Immobilized Microbial Cells with Polyacrylamide Gel and

13 Immobilized Microbial Cells with Polyacrylamide Gel and Carrageenan and Their Industrial Applications

Downloaded by GEORGE MASON UNIV on February 25, 2016 | http://pubs.acs.org Publication Date: August 16, 1979 | doi: 10.1021/bk-1979-0106.ch013

ICHIRO CHIBATA Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., 16-89, Kashima-3-chome, Yodogawa-ku, Osaka, Japan

Enzymes are very efficient and advantageous catalysts, and can catalyze specific reactions under mild conditions, in neutral aqueous solution at room temperature. However, they are not a l ways ideal catalysts for industrial application. In some cases, the above mentioned advantages of enzymes turn to be disadvantageous as catalysts. Namely, enzymes are generally unstable, and can not be used in organic solvents and at elevated temperatures. One of the approaches to prepare more superior catalysts for application purpose is immobilization of enzymes. Over the past 10 years, the immobilization of enzyme has been the subject of increased interest, and many papers on potential applications of immobilized enzymes and microbial cells have been published. However, practical industrial systems using immobilized enzymes and immobilized microbial cells have been very limited. In 1969, we [1, 2] succeeded in the industrial application of immobilized enzyme, i.e. immobilized aminoacylase, for the continuous production of L-amino acids from acetyl-DL-amino acids. This is the first industrial application of immobilized enzymes in the world. Since then we [3, 4, 5, 6, T\ also carried out the industrial applications of immobilized microbial cells for the continuous productions of L-aspartic acid and L-malic acid using immobilized microbial cells with polyacrylamide gel. For further improvement of these immobilization systems, we investigated many synthetic and natural polymers as a matrix for entrapping enzymes and microbial cells into gel lattice. As a result, we [8, _9] found that K-carrageenan is one of the most suitable polymer for immobilization of microbial cells. In this paper, the immobilization of microbial cells with polyacrylamide gel and K-carrageenan, and their industrial application are reviewed. tf

I. 1.

fl

Immobilization with Polyacrylamide Gel and Its Application Production of L-aspartic acid using immobilized EachQJbLckJjQL coti [3, 4, 5] 0-8412-0508-6/79/47-106-187$05.00/0 © 1979 American Chemical Society In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

IMMOBILIZED MICROBIAL CELLS

188

L - A s p a r t i c a c i d i s used f o r medicines and food a d d i t i v e s , and i t has been i n d u s t r i a l l y produced by fermentative or enzymic methods from ammonium fumarate using the a c t i o n of aspartase as shown i n the f o l l o w i n g r e a c t i o n . HOOC-CH=CH-COOH + NH

