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ICHIRO CHIBATA Tanabe Seiyaku Co., Ltd., Research Laboratory of Applied Biochemistry, 16-89, Kashima-3-chome, Yodogawa-ku, Osaka, Japan
Three immobilized enzyme or microbial cell systems currently used industrially in synthesis of chiral amino acids plus one presently under development are described. L-amino acids are produced by enzymatic hydrolysis of DL-acylamino acid with aminoacylase immobilized by ionic bind ing to DEAE-Sephadex. Escherichia coli cells immobilized by K-carrageenan crosslinked with glutaraldehyde and hexamethylenediamine are used to convert fumaric acid and ammonia to L-aspartic acid and Brevibacterium flavum cells similarly immobilized are used to hydrate fumaric acid to L-malic acid. The decarboxylation of L-aspartic acid by immobilized Pseudomonas dacunhae to L -alanine is currently under investigation. Enzymes are b i o l o g i c a l c a t a l y s t s and p a r t i c i p a t e i n many chemical
reactions occurring i n l i v i n g things.
Unlike
ordinary
chemical
c a t a l y s t s , enzymes have the a b i l i t y t o c a t a l y z e a r e a c -
t i o n under very m i l d c o n d i t i o n s i n n e u t r a l aqueous s o l u t i o n a t normal temperature and pressure, and with very high specificity.
substrate
They a l s o have c h i r a l s p e c i f i c i t y and c a t a l y z e
asymmetric r e a c t i o n s .
However, enzymes are produced by organ-
isms f o r t h e i r own requirements and, though e f f i c i e n t and e f -
0097-6156/82/0185-0195$05.00/0 © 1982 American Chemical Society
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
ASYMMETRIC
196
REACTIONS
A N D PROCESSES IN
f e c t i v e c a t a l y s t s , are not always i d e a l f o r p r a c t i c a l tions.
CHEMISTRY
applica-
Thus, i n order t o o b t a i n s u p e r i o r c a t a l y s t s f o r a p p l i -
c a t i o n s , t h a t i s , h i g h l y a c t i v e and s t a b l e c a t a l y s t s having appropriate s p e c i f i c i t y , m o d i f i c a t i o n o f enzymes has been c a r r i e d out.
Among such m o d i f i c a t i o n s , i m m o b i l i z a t i o n has been
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e x t e n s i v e l y s t u d i e d i n the p a s t f i f t e e n years.
I f a c t i v e and
s t a b l e immobilized enzymes are prepared, the expected advantages, compared t o s o l u b l e enzymes, are as f o l l o w s : s t a b i l i t y o f the enzymes i s improved, made f o r s p e c i f i c use,
(1)
(2) enzymes can be
tailor-
(3) enzymes can be reused, (4) continuous
operation becomes p r a c t i c a l ,
(5) r e a c t i o n s r e q u i r e l e s s space,
(6) b e t t e r c o n t r o l o f r e a c t i o n s i s p o s s i b l e , (7) higher p u r i t y and y i e l d o f products may be a t t a i n e d , and
(8) s a v i n g of r e -
sources with l e s s attendant p o l l u t i o n may be achieved. More r e c e n t l y , techniques o f d i r e c t i m m o b i l i z a t i o n o f whole m i c r o b i a l c e l l s have been developed; e i t h e r to avoid the need to e x t r a c t enzymes from m i c r o b i a l c e l l s or to u t i l i z e multi-enzyme systems.
1
Since the e a r l y I 9 6 0 s , we have s t u d i e d the immobili-
z a t i o n o f enzymes and m i c r o b i a l c e l l s f o r i n d u s t r i a l a p p l i c a t i o n s and have succeeded metric reactions.
i n the i n d u s t r i a l i z a t i o n o f three asym-
In t h i s r e p o r t , these already i n d u s t r i a l i z e d
systems and a p o t e n t i a l system f o r which we are p l a n n i n g indust r i a l i z a t i o n w i l l be d e s c r i b e d .
I.
IMMOBILIZED AMINOACYIASE —
Production o f L-Amino A c i d s
For the i n d u s t r i a l p r o d u c t i o n o f L-amino a c i d s , fermentation and chemical s y n t h e t i c methods appear to be promising.
However,
conventional chemical s y n t h e s i s leads t o a racemic mixture. Hence r e s o l u t i o n o f enantiomers a c t i v e L-amino a c i d s .
i s necessary t o o b t a i n o p t i c a l l y
Among many r e s o l u t i o n methods, the en-
zymatic method u s i n g mold aminoacylase developed by us proved to be one o f the most advantageous procedures.
