1984_(steverson) kinetics of the amylase system of saccharomycopsis fibuliger

7/23/2019 1984_(Steverson) Kinetics of the Amylase System of Saccharomycopsis Fibuliger

http://slidepdf.com/reader/full/1984steverson-kinetics-of-the-amylase-system-of-saccharomycopsis-fibuliger 1/6

inetics o f the am ylase sys tem of

a c h a r om c o p si s f i b u l i g er

E. Malone Steverson , Rog er A. Korus, W udneh Adm assu and Richard C. Heimsch~

D e p a r t m e n t o f C h e m i c a l E n g i n ee r in g a n d t D e p a r t m e n t o f B a c te r io l o g y a n d B io c h e m i s t ry , U n i v er si ty

o f Id a h o , M o s c o w , I d a h o 8 3 8 4 3 , U S A

(Rece ived 5 March 1984; rev i sed 12 September 1984)

The ex t race l lu lar amy lases produced by Saccharomycopsi s f ibu l ige r have been s tud ied wi th the

in ten t o f i den t i f y ing the k ine t i c mechani sm an d produc t d i s tr ibu t ion , and mode l l ing the produ c t ion

of D-g lucose dur ing s tarch hydro lys i s. H igh per formance l iqu id chroma tography was e f f ec t i ve ly used

to separate an d qu ant i fy the pr od uct ol igomers released, t~-Amylase rapidly hyd rolys ed the long

subs t ra te chains in to smal l e r o l igomers which became the subs t ra te for g lucoamylase in the produ c t ion

o f D-glucose. The form atio n o f a rate l imi t ing subs trate occurred late in the react ion. Glucoa mylase

and a-amylase ra te s were f i t t ed to Mich ae l i s -M enten mode l s wi th D-g lucose inh ib i t ion inc luded .

Keywords: Kinetics;Saccharomycopsis fibuliger;amylase; starch

Introduc t ion

A n e x t r a ce l l u la r a m y l o l y t i c e n z y m e sy s t e m i n Saccharomy-

c o p s is { E n d o m y c o p s i s f i b u l i g e r was f i r s t r epor t ed in 1944

a n d i t w a s d e t e r m i n e d t h a t t h e d o m i n a n t e n z y m e w a s

t~ -amylase ) Recent pape rs have repor t ed the presence of

saccha r i fy ing and dex t r in i z ing ac t iv i ty w i th the g luco-

a m y l a se a n d a - a m y l a se e n z y m e s p r o d u c e d .2 , a T h e e n z y m e

com plex exhib i t ed a s t rong debra r t ch ing ac t iv i ty , and no

t ransg lucos idase ac t iv i ty was de t ec t ed ,a Puri f ied gluco-

a m y l a se f r o m S. f ibul iger p r o d u c e d u p t o 9 5 % h y d r o l y s i s

of so lub le s t a rch and amylopec t in .2 ,a Mal tose and ma l to-

t r iose were hydro lysed l e ss rap id ly than h ighe r molecu la r

we ight o l igomers .3

S. f ibul iger h a s b e e n e x a m i n e d f o r th e p r o d u c t i o n o f

s ing le ce l l p ro t e in by cont inuous cu l tu re .4 Ce ll y i e lds f rom

a pota to s t a rch subs t ra t e were d imin i shed by a l imi t dex t r in

w h i c h w a s n o t h y d r o l y se d b y t h e a m y l a ses o f S fibuliger.

Also , S. f ibul iger h a s b e e n u se d i n c o n t i n u o u s c u l t u r e w i t h

Z y m o m o n a s m o b i l i s f o r t h e p r o d u c t i o n o f e t h a n o l , a n d

l imi t de x t r ins in the form o f o ligosaccha r ides were

prod uced , Th e purpose o f t h i s s tudy was to inves tiga t e

the k ine t i c s o f s t a rch hydro ly s i s by the ex t race l lu l a r amylase

c o m p l e x p r o d u c e d b y

S. f ibul iger

by de te rmin ing o l igo-

sa c c h ar i d e c o n c e n t r a t i o n s a s fu n c t i o n s o f r e a c t i o n

paramete rs .

Mater ia ls and m ethod s

Saccharomycops i s f i bu l iger was cu l tu red ba tchwise in a

M i c r o f e r m M F 1 0 5 c o n t i n u o u s f e r m e n t e r ( N e w B r u n sw i c k

S c i e n ti f ic C o . , U S A ) o n p o t a t o e x t r a c t m e d i u m a s r e p o r t e d

prev ious ly .6 Wh en the a my lo ly t i c ac t iv i ty reached 1 .20

u n i t sm 1 - 1 ( o n e u n i t is eq u i v a le n t to o n e m i c r o m o l e o f

D glucose re l eased f rom Ana la r s t a rch pe r minu te ) , 2 .3 l i tr e

*Approved by the D irector o f the Idaho Agricultural Experiment

Station as Research Paper no. 84 53.

of c u l tu re was rem oved , cen t r i fuged to rem ove ce ll s , passed

throu gh a Se i t z g rade C5 c l a r i fy ing f i l te r (Rep ubl i c F i l t e r

Corp . , U SA) , and f i l t e r s t e r il i zed th roug h a 0 .3 / am ce l lu lose

a c e t a t e m e m b r a n e f i l t e r ( G e l m a n I n s t r u m e n t C o . , U S A ) .

