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E L S E V IE R Carbohydrate Research 273 (1995) 235-242

CARBOHYDRATERESEARCH

Chitin deacetylation by enzymatic means:m onitoring o f deacetylat ion pro cesses

A . M a r t i n o u a,b , D . K a f e t z o p o u l o s a ,b , V . B o u r i o t i s a ,h ,,a Enzyme Technology Division, Institute o f Molecular B iology and Biotechnology, P .O. Bo x 1515, Heraklion711 10, Crete, Greece

b Department of Biology, Division of Applied B iology and Biotechnology, University of Crete, P.O. Box 14 70,Heraklion 711 10, Crete, GreeceReceived 1 Augu st 19 94; accepted in revised form 20 F ebruary 1995

A b s t r a c t

A metho d fo r m oni to r ing enzym at i c deace ty l a t ion p rocess es o f na tu ra l o r a r t i f ic i a l ch i t i nsubs t r a t es as we l l a s N-ac e ty l ch i too l igo sacch ar ides by the d i r ec t de t e rmina t ion o f t he ace t a t er e l eased i s desc r ibed . Fur the rmore , a new assay i s p r esen ted fo r t he de t e rmina t ion o f ch i t i nd e a c e t y l a s e a c t i v i ty e m p l o y i n g h e x a - N - a c e t y l c h it o h e x a o s e [ ( G I c N A c )6] as subs t r a te and m easur ingt h e a c e t a te r e l e a s e d e n z y m a t i c a l ly . T h e K m v a l u e f o r ( G I c N A c )6 h a s b e e n d e t e r m i n e d a s w e l l a st h e p H a n d t e m p e r a t u re d e p e n d e n c e o f a c t i v i ty a n d t h e t h e r m o s t a b i l it y o f t h e e n z y m e . F i n a l l y ,i n i t i a l s tud ies on the e f f ec t iveness o f t he enzyme on var ious ch i t i n and ch i tosan subs t r a t es a r ep resen ted .Keywords: Chitin; Chitosan; Chitin deacetylase; Mucor rouxii; Chitin deacetylation

1 . I n t r o d u c t i o n

C h i ti n , a h o m o p o l y m e r o f f l- (1 --* 4 ) - l i n k e d N - a c e t y l - o - g l u c o s a m i n e , is o n e o f t h em o s t a b u n d a n t , e a s i l y o b t a i n e d , a n d r e n e w a b l e n a t u r a l p o l y m e r s , s e c o n d o n l y t oc e l lu l o s e . It is c o m m o n l y f o u n d i n t h e e x o s k e l e t o n s o r c u t i c l e s o f m a n y i n v e r t e b ra t e s [1 ]a n d i n t h e c e l l w a l l s o f m o s t f u n g i a n d s o m e a l g a e [ 2] . C h i t i n e x i s t s in s e v e r a lZ y g o m y c e t e s s p e c i e s i n i t s d e a c e t y l a t e d fo r m a s c h i t o s a n [ 3,4 ].

* Corresponding author.0008-6215/95/$09.50 1995 Elsevier Science Ltd. A ll rights reservedS S D I 0 0 0 8 - 6 2 1 5 ( 9 5 ) 0 0 1 1 1 - 5

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236 A. Martinou et al. / Carbohydrate Research 273 (1995) 235-242C h i t o s a n i s a b i o p o l y m e r w i t h u n i q u e p r o p e r ti e s f a v o u r a b l e f o r a b r o a d v a r i e t y o f

indus t r i a l and b iomedica l app l i ca t i ons [5 -7] . P resen t l y , ch i t osan i s p roduced by t hethe rm och em ica l a lka l i ne deac e ty l a t i on o f c r ab ch i t i n . To deve lop an a l t e rna t ive , con-t ro l le d , n o n d e g r a d a t i v e a n d w e l l - d e f i n e d e n z y m a t i c p r o c e s s f o r c h i t o s a n p r o d u c t io n , as tudy of funga l ch i t i n deace ty l ases has been i n i t i a t ed . Fur the rmore , t he degree o fd e a c e t y l a t i o n o f c h i t o s a n h a s b e e n f o u n d t o i n f l u e n c e t h e p h y s i c a l a n d c h e m i c a lproper t i e s a s we l l a s t he b io log i ca l ac t i v i t ie s o f ch i t osan [8 ] . Thus , m oni to r ing o fdeac e ty l a t i on p rocesses i s o f pa r t i cu l a r impo r t ance .