3 3


Fumaric a c i d

N

v

_ HOOC·CH2-CH·COOH aspartase , (EC 4.3.1.1 ) NH L-Aspartic acid 2

T h i s r e a c t i o n has been c a r r i e d out by batch procedure, which has disadvantages f o r i n d u s t r i a l purpose. Thus, we s t u d i e d the continuous production of L - a s p a r t i c a c i d using a column packed w i t h immobilized aspartase. As the aspartase i s an i n t r a c e l l u l a r enzyme, i t was necessary to e x t r a c t the enzyme from m i c r o b i a l c e l l s before i m m o b i l i z a t i o n . E x t r a c t e d i n t r a c e l l u l a r enzyme i s g e n e r a l l y unstable, and most of the i m m o b i l i z a t i o n methods we t r i e d r e s u l t e d i n low a c t i v i t y and poor y i e l d . Although entrapment i n t o polyacrylamide g e l l a t t i c e gave r e l a t i v e l y a c t i v e imm o b i l i z e d aspartase, i t s o p e r a t i o n a l s t a b i l i t y was not s u f f i c i e n t , i . e . the h a l f - l i f e was 30 days a t 37°C [10]. Therefore, t h i s imm o b i l i z e d aspartase was not s a t i s f a c t o r y f o r the i n d u s t r i a l production o f L - a s p a r t i c a c i d . So we considered that i f the whole m i c r o b i a l c e l l s could be immobilized d i r e c t l y , these disadvantages might be overcome, and we s t u d i e d the i m m o b i l i z a t i o n of whole m i c r o b i a l c e l l s . Reports on i m m o b i l i z a t i o n of whole m i c r o b i a l c e l l s had been very scarce at that time, so we t r i e d v a r i o u s methods f o r i m m o b i l i z a t i o n of E. coLi c e l l s . Among the methods t e s t e d , the most a c t i v e immob i l i z e d E. coti c e l l s were obtained by entrapping the c e l l s i n t o polyacrylamide g e l l a t t i c e [ 3 ] . An i n t e r e s t i n g phenomenon was observed with these immobilized c e l l s . When the immobilized E. doti c e l l s were suspended a t 37°C f o r 24-48 hours i n s u b s t r a t e s o l u t i o n , i t s a c t i v i t y increased 10 times h i g h e r . The i n c r e a s e of enzyme a c t i v i t y was observed even i n the presence of chloramphenicol, i n h i b i t o r o f p r o t e i n synthesis. Therefore, t h i s a c t i v a t i o n was considered not t o be the r e s u l t of p r o t e i n s y n t h e s i s but to be due t o increased p e r m e a b i l i t y caused by a u t o l y s i s of E. coti c e l l s i n the g e l l a t t i c e . T h i s was a l s o confirmed by the e l e c t r o n micrographs of immobilized E. coLL c e l l s , which i n d i c a t e d that l y s i s of c e l l s had occurred. Even when l y s i s of the c e l l s d i d occur, the aspartase could not l e a k out from the g e l l a t t i c e , but the s u b s t r a t e , ammonium fumarate, and the product, L - a s p a r t a t e , passed e a s i l y through the g e l l a t tice. Using a column packed with immobilized E. coLL c e l l s , c o n d i t i o n s 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 from ammonium fumarate were i n v e s t i g a t e d 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 that f o r the immobilized aminoacylase system [ 2 ] . A s o l u t i o n of 1 M ammonium fumarate ( c o n t a i n i n g 1 mM M g C l 2 , pH 8.5) i s passed

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

13.

CHiBATA

Industrial Applications

189

through the immobilized E. coZl c e l l column at a flow r a t e of space velocity=0.6 h r " a t 37°C. The e f f l u e n t i s adjusted to pH 2.8 and then cooled a t 15°C. By t h i s simple procedure pure La s p a r t i c a c i d can be obtained without r e c r y s t a l l i z a t i o n i n very high y i e l d . The a c t i v i t y of immobilized c e l l s i s q u i t e s t a b l e , and i t s h a l f - l i f e i s 120 days at 37°C. This new system had been operating i n d u s t r i a l l y s i n c e 1973 i n Tanabe Seiyaku Co., L t d . The o v e r a l l production cost by t h i s system was reduced to above 60% of the conventional batch process using i n t a c t c e l l s because of the marked r e d u c t i o n i n cost f o r the p r e p a r a t i o n of c a t a l y s t s and of the r e d u c t i o n o f l a b o r cost by automation. Furthermore, the procedure employing immobilized c e l l s i s advantageous from the standpoint of waste treatment. This i s considered to be the f i r s t i n d u s t r i a l a p p l i c a t i o n o f entrapped m i c r o b i a l c e l l s i n the world.


1

2.

Production of L-malic a c i d using immobilized

Ifo&vlbcLCt&iium

ammoniagzneA [6, 7] In succession to the L - a s p a r t i c a c i d production, i n 1974 we succeeded i n the t h i r d i n d u s t r i a l a p p l i c a t i o n , i . e . the production of L-malic a c i d from fumaric a c i d by immobilized m i c r o b i a l c e l l s . L-Malic a c i d i s an e s s e n t i a l compound i n c e l l u l a r metabolism, and i s mainly used i n pharmaceutical f i e l d . L-Malic a c i d can be produced by fermentative or enzymatic methods from fumaric a c i d by the a c t i o n of fumarase as f o l l o w s . H00C-HC=CH-C00H + H 0 2

Fumaric

acid

^

^ fumarase (EC 4.2.1.2)

