s
t h a t was p lu g g ed w i t h g la s s wool and a llo w e d t o g r a v i t y pack f o r 2k h o u r s .
A fiv e m i l l i l i t e r
sample o f c ru d e enzyme e x t r a c t ,
a l l o w e d t o a b s o rb on t h e colum n.
F o rty m i l l i l i t e r s
added t o t h e column and f o u r - m i l l i l i t e r
e v e r y two f r a c t i o n s
b u f f e r o f pH 7 . 0 .
b u f f e r w ere
A fte r
w ith c itr ic -a c id -p h o s p h a te
The f r a c t i o n s c o l l e c t e d w ere checked f o r p r o t e i n by
(1 4 ),
in a Beckman Model
The e l u t i o n
o f pH 5 . 0
f r a c t i o n s w e re c o l l e c t e d .
t h e column was r e f i l l e d
t h e method o f K a lc k a r
measured
b u f f e r e d a t pH 5 , 0 , was
The o p t i c a l
d e n s i t i e s o f t h e f r a c t i o n s w ere
D U s p e c t r o p h o t o m e t e r a t 280 mu and 260 mu.
p a tte r n o f th e s e p a ra tio n
is g iv e n
in F i g u r e 13.
;
The f i r s t
F ra c tio n
peak was e l u t e d a t pH 5 . 0 and t h e second peak a t pH 5 , 6 ,
f i v e and f r a c t i o n
*D ,E ,A ,E , c e llu lo s e
-
f i f t e e n w ere checked f o r a c t i v i t y
d ie th y la m in o e th y l .c e llu lo s e
by m ix in g
o p tic a l
d e n s ity
- 29 -
10
20
F ig u r e
30
13.
40
—
50
60
70
80
90
volume in m i l l i l i t e r s
100
HO
120
—
G r a d ie n t E l u t i o n o f t h e Crude Enzyme E x t r a c t
130
- 30 t o g e t h e r one m i l l i l i t e r
fra c tio n ,
at
and s u b s t r a t e s o l u t i o n .
room t e m p e r a t u r e .
d e x tra n c o n ta in in g
s e lf.
o f each o f th e f o l l o w i n g :
These w e re th en
Two s u b s t r a t e s w ere t e s t e d ,
f o r 24 hours
t h e a c i d h y d r o ly z e d
T h is chromatogram is
in F i g u r e 14 .
T h is
in fo rm a tio n
i n d i c a t e d t h e p re s e n c e o f two enzymes.
is o m a lt o s e b u t n e i t h e r would h y d r o ly z e d e x t r a n .
chromatogram
i t was d i f f i c u l t
to t e l l
if
Both hy­
From th e
i s o m a l t o t r i o s e o r any o f th e
higher homologues w ere h y d r o ly z e d by e i t h e r o f t h e two f r a c t i o n s ,
it
it­
i n c u b a t i o n p e r i o d t h e samples w e re a p p l i e d t o
chromatogram p ap er w i t h an a p p r o p r i a t e c o n t r o l .
d ro ly z e d
in c u b a te d
e lu te d
is o m a lt o s e and h ig h e r homologues, and t h e d e x t r a n
A t t h e end o f t h e
d e p ic te d
b u f f e r pH 5 . 0 ,
d i d a p p e a r as though peak tw o ,
ly z e d some o f t h e
F i g u r e 13 , page 2 9 , m ig h t have h y d ro ­
is o m a lt o t r i o s e .
Some o f t h e e l u t i o n
shown in F ig u r e s
however,
15,
p a t t e r n s made on t h e same c ru d e e x t r a c t a r e
16, and 17.
These show how th e e l u t i o n
p attern s
changed under v a r io u s c o n d i t i o n s .
The d i f f e r e n c e between F ig u r e s
been r e p a c k e d ,
lo s t.
lite r
15 and 16 was t h a t t h e column had
thus the. r e s o l u t i o n o f t h e second and t h i r d
The e l u t i o n
p atte rn
peaks was
in F i g u r e 17 was o b t a in e d when a f i v e m i l l i ­
sample was chrom ato g rap h ed on t h e same column t h a t was used f o r
F i g u r e 16 .