T h e c r u d e e n z y m e so lu t i o n w a s s t o r e d a t - 6 0 C i n 8 0 m l

por t ions . Before use , enzyme was p l aced ove rn igh t i n a

f r e e z e r a t - 5 0 C a n d t h e n t h a w e d a t 5 0 C .

Reduc ing suga r was measured us ing 3 ,5-d in i t rosa l i cy l i c

ac id (DNS) reagent .7 D-Glucose solut ions were used to

ca l ib ra t e reduc ing suga r measurement s . Enzyme subs t ra t e s

were Ana la r s t a rch (BDH Chemica l s L td , Poole , England)

conta in ing 22% am ylose and 78% min or pec t in , pur i f i ed

amylose (S igma Chemica l Co . , S t Loui s , USA, type I I I ) ,

p u r i f i e d p o t a t o a m y l o p e c t i n ( S i g m a ) a n d m a l t o se ( S i g m a ,

grade 1) . The subs t ra t e s were d i sso lved in 0 .02M sodium

ace ta t e buf fe r , pH 4 .8 , by bo i l ing and s t i r r ing un t i l c l ea r .

T h e se w e r e f i l t er e d t h r o u g h W h a t m a n n o . 4 f i l te r p a p e r

( W h a t m a n L t d , U S A ) a n d a u t o c l a v e d a t 1 2 1 ° C f o r 3 0 m i n

in cu l tu re bo t t l e s . A l l subs t ra t e s conta in ing amylose

(amy lose and Ana la r s t a rch) were used wi th in 2 h o f

prepa ra t ion to min imize re t rograda t ion .

C a r b o h y d r a t e a n a l y s i s w a s d o n e w i t h a B e c k m a n h i g h

p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( h . p . l . c . ) sy s t e m c o n -

s is ti ng o f a mode l l l 0 A pum p, mode l 210 in j ec t ion va lve

a n d m o d e l 1 5 6 r e f r a c ti v e i n d e x ( R I ) d e t e c t o r . A H e w l e t t -

Packa rd mode l 3390A in t egra tor and a Supe lcos i l LC-NH 2

a m i n o p r o p y l s i l y l c o l u m n ( S u p e l c o , I n c . , U S A ) w e r e u se d .

T h e m o b i l e p h a se c o n s i s t e d o f a c e t o n i t r i l e - w a t e r ( 7 0 : 3 0 ) .

The f low ra t e was 1 ml ra in - t a t amb ien t t em pera tur e .

D-Glucose , ma l tose , an d m a l to t r iose w ere used a s s t anda rds .

In i t i a l r a t e expe r iment s t o de t e rmine the k ine t i c pa ra -

m e t e r s f o r t h e S. f ibul iger a m y l a se c o m p l e x w e re p e r f o r m e d

i n a c c o r d a n c e w i t h t h e d e s i g n p r o c e d u r e o f C l e l a n d .a

Amylase ac t iv i ty was de t e rmined by re l ease of D-g lucose

f r o m A n a l a r s t a r ch a n d a m y l o p e c t i n a s m e a su r e d b y D N S

reagent .7 '9 For these expe r iment s , subs t ra t e so lu t ions

c o n t a in i n g 1 . 1 25 , 2 . 2 5 , 4 . 5 a n d 9 . 0 m g m l - I o f A n a l a r

0141

0229 / 8 4 / 1 20549 06

0 3 . 0 0

© 1984 B u t te r wo rth & C o . (P u bl ish ers ) L td E n z ym e M i cr o b . T ec h n o l . , 198 4 , vo l . 6 , D ec em b er 5 49



Papers

s t a r c h o r a m y l o p e c t i n d i s s o l v e d i n 0 . 0 2 M s o d i u m a c e t a t e

b u f f e r , p H 4 . 8 , w e r e p r e p a r e d . A d d i t i o n a l t e s t s u b s t r a t e s

w e r e p r e p a r e d b y d i ss o lv i n g 2 . 0 a n d 4 . 0 m g D - g lu c o se

m 1- 1 in the above s ta r ch so lu t ions . The in i t i a l r a te expe r i -

m e n t s w e r e p e r f o r m e d b y a d d in g 0 .5 m l c r u d e e n z y m e

( 0 . 6 u n i t s ) t o 0 . 5 m l t e s t s u b s t r a t e a n d i n c u b a t i n g t h e

r e a c t i o n m i x t u r e f o r 5 . 0 m i n a t 3 2 C . A f t e r t h e 5 r a i n

i n c u b a t io n , e n z y m a t i c a c t iv i t y w a s s t o p p e d b y a d d i n g