H00C-CH -CH-C00H , OH L-Malic a c i d 2

In t h i s case, r e a c t i o n reaches an e q u i l i b r i u m when about 80% of fumaric a c i d are converted to L-malic a c i d . We i n v e s t i g a t e d continuous fumarase r e a c t i o n s using immobilized m i c r o b i a l c e l l s . Several microorganisms having high fumarase a c t i v i t y were immobilized by polyacrylamide g e l method and t h e i r a c t i v i t i e s were i n v e s t i g a t e d . 8. ammoviia.QZn2A showed the highest a c t i v i t y before and a f t e r i m m o b i l i z a t i o n . However, when immobilized 8. ammoyiiagznQA was used f o r the production of L-malic a c i d from fumaric a c i d , some by-products were formed. Besides remaining fumaric a c i d , c o n s i d e r a b l e accumulation of s u c c i n i c a c i d was observed i n the r e a c t i o n mixture. Although fumaric a c i d can be e a s i l y separated by a c i d i f y i n g the r e a c t i o n mixture, s e p a r a t i o n of s u c c i n i c a c i d from L-malic a c i d i s very d i f f i c u l t . Therefore, the p o i n t of success f o r i n d u s t r i a l production of pure L-malic a c i d i s the prevention of s u c c i n i c a c i d formation during the enzyme r e a c t i o n . So, we t r i e d various methods to suppress s u c c i n i c a c i d formation. Among the methods t e s t e d the detergents used as the s o l u b i l i z e r s f o r membrane- or p a r t i c l e - b o u n d enzymes, b i l e e x t r a c t , b i l e a c i d and deoxycholic acid,were found to be e f f e c t i v e to de-


190


crease s u c c i n i c a c i d formation i n immobilized 8. mmonuigmeA cells. Then the c o n d i t i o n s f o r continuous production of L-malic a c i d by a column packed w i t h t h i s b i l e e x t r a c t t r e a t e d immobilized c e l l s was s t u d i e d . When 1 M sodium fumarate (pH 7.0) i s passed through the column at 37°C at flow r a t e of space velocity=0.2 hr~ , the r e a c t i o n reaches an e q u i l i b l i u m . From the e f f l u e n t of the column, L-malic a c i d can be obtained by o r d i n a r y methods. Average y i e l d of pyrogen-free pure L-malic a c i d from consumed fumaric a c i d i s around 70% of the t h e o r e t i c a l . Tanabe Seiyaku Co., L t d . i s operating t h i s production system s i n c e 1974, and we are s a t i s f i e d both with the economical e f f i c i e n c y and w i t h the q u a l i t y of prod uct.


x

3.

Production of other u s e f u l compounds using immobilized c e l l s 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 of immobilized m i c r o b i a l c e l l s above mentioned, we s t u d i e d e f f i c i e n t continuous methods f o r the production of u s e f u l organic compounds using im m o b i l i z e d c e l l s as shown i n Table 1. Immobilization of r e s p e c t i v e microorganisms was c a r r i e d out by polyacrylamide g e l method. C i t r u l l i n e [11] used f o r medicine could be produced from a r g i n i n e i n higher y i e l d by a r g i n i n e deiminase a c t i v i t y of VaQudomoncu> pvutida immobilized with polyacrylamide g e l . H a l f - l i f e of the column i s around 140 days at 37°C. A l s o , urocanic a c i d [12], a sun screening agent i n pharmaceu t i c a l and cosmetic f i e l d , 6 - a m i n o p e n i c i l l a n i c a c i d [13], an im portant intermediate f o r s y n t h e t i c p e n i c i l l i n , NADP [_14, 15], glucose-6-phosphate [16] and g l u t a t h i o n e [17, 18] were found to be prepared by t h i s immobilized c e l l s using polyacrylamide method. These processes are considered to be more advantageous f o r the mass production of r e s p e c t i v e u s e f u l compounds than the batch method using e x t r a c t e d enzyme or m i c r o b i a l b r o t h . II.

Immobilization with Carrageenan and I t s A p p l i c a t i o n

1.