,
The f r a c t i o n s
fo r a c t iv it y
t h a t would a c c o u n t f o r
the t h i r d
peak w ere checked
in t h e same manner as peaks one and tw o .
a c t i v i t y w ith m a lto s e ,
p ro d u c in g g lu c o s e w h ic h
T h is peak e x h i b i t e d
in d ic a te d a m a lta s e .
- 31 -
G/M C o n t r o l
pk-1
„
pk-1
Dex
p k -2
p k-2
H ig h e r
Homologues
Is o m a lto se
M a lt o s e
G l ucose
G/M
C ontrol I
pk-1
pk-1 Dex
p k -2
p k -2 Dex
F ig u r e
g l u cose / m a lto s e
b u ffe r / s u b s tra te
F ig u r e 13,
peak I ,
F i g u r e 13,
peak I ,
F ig u r e 13,
peak 2 ,
F ig u r e 13,
peak 2 ,
14.
/ b o i l e d enzyme
b u f f e r and is o m a lt o s e s o l u t i o n
b u f f e r and d e x t r a n s o l u t i o n
b u f f e r and i soma I t o s e s o l u t i o n
b u f f e r and d e x t r a n s o l u t i o n
The A c t i v i t y E x h i b i t e d by Two F r a c t i o n s
E l u t e d from D . E . A . E . C e l l u l o s e Column
I
- 32 -
.28
. 26
.2 4
m illig ra m s o f p ro te in / m i l l i l i t e r
.22
I
.20
.1 8
.16
.1 4
.12
.10
.08
.06
.0 4
.02
70
80
90
100
HO
120 130
vo Iume in mi I I i I i t e r s
F ig u r e
15.
T h re e P r o t e i n s
S e p a ra te d from th e Crude E x t r a c t
140
- 33 -
I
L.
<D
-M
E
c
<D
M
O
U
Q.
MO
tn
E
ro
L-
CD
E
I
--
F ig u r e 16 .
vo Iume in mi 1 1 i I i t e r s —
The Change in E l u t i o n
P a t t e r n w i t h a New Column
- 34 -
-
. 20
m .12
= .10
20
30
50
—
F ig u r e
17,
60
80
90
volume in m i l l i l i t e r s
R e d u c tio n
100
--
1 10
120
130 140
in P r o t e i n a f t e r S u c c e s s iv e E l u t i o n s
-
35
-
Optiumum T e m p e ra tu re and Optimum pH
A p r e l i m i n a r y check was made on t h e c ru d e enzyme f o r
t e m p e r a t u r e and t h e optimum pH f o r a c t i v i t y
re s u lts
in d ic a te d
t h e optimum
o f t h e enzyme system s .
The
t h a t t h e optimum t e m p e r a t u r e was a p p r o x im a t e ly 7 0 ° C.
and t h e optimum pH was a p p r o x i m a t e l y 3 . 0 ,
A c lo s e r
i n v e s t i g a t i o n was th e n made on t h e two peaks
and 1 6 , pages 32 and 33)
t h a t had e x h i b i t e d a c t i v i t y .
of c itric
fo r
f i f t e e n m in u te s .
s o l u t i o n was a llo w e d t o e q u i l i b r a t e
o f the s u b s tra te
a c i d b u f f e r o f pH 3 . 5 and p l a c i n g
t h e sample in a w a t e r b a th o f a p p r o p r i a t e t e m p e r a t u r e .
a llo w e d t o e q u i l i b r a t e
15
For t h e t e m p e r a t u r e
s tu d y t h e samples w e re p re p a re d by m ix in g one m i l l i l i t e r
s o l u t i o n w i t h one m i l l i l i t e r
(F ig u re s
The samples were
One m i l l i l i t e r
in a s e p a r a t e tu b e f o r
o f t h e enzyme
f i v e m in u tes
th en t h e two s o l u t i o n s w e re m ixed and a l l o w e d t o r e a c t f o r one h o u r.
p er c e n t re d u c in g su g ar was measured a t
t h e end o f t h i s
tim e .
The
The enzyme
r e a c t i o n was sto p p ed by t h e a d d i t i o n o f t h e 3 , 5 d i n i t r o s a l i c y I i c a c i d and
b o ilin g
fo r
f i v e m in u te s .