1 . 0 m l D N S r e a g e n t t o t h e r e a c t i o n t u b e s . W e i g h t g a i n

b y w a t e r a d d i t i o n d u r i n g s t a r c h h y d r o l y s i s w a s i n c l u d e d

in r a te ca lcu la t ions . T im e cour se r eac t ions wer e ca r r i ed

out a t 32°C in s te r il i zed 100 m l f l a sks wi th in i t ia l enzy m e

c o n c e n t r a t i o n s o f 0 . 6 2 u n i ts m l - ~. T h e i n i ti a l s t a r c h c o n -

c e n t r a t i o n s w e r e d e t e r m i n e d b y a c id h y d r o l y s i s ) °

e s u l t s a n d d i s c u s s i o n

a Am ylase activity

S i n ce t h e a m y l o l y t i c sy s t e m p r o d u c e d b y S . f i b u l i g e r

c o n t a i n s b o t h a - a m y l a s e a n d g l u c o a m y l a s e , i n i ti a l r a t e

e x p e r i m e n t s t h a t e s t i m a t e t h e k i n e t i c p a r a m e t e r s f o r e a c h

e n z y m e w e r e d e s ig n e d w h e r e t h e r e a c t i o n r a t e i s g i v e n b y :

d P v m S

- - 1 )

d t S + K i n ( 1 + P / K i )

w h e r e : P = p r o d u c t ( r e d u c i n g s u g ar o r D - g l uc o s e, m g m l - 1 ) ,

vm = m a x i m u m r a te ( m g p r o d u c t m 1 - 1 m i n - 1 ) , S = s t a rc h

c o n c e n tr a ti o n ( m g m l - ~ ) , K m = M i c h a e l i s c o n s ta n t ( m g

m l - ~ ) a n d K i = i n h i b i t o r c o n s t a n t ( m g m l - 1 ) .

a - A m y l a s e i s k n o w n t o h y d r o l y s e t h e l i n ea r p o r t i o n s o f

s u b s t r a te c h a i n s a t r a n d o m w i t h t h e f o r m a t i o n o f p o l y -

s a c c h a r i d e s w i t h v a r y i n g d e g r e e s o f p o l y m e r i z a t i o n .7 T h i s

a c t i o n i n c re a s e s t h e n u m b e r o f a v a il a b le r e d u c in g e n d s ,

and the D NS p r oce dur e i s we l l su i t ed f o r in i t ia l k ine t ic

r a t e s t u d ie s f o r a - a m y l a s e , w A m y l a s e p r o d u c e s D - g lu co se

o n l y w h e n h y d r o l y s i n g s h o r t c h a i n o l i g o m e r s an d h a s t h e

h ighes t a f f in i ty f o r long cha ins . 7 ' 11 Ther e f or e , D- g lucose

p r o d u c t i o n b y a - a m y l a s e o n l y o c c u r s l a t e i n t h e h y d r o l y s i s

w h e n s m a l l o l i g o m e r s d o m i n a t e . P r e l i m i n a r y e x p e r i m e n t s

o n t h e h y d r o l y s i s o f s t a r c h b y t h e e n z y m e s p r o d u c e d b y

S . f i b u l i g e r showed a l inea r inc r ease in r educ ing power f o r

a p e r i o d o f ~ 1 0 m i n o n A n a l ar s t a rc h c o n c e n t r a t io n s u p

to 25 m g m l - ~. A lso , in the f i r s t 5 m in , l e s s tha n 0 . 5 m g

D- glucose m l - ~ was p r odu ced . S ince D- g lucose is the o n ly

p r o d u c t o f s t a r c h h y d r o l y s i s b y g l u c o a m y l a s e , t h e c h a n g e

i n t h e r e d u c i n g p o w e r o f t h e s u b s t r a t e i n t h e f ir s t 5 m i n o f

h y d r o l y s i s i s a l m o s t e n t i re l y d u e t o t h e a c t i o n o f a - a m y l a s e .

E s t i m a t e s o f K i n , vm a n d K i w e r e o b t a i n e d b y n o n - l i n e a r

" u

/

/ ®

. /

/

, , i J t i i , I I

- o ~ a - o . a - o . 4 - 0 . 2 o

o 2 0 4

o . a o . a i , o

I / S

F i g u r e 1 D o u b l e r e c i p ro c a l p l o t o f

S f i b u l i g e r

a m y l a s e i n i t i a l r a t e

d a t a f o r : o , A n a l a r s t a r c h ; a n d o , A n a l a r s t a r ch s u p p l e m e n t e d w i t h

2 . 0 g I - ~ a n d z x, 4 . 0 g I - ~ D - g l u c o s e . ( C i r c l e d p o i n t s w e r e n o t i n c l u d e d

i n d a t a a n a l y s i s )

6

I

4

2

- 0 . 4 - 0 . 2 0

/

/

/ ®

/

/

J

s~j

. / /

I ®

I Y

I I I

o l a 0 : 4 o , e o l e I o

I / S

F i g u r e 2 D o u b l e r e c i p r o c a l p l o t o f

S f i b u / i g e r

a m y l a s e i n i t i a l r a t e

d a t a f o r : o , a m y l o p e c t i n ; a n d D , a m y l o p e c t i n s u p p l e m e n t e d w i t h

2 . 0 g I - = a n d A , 4 . 0 g I - ~ D - g l u c o s e . ( C i r c l e d p o i n t s w e r e n o t i n c l u d e d

i n d a t a a n a l y s i s )

