Papp, Péter

Polyák, Béla

Szajáni, Béla

Ábrahám, Magdolna

English

Ábrahám Magdolna

Polyák Béla

Papp Péter

Szajáni Béla

University of Szeged

A c t a B i o l . S z e g e d . 35. pp. 231—205 (1989) SHORT COMMUNICATION AMINO ACID PRODUCTION ACCOMPANYING GLUTATHIONE SYNTHESIS BY IMMOBILIZED SACCHAROMYCES CEREVISIAE CELLS M . Á B R A H Á M . B . P O L Y Á K , P. P A P P A N D B. SZAJÁNI Department of Biochemistry. Attila József University. H—6701 Szeged. P. O. B. 533. Hungary ( R e c e v i e d : O c t o b e r 3 1 . 1988) Abstract T h e g l u t a t h i o n e s y n t h e s i s o f Saccharomyces cerevisiae ce l ls e n t r a p p e d i n p o l y a c r y l a m i d e gel w a s f o u n d t o be a c c o m p a n i e d b y a m i n o a c i d p r o d u c t i o n a s a c o n s e q u e n c e o f t h e i m m o b i l i z a t i o n , a p a r t i a l re lease o f t h e a m i n o a c i d p o o l w a s a l s o o b s e r v e d . Key words: i m m o b i l i z e d ce l ls , g l u t a t h i o n e p r o d u c t i o n , a m i n o a c i d Introduction Immobilized microbial cells containing the intact enzyme system of glycolysis are able to regenerate ATP for the synthesis of glutathione ( M U R A T A et al., 1978). The entrapment of Saccharomyces cerevisiae cells in polyacrylamide gel caused a change in the membrane permeability ( M U R A T A et al., 1981). Glutathione and NAD4 were released from the cells, but ATP was retained. By the addition of NAD*, the ATP regenerating system was completed and the ATP produced served as energy donor for the continuous production of glutathione. We have detected a significant quantity of different amino acids released together with glutathione owing to de novo synthesis, as well as a partial release of the amino acid pool. Materials and methods C U L T I V A T I O N A N D IMMOBIL IZATION. S. cerevisiae s t r a i n I F O 2044 w a s o b t a i n e d f r o m t h e I n s t i t u t e o f F e r m e n t a t i o n . O s a k a . J a p a n . C e l l s w e r e c u l t i v a t e d a n d e n t r a p p e d in p o l y a c r y l a m i d e ge l a c c o r d i n g t o M U R A T A et a l . ( 1 9 8 1 ) . ASSAY OF G L U T A T H I O N E A N D A M I N O A C I D PRODUCTION. 1.2 g o f gel p a r t i c l e s c o n t a i n i n g 0 . 5 g o f ce l l s w a s i n c u b a t e d in 2 0 m l o f t he c u l t u r e m e d i u m s u g g e s t e d b y M U R A T A et a l . ( 1 9 8 1 ) a t 3 0 C f o r 24 h o u r s w i t h c o n t i n u o u s s h a k i n g . A t a p p r o p r i a t e t i m e s . 0 .5 m l o f s a m p l e w a s r e m o v e d a n d the c o n c e n t r a t i o n s o f r e a c t a n t s a n d p r o d u c t s w e r e d e t e r m i n e d . G l u t a t h i o n e a n d a m i n o a c i d s w e r e m e a s u r e d b y q u a n t i t a t i v e t h i n - l a y e r c h r o m a t o g r a p h y o n K i e s e l g e l 6 0 F 2 5 4 c h r o m a t o p l a t e s ( M e r c k A G . D a r m s t a d t . F R G ) ( A B R A H A M et a l . . 1983) . T h e s p o t ' s w e r e d e t e c t e d i n T e l e c h r o m O H — 9 7 4 v i d e o d e n s i t o m e t e r ( C h i n o i n . B u d a p e s t , H u n g a r y ) . E t h a n o l w a s d e t e r m i n e d b y g a s c h r o m a t o g r a p h y , u s i n g a C h r o m 4 G C c h r o m a t o g r a p h ( L a b o r a t o r n i P r i s t r o j e P r a g u e . C z e c h o s l o v a k i a ) e q u i p p e d w i t h a flame i o n i z a t i o n d e t e c t o r a n d a P o r a p a k Q ( 8 0 — 1 0 0 m e s h ) c o l u m n . N i t r o g e n w a s used as a c a r r i e r gas a n d m e t h a n o l as a n i n t e r n a l s t a n d a r d . 