Properties of CGTases from Three Types of Bacillus and Production of

Chapter 10

Downloaded by STANFORD UNIV GREEN LIBR on August 1, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch010

Properties of CGTases from Three Types of Bacillus and Production of Cyclodextrins by the Enzymes Michikatsu Sato and Yoshiaki Yagi Central Research Laboratories, Sanraku Inc., 9-1, Johnan 4-Chome, Fujisawa 251, Japan Comparative studies of CGTases from Bacillus ohbensis, B. macerans and B. circulans and an enhanced production method of gamma-cyclodextrin (CD) are reported. Each CGTase was purified to a homogeneity. The CGTase from B. ohbensis was successfully crystallized. The enzyme from B. ohbensis had the smallest molecular weight (35,000) and had the lowest affinity to starch among the enzymes examined. The CGTase had similar enzymatic properties as the other enzymes, but i t exhibited high temperature and broad pH stability in the presence of substrates. CGTase from B. macerans had advantages for the production of alpha-CD but the reaction conditions had to be strictly controlled. CGTase from B. ohbensis can be used favorably for the production of beta- and gamma-CD. We elucidated an efficient gammaCD production system by addition of compounds with high affinity to the CD. Of the compounds tested, glycyrrhizic acid was the most effective for production of gamma-CD. But the addition had l i t t l e effect on gamma-CD production using CGTase from B. macerans and B. circulans.

C y c l o d e x t r i n (CD) f o r m i n g enzyme, c y c l o m a l t o d e x t r i n g l u c a n o t r a n s f e r ase (CGTase, a l p h a - 1 , 4 - g l u c a n 4 - g l y c o s y l t r a n s f e r a s e , EC.3.2.1.19) i s an enzyme which p r o d u c e s CDs from a l p h a - 1 , 4 - g l u c a n , such as m a l t o d e x t r i n , amylose, s t a r c h e s , e t c . T h e r e have been many r e p o r t s on CGTase p r o d u c i n g b a c t e r i a l strains (1~8). The enzymes a r e c l a s s i f i e d i n t o two groups a c c o r d i n g t o t h e i r major p r o d u c t ; one i s a group which m a i n l y produces alpha-CD, and t h e o t h e r i s one which m a i n l y p r o d u c e s beta-CD. P r i m a r y examples o f t h e alpha-CD p r o d u c i n g group and beta-CD group a r e t h e CGTase from B. macerans (1) and t h e enzyme from B. c i r c u l a n s (4) r e s p e c t i v e l y . We i s o l a t e d a s t r a i n p r o d u c i n g CGTase from a s o i l sample i n Ohba d i s t r i c t i n F u j i s a w a c i t y , Japan (5) and found t h a t t h e enzyme formed a l a r g e