T a b l e 1 K i n e t i c p a r a m e t e r s o f a - a m y l a s e f r o m

S f i b u l i g e r

l ) m

( m g r e d u c i n g K i

K m s u g a r ( r a g D - g l u c o s e

S u b s t r a t e ( r a g m 1 - 1 ) m 1 - 1 m i n - 1 ) m 1 - 1 )

A n a l a r s t a r c h 1 . 3 7 ± 0 . 3 9 a 1 . 2 0 + 0 . 1 0 a 1 . 8 9 + - 0 . 5 2 a

A m y l o s e 2 . 4 2 ± 0 . 4 9 a 1 . 4 0 ± 0 . 3 0 a -

A m y l o p e c t i n 2 . 5 8 -+ 0 . 6 4 0 . 9 9 + 0 . 1 0 a 1 . 7 8 -+ 0 . 2 8 a

a 9 5 c o n f i d en c e interval

T a b l e 2 K i n e t i c p a r a m e t e r s o f g l u c o a m y l a s e from

S. fibu/iger

V m

K m r a g D - g l u c o s e

S u b s t r a t e ( r a g m l - ] ) m 1 - 1 r n i n - t )

A n a l a r s t a rc h 0 . 3 4 ± 0 . 1 1 a 0 . 0 1 9 + 0 . 0 0 1 a

A m y l o s e 0 . 4 9 ± 0 . 2 0 a 0 . 0 1 6 ± 0 . 0 0 2 a

A m y l o p e c t i n 0 . 3 6 ± 0 . 1 8 a 0 . 0 1 6 ± 0 . 0 0 2 a

M a l t o s e 0 . 8 0 -+ 0 . 1 6 a 0 . 0 1 7 ± 0 . 0 0 1 a

a 9 5 c o n f i d e n c e i n t e rv a l

r egr es s ion of in i t i a l r a te da ta

F i g u r e s 1

a n d 2 ) a n d a r e

s h o w n i n T a b l e 1 . D a t a p o i n t s f o r S = 1 .1 m g m 1 - 1 w e r e

n o t i n c l u d e d i n p a r a m e t e r e s t i m a t i o n s f o r K i , s o t h e K i

e s t i m a t e s a r e o n l y v a l i d f o r S ~ 2 m g m 1 -1 .

Glucoamylase

P r e l i m i n a r y e x p e r i m e n t s s t u d y i n g D - g lu c o se p r o d u c t i o n

w i t h t i m e a t i n it i al s u b s t r a t e c o n c e n t r a t i o n s o f 2 . 2 5 m g m1 -1

i n d i c a t e d a l i n e a r i n c r ea s e i n D -g l u co s e f o r 1 2 0 r a i n b e f o r e

t h e r a t e s t a r t e d t o d e c l in e . S i n c e g l u c o a m y l a s e p r o d u c e s

D- g lucose a s i t s so le p r oduc t in s ta r ch hydr o lys i s and

a - a m y l a s e o n l y p r o d u c e s D - g lu c o se l a t e i n t h e r e a c t i o n ,

t h i s p r o d u c t i o n i s a l m o s t e n t i r e l y d u e t o g l u c o a m y l a s e .

As a r e su l t , in i ti a l r a te s tud ies wher e D- g lucose p r o du c t io n

r a t e w a s m e a s u r e d c a n b e u s e d t o e s t i m a t e K m a n d u m f o r

g l u c o a m y l a s e T a b l e 2 ) .

T h e v a l u e e s t i m a t e d f o r K m o n t h e s u b s t r a t e m a l t o s e

i n d i c a te s t h a t t h e a f f i n i t y o f g l u c o a m y l a s e f o r t h i s s u b s t r a te

i s l e s s than f o r the s ta r ch subs t r a te s . These and o the r

r e su l t s 12 '13 ind ica te a d ec r eased a f f in i ty o f g luco am y lase

f o r t h e s h o r t e r o l i g o m e r s . O u r d e t e r m i n a t i o n s o f K m f o r

s t a r c h s u b s t r a te s a r e i n c lo s e a g r e e m e n t w i t h d a t a o n

5 5 0 E n z y m e M i c r o b . T e c h n o l . , 1 9 8 4 , v o l , 6 , D e c e m b e r



6()0 860 10100 12100 14'00

T i m e ( M i n u t e s )

2 . 1 -

1 , 8 -

1 . 5 -

1.2-

o.g-

o.5-

0.3-

o.o-

o 2 6 0 4 ~ o

200 4( )0

600 800 10 '0 0 12w00 1400


m

0

m

0.7

0.6.

0,5 '

0.4.

0.3.

0.2.

0.1,

0.0.

0

Am ylase system k/net ics: E M Steverson et a l

E

CIJ

E

c

:o

@

¢ 4

O

0

_=

9

¢ P

e.

0

0 .7 -

0.6

0 ,5 -

0.4-

CO

(1. 0.3-

0.2-

i

0.1-

0.0-

0

0,25 -

0.20 -

0.15,

14")

a .