C h i t i n d e a c e t y l a s e ( C D A ) f r o m Mucor rouxii h a s b e e n p u r i f i e d t o h o m o g e n e i t y[ 9,1 0 ]. T h e e n z y m e i s an a c id i c g l y c o p r o t e i n o f ~ 7 5 k D a w i t h 3 0 % ( w / w ) c a r b o h y -dra t e con t en t . Fur the r b iochemica l cha rac t e r i za t i on r evea l ed t ha t t he enzyme has a ve rynar row spec i f i c i t y fo r ch i t i nous subs t r a tes . Fur the rmo re , a cDN A to t he Mucor rouxiim R N A - e n c o d i n g c h i ti n d e a c e t y l a s e w a s i s o la t e d , c h a ra c t e ri z e d , a n d s e q u e n c e d [1 1 ].P ro t e in sequence compar i sons r evea l ed s i gn i f i can t s imi l a r i t i e s o f t he funga l ch i t i nd e a c e t y l a s e t o r h i z o b i a l n o d B p r o te i n s , s u g g e s t i n g f u n c t io n a l h o m o l o g y o f t h e s e e v o l u -t i onary-d i s t an t p ro t e ins .

A r a d i o a c ti v e a s s a y e m p l o y i n g O - h y d r o x y e t h y l c h i t in ( g l y c o l c h i ti n ), r a d i o l a b e ll e d i nthe N-ace ty l g roup , a s subs t r a t e has been r epor t ed fo r t he de t e rmina t i on o f ch i t i ndeace ty l ase ac t i v i ty [12] . A l th oug h th i s me th od i s ve ry sens i t ive , i t is no t easy t oe v a l u a t e ( i ) t h e e x t e n t a n d d i s tr ib u t io n o f d e r i v a t iz a t io n ( O - h y d r o x y e t h y l g ro u p s ) i ng l y c o l c h i t i n c o m m e r c i a l l y a v a i l a b l e a n d (ii) t h e e f f e c t o f d e r i v a t i z a t i o n o n e n z y m ea c t iv i ty . F u r t h e rm o r e , t h is m e t h o d c a n n o t b e u s e d f o r m o n i t o r in g d e a c e t y l a t i o n p r o c e s se sof no nrad io l abe l l ed na tura l subs tr a t es.I n t h i s s t u d y w e r e p o r t a n e w m e t h o d f o r m o n i t o r i n g e n z y m a t i c d e a c e t y l a t i o nprocesses , us ing e i t he r na tu ra l o r a r t i f i c i a l ch i t i n subs t r a t es , and de t e rmin ing ch i t i ndeace ty l ase ac t iv i t y .

2. ExperimentalMaterials.--Enzymes a n d r e a g e n t s f o r a c e t a te d e t e r m i n a t io n w e r e o b t a i n e d f r o m

B o e h r i n g e r M a n n h e i m B i o c h e m i c a . N - A c e t y l c h i t o o li g o s a c c h a r i d e s w e r e p u r c h a s e d f r o mA c c u r a t e C h e m i c a l s ( U S A ) . G l y c o l c h i t o s a n w a s p u r c h a s e d f r o m S i g m a . A c e t y l a t e dc h i t o s a n s ( 2 8 % a n d 4 2 % ) f r o m Euphasia superba w e r e a g i f t f r o m D r B . F o c h e r(S t az ione Sper imenta l e Ce l lu losa , Car t a e F ibre Tess i l i , Mi l an , I t a l y ) . Amorphous andc r y s ta l li n e c h it in f r o m s h r im p a n d c r a b w e r e a g i f t f r o m D r K . M . V ~ tr um ( N o r w e g i a nB i o p o l y m e r L a b o r a t o ry , I n st it u te o f B i o t e c h n o l o g y , T r o n d h e i m , N o r w a y ) . H y a l u r o n a nw a s p u r c h a s e d f r o m S i g m a . Mucor rouxii ( A T C C N o 2 4 9 0 5 ) w a s o b t a i n e d f r o m t h eA m e r i c a n T y p e C u l t u r e C o l le c t io n . C h i t in d e a c e t y l a s e f r o m Mucor rouxii w a s g r o w ni n - h o u s e a n d p u r i fi e d t o h o m o g e n e i t y 1 [ 1 0 ] . A l l o t h e r c h e m i c a l s w e r e o f t h e h i g h e s tpur i t y commerc i a l l y ava i l ab l e .