An acyl-DL-amino
a c i d i s s e l e c t i v e l y hydrolyzed by aminoacylase t o give L-amino a c i d and unhydrolyzed acyl-D-amino a c i d .
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
CHiBATA
14.
Immobilized
DL-R-CH-COOH
197
Enzymes
L-R-CH-COOH
+ H 0 2
D-R-CH-COOH
+
aminoacylase
NHCOR' N-acyl-DLamino a c i d
.J
racemization
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Between 1954 and 1969,
t h i s enzymatic
r e s o l u t i o n method had
been employed by Tanabe Seiyaku Co., L t d . f o r the production o f s e v e r a l L-amino a c i d s .
In the 1960s we e x t e n s i v e l y s t u d i e d the
immobilization o f aminoacylase [1,2].
f o r continuous o p t i c a l r e s o l u t i o n
A v a r i e t y o f i m m o b i l i z a t i o n methods were t e s t e d f o r
i n d u s t r i a l purposes,
from which aminoacylase
i o n i c b i n d i n g t o DEAE-Sephadex was chosen. engineering s t u d i e s on aminoacylase
immobilized by Through chemical
columns we designed an en-
zyme r e a c t o r f o r continuous p r o d u c t i o n .
Since 1969, we have
been o p e r a t i n g s e v e r a l s e r i e s o f enzyme r e a c t o r s f o r the product i o n o f L-methionine,
L - v a l i n e , L-phenylalanine and so f o r t h .
With t h i s immobilized enzyme system, L-amino a c i d s can be p r o duced more economically compared t o the conventional batch s y s tem u s i n g n a t i v e enzyme as shown i n F i g . 1.
II. A.
IMMOBILIZED MICROBIAL CELLS Production o f L - A s p a r t i c A c i d We attempted
continuous production o f L - a s p a r t i c a c i d
fumaric a c i d and ammonia by immobilized Escherichia high aspartase a c t i v i t y
[3, 4, 5 ] .
coli
from
having
Various methods were t e s t e d
f o r the i m m o b i l i z a t i o n o f m i c r o b i a l c e l l s , and a s t a b l e and a c t i v e enzyme system was obtained by entrapping whole m i c r o b i a l c e l l s i n a polyacrylamide g e l l a t t i c e . HOOC-CH=CH-COOH + NH f 3
fumaric a c i d
aspartase
^ HOOC-CH -CH-COOH 2
NH
2
L-aspartic acid Using a column packed w i t h immobilized E. coli
c e l l s , condi-
t i o n s f o r continuous production o f L - a s p a r t i c a c i d were i n v e s t i -
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
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198
A S Y M M E T R I C REACTIONS A N D PROCESSES IN CHEMISTRY
•
DEAESephadex
Fuel
Labor
Aminoacylase
Conventional (batch )
Figure 1.
Immobilized (continuous )
Cost comparison
for production
Ac-DLamino a c i d
of L-amino
acids.
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
CHiBATA
14.
Immobilized
199
Enzymes
gated i n d e t a i l , and an aspartase r e a c t o r system was
designed.
The system i s e s s e n t i a l l y the same as t h a t f o r the immobilized aminoacylase
system and has been operated i n d u s t r i a l l y s i n c e
1973. By way
o f a f u r t h e r improvement o f the p r o c e s s , a new
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nique u s i n g κ-carrageenan was E. coli
[6, 7, 8 ] .
tech
discovered f o r immobilization of
K-Carrageenan i s a k i n d o f p o l y s a c c h a r i d e
prepared from seaweed and has the c h a r a c t e r i s t i c o f becoming a g e l under m i l d c o n d i t i o n s .
We compared the e f f i c i e n c y o f E.
coli
c e l l s immobilized with polyacrylamide and K-carrageenan i n p r o duction o f L - a s p a r t i c a c i d , and found t h a t E. coli
immobilized
with κ-carrageenan and then t r e a t e d with glutaraldehyde and hexamethylenediamine shows the h i g h e s t p r o d u c t i v i t y
(Table 1).
Therefore, we considered t h a t t h i s p r e p a r a t i o n i s most
advanta
geous f o r continuous p r o d u c t i o n of L - a s p a r t i c a c i d , and i n
1978
we r e p l a c e d the conventional polyacrylamide g e l method by the new
carrageenan method.