0 O . l O

0 . 0 5

0.00,

2 0 0 4 ~ o e o o 8 0 0 i o o o 1 2 o o 1 4 o o


0 2( )0 4; 0 5( )0 800 10 '00 12 '00 14 '00


a .

C)

0,25-

0.20-

¢O

O .

O

0.15-

o -ii

0 O0 ~ - - -- : •

i . - i i

0 200 460 600 800 1000


1 2 o o 1 4 o o

0.125,

0.100.

0.075.

0.050.

0.025,

0.00.

0

2 6 0 4 ~ 0 5 6 0 s 6 0 1 0 0 0 I i 0 0 1 4 0 0

T l me M l n u tea )

2.5-

2,0-

¢)

_=

¢0 1.5-

O~

._c

O 1,0-

1=

a)

n- o.5-

0 ,0 .

0 200 4 ( )0 c oo 8c 10 10 '00 12 '0o 14 '00


F i g u r e 3 C o n c e n t r a t i o n s o f o l i g o m e r s a n d r e d u c i n g s u g ar d u r in g t h e h y d r o l y s i s o f a m y l o s e w i t h a n in i t i a l c o n c e n t r a t i o n o f 2 . 5 m g m l - j b y

t h e S . f i b u l i g e r a m y l a s e s y s t e m a t p H 4 . 8 a n d 3 2 ° C

E n z y m e M i c r o b . T e c h n o l . , 19 8 4 , v o h 6 , D e c e m b e r 5 5 1



a p e r s

0,) 3'

0

0

:~ 2

0 2 ~ 0 4 ~ 0 3 ~ 0 1 ; 0 0 1 = 0 0 1 4 .0 0

T i m e M i n u t e s )

1.0-

o .8 -

( I) 0.6-

0

. . ,

0 . 4 -

0.2 -

0 .0 -

0

2 ~ o 4 6 o 5 ~ o 8 ~ o l O O O

1 = o o 1 4 o o


E

E

v

C

0

o

0

U

c

0

(,,3

0,8 ¸

0 . 8

0.7

0 . 6

03 0 .5 -

O.

0 .4 -

0.3 -

0 .2 -

0 .1 -

0 .0 .

0.21

0 . 1 8

0 , 1 5

0 . 1 2

u3

a .

o.oo

0.05

0.03

0 .00

2 ~ 0 4 ~ 0 8 ~ 0 8 ~ 0 1 0 . 0 0

1 = 0 0 1 4 0 0


200 4 ( )0 5 ( )0 800 10 '0 0 12 '0 0 14 '00

Time Minutes)

2 ~ o 4 ~ o 5 ; o 8 ; 0 lO O O 1 2 o o 1 4 o o


0 .4

0 . 3 -

(1. 0,2-

£3

0 . 1 -

0.0 -

; 2 ;0 4 ;0 8 ;0 8 0 0 1 0 00 1 = ' 0 0 1 2 00


0 .125

0 .100-

(D 0.075

a .

£3

0.050-

0 .025-

0 .0 0 0 .

a .

C~

0.125

0 . 1 0 0

0.075

0.050

0 .0 2 5

0 .0 0 0

5 ¸

m 4

0~

>

O) 3.

(3

0 2,

n -

1

2 ~ 0 4 ~ 0 8 6 0 8 ~ 0 1 0 0 0 1 2 0 0 1 4 0 0 0 2 ~ 0 , ,, ,~ 0 5 ~ 0 e ~ o 1 0 ~ 0 l = o o 1 4 ~ 0

T i m e ( M i n u t e s ) T i m e ( M i n u t e e )

F i g u r e 4 C o n c e n t r a t i o n s o f o l i g o m e r s a n d r e d u c i n g s u g a r d u r i n g t h e h y d r o l y s i s o f a m y l o p e c t i n w i t h a n in i t ia l c o n c e n t r a t i o n o f 5 . 9 m g m l -~ b y

t h e S . f i u l i g e r a m y l a s e s y s te m a t p H 4 . 8 a n d 3 2 ° C

5 5 2 E n z y m e M i c r o b . T e c h n o l . , 1 9 8 4 , v o l . 6 , D e c e m b e r



su b s t r a t e s w i t h d e g r e e s o f g l u c o se p o l y m e r i z a t i o n f r o m 3

to 10 .12 The va lues of Km repo r t ed for so lub le s t a rch a re

cons ide rab ly low er , a Thi s i nd ica t e s t h a t t he subs t ra t e fo r

g lucoamylase ac t iv i ty in our expe r iment s was shor t cha in

o l ig o m e r s w i t h d e g r e e o f p o l y m e r i z a t i o n ( D P ) < 1 0 , r a t h e r

than long cha in s t a rch molecu les . Our s tud ies were on an

e n z y m e c o m p l e x c o n t a i n i n g b o t h a - a m y l a se a n d g l u c o -

amylase , whereas o the r re su l ts 12 '13 re f l ec t t he ac t ion of

pur i f i ed g lucoamylase . The ve ry rap id and ea r ly ac t ion of

a - a m y l ase r e d u c e d t h e l o n g c h a in s o f a m y l a se a n d a m y l o -

pec t in in to much shor t e r o l igomers . These shor t o l igomers

w e r e d o m i n a n t e a r ly i n t h e h y d r o l y s i s an d w e r e t h e p r i m a r y

subs t ra t e s fo r g lucoamylase . T he K m va lues repor t ed he re

for s t a rch subs t ra t e s a re mo re ind ica tive of t he a f f in i ty o f

g lucoamylase for sho r t cha in o l igomers .

