9 Enzymatic Synthesis of Pantothenic Acid by Escherichia coli Cells
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Y. KAWABATA and A. L. DEMAIN Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, MA 02139
Pantothenic a c i d i s both a component o f coenzyme A and a v i t a min o f importance i n human and animal n u t r i t i o n . At present, i t i s produced commercially by chemical s y n t h e s i s . Pantothenic a c i d i s produced n a t u r a l l y by most m i c r o b i a l s p e c i e s , i t s formation being c a t a l y z e d by pantothenic a c i d synthetase (EC.6.3.2.1) (1,2). The enzyme-catalyzed r e a c t i o n i s as f o l l o w s :
CHo I HOH C - C - CH(OH)COOH + NH CH CH COOH + ATP 2
2
CH
2
3
pantoic acid
«
2
β-alanine
HOH C — C — CH(OH)CONHCH CH COOH + AMP + P P 2
2
CH
2
±
3
pantothenic a c i d Since pantothenic a c i d contains an asymmetric carbon atom, chemical s y n t h e s i s y i e l d s the racemic mixture. On the other hand, when enzymatic s y n t h e s i s i s used, only the b i o l o g i c a l l y a c t i v e form o f pantothenic a c i d i s produced. In the present work, the use o f E s c h e r i c h i a c o l i c e l l s , i n stead o f enzyme p r e p a r a t i o n s , as the source o f pantothenic a c i d synthetase was s t u d i e d . Success was achieved i n the p r o d u c t i o n o f the v i t a m i n by frozen-thawed cells. Pantothenate
Production by C e l l s of E. c o l i ATCC 9637
IS. c o l i ATCC 9637 was chosen f o r our s t u d i e s s i n c e a c o n s i d e r a b l e amount o f work has been done on the pantothenic a c i d syn thetase o f t h i s organism (_3,4_,5) . The c u l t u r e was grown i n a
0-8412-0508-6/79/47-106-133$05.00/0 © 1979 American Chemical Society In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
134
IMMOBILIZED MICROBIAL CELLS
medium c o n t a i n i n g glucose, ammonium l a c t a t e , enzyme- or a c i d hydrolyzed c a s e i n and yeast e x t r a c t ( a l l a t 2 g / l i t e r ) p l u s mine r a l s a l t s . Assay of the enzyme a c t i v i t y was c a r r i e d out i n a 2-ml volume c o n t a i n i n g 40 ymoles $-alanine, 40 ymoles potassium pantoate, 20 ymoles disodium ATP, 200 ymoles K C l , 20 ymoles MgS0 , 70 ymoles T r i s * H C l b u f f e r and 0.2 ml o f an enzyme source. Pantothenate was determined by a disk-agar d i f f u s i o n assay (6) u s i n g L a c t o b a c i l l u s plantarum ATCC 8014. In an e a r l y experiment, we learned t h a t the enzyme was produced d u r i n g the e x p o n e n t i a l growth phase and d e c l i n e d i n a c t i v i t y t h e r e a f t e r . T h i s experiment was done u s i n g c e l l s as enzyme source, since r e s t i n g c e l l s o f E. c o l i had been reported (_3) t o produce and excrete pantothenic a c i d . Since our c e l l s were washed twice with b u f f e r e d s a l i n e and stored overnight i n the r e f r i g e r a t o r before being t e s t e d , i t was reasonable t o assume t h a t t h e i r permea b i l i t y p r o p e r t i e s could have been a l t e r e d . T h i s assumption was confirmed by the o b s e r v a t i o n that such c e l l s were markedly h i n dered i n t h e i r a b i l i t y t o produce pantothenate when ATP was omitted from the r e a c t i o n mixture. T h i s dependency on exogenous ATP suggested t o us that c e l l p r e p a r a t i o n s t r e a t e d even more d r a s t i c a l l y would be more a c t i v e i n pantothenate p r o d u c t i o n . Indeed, we found t h a t f r e e z i n g and thawing the c e l l s markedly i n creased a c t i v i t y . A comparison of frozen-thawed c e l l s , acetone-dried c e l l s and a b u f f e r e x t r a c t o f acetone-dried c e l l s showed the order o f decreasing a c t i v i t y (on a v o l u m e t r i c b r o t h b a s i s ) t o be acetoned r i e d c e l l s > b u f f e r e x t r a c t > frozen-thawed c e l l s . Despite t h i s o b s e r v a t i o n , we decided t o use frozen-thawed c e l l s f o r our f u r t h e r s t u d i e s f o r the f o l l o w i n g reasons: a) the p r e p a r a t i o n of such an enzyme source i s very simple and convenient; b) i f the process e v e n t u a l l y assumes i n d u s t r i a l importance, the l a r g e amounts o f acetone and ether needed t o prepare acetone-dried c e l l s or ext r a c t s therefrom would be both expensive and a waste-treatment problem.