The p e r c e n t
is o m a lt o s e h y d r o ly z e d was c a l c u ­
l a t e d and p l o t t e d a g a i n s t t e m p e r a t u r e as shown in F i g u r e 18»
t e m p e r a t u r e was 6 5 ° C,
f o r b o th enzymes.
enzyme s o l u t i o n was a llo w e d
mixed w i t h
to re a c t
Here a g a in one m i l l i l i t e r
f i v e m in u te s t o a t t a i n
the b u ffe re d s u b s tra te s o lu t io n .
of
t e m p e r a t u r e and then
These samples w ere a llo w e d
f o r one hour and th e n t h e p e r c e n t re d u c in g su g ar was m easured.
The p e r c e n t
F ig u re
and 4 . 0
The optimum
is o m a lt o s e h y d r o ly z e d was c a l c u l a t e d and. p l o t t e d a g a i n s t pH.
19 shows an optimum pH o f 3 . 2 5 f o r t h e p r o t e i n o f peak one
f o r t h e p r o t e i n o f peak two ( F i g u r e
15,
page, 3 2 ) .
- 36 -
O - - - ^ ------------
F ra c tio n
F ra c tio n
from peak one
from peak two
— tem p eratu re
F ig u r e 18.
C --
The E f f e c t o f T e m p e ra tu re on A c t i v i t y o f t h e
ls o m a lta s e Enzymes
- 37 -
O ------- F r a c t i o n from peak one
^ ------ F r a c t i o n from peak two
100
<o
§
(/>
50
40
I
30
20
10
j __________ |__________ ;__________ j__________ I__________ I___________L
2
3
4
5
6
7
8
— pH u n i ts —
F ig u re
19.
The E f f e c t o f pH on t h e A c t i v i t y o f t h e
Enzyme Systems
Is o m a lta s e
- 38 The f a c t t h a t two peaks w ere o b s e rv e d from t h e Di E eA eE 1 s e p a r a t i o n
in d ic a te d
t h a t two enzymes w ere p r e s e n t .
v a lu e s w ere d e t e r m in e d s u p p o rte d t h i s .
The f a c t t h a t two optimum pH
A n o th e r f a c t t h a t
I ended s u p p o rt
JU
to the
idea o f two enzymes was t h a t two Q j q ' v a lu e s w e re d e t e r m in e d .
v a lu e s c a l c u l a t e d
fo r the
Qjq
The
o f t h e enzymes w ere ta k e n from F ig u r e 18,
page 3 6 ; t h e s e w ere 2 , 1 2 and 1 .8 5 f o r t h e p r o t e i n o f peak one and peak
two r e s p e c t i v e l y ,
R eversi b i l i t y
In o r d e r t o e s t a b l i s h t h e r e v e r s i b i l i t y
o f t h e e n z y m a t ic r e a c t i o n
a 2 . 6 8 m o la r s o l u t i o n o f g lu c o s e was p re p a re d by d i s s o l v i n g
f i v e grams
o f g lu c o s e
The m i x t u r e
in t e n m i l l i l i t e r s
was h e a te d t o 6 0 ° C.
o f c itra te
in a w a t e r b a t h .
b u f f e r o f pH 3 » 2 ,
Two m i l l i l i t e r s
o f c ru d e enzyme
s o l u t i o n was added and t h i s m i x t u r e was a l l o w e d t o r e a c t f o r 4 8 h o u rs .
A fte r
th is
t im e two 0 .0 1 m i l l i l i t e r
gram f o r q u a n t i t a t i v e d e t e r m i n a t i o n .
b o ile d
to
samples w ere a p p l i e d t o a chrom ato­
A s i m i l a r s o l u t i o n t h a t had been
i n a c t i v a t e t h e enzyme was used as a c o n t r o l .
The p h e n o l - s u l f u r i c a c i d method o f Dubois e t a l .
s e n s itiv e
in t h e ran g e o f
10 ug t o 80 ug o f g lu c o s e was used f o r t h e
d e t e r m i n a t i o n o f su g ar p r e s e n t .
d e fin ite ly
( 6 ) w h ic h is
The chromatogram d e p i c t e d
in F ig u r e 20
showed t h a t t h e r e a c t i o n was r e v e r s i b l e b u t t h e amount o f
is o m a lt o s e o b t a i n e d was belo w t h e d e t e c t i o n
a c i d s u g ar a n a l y s i s .
l i m i t o f the p h e n o l-s u lfu r ic
C o n s e q u e n t ly , an e q u i l i b r i u m
c o n s t a n t was not
o b ta in e d .