0097-6156/91/0458-0125$06.00/0 © 1991 American Chemical Society

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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amount o f beta-CD and d i d n o t produce alpha-CD under c o n v e n t i o n a l r e a c t i o n c o n d i t i o n s f o r t h e enzyme. T h e r e f o r e , we examined t h e p r o p e r t i e s o f t h e enzyme and compared them w i t h t h o s e o f o t h e r CGTases o f B a c i l l u s o r i g i n . CD p r o d u c i n g s t r a i n s , B. macerans I AM 1227 and B. c i r c u l a n s IFO 3329 from o u r l a b o r a t o r y ' s c u l t u r e c o l l e c t i o n , were s t u d i e d and compared t o t h e CGTases from B. o h b e n s i s , B. macerans and B. c i r c u l a n s ( 9 ) . R e c e n t l y , we d e v i s e d a new gammaCD p r o d u c t i o n system by a d d i n g a w e l l f i t t e d g u e s t compound f o r gamma-CD i n t h e r e a c t i o n m i x t u r e s ( 1 0 ) . We r e p o r t h e r e c o m p a r a t i v e s t u d i e s o f t h e CGTases and a new method f o r enhanced p r o d u c t i o n o f gamma-CD by t h e enzyme from B. o h b e n s i s . P u r i f i c a t i o n and P r o p e r t i e s o f 5 K i n d s o f CGTases CGTase from B. macerans i s known as a C D - p r o d u c i n g enzyme (_1) and F r e n c h e t a l . (11) r e p o r t e d d e t a i l e d r e s e a r c h on t h e enzyme i n 1957. The CGTase p r e d o m i n a n t l y produces alpha-CD and t h e r e was no r e p o r t on o t h e r t y p e s o f CGTases u n t i l 1972. Okada e t a l . ( 3 ) f i r s t r e p o r t e d t h e CGTase o f B. megaterium, a beta-CD t y p e CGTase. Since t h e n many CGTases o f B a c i l l u s o r i g i n have been r e p o r t e d , and we a l s o were a b l e t o i s o l a t e a beta-CD t y p e CGTase p r o d u c i n g b a c t e r i u m (5). S i n c e t h e enzyme d i d n o t form alpha-CD under normal enzyme r e a c t i o n c o n d i t i o n s , we examined t h e p r o p e r t i e s o f t h e enzyme and compared them with the other CGTases from d i f f e r e n t Bacillus strains. P u r i f i c a t i o n o f CGTases from B. o h b e n s i s The CGTase from B. o h b e n s i s was p u r i f i e d t o homogeneity {12} by a c e t o n e p r e c i p i t a t i o n , s t a r c h a d s o r p t i o n (125) w i t h ammonium s u l f a t e , D E A E - c e l l u l o s e column chromatography and c r y s t a l l i z a t i o n (14). The s p e c i f i c activity was e l e v a t e d 15-fold from the acetone precipitation and t h e h e x a g o n a l c r y s t a l w i t h a p r o t r u d i n g c e n t e r was formed

· P u r i f i c a t i o n o f CGTase from B. macerans B. macerans I AM 1227 was c u l t u r e d and t h e s u p e r n a t a n t o f t h e c u l t u r e d b r o t h was a d s o r b e d o n t o c o r n s t a r c h and a c t i v e f r a c t i o n s were r e c o v e r e d by e l u t i n g w i t h warm water (50°C). The s t a r c h a d s o r p t i o n was e f f e c t i v e f o r d e c o l o r i n g and i n c r e a s i n g t h e s p e c i f i c a c t i v i t y o f t h e CGTase. The a c t i v e f r a c t i o n s were combined, s u b j e c t e d t o t w i c e r e p e a t e d DEAE-cellulose column chromatography and Sephadex G-100 column chromatography. The a c t i v i t y was i n c r e a s e d 150-fold from t h e c u l t u r e b r o t h and t h e enzyme was p u r i f i e d as an almost single protein (9). P u r i f i c a t i o n o f CGTase from B. c i r c u l a n s B. c i r c u l a n s IFO 3329 was c u l t u r e d and t h e CGTase was p u r i f i e d by s t a r c h adsorption. The e l u a t e from t h e s t a r c h was c o n c e n t r a t e d by u l t r a f i l t r a t i o n . The enzyme was f u r t h e r p u r i f i e d by ammonium s u l f a t e p r e c i p i t a t i o n , DEAE-column chromatography, Sephadex G-100 column chromatography and g e l i s o e l e c t r i c f o c u s i n g . In the i s o e l e c t r i c f o c u s i n g e l e c t r o p h o r e s i s , 2 a c t i v e bands appeared. The major band's i s o e l e c t r i c p o i n t ( I p ) was 8.8 and t h a t o f t h e o t h e r band was 8.5. The enzyme o f t h e major band was used f o r f u r t h e r e x p e r i m e n t s . The Ip 8.8 enzyme was p u r i f i e d 2 , 2 4 0 - f o l d from t h e c r u d e enzyme p r e p a r a t i o n (9).


10. SATO & YAGI Comparison

Properties of CGTasesfromThree Types of Bacillus

o f P r o p e r t i e s o f t h e CGTases

E n z y m a t i c p r o p e r t i e s o f t h e r e s p e c t i v e CGTases such as optimum pH, pH s t a b i l i t y , optimum t e m p e r a t u r e , t h e r m a l s t a b i l i t y , b e h a v i o r on s t a r c h a d s o r p t i o n , m o l e c u l a r weight by SDS-PAGE and i s o e l e c t r i c p o i n t were compared. The r e s u l t o f t h e s t a r c h a d s o r p t i o n i s shown i n Table I (9).


Table I.