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4
P r o p e r t i e s of Frozen-thawed
Cells
Using frozen-thawed c e l l s , we next determined the importance of each component of the r e a c t i o n mixture. Omission of 3-alanine, K-pantoate, ATP, MgSO^ o r enzyme e l i m i n a t e d v i r t u a l l y a l l a c t i v i t y . The product found i n the e x t r a c e l l u l a r f l u i d i n the presence o f a l l components was pantothenate, as i d e n t i f i e d by bioautography on paper. Although p e r m e a b i l i t y m o d i f i c a t i o n i s c e r t a i n l y important f o r producing pantothenate by c e l l p r e p a r a t i o n s , the a l t e r a t i o n produced by f r e e z i n g and thawing must be r a t h e r s u b t l e , since pantot h e n i c a c i d synthetase was found not t o leak out o f the f r o z e n thawed c e l l s (Figure 1). T h i s experiment was done by allowing a r e a c t i o n t o proceed f o r 20 hours, a t which time the c e l l s were removed by c e n t r i f u g a t i o n . L i t t l e t o no pantothenate was p r o duced d u r i n g the next 28 hours i n the presence o f the supernatant
In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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AND DEMAIN
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Synthesis of Pantothenic Acid
fluid. However, i n a c o n t r o l r e a c t i o n mixture t h a t was not cen t r i f u g e a , pantothenic a c i d was produced i n a l i n e a r f a s h i o n throughout the 48-hour i n c u b a t i o n p e r i o d . The optimum pH f o r pantothenate formation by frozen-thawed c e l l s was found t o be 8.0 and the optimum temperature about 50°C. Maas (_3) reported t h a t e x t r a c t s of acetone-dried c e l l s were optimal a t pH 8.5 t o 9.0, and r e s t i n g c e l l s a t 7.0 t o 7.5. Miyatake e t a l . (4^,5) found the optimum f o r pure enzyme t o be 10.0. Frozen-thawed c e l l s apparently behave w i t h respect t o pH i n an intermediate manner r e f l e c t i n g t h e i r nature as being n e i t h e r c e l l f r e e nor i n t a c t . Of importance i s the f i n d i n g o f Maas {3) t h a t e x t r a c t s of acetone-dried c e l l s have an optimum temperature of 25°C, are s t a b l e a t 35°C, but lose a c t i v i t y a f t e r one hour a t 45°C. Miyatake e t a l . i^,5) found 30°C t o be optimal f o r pure enzyme and a 10-minute i n c u b a t i o n a t 60°C t o i n a c t i v a t e the enzyme almost completely. In c o n t r a s t t o the reported l a b i l i t y of e x t r a c t s and pure enzymes, our optimum temperature of 50°C f o r frozen-thawed c e l l s was determined i n a prolonged experiment t h a t l a s t e d 24 hours. I t i s thus obvious t h a t frozen-thawed c e l l s have a s t a b i l i t y advantage over both e x t r a c t s and pure enzymes. Immobilization of Frozen-thawed
Cells
Immobilization was s t u d i e d as a p o t e n t i a l means t o increase s t a b i l i t y f u r t h e r and t o f a c i l i t a t e re-use o f the p r e p a r a t i o n . Agar was chosen as the support to be t e s t e d because of convenience and the m i l d c o n d i t i o n s necessary f o r i m m o b i l i z a t i o n . A 3% agar s o l u t i o n at 40-50°C was mixed with a concentrated suspension o f frozen-thawed c e l l s h e l d a t 30-40°C. The suspension was poured i n t o 1-ml aluminum cups and allowed t o s o l i d i f y . A c y l i n d e r o f agar (12 mm diameter χ 10 mm) was removed from each cup and used as the enzyme source. The enzyme a c t i v i t y of t h i s immobilized p r e p a r a t i o n i s shown i n F i g u r e 2; the requirement f o r ATP i s e a s i l y seen. Such p r e p a r a t i o n s have a h a l f - l i f e o f about 100 hours a t 37°C when incubated i n the presence of r e a c t i o n mixture. About 1000 hours are necessary t o i n a c t i v a t e such a p r e p a r a t i o n completely. Discussion Although i t