Table 1 COMPARISON OF PRODUCTIVITY OF E. coli IMMOBILIZED WITH POLYACRYLAMIDE AND WITH CARRAGEENAN FOR PRODUCTION OF L-ASPARTIC ACID
Immobilization method
Aspartase Stability Relative activity a t 37°C productivity* (unit/g c e l l s ) ( h a l f - l i f e , days)
Polyacrylamide
18,850
120
100
Carrageenan
56,340
70
174
Carrageenan (GA)
37,460
240
397
Carrageenan (GA+HMDA)
49,400
680
GA:glutaraldehyde,
1,498
HMDA:hexamethylenediamine
* Productivity = | Q O E
exp(-k^»t)dt
E o = i n i t i a l a c t i v i t y , k =decay constant, t = o p e r a t i o n a l p e r i o d d
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
ASYMMETRIC
200 B.
REACTIONS
A N D PROCESSES IN
CHEMISTRY
Production o f L - M a l i c A c i d In
1974 we succeeded
i n the i n d u s t r i a l p r o d u c t i o n o f L-malic
a c i d from fumaric a c i d by Brevibacteriurn
ammoniagenes c e l l s immo
b i l i z e d by the polyacrylamide g e l method [9, 10]. The asymmetric r e a c t i o n c a t a l y z e d by the fumarase a c t i v i t y o f the c e l l s i s shown Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 18, 2014 | http://pubs.acs.org Publication Date: April 28, 1982 | doi: 10.1021/bk-1982-0185.ch014
below. H00C-CH=CH-C00H + H 0 2
fumaric a c i d
N
v
_ HOOC-CH -CH-COOH fumarase , OH L-malic a c i d 2
As i n the case o f L - a s p a r t i c a c i d p r o d u c t i o n , we i n v e s t i gated the carrageenan method t o improve the p r o d u c t i v i t y f o r L-malic a c i d .
A f t e r s c r e e n i n g v a r i o u s microorganisms
fumarase a c t i v i t y , Brevibacterium
f o r maximal
flavum was found t o show a
higher enzyme a c t i v i t y a f t e r i m m o b i l i z a t i o n with K-carrageenan than the formerly used B. ammoniagenes, as shown i n Table 2 [11]. Therefore, t h i s polyacrylamide method was a l s o changed t o the carrageenan method i n 1977.
The new method gives s a t i s f a c t o r y
r e s u l t s f o r i n d u s t r i a l p r o d u c t i o n o f L-malic a c i d .
Table 2 COMPARISON OF PRODUCTIVITIES OF Brevibacterium ammoniagenes AND Brevibacterium flavum IMMOBILIZED WITH POLYACRYLAMIDE AND WITH CARRAGEENAN FOR PRODUCTION OF L-MALIC ACID Β . flavum B. ammoniaqenes Immobi Activity H a l f - l i f e Relative Activity H a l f - l i f e Relative lization (unit/ a t 37°C produc(unit/ a t 37°C producmethod g cells) (day) tivity g cells) (day) tivity Polyacryl amide
5,800
53
100
6,680
94
204
Carra geenan
5,800
75
142
9,920
160
516
* a c t i v i t y a f t e r treatment with b i l e e x t r a c t P r o d u c t i v i t y = J^Eo e x p ( - k ^ * t ) d t E = i n i t i a l a c t i v i t y , kd=decay constant, t = o p e r a t i o n a l p e r i o d 0
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
14.
CHiBATA
C.
Immobilized
201
Enzymes
Production o f L-Alanine and D-Aspartic A c i d Besides these two i n d u s t r i a l a p p l i c a t i o n s o f immobilized
m i c r o b i a l c e l l s f o r asymmetric r e a c t i o n s , we are p r e s e n t l y studying the continuous production o f L-alanine from L - a s p a r t i c acid.
A t present, L - a l a n i n e i s produced by a batch process.
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A continuous production system u s i n g immobilized Pseudomonas dacunhae c e l l s with high L-aspartate 3-decarboxylase i s c u r r e n t l y under i n v e s t i g a t i o n
activity
[ 1 2 ] . The r e a c t i o n proceeds
as shown below. L-H00C-CH2-CH-C00H
—
, NH L-aspartic acid
> L-CH3-CH-COOH +
L-aspartate β-decarboxylase
2
In t h i s continuous
2
2
system u s i n g immobilized
are problems a s s o c i a t e d with e v o l u t i o n o f C0 reaction.