The ~ 'rn va lues e s t ima ted on the va r ious subs t ra t e s

ind ica t e tha t t he ra t e o f D-g lucose prod uc t io n is i ndepen-

den t o f t he subs t ra t e . Thi s cont ra s t s w i th prev ious re su l ts

which showed a pos i t i ve cor re l a t ion be tween reac t ion ra t e

a n d t h e d e g r ee o f p o l y m e r i z a t i o n o f t h e su b s t r a te ,a'12-14

T h e l a ck o f a g r e e m e n t p r o b a b l y r e f le c t s t h e d i f f e r e n c e

b e t w e e n h y d r o l y s i s b y p u r i f i e d g l u c o a m y l a se a n d h y d r o -

l y si s b y a n a m y l o l y t i c c o m p l e x c o n t a i n i n g b o t h g l u c o am y l a se

and a -amylase .

T i m e c o u r s e r e a c ti o n s

Analyses of t ime course samples us ing h .p . l . c , p rov ided

p r o d u c t d i s t r i b u t i o n p r o f i l e s o f s t a r c h h y d r o l y sa t e s

versus

t ime . The hydro lys i s o f so lub le s t a rch amylose and amylo-

p e c t i n b y t h e a m y l o l y t i c sy s t e m o f

S. f ibuliger

was s tud ied

b y f o l lo w i n g t h e r e l a ti v e a m o u n t s o f t h e o l ig o m e r s d e t e c t e d

( D P 1 - 8 ) a n d t h e r e d u c in g p o w e r f o r 2 4 h .

A p lo t o f reduc ing va lue and re l a tive am oun t s o f D-g lucose

and o l igomers

versus

t i m e f o r t h e h y d r o l y s i s o f a m y l a se i s

sh o w n i n

Figure 3.

A f t e r 2 4 h , 7 6 % o f t h e t o t a l su b s t r a te

had been hydro lysed to D-g lucose , a l lowing for t he we ight

g a in d u r i n g h y d r o l y s i s b y t h e a d d i t i o n o f w a t e r . T h e r e d u c -

ing va lue a f t e r 24 h was 88% of the to t a l r edu c ing va lue

( o n a c id h y d r o l y s e d su b s t ra t e ), i n d i ca t in g t h a t - 8 8 % o f

the av ai lable a-( 1,4)-gluco sidic linkages had b een h yd roly sed .

A p l o t o f r e d u c i n g v a l u e s a n d t h e r e l a t i v e a m o u n t s o f

D-glucose and ol igomers versus t i m e f o r t h e h y d r o l y s i s o f

a m y l o p e c t i n i s sh o w n i n

Figure 4.

T h e c o n c e n t r a t i o n s o f

D P 4 , D P 5 , D P 6 a n d D P 7 w e n t t h r o u g h a n e a r l y m a x i -

m u m , d e c l i n e d a n d t h e n i n c re a se d. T h e c o n c e n t r a t i o n s o f

DP 6 and DP 7 dec reased to sma ll am oun t s a f t e r 24 h , bu t

t h e c o n c e n t r a t i o n s o f D P 4 a n d D P 5 r e m a i n e d h ig h .

A p p r o x i m a t e l y 7 0 % o f t h e t o t a l su b s t r a te h a d b e e n h y d r o -

lysed to D-g lucose a f t e r 2 4 h of hydro lys i s . The reduc ing

va lue a t 24 h in to the rea c t ion ind ica t ed th a t 83% of the

a - (1 ,4) l i nkages ava il ab le had been h ydro lysed .

The produc t d i s t r ibu t ions of t he th ree d i f fe ren t sub-

s t ra t e s a l l showed the rap id form a t ion o f smal l cha in

o l igomers ea r ly in the hydro lys i s which i s cha rac t e r i s t i c

o f t h e a c t i o n o f a - a m y l a se . T h e se o l ig o m e r s b e c a m e t h e

subs t ra t e fo r t he g lucoamylase enzym e presen t i n the

a m y l o l y t i c sy s t e m . L a t e r i n t h e r e a c t i o n t h e d o m i n a n t

o l i g o m e r s w e r e m a l t o se a n d m a l t o t r i o se , w h i c h w e r e

h y d r o l y se d s l o w ly d u e t o t h e l o w e r a f f i n i ty o f g l u c o a m y l a se

for t hese shor t o l igomers , a -Amylase , when work ing on

branched subs t ra t e s , f i r s t hydro lysed the pe r iphe ra l l i nea r

cha ins and then the l i nea r sec t ions be tween branch po in t s .

T h e p r o d u c t s o f t h e h y d r o l y s i s b e t w e e n b r a n c h p o i n t s

were shor t o l igomers w i th an a - ( l ,6 ) l i nkage a t t he non-

reduc ing end ( see

Figure 5).