was known almost 30 years ago (3_ 7) t h a t r e s t i n g c e l l s of E. c o l i could produce pantothenate from pantoate and 3-alanine, only i n the present work was i t found t h a t c e l l s r e spond t o exogenous ATP. We were pleased to f i n d t h a t f r o z e n thawed c e l l s responded to an even greater degree, and t h a t such p r e p a r a t i o n s were more s t a b l e than crude e x t r a c t s of acetoned r i e d c e l l s or pure enzyme. These f i n d i n g s p o i n t t o the use o f immobilized c e l l s f o r the p r o d u c t i o n o f pantothenic a c i d and, indeed, we found t h a t agar-immobilized c e l l s are a c t i v e . I t f
In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
IMMOBILIZED MICROBIAL CELLS
136
• cΕ 600L
-
5L
o
400L-
/CONTROL CENTRIFUGATION
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Lu
/
%/
< LU Χ
r^SUPERNATANT
200
Ζ
2
I
24 TIME (hours)
1
48
Figure 1. Nonleakage of pantothenic acid synthetase from frozen-thawed cells. After 20 hr of reaction, the cells were removed from the experimental reaction
0 2 4
6
24 TIME (hours)
Figure 2. Effect of ATP on pantothenic acid synthetase activity of frozenthawed cells immobilized in agar. The experiment was carried out at 30°C and pH 8.0
In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
9.
KAWABATA
AND DEMAIN
Synthesis of Pantothenic Acid
137
should be noted that a p o s i t i v e response t o ATP by c e l l u l a r prep a r a t i o n s has a l s o been observed i n other systems (8,9,10/11/12).
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Abstract Pantothenic acid, a vitamin of importance in human and animal nutrition, is produced commercially by chemical synthesis. We investigated its bioconversion from β-alanine, potassium pantoate and ATP by various types of Escherichia coli ATCC 9637 cell prep arations in Tris buffer containing KCl and MgSO . We found ex ponential phase cells to be most capable of carrying out this reaction. ATP had a marked stimulatory effect on the reaction even though cells were used. Pantothenate production was also observed with acetone-dried cells and crude extracts. A marked increase in pantothenate production by cells was effected by freezing and thawing. No enzyme activity leaked from the cells during 20 hours of reaction. Production by frozen-thawed cells was considerably more stable to heat than that reported for crude ex tracts or pure enzyme. Immobilization of frozen-thawed cells in agar yielded active preparations, which required 1000 hours at 37°C for complete inactivation. 4
Acknowledgment ; The authors are indebted t o Toray I n d u s t r i e s , Inc., f o r f i n a n c i a l support o f Y. K. during h i s stay a t M. I . T. Y. K. *s permanent address i s : Basic Research L a b o r a t o r i e s , Toray I n d u s t r i e s , Inc., Kamakura, Japan.
Literature Cited 1.
Brown, G.M. Biosynthesis of pantothenic acid and coenzyme A. In: "Comprehensive Biochemistry," vol. 21 (M. Florkin and E.H. Stotz, eds.), p. 75. Elsevier, Amsterdam, 1971. 2. Matsuyama, A. Bull. Agr. Chem. Soc. (Japan) (1957) 21, 47. 3. Maas, W.K. J. Biol. Chem. (1952) 198, 23. 4. Miyatake, K., Nakano, Y. and Kitaoka, S. Agr. Biol. Chem. (1973) 37, 1205. 5. Miyatake, Κ., Nakano, Y. and Kitaoka, S. J. Nutr. Sci. Vitaminol. (1978) 24, 243. 6. Kojima, Η., Matsuya, Υ., Ozawa, Η., Konno, M. and Uemura, T. J. Agr. Chem. Soc. (Japan) (1958) 32, 33. 7. Maas, W.K. J. Bacteriol. (1950) 60, 734. 8. Ogata, Κ., Shimizu, S. and Tani, Y. Agr. Biol. Chem. (1970) 34, 1757. 9. Shimizu, S., Miyata, Κ., Tani, Y. and Ogata, K. Biochim. Biophys. Acta (1972) 279, 583. 10. Shimizu, S., Tani, Y. and Ogata, K. Agr. Biol. Chem. (1972) 36, 370. 11. Shimizu, S., Morioka, Η., Tani, Y. and Ogata, K. J. Ferm. Technol. (1975) 53, 77. 12. Uchida, T., Watanabe, T., Kato, J. and Chibata, I. Biotech. Bioeng. (1978) 20, 255. RECEIVED
February 15, 1979.
In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