(NH ) S0 cone. {% s a t u r a t i o n ) 4

2

4

0 10 20

A d s o r p t i o n o f CGTase on S t a r c h

B. o h b e n s i s adsorption(%) + 3.4 64 74

92 18 8.6

B. macerans adsorption(%) + 36 30 24

61 60 63

B. c i r c u l a n s adsorption(%) + 53 52 53

23 20 17

+: p e r c e n t adsorbed; -: p e r c e n t n o t a d s o r b e d .

The enzyme from B. o h b e n s i s had l e s s a f f i n i t y f o r s t a r c h than t h e o t h e r enzymes. The CGTase was o n l y adsorbed by a d d i n g ammonium s u l f a t e t o 20 % s a t u r a t i o n . The enzyme from B. macerans had a h i g h a f f i n i t y f o r s t a r c h , and i t d i d n o t need any a d d i t i o n o f ammonium s u l f a t e . The CGTase from B. c i r c u l a n s showed moderate a f f i n i t y and t h e r e was no dependence on ammonium s u l f a t e . The p r o p e r t i e s o f t h r e e enzymes a r e summarized i n T a b l e I I ( 9 ) .

Table I I .

P r o p e r t i e s o f CGTases

B. o h b e n s i s

Main p r o d u c t beta-CD Optimum pH 5.5 pH s t a b i l i t y 6.5-9.5 Optimum temp.(°C) 60 Thermal s t a b i l i t y ( °C)* 55 M o l e c u l a r weight 35,000 I s o e l e c t r i c point alpha-CD 6.0 6.0-9.5 55 55 (200,000) 8.5, 8.8

+++

++

*The temperature which showed the r e s i d u a l a c t i v i t y (>80 %) a f t e r exposure f o r 60 min a t each temperature a t pH 6.0.

E n z y m a t i c a c t i v i t i e s o f t h e enzymes were, s i m i l a r , b u t t h e m o l e c u l a r w e i g h t , i s o e l e c t r i c p o i n t and a f f i n i t y t o s t a r c h were d i f f e r e n t . Of g r e a t e s t n o t e i s t h e m o l e c u l a r weight; t h e s i z e o f t h e enzyme from B. o h b e n s i s i s t h e s m a l l e s t t h a t has e v e r been r e p o r t e d from


