by a strain of Bacillus subtilis, designated MIR-5 which we had isolated through ... sugars were determined using the 3,5-dinitrosalicylic reagent. (Bruner, 1964) ...
Biotechnology Letters Vol 14 No 1 Received as revised 13th December
49-54
(1992)
SIMULTANEOUS PRODUCTION OF ALPHA AND BETA AMYLASES BY BACILLUS
SUBTILIS M I R - 5 I N BATCH AND CONTINUOUS CULTURE = .
G u i l l e r m o R. C a s t r o ~.2C~), M a r c e l a A b a t e =, B e a t r i z S. M ~ n d e z ~, and
A. F e r r e r o =, C a r l o s F a u s t i n o S i ~ e r i z 2.
M.
~Dto. Qulmica B i o l b g i c a , Fac. Cs. Exactas y N a t u r a l e s , UBA, Cdad. U n i v e r s i t a r i a , 1 4 2 8 Buenos A i r e s , and =Catedra de M i c r o D i o [ o g i a S u p e r i o r , Fac. B i o q c a . , Qca. y Farm., UNT-PROIMIMIRCEN, Avda. Belgrano y P j e . Caseros, 4000 Tucum~n, A r g e n t i n a .
SUMMARY
Simultaneous p r o d u c t i o n o f a- and R-amylase was s t u d i e d i n batch and c o n t i n u o u s c u l t u r e u s i n g s t a r c h as s u b s t r a t e i n B. s u b t i l i s MIR-5. By m a n i p u l a t i n g t h e c u l t u r a l c o n d i t i o n s , both enzymes could then be produced by the same s t r a i n . INTRODUCTION
The p r o d u c t i o n o f sweeteners by enzymatic c o n v e r s i o n o f s t a r c h i n v o l v e s l i q u e f a c t i o n and s a c c h a r i f i c a t i o n o f the s u b s t r a t e . The enzymes are u s u a l l y an ~-amylase i n t h e f i r s t s t a g e , and a m y l o g l u c o s i d a s e i n a second s t a g e ; a l t e r n a t i v e l y i t i s p o s s i b l e t o use p u l l u l a n a s e w i t h B-amylase f o r t h e s a c c h a r i f i c a t i o n s t a g e ( L u e n s e r , 1983). G e n e r a l l y , these enzymes a r e o b t a i n e d from d i f f e r e n t n a t u r a l s o u r c e s . I n d u s t r i a l l i q u e f y i n g t h e r m o s t a b l e ~-amylase i s o b t a i n e d from s e v e r a l s t r a i n s o f the genus B a c i l l u s and B-amylase i s e x t r a c t e d from p l a n t s (sweet p o t a t o , b a r l e y , e t c . . . ) in a tedious process. More r e c e n t l y , t h e s i m u l t a n e o u s presence o f d i f f e r e n t enzymes i n some B a c i l l u s spp. has been d e s c r i b e d : a-amylase and p u l l u l a n a s e i n B. s u b t i l i s ( T a k a s a k i , 1987), ~- and B-amylases i n B. megaterium ( S t a r k e t a l . , 1982) or i n B a c i l l u s polymyxa (Uozumi e t a l . , 1989). However, t h e l i t e r a t u r e c o n c e r n i n g the p r o d u c t i o n o f these enzyme i s very s c a r c e ( P r i e s t e t a l . , 1985; A n t r a n i k i a n e t a l . , 1987), and no r e p o r t i s a v a i l a b l e t o our knowledge, d e a l i n g w i t h the s i m u l t a n e o u s p r o d u c t i o n o f b a c t e r i a l ~- and 8-amylases w i t h a comparison and t h e comparisson o f batch and c o n t i n u o u s c u l t u r e t e c h n i q u e s , The aim o f t h i s work was t o study t h e p r o d u c t i o n o f e x t r a c e l l u l a r ~- and B-amylases i n batch and c o n t i n u o u s c u l t u r e by a s t r a i n o f B a c i l l u s s u b t i l i s , d e s i g n a t e d MIR-5 which we had i s o l a t e d t h r o u g h a s c r e e n i n g program from n a t u r a l s o u r c e s . MATERIALS AND METHODS
S t r a i n c h a r a c t e r i z a t i o n . The c h a r a c t e r i z a t i o n was done following international criteria ( P r i e s t e t a l . , 1988; Sneath, 19~6)
..........................