New matrix,κ-carrageenan, f o r i m m o b i l i z a t i o n of m i c r o b i a l cells As d e s c r i b e d above, the polyacrylamide g e l method i s advan tageous f o r i m m o b i l i z a t i o n of m i c r o b i a l c e l l s and f o r i n d u s t r i a l a p p l i c a t i o n . However, there are some l i m i t a t i o n s i n t h i s method. That i s , some enzymes are i n a c t i v a t e d during i m m o b i l i z a t i o n pro cedure by the a c t i o n of acrylamide monomer, $-dimethylaminop r o p i o n i t r i l , potassium p e r s u l f a t e or heat of the p o l y m e r i z a t i o n r e a c t i o n . Therefore, t h i s method has l i m i t a t i o n i n a p p l i c a t i o n f o r i m m o b i l i z a t i o n of enzymes and m i c r o b i a l c e l l s . Thus, to f i n d out more general i m m o b i l i z a t i o n technique and to improve the pro d u c t i v i t i e s of immobilized m i c r o b i a l c e l l systems we s t u d i e d new immobilization techniques. As the r e s u l t s , we have found out Kcarrageenan i s very u s e f u l f o r i m m o b i l i z a t i o n of c e l l s [ 8 ] . κ— Carrageenan, which i s composed of u n i t s t r u c t u r e of 3-D-galactose


In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 14 15 16

20 (37°C) 15 (36.5°C) 20 (37°C) 20 (30°C)

NADP, ADP NADP, ( P i ) n - i Glucose-6phosphate Glutathione

NAD, ATP NAD, ( P i ) n Glucose, ( P i ) n

KchAomoboicZzn. buutysii

Saccka/tomyczà L-Glutamic a c i d , L-Cysteine, Glycine

Polyphosphateglucose kinase

Glutathione synthetase

6-Aminopenicillanic acid

Ifazvlbact&Uum awnoYUagmoA

PolyphosphateNAD kinase

*

Ach&omobacteA

NAD-Kinase

17

13

12

42 (30°C)

180 (37°C)

6-APA*

coti

acid ,

Penicillin

Penicillin amidase

3

L-Histidine

AchfiomobacteA liqiildum

L-Histidine ammonia-lyase

3

Urocanic NH

L-Arginine

PAmdomowu pvutida

L-Arginine deiminase

Substrate

Microbial cell

Enzyme system

Operational Referstability Product (Half-life, ence day, Temp.°C) 11 140 L-Citrulline, (37°C) NH

TABLE 1 CONTINUOUS PRODUCTION USING IMMOBILIZED MICROBIAL CELLS PREPARED BY POLYACRYLAMIDE GEL METHOD




192

s u l f a t e and 3,6-anhydro-a-D-galactose, i s a r e a d i l y a v a i l a b l e polysaccharides i s o l a t e d from seaweeds, and i s non-toxic compounds widely used as food a d d i t i v e s , κ-Carrageenan has c h a r a c t e r i s t i c s that i t becomes g e l under the m i l d c o n d i t i o n s as f o l l o w s . I t becomes g e l by c o o l i n g as i n the case of agar. G e l a t i o n occurs by contact with an aqueous s o l u t i o n c o n t a i n i n g metal ions such as a l k a l i metal i o n s , a l k a l i n e earth metal ions and other b i - o r t r i v a l e n t metal i o n s . I t becomes g e l by contact with an aqueous s o l u t i o n c o n t a i n i n g ammonium i o n or amines such as a l i p h a t i c or aromatic diamines and amino a c i d de r i v a t i v e s . G e l a t i o n a l s o occurs by the contact with waterm i s c i b l e organic s o l v e n t s . Therefore, t a k i n g i n t o c o n s i d e r a t i o n of c h a r a c t e r i s t i c s of the enzyme-protein and the k i n d of s u b s t r a t e and product, we can choose the most s u i t a b l e procedure f o r i m m o b i l i z a t i o n of the microbial c e l l s . In our experiences, the procedures c o o l i n g and/or c o n t a c t i n g with an aqueous s o l u t i o n c o n t a i n i n g K or NR\ are very e a s i l y c a r r i e d out f o r g e l a t i o n , and as these c o n d i t i o n s are very m i l d , the immobilized p r e p a r a t i o n having high a c t i v i t y can be obtained [9]. The i m m o b i l i z a t i o n procedure of m i c r o b i a l c e l l s using κcarrageenan i s as f o l l o w s . A c e l l suspension i s warmed at 37°^ 50°C, and κ-carrageenan d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e i s warmed at 37°^60°C. The both are mixed, and the mixture i s cooled and/or contacted with an aqueous s o l u t i o n c o n t a i n i n g a g e l i n ducing agent. A f t e r t h i s treatment, the g e l i s granulated i n s u i t a b l e p a r t i c l e s i z e , and the immobilized c e l l s can be obtained. I f the o p e r a t i o n a l s t a b i l i t y of immobilized c e l l s i s not s a t i s f a c t o r y , the immobilized c e l l s are t r e a t e d with hardening agents such as hexamethylenediamine and glutaraldehyde. As the r e s u l t s , the s t a b l e immobilized c e l l s can be obtained [9]. By t h i s simple procedure many kinds of m i c r o b i a l c e l l s are s u c c e s s f u l l y entrapped i n t o g e l - l a t t i c e . Further, anogher advan tage of t h i s method using κ-carrageenan i s that v a r i o u s shapes of immobilized p r e p a r a t i o n s , such as cubic, bead and membrane types, can be e a s i l y t a i l o r e d f o r p a r t i c u l a r a p p l i c a t i o n purposes. For instance, bead type can be r e a d i l y prepared by dropping the mix ture of c e l l suspension and κ-carrageenen s o l u t i o n i n t o s t i r r e d s o l u t i o n c o n t a i n i n g a g e l inducing reagent. +