127


128

BIOTECHNOLOGY OF AMYLODEXTRIN

OLIGOSACCHARIDES

genus B a c i l l u s . The m o l e c u l a r weight was measured by SDS-PAGE and c o n f i r m e d by u l t r a - c e n t r i f u g a t i o n . We a l s o c a r r i e d o u t g e l p e r m e a t i o n column chromatography to elucidate further information on t h e m o l e c u l a r w e i g h t , b u t t h e v a l u e showed about 70,000~80,000. The r e a s o n i s n o t known, b u t i t may have been by a g g r e g a t i o n o f t h e enzyme m o l e c u l e s . Amino a c i d c o m p o s i t i o n s o f t h e CGTases from 3 k i n d s o f B a c i l l u s were examined ( 9 ) . None o f t h e t h r e e enzymes c o n t a i n e d c y s t e i n e , and t h e amounts o f n e u t r a l and b a s i c amino a c i d s were s i m i l a r . A r e l a t i v e l y l a r g e amount o f a c i d i c amino a c i d s was c o n t a i n e d i n t h e CGTase from B. o h b e n s i s , which may be r e l a t e d t o t h e a c i d i t y o f the enzyme. C y c l o d e x t r i n F o r m a t i o n by t h e CGTases We performed c o m p a r a t i v e s t u d i e s o f t h e 3 k i n d s o f enzymes on CD production. The s u b s t r a t e s p e c i f i c i t i e s , i n f l u e n c e o f i n c u b a t i o n t i m e , pH, temperature and q u a n t i t y o f t h e CGTase produced under v a r i o u s c o n d i t i o n s were examined. Substrate S p e c i f i c i t y The s u b s t r a t e s p e c i f i c i t y o f t h e CGTases was examined. I n c u b a t i o n c o n d i t i o n s were as f o l l o w s : s u b s t r a t e c o n c e n t r a t i o n , 1 % (w/v); i n c u b a t i o n t e m p e r a t u r e , 50'C; i n c u b a t i o n t i m e , 20 h r s . The r e s u l t s a r e shown i n F i g . 1. The CGTase from B. o h b e n s i s produced t h e l a r g e s t amount o f beta-CD o f a l l t h e enzymes. The enzyme from B. macerans was a t y p i c a l alpha-CD t y p e CGTase; alpha-CD was t h e dominant p r o d u c t i n any o f i t s s u b s t r a t e s , and t h e amount o f alpha-CD was t h e l a r g e s t i n t h e CGTases examined. CGTase from B. c i r c u l a n s was a beta-CD t y p e enzyme b u t i t a l s o formed a r e l a t i v e l y l a r g e amount o f alpha-CD. The p r o p e r t i e s o f CGTase from t h e s t r a i n IFO 3329 was d i f f e r e n t from t h a t r e p o r t e d by Okada e t a l . ( 4 ) . The CGTase from B. o h b e n s i s d i d n o t form any amount o f alpha-CD i n t h e r e a c t i o n c o n d i t i o n s and t h e CGTase was found t o be u n i q u e o f a l l t h e beta-CD t y p e enzymes which have been reported. The enzyme from B. o h b e n s i s produced t h e l a r g e s t amount o f gamma-CD o f t h e enzymes, and t h e amount (8-10 % t o s u b s t r a t e ) may be t h e l a r g e s t among t h e CGTases r e p o r t e d ( 1 5 ) . Time Course o f CDs F o r m a t i o n by t h e CGTases We examined t h e time c o u r s e o f CD f o r m a t i o n by t h e enzymes. The i n c u b a t i o n c o n d i t i o n s were as f o l l o w s : s u b s t r a t e , 5 % (w/v) o f s o l u b l e s t a r c h ; i n c u b a t i o n pH, 6.0; i n c u b a t i o n temperature, 5 0 C . The r e s u l t s a r e shown i n F i g . 2. The CGTase from B. o h b e n s i s produced beta-CD from t h e b e g i n n i n g o f enzyme r e a c t i o n and gamma-CD was formed l a t e r , but alpha-CD was n o t produced throughout t h e i n c u b a t i o n p e r i o d . The CGTase from B. macerans began t o produce alpha-CD a t f i r s t , but t h e f o r m a t i o n c e a s e d a f t e r 10 h o u r s o f i n c u b a t i o n . Beta-CD f o r m a t i o n was a l i t t l e d e l a y e d b u t i t c o n t i n u e d even a f t e r 24 hours o f i n c u b a t i o n . T h e r e f o r e , t h e r a t i o o f alpha-CD/beta-CD d e c r e a s e d i n t h e l a t e r phase o f enzyme r e a c t i o n . In the case o f B. c i r c u l a n s enzyme, beta-CD was d o m i n a n t l y produced and alpha-CD was a l s o produced from t h e e a r l y s t a g e o f t h e enzyme r e a c t i o n . e

E f f e c t o f pH on CD F o r m a t i o n by t h e CGTases The enzyme r e a c t i o n c o n d i t i o n s were t h e same as t h e p r e v i o u s s e c t i o n . The r e a c t i o n pH


10. SATO & YAGI

Properties of CGTases from Three Types of Bacillus

B. ohbensis

Substrate

Β. c i r c u l a n s

Β. macerans

Glucose Maltose


Maltotrlose Maitotetraose

• 1

Ώ

Maltopentaose

•

Maitohexaose Maltoheptaose

•

1 1

•

Amylose

Soluble

i—:

1

•

•

^

— •• O "1 ρ Ko -•' - • '—^-J •

Amylopectin Glycogen

•

= s J

S 3

I

tt

ruSir?

Ρ

•

starch

Potato s t a r c h ι

Sweet potato

1

Corn starch

• •

==-•=1

I 20

ι 40

g

— J

•

ι

ι

ι

I

I

ι

60

20

40

60

20

40

1

60

Cyclodextrins 7.(wt/wt) Figure 1. Substrate s p e c i f i c i t y of CGTases on CD formation. Incubation conditions were described i n the t e x t . (Reproduced with permission from Ref. 9. Copyright 1986 M. Nakamura.)