1 The ch i t i n deace ty l a se t echno logy i s p ro t ec t ed by pa t en t s i s sued to t he Ins t i t u t e o f Molecu la r B io lo gy andB i o t e c h n o l o g y .

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A. Ma rt inou e t a l. / Carbohydrate Research 273 (1995) 235 -24 2 23 7

Enzym e activity assays.--Acetate r e l e a s e d b y t h e a c t i o n o f c h i t i n d e a c e t y l a s e o nv a r i o u s c h i t in o u s s u b s t r a te s w a s d e t e r m i n e d b y t h e e n z y m a t i c m e t h o d o f B e r g m e y e r [ 13 ]v i a t h r e e c o u p l e d e n z y m e r e a c t i o n s :

CD A A c e t a t e )Chi t in - -- , Ch i tosa n +AC SA c e t a t e + A T P + C o A - --, A c e t y l - C o A + A M P + P P i

CSA c e t y l - C o A + O x a l o a c e t a t e + H 2 0 - - - , C i t r a te + C o A+ MDHM a l a t e + N A D ~ O x a l o a c e t a te + N A D H + H +

w h e r e C D A = c h i t i n d e a c e t y la s e , A C S = a c e t y l - C o A s y n t h e ta s e , C S = c i tr a t e s y n th a s e ,M D H = m a l a t e d eh y d r o g e n a se .U n i t s o f c h i t i n d e a c e t y l a s e a c t i v i t y w e r e e s t i m a t e d u s i n g 1 6 6 n m o l o f h e x a - N -a c e t y l c h it o h e x a o s e i n a t o ta l v o l u m e o f 5 0 0 / x L o f 2 5 m M s o d i u m g l u ta m a t e b u f f e r ( p H4 . 5) . I n c u b a t i o n t i m e w a s 1 5 m i n a t 5 0 C , a n d t h e r e a c t i o n w a s t e r m i n a t e d b y h e a t i n g a t1 0 0 C p r i o r t o a c e t a t e d e t e r m i n a t io n .

W e d e f i n e o n e u n i t o f c h i t i n d e a c e t y l a s e a c t i v i t y a s t h e a m o u n t o f t h e e n z y m er e q u i r e d to p r o d u c e 1 / z m o l o f a c e ta t e p e r m i n w h e n i n c u b a t e d w i t h h e x a - N -a c e t y l c h i t o h e x a o s e a s d e s c r i b e d a b o v e . A s a c o m p r o m i s e b e t w e e n s e n s i t i v i t y a n d s p e e d ,5 0 C a n d 1 5 - m i n i n c u b a t i o n w e r e c h o s e n a s t h e s t a n d a r d a s s a y c o n d i t i o n s f o r c h i t i nd e a c e t y l a s e .

Protein concentration.--The p r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y t h e m e t h o d o fL o w r y e t a l . [ 1 4 ] u s i n g b o v i n e s e r u m a l b u m i n ( B S A ) a s s t a n d a r d .Preparation of artificial chitin derivatives.--Carboxymethylchitin w a s p r e p a r e d a sp r e v i o u s l y d e s c r i b e d [ 1 5 ] , w h i l e g l y c o l c h i t i n w a s p r e p a r e d a c c o r d i n g t o A r a k i a n d I t o[12].Determination of acetamido group s.--The N - a c e t y l g r o u p c o n t e n t o f w a t e r - s o l u b l ec h i t in s u b s t ra t e s a n d N - a c e t y l c h i t o o l ig o s a c c h a r i d e s w a s m e a s u r e d b y t h e s p e c t r o p h o t o -m e t r i c m e t h o d o f M u z z a r e l l i a n d R o c c h e t t i [ 1 6 ] .

3 . R e s u l t s a n d d i s c u s s i o n

M onitoring o f deacetylation processes of chitin subs trates.--B y t h e d i r e c t d e t e r m i n a -t i o n o f t h e a c e t a t e r e l e a s e d i t is p o s s i b l e t o m o n i t o r d e a c e t y l a t i o n p r o c e s s e s u s i n g e i t h e rn a tu ra l o r a r t i f i c i a l ch i t in o u s su b s t r a t es .