2.

Production of L - a s p a r t i c a c i d using immobilized

coli

+

EaeÂ&uchUl

Since κ-carrageenan was found to be a u s e f u l matrix f o r im m o b i l i z a t i o n of m i c r o b i a l c e l l s , we e x t e n s i v e l y s t u d i e d t h i s carrageenan method to improve the i n d u s t r i a l production of La s p a r t i c a c i d u s i n g immobilized E. cotL c e l l s prepared by p o l y acrylamide g e l method [18]. The aspartase a c t i v i t y and the o p e r a t i o n a l s t a b i l i t y of im m o b i l i z e d E. coLL c e l l s with κ-carrageenan were compared with that of immobilized one with polyacrylamide. As shown i n Table 2,



CHIBATA

Industrial

193

Applications

TABLE 2 HARDENING TREATMENT FOR STABILIZATION OF ASPARTASE ACTIVITY OF IMMOBILIZED EAck&UcktCL COlL Hardening treatment Aspartase a c t i v i t y after activation Reagent and Temp. Time (unit/g c e l l s ) f i n a l cone. 56,340 None 54,500 37° 2 hr Persimmon tannin (0.02%) 15 min GA (mM) 4° 39,690 2.5 37,460 5.0 28,040 10.0 30 min HMDA GA 4° (mM) (mM) 50,210 85.0 1.7 49,900 85.0 17.0 49,400 85.0 85.0 36,120 85.0 17.0 Polyacrylamide GA:

glutaraldehyde;

18,850

Half-life at 37°C (day) 70 86

113 240 252

75 108 680 443 120

HMDA: hexamethylenediamine



194


enzyme a c t i v i t y of immobilized p r e p a r a t i o n with κ-carrageenan was much higher, but the s t a b i l i t y of immobilized p r e p a r a t i o n with κ— carrageenan was r a t h e r lower than that with polyacrylamide. Thus, i n order to i n c r e a s e o p e r a t i o n a l s t a b i l i t y of t h i s immobilized p r e p a r a t i o n , hardening treatments were c a r r i e d out. As shown i n Table 2, aspartase a c t i v i t i e s of immobilized E. coLL c e l l s were somewhat lowered by t h i s hardening treatment, but t h e i r o p e r a t i o n a l s t a b i l i t i e s were markedly enhanced. E s p e c i a l l y , i n the case of 85 mM hexamethylenediamine and 85 mM glutaraldehyde, the h a l f l i f e was extended to 680 days, around two years. Thus, we compared the p r o d u c t i v i t y of E. coti immobilized with polyacrylamide and κ-carrageenan f o r i n d u s t r i a l production of L - a s p a r t i c a c i d (Table 3). The p r o d u c t i v i t y was c a l c u l a t e d from the equation shown i n Table 3. When the p r o d u c t i v i t y of im m o b i l i z e d p r e p a r a t i o n with polyacrylamide was taken as 100, that of immobilized c e l l s with κ-carrageenan and hardened with g l u t a r aldehyde and hexamethylenediamine was 1500. TABLE 3 COMPARISON OF PRODUCTIVITY OF Εό okVLLokid COti IMMOBILIZED WITH POLYACRYLAMIDE AND CARRAGEENAN FOR PRODUCTION OF L-ASPARTIC ACID Immob i l i z a t i o n method Polyacrylamide