Q] 0
is t h e
in c r e a s e
in r a t e
f o r a t e n d e g re e
in c r e a s e
in t e m p e r a t u r e .
- 39 -
Equi l i b r i u m
M ix tu re
C o n tro l
I soma I t o s e
G lucose
C o n tro l - g lu c o s e /
^ E q u i l i b r i u m M i x t u r e - g lu c o s e /
F ig u r e 2 0 .
enzyme b o i l e d f i v e m in u te s
enzyme in c u b a te d 48 hours
R e v e rs ib ility
o f t h e E n zy m a tic R e a c t io n
- 40 -
i n h i b i t o r s o f C a rb o h y d ra s e Enzymes
A number o f
in h ib ito rs
t h a t a r e g e n e r a l l y used f o r d e t e r m in in g thb
r e q u ir e m e n t s o f enzymes b e lo n g in g t o t h e g e n e r a l
was
in c u b a te d w i t h
t h e enzyme s y s te m s »
w e re p re p a r e d by m ix in g one m i l l i l i t e r
(See T a b le
o f the
_3
2 x I O M c o n c e n t r a t i o n , w i t h one m i l l i l i t e r
s o lu tio n
t h a t was b u f f e r e d a t pH 3 . 2 ,
o f w a t e r and one m i l l i l i t e r
tre a te d
in
id e n tic a l
1 1 1 .)
The samples
in h ib ito r s o lu tio n , o f
o f an e n z y m e - s u b s t r a t e
These m ix t u r e s w e re th en
in a w a t e r b a th f o r one hour a t 6 0 ° C .
lite r
c la s s o f c a rb o h yd rase s
in c u b a te d
A s o l u t i o n c o n t a i n i n g one m i l l i ­
o f t h e e n z y m e - s u b s t r a t e s o l u t i o n was
f a s h i o n t o be used as a c o n t r o l .
A t t h e end o f t h e
i n c u b a t i o n p e r i o d t h e samples and c o n t r o l w e re a n a ly z e d f o r r e d u c in g
sugar.
tro l
The com parison o f t h e a c t i v i t y
d e fin ite ly
te s te d and,
s m all
showed no d e c re a s e in a c t i v i t y
in f a c t ,
in a c t i v i t y .
The i n c r e a s e
but re p ro d u c ib le .
fa c t,
in a c t i v i t y
However,
t h e Fe
as t h e c o n t r o l
From t h e r e s u l t s o f t h i s
n e c e s s i t y o f a m e ta l
stu d y
t r e a t e d sample showed a p p r o x i ­
in s e v e r a l
te s ts .
In view o f
f o r t h e enzyme system .
g roups.
ion even though f e r r o u s
T h e re was no a p p a r e n t
ion enhanced t h e a c t i v i t y .
f o r c a r b o h y d r a t e h y d ro la s e s a r e Ca"H " and Cl
These ions w e re checked f o r a c t i v a t i n g
duce an i n c r e a s e
and Mn+J_ was
i t was a p p a r e n t t h a t t h e a c t i v e s i t e s
in c lu d e s u lfh y d r y I
Two common a c t i v a t o r s
FeSOj1 showed an in c r e a s e
in t h e cases o f C o ^
Fe+4" was c o n s id e r e d an a c t i v a t o r
o f t h e enzyme d id n o t
f o r any o f t h e substances
CoC l 2 » MnSOj1., and e s p e c i a l l y
m a te ly tw ic e th e a c t i v i t y
th is
o f t h e samples t o t h a t o f t h e con­
in a c t i v i t y .