C0
, NH L-alanine
2
c e l l s , there
gas d u r i n g the
I t i s d i f f i c u l t t o maintain the plug-flow o f the
substrate s o l u t i o n under normal pressure, and t o keep a constant pH o f r e a c t i o n mixture vescence.
i n the r e a c t o r because o f the C0
2
effer
We t h e r e f o r e designed a c l o s e d column r e a c t o r which
performs the enzyme r e a c t i o n a t an e l e v a t e d pressure such as 2
10Kg/cm .
Using t h i s r e a c t o r , s i n c e l i b e r a t e d C0
2
gas i s melded
i n t o r e a c t i o n mixture, the complete plug-flow o f the s u b s t r a t e s o l u t i o n i s maintained a p p r e c i a b l y changed.
and the pH o f r e a c t i o n mixture
i s not
Therefore, as shown i n Table 3, the e f f i
ciency o f immobilized c e l l s f o r p r o d u c t i o n o f L-alanine i n the c l o s e d column system (at high pressure) i s much higher than t h a t i n the conventional column system a t normal p r e s s u r e . The decarboxylase
enzyme shows h i g h enantiomer s e l e c t i v i t y
r e a c t i n g only with L - a s p a r t i c a c i d .
Thus, L-alanine and D-
a s p a r t i c a c i d can be produced from DL-aspartic a c i d a t the same time. D-Aspartic a c i d i s used as an important intermediate f o r s y n t h e t i c p e n i c i l l i n , whose s y n t h e s i s has been developed by Tanabe Seiyaku Co.
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
ASYMMETRIC
202
REACTIONS A N D PROCESSES IN
CHEMISTRY
Table 3 COMPARISON OF EFFICIENCIES AND STABILITIES OF CONVENTIONAL AND CLOSED COLUMN REACTORS FOR L-ALANINE PRODUCTION
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Closed Conventional (normal pressure) (high pressure) r Efficiency Λ ymole/hr/ml o f r e a c t o r ^ a t 99% conveneion ' f ^
Stability h a l f - l i f e , days a t 37°C
250
360
46
46
} '
We are p l a n n i n g i n d u s t r i a l i z a t i o n o f these continuous L-alanine and D - a s p a r t i c a c i d p r o d u c t i o n systems u s i n g immobi l i z e d P. dacunhae i n the near f u t u r e .
Literature Cited 1.
Tosa, T.; Mori, T.; Fuse, N.; Chibata, I. Enzymologia 1966, 31, 214-224. 2. Chibata, I.; Tosa, T.; Sato, T.; Mori, T.; Matuo, Y. "Fermentation Technology Today (G. Terui ed.), P.383, Soc. Ferment. Technol., Osaka, Japan (1972). 3. Chibata, I.; Tosa, T.; Sato, T. Appl. Microbiol. 1974, 27, 878-885. 4. Tosa, T.; Sato, T.; Mori, T.; Chibata, I. Appl. Microbiol. 1974, 27, 886-889. 5. Sato, T.; Mori, T.; Tosa, T.; Chibata, I.; Furui, M.; Yamashita, K.; Sumi, A. Biotechnol. Bioeng. 1975, 17, 1797-1804. 6. Tosa, T.; Sato, T.; Mori, T.; Yamamoto, K.; Takata, I.; Nishida, Y.; Chibata, I. Biotechnol. Bioeng. 1979, 21, 133-145. 7. Nishida, Y.; Sato, T.; Tosa, T.; Chibata, I. Enzyme Microb. Technol. 1979, 1, 95-99.
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 18, 2014 | http://pubs.acs.org Publication Date: April 28, 1982 | doi: 10.1021/bk-1982-0185.ch014
14.
CHIBATA
Immobilized
Enzymes
203
8. Sato, T.; Nishida, Y.; Tosa, T.; Chibata, I. Biochim. Biophys. Acta 1979, 570, 179-186. 9. Yamamoto, K.; Tosa, T.; Yamashita, K.; Chibata, I. Europ. J. Appl. Microbiol. 1976, 3, 169-183. 10. Yamamoto, K.; Tosa, T.; Yamashita, K.; Chibata, I. Biotechnol. Bioeng. 1977, 19, 1101-1114. 11. Takata, I., Yamamoto, K.; Tosa, T.; Chibata, I. Europ. J. Appl. Microbiol. Biotechnol. 1979, 7, 161-172. 12. Yamamoto, K.; Tosa, T.; Chibata, I. Biotechnol. Bioeng. 1980, 22, 2045-2054. RECEIVED January 4, 1982.
In Asymmetric Reactions and Processes in Chemistry; Eliel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.