Incubation period (hr) Figure 2. Time course of CD formation. Incubation conditions were described i n the t e x t . (Reproduced with permission from Ref. 9. Copyright 1986 M. Nakamura.)


129

130


was a d j u s t e d w i t h 100 mM o f v a r i o u s b u f f e r s : pH 4 t o 8 w i t h M a c l l v a i n e b u f f e r , pH 8 t o 11 w i t h b o r a t e - N a C 0 b u f f e r and pH 11 t o 12 w i t h Na HP0 -NaOH b u f f e r . The r e s u l t s a r e shown i n F i g . 3. The enzyme from B. o h b e n s i s produced CDs i n a wide range o f pH (pH 5 t o 1 0 ) . As f o r t h e CGTase from B. macerans, CD p r o d u c t i o n r a t e was changed by t h e i n c u b a t i o n pH. I n t h e a c i d i c pH range (pH 5 ~ 6 ) the r a t i o o f alpha-CD was h i g h w h i l e i n t h e n e u t r a l t o a l k a l i n e range, i t became low. The CGTase from B. c i r c u l a n s had a narrow pH a c t i v i t y range and was a c t i v e o n l y from pH 5 t o 7. 2

2

3

4


t

E f f e c t o f I n c u b a t i o n Temperature on CDs F o r m a t i o n The I n c u b a t i o n pH was a d j u s t e d t o pH 6.0 w i t h 100 mM M a c l l v a i n e b u f f e r and o t h e r i n c u b a t i o n c o n d i t i o n s were t h e same as t h e p r e v i o u s e x p e r i m e n t . The r e s u l t s a r e shown i n F i g . 4. The enzyme r e a c t i o n p r o c e e d e d b e s t i n t h e range o f 40°C t o 70°C f o r t h e CGTase from B. o h b e n s i s . In the enzyme from B. c i r c u l a n s , t h e r e a c t i o n p r o c e e d e d b e s t from 40°C t o 80°C. B u t t h e CGTase from Β . macerans had a v e r y narrow t e m p e r a t u r e a c t i v i t y range around 50°C. E f f e c t o f S u b s t r a t e C o n c e n t r a t i o n on CD F o r m a t i o n Soluble starch was used as t h e s u b s t r a t e . The enzyme r e a c t i o n was c a r r i e d o u t a t pH 6.0 and 50°C and f o r 24 h r s . The r e s u l t s a r e shown i n F i g . 5. G e n e r a l l y , the y i e l d decreased with i n c r e a s e d substrate concentra tion. The d e c r e a s e was s i g n i f i c a n t i n t h e enzyme from B. macerans and i t had t o be i n c u b a t e d w i t h r e l a t i v e l y low c o n c e n t r a t i o n o f starch. E f f e c t o f CGTase C o n c e n t r a t i o n on CD F o r m a t i o n The i n c u b a t i o n c o n d i t i o n s were as f o l l o w s : s u b s t r a t e c o n c e n t r a t i o n , 5 %; i n c u b a t i o n pH, 6.0; i n c u b a t i o n time, 24 h r s . One u n i t o f enzyme was defined as t h e amount o f enzyme which produces 1 mg o f CD/hr a t 50°C, pH 6.0 w i t h 5 % o f s o l u b l e s t a r c h as s u b s t r a t e . Enzyme u n i t s were d e t e r m i n e d by HPLC u s i n g t h e method o f S a t o e t a l . ( 1 6 ) . The r e s u l t s a r e shown i n F i g . 6. As t h e amount o f enzyme i n c r e a s e d , the amount o f CD produced i n c r e a s e d and then d e c r e a s e d . With B. o h b e n s i s , t h e optimum amount o f enzyme f o r p r o d u c t i o n o f b e t a - and gamma-CD was a p p r o x i m a t e l y t h e rsame, b u t p r o d u c t i o n o f alpha-CD d i d n o t s t a r t u n t i l t h i s o p t i m a l amount o f enzyme was u s e d . As t h e amount o f enzyme i n c r e a s e d , t h e p r o d u c t i o n o f alpha-CD was i n c r e a s i n g as t h e amount o f b e t a - and gamma-CD was d e c r e a s i n g . An i n c r e a s e and then d e c r e a s e i n CD p r o d u c t i o n w i t h B. c i r c u l a n s enzyme was a l s o found as enzyme c o n c e n t r a t i o n was i n c r e a s e d . The o p t i m a l enzyme c o n c e n t r a t i o n f o r a l l CDs was a p p r o x i m a t e l y t h e same. With B. macerans enzyme, CD p r o d u c t i o n a g a i n i n c r e a s e d and then d e c r e a s e d w i t h i n c r e a s i n g enzyme c o n c e n t r a t i o n . However, t h e o p t i m a l enzyme c o n c e n t r a t i o n f o r t h e maximum p r o d u c t i o n o f each CD was d i f f e r e n t . The d e c r e a s e i n CD p r o d u c t i o n seemed t o be due t o a n o t h e r a c t i o n o f CGTase, such as d i s p r o p o r t i o n a t i o n . I f we employ h i g h enzyme c o n c e n t r a t i o n , i t i s p o s s i b l e t o p r o d u c e alpha-CD by CGTase from B. o h b e n s i s . I n t h e B. macerans enzyme, beta-CD r a t i o i n c r e a s e d w i t h t h e i n c r e a s e d c o n c e n t r a t i o n o f t h e enzyme, s o , t h e p r o p e r amount o f enzyme must be s e l e c t e d t o p r o d u c e alpha-CD. As f o r t h e CGTase from B. c i r c u l a n s , t h e CD p r o d u c t i o n r a t i o was n o t changed w i t h t h e v a r i o u s enzyme c o n c e n t r a t i o n s . The