p r o p e r t i e s b u t n e i t h e r would p r o ­
- 4l -
TABLE ! 11In h ib i to rs
Used w i t h t h e
(
Is o m a lt a s e System
% R e la tiv e A c tiv ity
T ria l-1
T ria l-2
In h ib ito r
106
PCMB1
AgNO3
CoC 12
MnSOz+
FeSO4
KCN
103
120
113
203
102
122
118
185
' PCMB - p a r a c h l o r o m e r c u r i b e n z o a te
TABLE IV
S p e c i f i c i t y o f Enzyme Systems
C arb o h yd rate te s te d
Is o m a lt o s e
Amy Io s e
Glycogen
- L im it D e x trin
D e x tra n
Is o m a lto trio s e
Panose
M a lt o s e
G e n t io b io s e
C e llo b io s e
L a c to s e
S ucrose
Lamin aran
Key:
Enzym e-I
ErizymereZ . -
p ro te in
p ro te in
H y d r o ly s is O ccu rred
E n zym e-I
Enzyme-2
-
-
-
from peak o n e , F i g u r e
from peak tw o , F i g u r e
15» page 32
15» page 32
- 42 S p e c i f i c i t y o f t h e Enzyme Systems
In o r d e r t o e s t a b l i s h
t h e s p e c i f i c i t y o f th e enzyme system a number
o f c a r b o h y d r a t e s w e re used in p la c e o f t h e usual
w ere p r e p a r e d by m ix in g one m i l l i l i t e r
are
o f enzyme s o l u t i o n .
in c u b a te d a t 6 0 ° C . f o r one h o u r .
mined b e f o r e and a f t e r
lis te d
in T a b l e
the
re fe rre d
of
These sam­
The re d u c in g su g ar was d e t e r ­
i n c u b a t io n p e r i o d .
The r e s u l t s o f t h i s stu d y
I V, page 4 1 ,
The enzyme c o r r e s p o n d in g t o t h e f i r s t
(h e re a fte r
The samples
o f b u f f e r and one m i l l i l i t e r
c a r b o h y d r a t e s o l u t i o n w i t h one m i l l i l i t e r
p le s w e re
su b s tra te .
t o as
peak o f F i g u r e 15» page 3 2 , .
i soma I t a s e - 1 ) has v e ry s p e c i f i c
re q u ire m e n ts
f o r a s u b s t r a t e from t h e f a c t t h a t t h e o n l y c a r b o h y d r a t e h y d r o ly z e d was
is o m a lto s e .
(h e re a fte r
trio s e
of
The p r o t e i n o f t h e second peak shown in F i g u r e 15,
re fe rre d
in a d d i t i o n
t o as
to
i soma I t a s e - 2 )
is o m a l t o s e .
h y d r o ly z e d panose and i s o m a l t o -
The s p e c i f i c a c t i v i t y
is o m a lt o s e was g r e a t e r th a n f o r t h e h y d r o l y s i s o f
fo re
i somaI t o t r i o s e
le s s s p e c i f i c i t y
is n o t t h e n a t u r a l
than
i soma I t a s e - 1
page 32,
s u b s tra te ,
fo r the h y d ro ly s is
i soma I t o t r l o s e ,
th e re ­
Is o m a lt a s e - 2 has much
in r e g a r d t o t h e re d u c in g end o f t h e
m o le c u le .
T h is
t o t r lo s e .
T h e r e ap p e a re d t o be a r e q u ir e m e n t t h a t t h e n o n red u cin g m o ie ty
must be a t t a c h e d
f a c t was e v id e n c e d by t h e a c t i v i t y
on panose and i soma I -
in a d e f i n i t e s t e r i c way t o t h e number s i x c a rb o n .
F a i l u r e o f t h e enzyme t o h y d r o l y z e g e n t i o b i o s e s u p p o rte d t h i s
The enzyme a p p a r e n t l y a t t a c k e d
c o n c lu s io n .
from t h e n o n re d u c in g end o f t h e m o le c u le
and t h e r e p ro b a b ly was a p o i n t o f a t t a c h m e n t on t h e re d u c in g end o f th e
m o le c u le .
T h is
r e q u ir e m e n t was su g g ested by t h e f a c t t h a t t h e enzyme
c o u ld n o t h y d r o l y z e t h e d e x t r a n b u t c o u ld h y d r o ly z e panose and i soma I t o t r i o s e ,
- 43 A th o ro u g h k i n e t i c s tu d y o f co m p eti ve i n h i b i t i o n w i t h
t h e v a r io u s
su g ars and s u g ar d e r i v a t i v e s must be c o m p leted b e f o r e t h e s t e r i c
ments can be f u l l y
re q u ire ­
d e s c rib e d .