P r e s e n t l y , c h i t o s a n i s p r o d u c e d f r o m c h i t i n o f c r u s t a c e a n s h e l l s b y t h e r m o c h e m i c a la l k a l in e d e a c e t y l a t i o n [ 17 ]. T h i s p r o c e s s l e a d s t o a p r o d u c t h a v i n g a b r o a d r a n g e o fm o l e c u l a r w e i g h t s a n d a h e t e r o g e n e o u s e x t e n t o f d e a c e t y l a t i o n a s w e l l . H o w e v e r , f o rm a n y h i g h - v a l u e b i o m e d i c a l a p p l ic a t i o n s , u n i f o r m m a t e r i a l w i t h s p e c i f i c p h y s i c a l a n dc h e m i c a l p r o p e r t i e s i s r e q u i r e d . F u r t h e r m o r e , t h e d e g r e e a n d d i s t r i b u t i o n o f d e a c e t y l a -t i o n o f c h i to s a n h a s b e e n f o u n d t o i n f l u e n c e t h e p h y s i c a l a n d c h e m i c a l p r o p e r t i e s a s w e l l

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238 A. Martinou et al. / Carbohydrate Research 273 (1995) 235-242I0 0

90

~ ' 708: 6O

, , , I , , , I , , , I , , , i , i ,200 400 600 800 1000T t m e ( m t n )

Fig . 1 . T im e course o f deace ty l a t i on o f pa r t ia l l y ace ty l a ted ch i tosans (42%, O ; 28%, [ ] ) by ch i t i n deace ty l a se .T h e e n z y m a t i c r e a c ti o n s w e r e p e r f o r m e d u s i n g 1 m L o f 0 . 5 % c h i t o s a n s o lu t i o n in g l u t a m a t e b u f f e r ( p H 4 . 5 )a n d t h e m i x t u r e s e a c h c o n ta i n i n g 3 m U o f C D A w e r e i n c u b a t e d at 5 0 C . A l i q u o t s o f 0 .1 m L w e r e t a k e n o u t a td i f f e re n t t i m e i n t e r va l s . S a m p l e s w e r e m e a s u r e d i n d u p l ic a t e. F r o m t h e a m o u n t o f a c e t at e r e le a s e d , w h i c h w a sde t e rmined enzymat i ca l l y , t he % degree o f deace ty l a t i on was e s t ima ted .

as b io log i ca l ac t i v it i e s o f t h i s po lym er , fo r exam ple , t he adsorp t i on ab i l i ty o f me ta l i ons[18], t he t ens i l e s t r eng th o f ch i t osan f i lms [19] , t he e nzy m e b ind ing ab i l i t y [20] , andi m m u n o l o g i c a l a c t i v it y [ 2 1 ]. T h u s , m o n i t o r in g t h e d e g r e e o f d e a c e t y l a t io n o f c h i t o s a nsubs t r a t es i s o f pa r t i cu l a r impor t ance . The deve lopment o f a con t ro l l ab l e p rocess us ingthe enzymat i c deace ty l a t i on o f ch i t i nous subs t r a t es p resen t s an a t t r ac t i ve a l t e rna t i ve ,w h i le i t c a n p o t e n t i a ll y re s u l t in t h e p r e p a r a t io n o f n o v e l c h i to s a n p o l y m e r s .In o rde r t o t e s t ch i t i n deace ty l ase e f f ec t i veness i n deace ty l a t i ng ch i t i n and ch i t osansubs t r a t es , two c rys t a l l i ne and two amorphous ch i t i n samples , a s we l l a s two ch i t osans a m p l e s o f d i f fe r e n t d e a c e ty l a t io n d e g re e s , w e r e i n c u b a t e d w i t h t h e e n z y m e u n d e r t h es t a n d a rd a s s a y c o n d i t i o n s . T h e e n z y m e d e a c e t y l a t e d a 2 8 % a c e t y l a te d c h i to s a n w i t h i n 6h a n d a 4 2 % a c e t y l a te d c h i t o s a n w i t h i n 1 3 h a l m o s t c o m p l e t e l y ( 9 7 a n d 9 8 % ,r e s p e c t i v e l y ) , d e m o n s t r a t i n g t h e e n z y m e ' s e f f e c t i v e n e s s i n d e a c e t y l a t i n g a l m o s t a l lG l c N A c u n i t s o f t h e p o l y m e r s ( F i g . 1 ) . T h e d e g r e e s o f d e a c e t y l a t i o n o f t h e f i n a lp r o d u c t s d e t e r m i n e d w i t h t h i s m e t h o d w e r e i n g o o d a g r e e m e n t w i t h t h e v a l u e s o b t a in e db y t h e s p e c t r o p h o t o m e t r i c m e t h o d o f M u z z a r e l l i a n d R o c c h e t t i [ 1 6 ] ( 9 5 a n d 9 6 % ,respec t i ve ly ) . The method g ives r eproduc ib l e r e su l t s , and hence wi l l be use fu l fo r ane a s y a n d r a p i d d e t e r m i n a t i o n o f t h e d e g r e e o f d e a c e t y l a t i o n o f c h i t i n a n d c h i t o s a np o l y m e r s d u r i n g d e a c e t y l a t io n p r o ce s s e s. W h e n c r y s ta l li n e c h i ti n a n d i ts c h e m i c a l lym o d i f i e d f o r m , a m o r p h o u s c h i t i n , w e r e i n c u b a t e d w i t h t h e e n z y m e f o r 2 0 h , a p p r o x i -m a t e l y 0 . 5 % a n d 9 . 5 % d e a c e t y l a ti o n w a s a c h i e v e d r e s p e c ti v e ly ( T a b l e 1 ). T h i s i n d i c a t e stha t t he enz ym e i s no t ve ry e f f ec t i ve i n dea ce ty l a t i ng i nso lub l e ch i t i n subs t ra t es and t ha tp re t r ea tment o f c rys t a l l i ne ch i t i n subs t r a t es p r io r t o enzyme add i t i on i s necessa ry i no r d e r t o i m p r o v e t h e a c c e s s i b i l i t y o f t h e a c e t y l g r o u p s t o t h e e n z y m e a n d t h e r e f o r ee n h a n c e t h e y i e l d a n d r a t e o f t h e d e a c e t y l a t i o n r e a c ti o n . T h e m e c h a n i s m o f a c t i o n o f t h ee n z y m e o n c h i t in o u s p o l y m e r s u b s tr a te s i s u n d e r i n v e s t ig a t io n .