enzyme

from

B.

macerans

i s t h e most

advantageous f o r


10. SATO & YAGI

Properties of CGTasesfromThree Types of Bacillus


B. ohbensis

4

6

8

B. macerans

10

12

6

4

8

10

B.

12

4

cireulans

6

8

10

12

Incubation pH Figure 3. Effect of pH on CD formation. Incubation conditions were described i n the text. (Reproduced with permission from Ref. 9. Copyright 1986 M. Nakamura.) B. ohbensis

0

20

40

60

B. macerans

80

20

40

60

B.

80

circulans

20

40

60

80

Incubation temp. (°C) Figure 4* Effect of temperature on CD formation. Incubation conditions were described i n the text. (Reproduced with permission from Ref. 9. Copyright 1986 M. Nakamura.) B, ohbensis

0

5

10

B. macerans

15

5

10

B.

15

circulans

5

10

15

Substrate concentration (%, w/v) Figure 5. Effect of substrate concentration on CD formation. Incubation conditions were described i n the text. In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

131

132


p r o d u c i n g alpha-CD, b u t t h e i n c u b a t i o n c o n d i t i o n s must be r e l a t i v e l y strictly controlled. CGTase from B. o h b e n s i s i s now b e i n g u s e d f o r the i n d u s t r i a l production o f beta-CD and a wide range o f t e m p e r a t u r e and pH c a n be used f o r t h e p r o d u c t i o n o f beta-CD. F o r t h e p r o d u c t i o n o f gamma-CD, t h e enzyme from B. o h b e n s i s i s a l s o f a v o r a b l e and i s b e i n g used because t h e enzyme does n o t produce alpha-CD under normal enzyme r e a c t i o n c o n d i t i o n s , a l l o w i n g easy e l i m i n a t i o n o f beta-CD from t h e r e a c t i o n b r o t h .