P u r i t y o f t h e Enzymes E l u t e d from t h e D . E . A . E , C e l l u l o s e Column
The enzymes t h a t w ere e x t r a c t e d
t h e pH t o 8 . 0
had a r e l a t i v e l y
from t h e y e a s t c e l l s
h ig h s p e c i f i c a c t i v i t y
by a d j u s t i n g
o f 6 .4 5 m illig ra m s
o f su g ar h y d r o ly z e d p er m i l l i g r a m o f p r o t e i n p er h o u r.
T h is v a lu e was
in c r e a s e d t o
1 5 . 4 m i l l i g r a m s o f su g ar p er m i l l i g r a m o f p r o t e i n
is o m a lt a s e - 1
and t o 2 5 . 8 m i l l i g r a m s o f su g ar p er m i l l i g r a m o f p r o t e i n
fo r
i s o m a l t a s e - 2 by f r a c t i o n a t i n g
lo s e colum n.
f o r th e
t h e c ru d e e x t r a c t on a D . E . A . E . c e l l u ­
The s p e c i f i c a c t i v i t y
o f th e p ro te in s a f t e r
s e p a r a t i o n on
t h e D . E . A . E . c e l l u l o s e column compared f a v o r a b l y w i t h t h e enzyme m y r o s in ase.
M y r o s in a s e 9 a f t e r a p u r i f i c a t i o n o f a p p r o x im a t e ly 4000 f o l d ,
a s p e c ific a c t iv it y
enzymes w h ich w e re
d id n o t
had
a p p r o x i m a t e l y s i x tim e s g r e a t e r th a n t h a t o f th e
is o la te d
in t h i s
stu d y.
A d d itio n a l
fra c tio n a tio n s
improve t h e s p e c i f i c a c t i v i t y .
One s e r i o u s d i f f i c u l t y
t h e e x t r e m e ly s m all
m illig ra m s o f the
in p e r f o r m in g a d d i t i o n a l
p u r i t y assays was
q u a n t i t i e s o f t h e enzymes o b t a i n e d .
is o m a lt a s e - 1 w ere o b t a i n e d
t h e D 1E i A . E i colum n.
The f a i l u r e
to
Only t w e lv e
from e i g h t s e p a r a t i o n s on
im prove t h e s p e c i f i c a c t i v i t y o f t h e
enzymes by su b seq u en t s e p a r a t i o n on t h e D . E . A . E . c e l l u l o s e column was r e a ­
s o n a b le a s s u ra n c e t h e enzyme systems w e re r e l a t i v e l y
pure.
An e l e c t r o p h o r e t i c s e p a r a t i o n was made w i t h a Model
paper e l e c t r o p h o r e s i s .
A p p r o x im a t e ly
E- 8 0 0 - 2 Reco
150 micrograms o f p r o t e i n was
- 44 a p p lie d
t o Whatman number one f i l t e r
was c i t r i c
p ap er
in a s t r i p .
The b u f f e r used
a c i d - p h o s p h a t e b u f f e r o f pH 5 . 0 and i o n i c s t r e n g t h 0 . 0 5 .
s t r i p s w ere s u b je c t e d t o 250 v o l t s
The
f o r s i x hours f o r c o m p le te s e p a r a t i o n .
The d e v e lo p in g s o l u t i o n and s t a i n i n g p r o c e d u re was t h a t used by B lock
et a l.
(I).
An e l e c t r o p h o r e s i s
F i g u r e 21
s trip
is d e p i c t e d
in F ig u r e 2 1 .
i n d i c a t e t h a t t h e c ru d e enzyme m a t e r i a l
The bands
c o n t a in s a t
in ,
le a s t th re e
com ponents.
The e l e c t r o p h o r e s i s o f
one band in, each s a m p le .
in F i g u r e 21»
te in
u sed .
is o m a lt a s e - 1
and i soma I t a s e - 2
showed o n ly
T h is band c o rresp o n d s t o t h e c e n t e r band shown
The o t h e r two bands w ere m a l t a s e and some e x t r a n e o u s p r o ­
t h a t c o u ld n o t be e l u t e d
from t h e D . E . A . E . column under t h e c o n d i t i o n s
- 45 -
P o s i t i v e Pole
Origin
Ne ga ti ve Pole
F ig u re 21 .