= F o o t n o t e : d e d i c a t e d t o 75 ~
a n i v e r s s a r y o f Dr. Rabl E. T r u c c o . 9
Medium. The minimal s a l i n e medium by Zhang e t a l . (1983), w i t h m o d i f i c a t i o n s was used; i t contained i n g / l : (NH4)=S04, I ; K=HP04, 6; KH=P04, 3; ZnSO4.7H=O, 0.001; MgSO4.4H=O, 0 . 0 1 ; CaCl=.2H=O~ 0 . 0 5 ; FeSO4.7H=O, 0.001; N a = C i t r a t e , 1 . 0 ; s o l u b l e starch~ 7 . 0 , and the pH was a d j u s t e d t o 7 . 0 . The r e a g e n t s were o f a n a l y t i c a l o r b a c t e r i o l o g l c a l grade from Merck (Darmstadt, Germany). Batch and chemostat c ~ l t u r e . The s e l e c t e d s t r a i n was grown i n a Gallenkamp f e r m e n t o r (London, UK) w i t h a working volume of 400 ml, pH 7.0 and 45 "C. A e r a t i o n was s e t t l e d a t 1.0 • 0.2 v / v min. and a g i t a t i o n a t 180 rpm. Growth was measured as o p t i c a l d e n s i t y a t 560 nm d i l u t i n g w i t h 0.85% NaC1 when n e c e s s a r y . Assays. P r o t e i n s were measured by the neocuproine method ( C a s t r o e t a l . , 1991a) using Bovine Serum Albumin f r a c t i o n V as s t a n d a r d . Starch was measured by the Blue index method ( B e r n f e l d , 1 9 5 5 ) using s o l u b l e s t a r c h as s t a n d a r d . Reducing sugars were determined using the 3 , 5 - d i n i t r o s a l i c y l i c reagent ( B r u n e r , 1964), using maltose as s t a n d a r d . Glucose was determined e n z y m a t i c a l l y using the o x i d a s e - p e r o x i d a s e system ( T e s t Combination g l u c o s e , Boehringer-Mannheim GmbH D i a g n o s t i c a , Germany). Enzymatic assays. E x t r a c e l l u l a r a c t i v i t i e s were determined by s p i n n i n g down the c u l t u r e samples f o r 20 min a t IO,000 x g i n the cold (2-4 "C). D e x t r i n i z i n g a c t i v i t y a-amylase ( E . C . 3 . 2 . 1 . I ) was assayed using the i o d i n e reagent and s o l u b l e s t a r c h , 0.5 g/1 i n I00 mM phosphate/15 mM NaC1 b u f f e r , pH 7 . 0 . The a-amylase u n i t i s d e f i n e d as t h e amount o f enzyme r e q u i r e d t o degrade I 0 ~g o f s t a r c h i n 30 min a t 45 "C. B-amylase ( E . C . 3 . 2 . 1 . 2 ) was assayed w i t h 3,5 d i n i t r o s a l i c y l i c using the same m i x t u r e as b e f o r e but a t pH 6 . 0 . The B-amylase u n i t i s d e f i n e d as the amount o f enzyme r e q u i r e d t o produce I ~mol o f reducing sugars (as m a l t o s e ) p e r min. a t 45 "C. RESULTS The screening procedure d e s c r i b e d i n p r e v i o u s work ( C a s t r o e t a l . , 1991b) allowed the s e l e c t i o n o f t h e s t r a i n MIR-5. I t was i d e n t i f i e d as B a c i l l u s s u b t i l i s ( d a t a not shown). The r e s u l t s o b t a i n e d i n batch i n e x p e r i m e n t s using minimal s a l i n e medium w i t h s t a r c h as s u b s t r a t e are presented i n F i g . I . a-Amylase showed a maximum o f 8,400 U / l i n the l a t e e x p o n e n t i a l growth phase, however o n l y 19 % was e x t r a c e l l u l a r and t h e ' c o n c e n t r a t i o n remained more or l e s s c o n s t a n t . The maximum o f Bamylase occurred e a r l i e r i n the e x p o n e n t i a l growth phase w i t h an a c t i v i t y o f 522 U / l , 87 % being e x t r a c e l l u l a r ; the a c t i v i t y decreased a f t e r w a r d s . The b e h a v i o r o f s t r a i n MIR-5 was s t u d i e d i n continuous c u l t u r e a t d i l u t i o n r a t e s i n the range 0 . I t o 0.45 h-~ using m i n e r a l s a l i n e medium w i t h s t a r c h as s u b s t r a t e ( F i g . 2 ) . The range w a s s e l e c t e d t a k i n g i n t o account t h a t the c r i t i c a l d i l u t i o n r a t e o b t a i n e d by the washout method was 0.47 h-~; lower d i l u t i o n r a t e s were not used i n o r d e r to s t a y over the l i m i t (10-15 % o f ~max.) under which chemostat t h e o r y does not a p p l y ( P i t t , 1972). The p r o t e i n c o n c e n t r a t i o n i n the s u p e r n a t a n t was h i g h e s t a t D= 0 . 1 5 - 0 . 2 5 h- ~ , the same range a t which e x t r a c e l l u l a r a-amylase c o n c e n t r a t i o n was maximal.
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