Enhanced F o r m a t i o n Guest Compound

o f Gamma-CD

by t h e A d d i t i o n

o f High

Affinity

I t i s v e r y c o s t l y t o p r o d u c e gamma-CD because o f t h e s m a l l amount p r o d u c e d by t h e enzymes. Here, we r e p o r t a new system f o r p r o d u c i n g gamma-CD by e m p l o y i n g a g u e s t compound which h a s a h i g h a f f i n i t y f o r gamma-CD. S c r e e n i n g o f Guest Compounds w i t h A c c o r d i n g t o T h e i r A f f i n i t y t o Gamma-CD I n s t u d i e s t o mask t h e b i t t e r n e s s o f s t e v i o s i d e , a n a t u r a l sweetener, gamma-CD was found t o be e f f e c t i v e t o e l i m i n a t e t h i s b i t t e r n e s s and t o complex t h e s t e v i o s i d e v e r y w e l l ( 1 7 ) . F i g . 7 shows a phase s o l u b i l i t y diagram o f C D - s t e v i o s i d e system by t h e method o f H i g u c h i e t a l . ( 1 8 ) . O n l y gamma-CD c o u l d form an i n c l u s i o n complex v e r y w e l l w i t h t h e guest compound. The s t a b i l i t y c o n s t a n t was 11,200/M. From t h e e x p e r i m e n t a l r e s u l t s , we examined t h e e f f e c t o f t h e a d d i t i o n o f s t e v i o s i d e on gamma-CD f o r m a t i o n . The i n c u b a t i o n c o n d i t i o n s were as f o l l o w s : a d d i t i o n a l amount o f s t e v i o s i d e , 0.2-3.0 % (w/v); s u b s t r a t e , 5 % (w/v) o f s o l u b l e s t a r c h ; incubation pH, 6.5 w i t h 100 mM M a c l l v a i n e buffer; incubation t e m p e r a t u r e , 50°C; i n c u b a t i o n time, 20 h r s . As was had e x p e c t e d , gamma-CD i n c r e a s e d p r o p o r t i o n a l l y w i t h t h e i n c r e a s e d c o n c e n t r a t i o n o f s t e v i o s i d e from 0.2 t o 1.0 % ( F i g . 8 ) . As t h e r e had been no r e p o r t on enhanced gamma-CD p r o d u c t i o n by a d d i t i o n o f a s e l e c t i v e g u e s t compound, we s c r e e n e d t h e compounds by i t s a f f i n i t y t o gammaCD. We e s p e c i a l l y f o c u s e d on t h e guest compounds which were n a t u r a l products because they may be s a f e r than c h e m i c a l l y synthesized compounds. The s c r e e n i n g system was as f o l l o w s : c o n c e n t r a t i o n o f g u e s t compound t o be added, 1 % (w/v); enzyme used, CGTase from B. o h b e n s i s ; s u b s t r a t e , 5 % (w/v) o f s o l u b l e s t a r c h ; i n c u b a t i o n pH, 7.5; i n c u b a t i o n t e m p e r a t u r e , 50°C; i n c u b a t i o n time, 20 h r s . Some examples o f e f f e c t i v e guest compounds a r e l i s t e d i n F i g . 9. Glycyrrhizic and g l y c y r r h e t i c a c i d s were found t o be s u p e r i o r compounds t o enhance gamma-CD f o r m a t i o n . G l y c y r r h i z i c a c i d was s e l e c t e d as t h e guest compound f o r f u r t h e r s t u d i e s t o enhance gammaCD f o r m a t i o n . E f f e c t s o f G l y c y r r h i z i c A c i d on t h e F o r m a t i o n o f Gamma-CD Incubat i o n pH, s u b s t r a t e c o n c e n t r a t i o n and g l y c y r r h i z i c a c i d c o n c e n t r a t i o n were s t u d i e d . When t h e s u b s t r a t e c o n c e n t r a t i o n was i n c r e a s e d i n the p r e s e n c e o f g l y c y r r h i z i c a c i d , gamma-CD y i e l d p e r g o f s u b s t r a t e was c o n s t a n t up t o t h e s u b s t r a t e c o n c e n t r a t i o n o f 20 % ( d a t a n o t shown). Incubation pH a f f e c t e d t h e gamma-CD f o r m a t i o n . The a d d i t i o n o f g l y c y r r h i z i c a c i d was n o t v e r y e f f e c t i v e f o r e n h a n c i n g t h e gamma-CD p r o d u c t i o n below pH 6.0, because t h e guest compound i s n o t water s o l u b l e i n a c i d i c c o n d i t i o n s . The optimum pH f o r


10.

SATO & YAGI

Properties of CGTasesfromThree Types of Bacillus 133


B. ohbensis

CGTase cone,

£. macerans

£.

circulans

(units/g of substrate)

Figure 6. Effect of CGTase concentration on CD formation. Incubation conditions were described i n the text. (Reproduced with permission from Ref. 9. Copyright 1986 M. Nakamura.)


134


40

-


5

J? w C U 4J

γ-CD

-o-o-

20

X

Properties of CGTases from Three Types of Bacillus and Production of

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