E l e c t r o p h o r e s i s S e p a r a t i o n o f t h e Crude E x t r a c t
SUMMARY
Is o m a lt o s e has been i s o l a t e d and i d e n t i f i e d as t h e system
re s p o n s ib le fo r th e fe rm e n ta tio n o f
u tiI is .
In a d d i t i o n
d r o l y z e d pan o se,
it
to t h i s ,
Candida
a second enzyme was i s o l a t e d w h ich hy­
I s o m a l t o t r l o s e , and i s o m a lt o s e .
is n o t common t o f i n d
T h is was unusual
s in c e
two enzymes from t h e same s o u rc e t h a t have t h e
same f u n c t i o n on a s u b s t r a t e .
The o n l y o t h e r
i n v e s t i g a t o r was t h e m u l t ! m o l e c u l a r
H. 0 .
is o m a lt o s e by t h e y e a s t ,
in c id e n c e known t o t h e
forms o f p e c t i n e s t e r a s e
i s o l a t e d by
H u l t i n and A . S. L e v in e ( 1 2 ) .
The l o c a t i o n o f t h e enzyme was d e m o n s tra te d t o be p r i m a r i l y a s s o c i ­
ated w ith
c e ll.
t h e c e l l membranes under c o n d i t i o n s u s u a l l y
T h e r e was a s m all amount o f enzyme in t h e
s in c e p r o te in s y n th e s is
f i n d some a c t i v i t y
found
in t h e y e a s t
in tra c e llu la r
flu id
but
ta k e s p la c e on t h e ribosomes one w ould e x p e c t t o
in s id e th e c e l l .
o f t h e enzyme was e x t r a c t e d
A p p r o x im a t e ly s e v e n t y - f i v e per c e n t
from t h e membranes by m e r e ly a d j u s t i n g
th e
pH t o 8 . 0 .
Due t o t h e s m all
amount o f enzyme t h a t c o u ld be o b t a i n e d ,
p u rific a ­
t i o n o f t h e enzymes was l i m i t e d t o D4E oA 4E 0 c e l l u l o s e c o lu m n , chrom ato­
graphy .
The h ig h s o l u b i l i t y o f t h e enzyme p re v e n te d p u r i f i c a t i o n
means o f s a l t
a c t i v ity o f
hour.
fra c tio n a tio n .
1 5 .4 m ill!g ra m s o f
The p u r i f i e d
i soma I t a s e - 1
by
had a s p e c i f i c
i somaI t o s e p e r m i l l ! g r a m o f p r o t e i n per
The second enzyme i s o l a t e d ,
isbmal tase%:2,
had a s p e c i f i c a c t i v i t y
o f 2 5 , 6 m i l l i g r a m s o f sugar p e r m i l l i g r a m o f p r o t e i n p er h o u r „
The i soma I t a s e - 1
4 .0
and i soma I t a s e - 2 had optimum pH v a lu e s o f 3 . 2 and
r e s p e c t i v e l y and b o th had an optimum t e m p e r a t u r e o f 6 5 ° C.
The
J
- 47 h y d ro ly s is o f
e q u ilib riu m
is o m a lt o s e was shown t o be r e v e r s i b l e ,
c o n s t a n t was n o t c a l c u l a t e d .
The i s o m a lta s e -1 was s p e c i f i c
h y d r o ly z e d
fo r
is o m a l t o s e ,
is o m a lt o s e and panose in a d d i t i o n
t h e common i n h i b i t o r s
how ever,
h o w ever, an
Fe+*
From t h e
to
Is o m a lt a s e - 2
i s o m a lt o s e .
None o f
im p a ire d t h e a c t i v i t y o f e i t h e r o f t h e enzymes,
a p p r e c i a b l y enhanced t h e a c t i v i t y
in fo rm a tio n g a th ered
c h a ra c te riz a tio n o f yeast
in t h i s
is o m a lt a s e w i l l
o f t h e enzymes.
in v e s tig a tio n
t h e co m p lete
be a s i m p l e r t a s k .