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A. Martinou et al. / Carbohydrate Research 273 (1995) 235-242Table 1Deacetylation o f chitin substrates by CDA a

23 9

Subs(rate Acetate released Deacetylation (%)(nmol)Crystalline chitin (shrimp) 12 6+ 2 0.50 +0.0 1Amorphous chitin (shrimp) 23 83 +3 0 9.5 +0 .1Crystalline chitin (crab) 12 0+ 2 0.48 +0.0 1Amorphous chitin (crab) 23 47 +2 9 9.4 +0 .1a Duplicate enzyme incubations (plus control reactions) were carded out in a final volume of 2 mL including 5mg of subs(rates, 25 m M glutamate buffer (pH 4.5), and 123 mU of C DA . Reactions were performed at 50Cfor 22 h and stopped by heat inactivation (10 min at 100C).

F i n a l l y , fo r e x t e n s i v e d e a c e t y l a t i o n p r o c e s s e s t h e s t a b i li t y o f c h i ti n d e a c e t y l a s e t ot h e r m a l d e n a t u r a t i o n i s a n i m p o r t a n t fa c to r . A f t e r i n c u b a t i o n o f th e e n z y m e a t 5 0 C f o r2 3 h , le s s t h a n 3 0 % d e c r e a s e i n a c t i v i t y w a s o b s e r v e d ( F i g. 2 ) . T h e r e f o r e , t h e e n z y m e i sq u i t e s t a b le a t i ts i n c u b a t i o n te m p e r a t u r e a n d c o u l d p o t e n t i a l l y b e e m p l o y e d f o r l o n gi n c u b a t i o n s w i t h v a r i o u s c h i t i n o u s s u b s (r a t es .

S u b s t r a t e s p e c i f ic i t y a n d k i n e t ic p r o p e r t i e s . - - D e a c e t y l a t e d N - a c e t y l c h i t o o l i g o m e r se x h i b i t u n i q u e p r o p e r t i e s ( w a t e r - s o l u b l e , e a s i l y d e r i v a t i s a b l e , b i o l o g i c a l l y a c t i v e )f a v o u r a b l e f o r m a n y b i o m e d i c a l a n d i n d u s t r i a l a p p l i c a t io n s . I n o rd e r t o t e st t he e n z y m e ' se f f e c t i v e n e s s i n d e a c e t y l a t i n g N - a c e t y l c h i t o o l i g o s a c c h a r i d e s u b s t r a t e s w e o v e r i n c u b a t e ds e v er a l N - a c e t y l c h i t o o l i g o m e r s w i th t h e e n z y m e ( T a b l e 2 ) . W h e n h e x a - N - a c e ty l c h i t o -h e x a o s e [ ( G l c N A c ) 6 ] w a s u s e d a s s u b st r a t e a n i n c r e a s e d r at e o f d e a c e t y l a t i o n w a so b s e r v e d i n c o m p a r i s o n t o ( G I c N A c ) 4 a n d ( G l c N A c ) 5, w h i l e t h e e n z y m e w a s i n a c t iv e o nN - a c e t y l - o - g l u c o s a m i n e , ( G l c N A c ) 2 , a n d ( G l c N A c ) 3. T h e m e c h a n i s m o f a c t io n o f t h ee n z y m e o n N - a c e t y l c h i t o o l i g o m e r s is u n d e r i n v e s t ig a t i o n .