Since g lucoamylase was an exo-

e n z y m e t h a t w o r k e d o n l y f r o m t h e n o n - r e d u c i n g e n d , t h e

A m y l a s e s y s t e m k i n e t i c s : E 114 S t e v e r s o n e t a l

Par t i a l l y degrade d • _ -~ = - - - - e e - - = - - _ - = -=

branched substrate

a - A m y l a s e h y d r o ly s e s ( I - 4 ) li n k a g e • . - ¢ e e +

of chain between bronchpoints - ¢ e = _ _ _ e -=

l g l u c ~ m y l s e

e

Ra te l im i t i ng de x t r i n s ~ ~ . ~ . - - + ~=- = . - - ÷ 14o -

• Glucoseunit

- - ( I - 4 ) Linkage

( I - 6 ) Linkage

e Reducing end

F i g u r e 5 S c h e m a t i c r e p r e s e n t a t i o n o f t h e f o r m a t i o n o f a r a te

l i m i t i n g s u b s t r a te d u r i n g t h e h y d r o l y s i s o f a m y l o p e c t i n

T a b l e 3 K i n e t i c p a r a m e t e r s f o r D g l u c o s e p r o d u c t i o n

k o

k o pb ( r a g m l i

S u b s t r a t e ( m i n - 1) ( m g / m l ) ( m g m l - l ) m i n - 1)

A n a l a r 0 . 0 0 1 9 - + 0 . 0 0 0 1 a 1 0 9 . 2 0 . 0 1 9 0

s t a r c h

A m y l a s e 0 . 0 0 9 8 ~ 0 . 0 0 0 6 a 2 . 5 2 .1 0 . 0 2 4 5

A m y l o - 0 . 0 0 3 1 + 0 . 0 0 0 2 a 5 . 9 4 . 5 0 . 0 1 8 3

p e c t i n

a 9 5 % c o n f i d e n c e i n t e rv a l

b D - G l u c o s e c o n c e n t r a t i o n a f t e r 2 4 h

a - ( l , 4 ) l i n k a g e s i n t h e se p r o d u c t s w e r e n o t i m m e d i a t e l y

avai lab le for hydro lys i s . The ra t e o f hyd ro lys i s o f a - ( l ,6 )

l inkages b y g lucoamylase was ve ry low re l a t ive to th e ra t e

on a - ( l ,4 ) l i nkages a so these p rodu c t s were ra t e limi t ing

subs t ra t e s , and th ey remained in the hydro lysa t e un t i l t he i r

a - ( l ,6 ) l i nkage was hy dro ly sed . Seve ra l work e rs have ind i-

ca t ed the presence of limi t dex t r ins when

S. f ibuliger

a m y l a se s w e r e u se d f o r s t a r ch h y d r o l y s i s in c o n t i n u o u s

processes , ' 6 ' l s The DP 4 , DP 5 and DP 6 o l igomers f rom

t h e h y d r o l y s i s o f a m y l o p e c t i n c o r r e sp o n d e d t o t h e se r a t e

l imi t ing dextr ins.

G lucot ransfe rase ac t iv i ty was obse rved dur ing the hyd ro-

lys i s o f ma l tose . Wi th an in i t i a l ma l tose concent ra t ion of

2 0 m g m l - ~, m a l t o t r i o se ( D P 3 ) c o n c e n t r a t i o n r e a c h e d

1 . 0 m g m 1 - 1 w h e n m a l t o se a n d g lu c o se c o n c e n t r a t i o n s

w e r e a p p r o x i m a t e l y 9 . 5 m g m 1 -1 . T h e m a l t o t r i o se c o n c e n -

t ra t ion then dec l ined s lowly wi th t ime .

T i m e c o u r s e m o d e l l i n g o l D - g l u c o s e p r o d u c t i o n

Time ~ourse d a t a fo r D-g lucose produ c t ion

Figures 3

and 4) were f i t t ed to equa t ion (1) and to the f i r s t -o rde r

k ine t i c ra t e express ion

d P /d t = k S

(2)

us ing non- l inea r regress ion of t he in t egra t ed equa t ions for

pa ramete r e s t ima t ions . The in i t i a l r a t e expe r iment s on the

f o u r su b s t r a te s t e s t e d sh o w e d t h a t t h e p r o d u c t i o n o f

D-glucose f rom s t a rch by the am ylo ly t i c sys t em of

S. f ibuliger

w a s w e l l d e sc r i b e d b y M i c h a e l i s - M e n t e n

kine t i c s . However , equa t ion (1) d id no t g ive a good f i t

w i t h t i m e c o u r se d a t a f o r a t h r e e p a r a m e t e r m o d e l . T h e

D-glucose da t a were be t t e r mo de l l ed by e qua t io n (2) .