SUGGESTIONS FOR FUTURE RESEARCH
A c o m p le te c h a r a c t e r i z a t i o n o f t h e two enzymes
t h e s i s w ould p ro ve o f
i n t e r e s t s in c e
it
is
r a r e to f in d
t h e same s o u rc e h a v in g t h e same s u b s t r a t e s .
i s o l a t e d had a g r e a t e r a c t i v i t y w i t h
th e re fo re ,
in t h i s
two enzymes from
The second enzyme w hich was
is o m a lt o s e th a n w i t h
is o m a lt o s e a p p e a re d t o be t h e n a t u r a l
s t u d i e s u s in g c o m p e t i t i v e
id e n tifie d
i soma I t o t r l o s e ;
s u b s tra te .
i n h i b i t i o n w ould c l a r i f y
K in e tic
t h e c h a r a c t e r o f th e
second system o b t a i n e d .
The mechanism in v o lv e d
c o n tro v e rs ia l
is
in d ire c t,
su g ar t h a t
in t h e u t i l i z a t i o n
s u b j e c t f o r many y e a r s .
i.e .,
g lu c o s e
o f m a lt o s e has been a
Some w o rk e rs f e e l
the u t i l i z a t i o n
is produced by a m a l t a s e , and g lu c o s e
is ta k e n up by t h e y e a s t .
On t h e o t h e r hand s e v e r a l
is th e
in v e s ti­
g a t o r s h a v e .f o u n d c o n t r a r y e v id e n c e w h ich would su g g est d i r e c t u t i l i z a t i o n
by a m a lt o s e p h o s p h o ry l.ase.
From t h e s tu d y o f t h e u t i l i z a t i o n
in t h e p re s e n c e o f g lu c o s e w h ich was made in t h i s
mechanism seemed t o be a l o g i c a l
o f m a lt o s e
i n v e s t i g a t i o n , a m a lt a s e
a n s w e r.
S e e k in g t h e answer t o t h i s q u es­
s u b j e c t to d a y
is t h a t o f permeases and c a r r i e r s
t i o n w ould be a w o r t h w h i l e s t u d y .
A n o th e r c o n t r o v e r s i a l
b e in g r e q u i r e d
f o r t h e u p ta k e o f some n u t r i e n t s .
a permease o r c a r r i e r
has n o t been i s o l a t e d
a c c o m p lis h e d ,
c e lls
as compared t o
w ould c e r t a i n l y
I f a permease o r c a r r i e r
t h e c o n t r o v e r s y w ould be ended.
th e n an i n t r i c a t e
in ta c t,
If
th is
can n o t be
stu d y o f t h e k i n e t i c s o f u p ta k e by l i v i n g
n o n liv in g c e lls
be e n l i g h t e n i n g .
know ledge,
b u t e v id e n c e o f t h e i r p resen ce
was based on t h e k i n e t i c s o f u p ta k e phenomena.
c o u ld be i s o l a t e d ,
To t h e a u t h o r ' s
or a s u ita b le s u b s titu te ,
- 49 In t h e case o f a d a p t i o n o f c e l l s
t o new compounds, g e n e t i c i s t s
t h a t t h e compound must be in t h e p re s e n c e o f t h e g e n e t i c m a t e r i a l
the c e l l s
can a d a p t t o t h e new s u b s t r a t e .
t h e u p ta k e o f a s u b s t r a t e th e n
propose
b e fo re
I f a permease is n e c essa ry f o r
in t h e case o f a new s u b s t r a t e a permease
w ould have t o be p r e s e n t t o t r a n s p o r t t h e new compound i n s i d e t h e c e l l
fo r a d a p ta tio n .
a d a p ta tio n .
The l a r g e r a t e o f u p ta k e does not o c c u r u n t i l
T h e re fo re e it h e r
r a t e o f u p ta k e o r
(2 )
(I)
a permease
a fte r
a permease is n o t t h e cause o f t h e l a r g e
is n o t r e q u i r e d f o r e n t r a n c e t o a c e l l .
LITERATURE CITED
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i r 73
CO
CO
762
§
111111III 111111III
»
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R5?6
c o p .2
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The l o c i i t i o n an d i d e n t i f i c a tio n of,
R D o n sib
AiAMK A N D A O D h K S S
.
.
^
-
(C
IT