100.0._. 80.0

60.04 0 . 0

o 20.0ae0 .0 . . . . , . . . . , . . . . , . . . . , . . . . ,0 5 10 15 20 25

T i m e ( h o u r s )Fig. 2. Thermal stability of C D A. Thermo s(ability of CD A was tested after preincubation of 2 mU o f theenzyme for different time periods at 37, 50, and 60C. After cooling on ice, each sample was assayed understandard conditions to determine the remaining activity.

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2 4 0 A. Martinou et al. / Carbohydrate Research 273 (1995) 235-242T a b l e 2D e a c e ty l a ti o n o f N - A c e t y l c h i to o l i g o s ac c h a r i d es b y C D A aS u b s t r a t e A c e t a t e r e l e a s e d D e a c e t y l a t i o n ( % )

( n m o l )N - A c e t y l - D - g l u c o sa m i n e 0 0( G l c N A c ) 2 0 0( G I c N A c ) 3 0 0( G l c N A c ) 4 152 + 2 23 . 0 _ 0 . 3( G I c N A c ) 5 3 3 0 5 : 4 4 0 . 0 + 0 . 5(GIc NA c) 6 480 + 6 48 . 5 - t- 0 . 6a D u p l i c a t e e n z y m e i n c u b a t i o n s ( p l u s c o n t r o l r e a c t i o n s ) w e r e c a r d e d o u t w i t h t h e f o l l o w i n g c o m p o n e n t s i n af i n al v o l u m e o f 2 0 0 p .L : 1 6 5 n m o l o f s u b s tr a t es , 2 5 m M g l u t a m a t e b u f f e r (p H 4 .5 ) , a n d 3 . 6 m U o f C D A .R e a c t i o n s w e r e p e r f o r m e d a t 5 0 C f o r 4 8 h a n d s t o p p e d b y h e a t i n a c t i v a ti o n ( 1 0 m i n a t 1 0 0 C ) .

W h e n p u r i f ie d n o d B , a n N - a c e t y l c h i t o o l i g o s a c c h a r i d e - s p e c i f i c d e a c e t y l a s e e n c o d e db y t h e n o d B g e n e s o f R h i z o b i a , w a s i n c u b a t e d w i t h v a r i o u s N - a c e t y l c h i t o o l i g o m e r s i tw a s o b s e r v e d t h a t o n l y t h e n o n r e d u c i n g r e s i d u e s o f ( G l c N A c ) 2 , ( G l c N A c )3, and( G I c N A c )4 w e r e d e a c e t y l a t e d w h i l e N - a c e t y l - o - g l u c o s a m i n e w a s n o t m o d i f i e d [2 2] .C h i t i n d e a c e t y l a s e f r o m M u c o r r o u x i i a n d n o d B , t w o e v o l u t i o n a r i l y d i s ta n t p r o t e i n s ,sh o w s ig n i f i can t p ro te in sequ en ce s imi l a r i t i e s [1 1 ] . T h e s imi l a r i t i e s an d d i f f e ren cesb e t w e e n t h e s e e n z y m e s m a k e c h i t i n d e a c e t y l a s e s a n a t t r a c t i v e s y s t e m f o r s t u d y i n gs t ru c tu re an d fu n c t io n r e l a t io n sh ip s in su b s t r a t e r eco g n i t io n an d ca t a ly s i s . C o mp ara t iv ea n a l y s i s o f s e q u e n c e s i m i l a r i t i e s b e t w e e n t h e s e t w o p r o t e i n s c a n p r o v i d e t h e b a s i s f o rd e v e l o p i n g a p r o t e i n e n g i n e e r i n g s t r a te g y t o m o d i f y t h e s p e c i f i c i t y o f c h i t i n d e a c e t y -lases .S i n c e a n i n c r e a s e d r a t e o f d e a c e t y l a t i o n w a s o b s e r v e d w i t h ( G I c N A c ) 6 , a m o r es y s t e m a t i c s t u d y w a s p e r f o r m e d w i t h t h i s N - a c e t y l c h i t o o l i g o m e r . A c e t a t e w a s r e l e a s e dl i n e a r l y u p t o a n i n c u b a t i o n t i m e o f 3 0 m i n . A l l s u b s e q u e n t i n c u b a t i o n s w e r e p e r f o r m e dw i t h i n t h i s l i n e a r t i m e r a n g e . L i n e a r i t y o f t h e m e t h o d w a s o b s e r v e d a s a c o r r e l a t i o nb e t w e e n e n z y m e a c t i v it y an d c h i t i n d e a c e t y l a s e c o n c e n t r a t i o n u p t o 0 . 6 / xg o f C D A p e rm L . I n c u b a t i o n o f c h i t in d e a c e t y l a s e w i t h v a r i o u s c o n c e n t r a t io n s ( 5 X 1 0 - 5 t o 3 X 1 0 - 3M ) o f s u b s t r a te ( F ig . 3 ) s h o w e d t h a t t h e K m v a l u e f o r ( G l c N A c ) 6 , u s i n g t h eL i n e w e a v e r - B u r k m e t h o d , is 1 .3 m M .