Aspartase Stability activity at 37°C (unit/β c e l l s ) ( h a l f - l i f e , day) 120 18,850

Relative productivity 100

Carrageenan

56,340

70

174

Carrageenan (GA)

37,460

240

397

Carrageenan (GA+HMDA)

49,400

680

1,498

GA:

glutaraldehyde; HMDA:

hexamethylenediamine

t

Productivity=J E exp(-kd«t)dt 0

E o = 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 As t h i s K-carrageenan method i s apparently more advantageous than the polyacrylamide method, we have changed conventional p o l y acrylamide method to t h i s new κ-carrageenan method f o r i n d u s t r i a l production of L - a s p a r t i c a c i d . T h i s new method g i v e s us very satisfactory results. 3.

Production of L-malic a c i d u s i n g immobilized

BSL&vZbacteMlum

Succeeding to the p r o d u c t i o n of L - a s p a r t i c a c i d , we t r i e d to apply t h i s κ-carrageenan method to improve the p r o d u c t i v i t y f o r L-malic a c i d [19].


CHiBATA


13.

195


In t h i s case, we screened again the microorganisms having higher fumarase a c t i v i t y . Compared with p r e v i o u s l y employed ZKdvibdoXzAÀjm ammonigameA, JfozvlbactznAJum ^lavum was found to have higher fumarase a c t i v i t y and showed 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 carrageenan. For i n d u s t r i a l purpose, the o p e r a t i o n a l s t a b i l i t y i s a l s o very important. Thus, the s t a b i l i t i e s were compared, and the p r o d u c t i v i t i e s of immobilized preparations f o r L-malic a c i d were c a l c u l a t e d . As shown i n Table 4, i t i s evident that κ-carrageenan method i s more advantageous than the conventional polyacrylamide method. So, we changed the polyacrylamide method to the K-carrageenan method i n 1977. T h i s new method a l s o gives us s a t i s f a c t o r y r e s u l t f o r i n d u s t r i a l production of L-malic a c i d . TABLE 4 COMPARISON OF PRODUCTIVITY OF IfozvlbatâQAÂum ammoYviouQm^ AND BKOVlbCLCtQAium ulavum IMMOBILIZED WITH POLYACRYLAMIDE AND CARRAGEENAN FOR PRODUCTION OF L-MALIC ACID

8. cmmovyLoLgmoA

Immobilization method

Activity H a l f - l i f e Relative ( u n i t / ^ at 37°C producg c e l l s ) (day) tivity P o l y a c r y l 5,800 53 100 amide (60%) Carra geenan *

5,800 (60%)

75

142

8. AZcLvum

A c t i v i t y H a l f - l i f e Relative (unit/ at 37°C producg cells) (day) 6,680 152 72 (34%) Λ

9,920 (51%)

70

226

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

Productivity=j^E exp(kd·t)dt 0

E = i n i t i a l a c t i v i t y , kd=decay constant, t=operational p e r i o d Q

Summary of K-carrageenan method Besides these immobilized m i c r o b i a l c e l l s , we a p p l i e d t h i s carrageenan method to immobilize some kinds of e x t r a c t e d enzymes. These r e s u l t s are summarized and compared with those of immo b i l i z e d p r e p a r a t i o n s using polyacrylamide (Table 5 ) . On the p o i n t of enzyme a c t i v i t y , a l l t e s t e d m i c r o b i a l c e l l s and enzymes could be immobilized i n higher a c t i v i t y by carrageenan method compared with polyacrylamide g e l method. H a l f - l i f e of im m o b i l i z e d p r e p a r a t i o n s with K-carrageenan was i n c r e a s e d by harden i n g treatment, and i t was longer than that of polyacrylamide g e l method. The reason of higher s t a b i l i t y of immobilized p r e p a r a t i o n with κ-carrageenan i s not c l e a r , but by a s e v e r a l experiments we found that "κ-carrageenan i n l i q u i d - s t a t e " does not show any s t a b i l i z i n g e f f e c t and "κ-carrageenan i n g e l - s t a t e " i s e s s e n t i a l f o r s t a b i l i z a t i o n of enzyme a c t i v i t y . T h i s r e s u l t suggests that gel-matrix of κ-carrageenan may p l a y an important r o l e f o r t h i s 4.