D u r i n g t h e t i m e c o u r se e x p e r i m e n t s , e n z y m e a c t i v i t y

w a s m e a su r e d a s a f u n c t i o n o f t i m e o n e n z y m e su b j e c te d

to s imi l a r condi t ions in a cont ro l r eac tor (enzyme wi thout

subs t ra t e ). Th e ac t iv i ty o f t he enz ym e dec reased wi th a

f i r s t -o rde r deca y cons t an t o f 2 .9 × 10 -4 min -1 . Thi s ac t iv i ty

E n z y m e M i c r o b . T e c h n o l . 1 9 8 4 v o l . 6 D e c e m b e r 5 5 3



apers

c h a n g e w a s n o t i n c l u d e d i n d e t e r m i n a t i o n s o f t h e f i r s t-

o r d e r r a t e c o n s t a n t s , k . V a l u e s d e t e r m i n e d f o r k a r e s h o w n

in Table 3. T h e i n i t i al r a t e s o f s t a r c h h y d r o l y s i s , kS o,

a r e s i m i l ar t o t h e m a x i m a l r a t e s, v m , d e t e r m i n e d f r o m

i n i t i a l r a t e d a t a Table 2).

onc lu s i on s

T h e h y d r o l y s i s o f st a r c h b y t h e e x t r a c e l l u l a r e n z y m e s

p r o d u c e d b y S. fibuliger f o l l o w e d M i c h a e l i s - M e n t e n

k i n e t i c s w i t h o - g l u c o s e i n h i b i t i o n , a n d h . p . l . c , w a s a n

e x c e l l e n t a n a l y t i c a l te c h n i q u e f o r s t u d y i n g t h e s e r e a c t i o n s .

T h e a c t i o n o f a - a m y l a se w a s v e r y r a p i d a nd d o m i n a n t i n

t h e e a r l y s t a g e s o f h y d r o l y s i s , p r o d u c i n g s h o r t o l i g o m e r s

w h i c h b e c a m e t h e s u b s t r a t e f o r g h c o a m y l a s e . R a t e li m i t -

i n g d e x t r in s w e r e p r o d u c e d w h e n b r a n c h e d s u b s t ra t e s

w e r e h y d r o l y s e d . T h e s e d e x t r i n s w e r e h y d r o l y s e d v e r y

s l o w l y p r o b a b l y d u e t o t h e p r e s e n c e o f a n o r - 1 ,6 ) l i n k a g e

o n t h e i r n o n - r e d u c i n g e n d .

cknowledgements

T h i s m a t e r i a l i s b a s e d u p o n w o r k s u p p o r t e d b y t h e

N a t i o n a l S c i e n c e F o u n d a t i o n u n d e r G r a n t n o . C P E - 8 1 0 -

8 1 0 4 7 0 9 .

Re f e r enc e s

1 Wickerham, L . J . Lockwood, L . B. , Pe t t i john , O. G. and

Ward , G. E . Z Bacteriol. 1 94 4 , 4 8 , 4 1 3 -4 2 7

2 Ueda, S. and Saha, B. C. Enzyme Microb. Technol. 1983, 5,

1 9 6 - 1 9 8

3 Sukhumavasi, J . , Ka to, K. and Harada, T. J .

Ferment. Technol.

1 9 7 5 , 5 3 , 5 5 9 - 5 6 5

4 Adm assu, W., Koru s, R. A. and Heimsch, R. C.

Biotechnol.

Bioeng. 1983 , 25 , 2641-2651

5 Dostfilik, M. and H ~iggstr/Sm, M. H. Eur. J. Appl. Microbiol.

Biotechnol. 1 9 8 3 , 1 7 , 2 6 9 -2 7 4

6 Adm assu, W., Koru s, R. A., Heimsch, R. C. and Lemm el, S. A.

Biotechnol. Bioeng.

1981 , 23 , 2361-2371

7 Bernfield , P. Adv. Enzymol. 1 9 5 1 , 1 2 , 3 7 9 - 4 2 7

8 Cle land , W. W.Adv.

Enzymol.

1967 , 29 , 1 -32

9 Beta Amylase, Worth ing ton Enzyme Manua l , Worth ing ton

Biochemica ls , F reeh o ld , NJ , 1977, pp . 177 -17 8

10 Pitt , S. J . and Whelan, W. J. J. Sci. Food Agric. 1951, 2,

2 2 4 - 2 2 8

11 Henriksn~s, H. and LOvgren, T.

Biotechnol. Bioeng.

1978, 20,

1 3 0 3 - 1 3 0 7

12 Lee, D. D., Lee, G. K., ReiUy, P. J . and Lee, Y. Y.

Biotechnol.

Bioeng. 1980 , 22 , 1 -17

13 Kus unoki, K. , Kawak ami, K., Shiraishi, F. , Ka to, K. and Kai,

M. Biotechnol. Bioeng. 1982 , 24 , 347-354

14 Ono , S. , Hirom i, K. and Zinbo , M. J . Biochem. 1964, 55,

3 1 5 - 3 2 0

15 Lem mel, S. A., Heimsch, R. C. and Korus, R. A. Appl. Environ.

Microbiol.

1980 , 39 , 387-393

5 5 4 E n z y m e M i c r o b . T e c h n o l . 1 9 8 4 v o l . 6 D e c e m b e r

1984_(steverson) kinetics of the amylase system of saccharomycopsis fibuliger

Documents