F i n a l l y , c h i t i n d e a c e t y l a s e f r o m M u c o r r o u x i i a p p e a r s t o e x h i b i t a v e r y s t r i n g e n ts p e c i f i c i ty a c ti n g o n l y o n c h i t i n o u s s u b s tr a te s . T h e e n z y m e w a s n o t a c t i v e o n h y a l u r o -n a n , a c o p o l y m e r o f a l t e r n a ti n g / 3 - o- g l u c ur o n i c a c i d a n d / 3 - N - a c e t y l -o - g l u c o s a m i n er e s i d ue s . F u r t h e r m o r e , i n it ia l s t u d ie s h a v e s h o w n t h a t t h e e f f e c t i v e n e s s o f t h e e n z y m e i nd e a c e t y l a t i n g v a r i o u s c h i t i n o u s s u b s tr a t e s w a s i n t h e f o l l o w i n g o r d e r : c a r b o x y m e t h y l -ch i t in > g ly c o l ch i t in > am o rp h o u s ch i t in > c ry s t a l l i n e ch i t in (d a ta n o t sh o w n ) , i n d ica t -i n g d i f f e r e n t s u b s t ra t e s p e c i f i c i ty a n d / o r s u s c e p t ib i l it y o f c h i ti n s u b s tr a t e s t o e n z y m a t i cd e a c e t y l a t i o n .

T h r e e f a c t o r s c o u l d m a i n l y a f f e c t t h e e n z y m a t i c d e a c e t y l a t i o n o f i n s o l u b le c h i t i n a n dc h i t i n d e r i v a t i v e s : t h e p r o p e r t i e s a n d m e c h a n i s m o f a c t i o n o f C D A , t h e s t r u c t u r a lp r o p e r t i e s o f c h i t i n , a n d t h e m o d e o f i n t e r a c t i o n b e t w e e n t h e e n z y m e a n d t h e c h i t i nm o l e c u l e s . P r e t r e a t m e n t o f c h i t i n m a t e r i a l s c a n m o d i f y t h e c o n f o r m a t i o n o f t h e p a r e n t

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A. Ma rt inou e t a l. / Carbohydrate Research 273 (1995) 235 -24 2 24 11 .2E1 .00 . 8

~ 0 . 6~ 0 . 4"~ 0.2

. , , I . , . , I , , , . I . , , , | . . , , I , , , , I , , , ,5 . 1 0 - 4 1 0 - 3 2 . 1 0 - 3 3 . 1 0 - 3Hexa-IL,-ace ty l ch t tohexaose concen t ra t i on (M)

Fig . 3 . E f fec t o f subs t ra t e (hexa -N -ace ty l ch i tohexaose ) concen t ra t i on on C DA ac t iv it y . Incuba t ions werep e r f o r m e d i n a f i n a l v o lu m e o f 0 . 5 m L c o n t a i n i n g 0 .3 m U o f C D A , ( G l c N A c ) 6 (5 1 0 - 5 t o 3 X 1 0 - 3 M ) , a n d2 5 m M s o d i u m g l u t a m a t e b u f f e r ( p H 4 . 5 ) a t 5 0 C f o r 1 5 m i n . S a m p l e s w e r e m e a s u r e d i n t r ip l ic a te .