IMMOBILIZED


196

MICROBIAL

CELLS

TABLE 5 SUMMARY OF ENZYME ACTIVITY AND STABILITY OF IMMOBILIZED MICROBIAL CELLS AND IMMOBILIZED ENZYMES Enzyme and microorganism (enzyme)

E. coti (Aspartase)

Operational s t a b i l i t y Enzyme a c t i v i t y (unit/g c e l l s H a l f - l i f e (day) or mg p r o t e i n ) Temp. P o l y a c r y l - CarraP o l y a c r y l - Carra- (°C) None amide amide geenan 18,900 49,400* 680* 120 10 37 (73%) (49%)

4,160 St. phcLZOcM/iomoamoA (37%)

4,310* (59%)

60

-

150

(Fumarase)

5,800 (60%)

5,800 (60%)

37

6

53

75

(Fumarase)

6,680 (34%)

9,920 (51%)

37

-

72

70

Aminoacylase

10 (50%)

10 (50%)*

37

-

30

60*

Aspartase

190 (29%)

300 (46%)

37

-

20

-

Glucose isomerase

660 (12%)

4,640* (69%)

60

-

5

(Glucose

isomerase)

B. ammowLoLQQyiQA

*

hardening with glutaraldehyde

and hexamethylenediamine


289*

120*


13.

CHD3ATA


197

stabilization. Here, we summarize c h a r a c t e r i s t i c s and advantages of t h i s new i m m o b i l i z a t i o n method using K-carrageenan. (1) T h i s i m m o b i l i z a t i o n method i s a p p l i c a b l e to many kinds of en zymes and m i c r o b i a l c e l l s . (2) A c t i v i t y y i e l d of immobilized enzymes and m i c r o b i a l c e l l s i s high. (3) Various shapes of immobilized enzymes and m i c r o b i a l c e l l s s u i t a b l e f o r t h e i r a p p l i c a t i o n purposes can be e a s i l y t a i lored. (4) O p e r a t i o n a l s t a b i l i t y of immobilized enzymes and m i c r o b i a l c e l l s i s high, e s p e c i a l l y of that t r e a t e d with a p p r o p r i a t e hardening agents. (5) Number of l i v i n g immobilized c e l l s can be e a s i l y counted, as entrapped c e l l s are r e a d i l y converted to c e l l suspension by removing g e l inducing agents. Thus t h i s κ-carrageenan method i s considered to be more ad vantageous f o r i n d u s t r i a l purpose than the conventional p o l y a c r y l amide g e l method. III.

L i v i n g Immobilized M i c r o b i a l C e l l s Above described continuous enzyme r e a c t i o n s using immobilized m i c r o b i a l c e l l s f o r production purposes are p r i m a r i l y c a t a l i z e d by a s i n g l e enzyme and the immobilized c e l l s are i n dead s t a t e , though the enzyme i s i n a c t i v e s t a t e . However, many u s e f u l com pounds e s p e c i a l l y produced by fermentation method are u s u a l l y formed by m u l t i - s t e p r e a c t i o n s c a t a l y z e d with many kinds of en zymes i n l i v i n g m i c r o b i a l c e l l s . A l s o , these r e a c t i o n s o f t e n r e q u i r e generation of ATP and other coenzymes. I f immobilized c e l l s are kept i n l i v i n g s t a t e , they may be a p p l i e d f o r these m u l t i enzyme r e a c t i o n s . We a p p l i e d κ-carrageenan method to the l i v i n g immobilized yeast c e l l s and b a c t e r i a l c e l l s f o r productions of ethanol and L - i s o l e u c i n e , r e s p e c t i v e l y . 1.

Production of ethanol using immobilized yeast c e l l s The i m m o b i l i z a t i o n of yeast, Saccha/iomycZA (LOXJUb

Immobilized Microbial Cells with Polyacrylamide Gel and

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