232 M . A b r a h a m . B. P o l y a k , P Papp a n d B. S z a j a m Results and discussion A mixture containing the glutathione constituent amino acids (i. e. glycine, glutamic acid and cysteine) in equimolar quantities was incubated with S. cerevisiae cells entrapped in polyacrylamide gel. Besides glutathione and the glutathione constituent amino acids, aspartic acid, alanine, glutamine and serine were detected in the reaction mixture (Table 1). luble I. Release o f m e t a b o l i t e s a n d a m i n o a c i d p o o l f r o m S. cerevisiae ce l l s i m m o b i l i z e d i n p o l y a c r y l a m i d e gel I n c u b a t i o n E t h a n o l G l u t a t h i o n e A s p A l a G i n S e r t i m e ( h r ) ( u m o l ) ( u m o l ) ( u m o l ) ( u r a o ! ) ( u m o l ) ( u m o l ) 0 53 0 4 0 0 0 — 2 174 2 56 56 6 0 6 4 347 9 3 4 100 110 12 8 1346 13 34 132 136 4 0 24 5646 14 34 192 176 6 0 On this basis it was assumed that the cell membrane became permeable for glutathione and for certain amino acids as a consequence of the immobilization. Aspartic acid was released before the start of fermentation. Its concentration in the effluent remained relatively high and constant during a fairly long period. The concentrations of glutathione, alanine, glutamine and serine increased in parallel with the concentration of ethanol, showing the connection between the synthesis as energy consumer and the glycolysis as energy supplier. The synthesis of alanine, glutamine and serine presumably requires a lower ATP level than does the peptide synthesis. Aspartic acid was released from the amino acid pool of the damaged cells, but its synthesis could not be excluded. Acknowledgements We wish to thank Reanal Laboratory Chemicals (Budapest. Hungary) for financial support of this work. References A B R A H A M . M . . POLYAK. B. . S / A J A N I . B . a n d BOROSS, L . ( 1 9 8 3 ) : A n a l y t i c a l S e p a r a t i o n o f R e d u c e d a n d O x i d i z e d F o r m s o f G l u t a t h i o n e f r o m A m i n o A c i d M i x t u r e s b y O v e r p r c s s u r e d T h i n - L a y e r C h r o m a t o g r a p h y . — J. L i q u i d C h r o m . 6, 2 6 3 5 — 2 6 4 5 . M i ; R A T A . K . . T A N I . K . . K A T O . J. a n d CHIBATA. I . . (1978) : C o n t i n u o u s P r o d u c t i o n o f G l u t a t h i o n e b y I m m o b i l i z e d S a c c h a r o m y c e s c e r e v i s i a e Ce l l s . — E u r . J . A p p l . M i c r o b i o l . B i o t e c h n o l . ft. 2 3 — 2 7 . M L R A T A . K . . T A N I . K . . K A T O , J. a n d C H I B A T A . I . ( 1 9 8 1 ) : G l u t a t h i o n e P r o d u c t i o n b y I m m o b i l i z e d Saccharomyces cerevisiae C e l l s C o n t a i n i n g a n A T P R e g e n e r a t i o n S y s t e m . F.ur . J A p p l . M i c r o b i o l . B i o t e c h n o l . I I , 7 2 — 7 7 . 

Amino acid production accompanying glutathione synthesis by immobilized Saccharomyces cerevisiae cells

http://acta.bibl.u-szeged.hu/22140/1/biologica_035_231-232.pdf

Amino acid production accompanying glutathione synthesis by immobilized Saccharomyces cerevisiae cells

Abstract

Similar works

Full text

Available Versions

University of Szeged