p o l y m e r . A s a r e s u lt , t h e i n t e r a c ti o n b e t w e e n e n z y m e a n d s u b s tr a t e , a s w e l l a s t h es u b s e q u e n t d e a c e t y l a t i o n r e a c t io n , c o u l d b e a f f e c t e d . T h e s u s c e p t i b il i ty o f c h i t i n d e r i v a -t i v e s t o e n z y m a t i c d e a c e t y l a t i o n m a y v a r y w i t h t h e t y p e o f s u b s t i t u e n t g r o u p a s w e l l a st h e p a t t e rn o f s u b s ti tu t io n . T h e m e c h a n i s m o f e n z y m a t i c d e a c e t y l a t io n o f s o l u b le c h i t i nd e r i v a t i v e s a s c o m p a r e d t o i n s o l u b l e c h it i n is s i m p l e r b e c a u s e o f t h e h o m o g e n e i t y o f t h ereac t io n , b u t t h e su b s t i t u t io n p a t t e rn an d th e d eg ree o f su b s t i t u t io n o f t h e su b s t r a t e mu s tb e a d e q u a t e l y t a k e n i n t o a c c o u n t .C o r r e l a t i o n b e t w e e n t h e s t r u c t u r a l f e a t u r e s o f c h i t i n o u s s u b s t r a t e s a n d e x t e n t o fe n z y m a t i c d e a c e t y l a t i o n i s r e q u i r e d f o r a b e t t e r u n d e r s t a n d i n g o f t h e r e a c t i o n m e c h a -n i s m . I d e n t i f ic a t i o n o f t h e a p p r o p r i a t e s u b s tr a t e p r e t r e a t m e n t r e q u i r e m e n t s c o u l d p o t e n -t i a ll y r e s u l t in t h e p r o d u c t i o n o f n o v e l c h i t o s a n p o l y m e r s f o r v a r i o u s a p p l i c a t io n s .Assay of chitin deacetylase.--Determination o f c h i t i n d e a c e t y l a s e a c t i v i t y w a s p e r -f o r m e d u s i n g t h e e n z y m a t i c m e t h o d o f B e r g m e y e r [ 13 ]. A c e t a t e r e l e a s e d a f t e r th ee n z y m a t i c d e a c e t y la t i on o f h e x a - N - a c e t y lc h i t o h e xa o s e w a s m e a s u r e d v i a t h re e c o u p l e de n z y m e r e a c t i o n s a c c o r d i n g t o t h e a b o v e m e t h o d . S e n s i t i v i t y o f t h e a s s a y i s a p p r o x i -m a t e l y 0 .5 /~ g o f a c e t a t e p e r s a m p l e v o l u m e .

W h e n ( G l c N A c ) 6 w a s u s e d a s s u b s t r a te a n i n c r e a s e d r a t e o f d e a c e t y l a t i o n w a so b s e r v e d i n c o m p a r i s o n t o ( G I c N A c )4 a n d ( G I c N A c )5 ( T a b l e 2 ) . F u r t h e r m o r e , s i n c e( G I c N A c ) 7 is n o t c o m m e r c i a l l y a v a i la b l e a n d N - a c e t y l c h i t o o l i g o m e r s o f h i g h e r c h a i nl e n g t h a re i n s o l u b le i n a q u e o u s s o l u ti o n s w e h a v e e m p l o y e d ( G I c N A c ) 6 as a n o v e ls u b s t r a t e f o r t h e d e t e r m i n a t i o n o f c h i t i n d e a c e t y l a s e a c t i v i t y . T h e t e m p e r a t u r e w h e r em a x i m u m e n z y m e a c t iv i ty w a s o b s e r v e d w i t h o u t a n y s i g n if i ca n t e n z y m e d e n a t u ra t io n i s5 0 C . A t e m p e r a t u r e r a n g e b e t w e e n 3 7 a n d 6 0 C w a s e x a m i n e d . A n o p t i m u m p H o f 4 . 5w a s o b s e r v e d b y m e a s u r i n g t h e r a t e o f h e x a - N - a c e t y l c h i t o h e x a o s e d e a c e t y l a t i o n at a p Hr a n g e b e t w e e n 3 . 5 a n d 8 . 0 .

T h e n e w m e t h o d p r e s e n t e d i s n o n r a d i o a c t i v e , f a s t, s i m p l e , a n d r e p r o d u c i b l e , a n d c a nb e t h e r e f o r e u s e d r o u t i n e l y f o r e s t im a t i n g c h i t i n d e a c e t y l a s e a c t i v i ty .

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