有机介质中酶促_R_及_S_苯甘氨酸甲酯的氨解反应_英文_

有机介质中酶促_R_及_S_苯甘氨酸甲酯的氨解反应_英文_ ( )华 南 理 工 大 学 学 报 自 然 科 学 版 第 29 卷 第 10 期 V ol . 29 N o . 10 J o u r n a l of S o ut h C hi n a U ni ve rsi t y of Te c h n ol o g y ( )2001 年 10 月O c t o be r 2001 N a t u r a l Sci e nc e Edi t i o n ) ( Ar t icle I D : 1000 - 565 X 200110 - 0014 - 04 ( ) ( ) Enz y m a t ic A m monol ys is of R- a n d S - P he n yl gl yci ne 3O r ga nic S ol ve nt Met h yl Es t e r i n D u Wei L i H u i- qi ng Zo ng Mi n- h u a G u o Yo ng ( )D ep t . of B i oe n gi ne e ri ng , S o ut h C hi n a U ni v . of Tec h . , Gua n gz h o u 510640 , C hi n a Abs t r a ct : L ip as e N o v oz y m435 w as f o u n d t o s h ow hi g h ac t i vi t y a n d e n a n t i os elec t i vi t y t ow a r d t h e a m2 m o n ol ysis of p h e n yl gl yci ne me t h yl es t e r . Ef f ec ts of t h e c o nce n t r a t i o n of c a r ba m a t e , t e mp e r a t u r e , s ol2 ve n ts a n d i ni t i al w a t e r ac t i vi t y o n t h e a m m o n ol ysis w e r e s t u die d i n t his p ap e r . Key wor ds : lip as e ; a m m o n ol ysis ; p h e n yl gl yci ne me t h yl es t e r D oc u me nt code : A CL C n u m be r : Q 556 cl assi c a l r es ol ut i o n vi a di as t e r e o m e ri c s a l t c r ys t a l2 0 I nt r od uct i on liz a t i o n o r e n a n t i os el e c t i ve h y d r ol ysis of t h e c o r 2 [ 3 ] N o w a d a ys t h e r e is a n i nc r e asi n g de m a n d f o r r esp o n di n g h y d a n t oi n . ef f i ci e n t p r oc ess es f o r t h e s y n t h esis of op t i c a ll y I n t his p ap e r , w e i n t e n d t o p r o be i n t o t h e a c t i ve c o m p o u n ds . F o r t his p u rp os e , bi oc a t a l ys ts p ossi bili t y of t h e e n a n t i os el e c t i ve a m m o n ol ysis of h a ve be e n w i del y ut iliz e d . D es p i t e of t h e f a c t t h a t r a c e mic p h e n yl gl yci n e m e t h yl es t e r . Ef f e c ts of m os t of t h e bi o t r a nsf o r m a t i o ns occ u r i n a q ue o us t h e a m o u n t of c a r b a m a t e , t e m p e r a t u r e , s ol ve n ts , i m e di u m , i t is a w ell es t a blis h e d f a c t t h a t m a n y ( ) ni t i a l w a t e r a c t i vi t y o n t h e a m m o n ol ysis of R - a e nz y m es a r e a c t i ve i n o r ga ni c s ol ve n ts a n d c a n be ( )n d S- p h e n yl gl yci n e m e t h yl es t e r a r e a ls o s t u d2 i e [ 1 ] us e d f o r e n a n t i os el e c t i ve t r a nsf o r m t i o ns . A d . To o u r bes t k n o w l e d ge , t h e r e is n o d o m es t i c n o vel e nz y m a t i c r e a c t i o n of c a r b o x yli c es t e rs w i t h r es e a r c h i n t his a r e a . ( ) a m m o ni a a m m o n ol ysis w as disc o ve r e d i n t h e mi d of 1990s . T his r e a c t i o n p r o vi des a mil d p r o2 1 M a t e ri a ls a n d Met h ods c e d u r e f o r t h e p r e p a r a t i o n of c a r b o x ylic a mi des as 1 . 1 M a t e ri a l s w ell as a n a t t r a c t i ve m e t h o d f o r t h e r es ol ut i o n of A ll s ol ve n ts a n d r e a c t a n ts w e r e of r e a ge n ts [ 2 ] c hi r a l a ci ds , a l c o h ols a n d es t e rs .g r a de . L ip as e O F a n d L i p as e M Y w e r e ge n e r o us D- p h e n yl gl yci n e a n d i ts 4- h y d r o x y de ri va 2 ( ) C o . L t d J a p a n . gif ts f r o m M ei t o S a n gy o t i ves a r e k e y i n t e r m e di a t es i n t h e m a n uf a c t u r e of ( ) N o v oz y m871 lip as e f r o m Ca n di d a a n t a rct ic a , p e nicilli n a n d c e p h a l osp o ri n a n t i bi o t ics . I n t h e ( ( ) L ip oz y m e lip as e f r o m M uc o r m ie hei , C RL li2 c h e mic a l p r oc e d u r e , t w o p r oc ess es a r e us e d f o r ) p as e f r o m Ca n di d a a n t a rct ic a a n d N o v oz y m 435 t h e c o m m e r ci a l p r o d uc t i o n of t h es e a mi n o a ci ds : () lip as e f r o m Ca n di d a a n t a rct ic a B w e r e ki n dl y ( ) d o n a t e d b y N o v o N o r dis k I n d us t ri es D e n m a r k . Recei ve d d a t e : M a r c h 12 , 2001 E n a n t i op u r e p h e n yl gl yci n e m e t h yl es t e r a n d a m2 3 Fou n d a t i on i t e m : N a t u r al Scie nce F o u n da t i o n of Gua n g2 m o ni u m c a r b a m a t e w e r e p u r c h as e d f r o m Si g m a . ( )d o n g Pr o vi nce 980543 ) ( Bi ogr ap h y : D u W ei b o r n i n 1975, f e m ale , P h . D . c a n2 A ll o t h e r c h e mic a ls w e r e o b t a i n e d c o m m e r ci a ll y di da t e , m ai nl y r es e a r c hs o n n o n- a q ue o us e nz y m a t ic c a t al2 a n d of a n a l yt ic a l g r a de . ysis . S ol ve n ts w e r e de h y d r a t e d bef o r e us e w i t h m ol e c ul a r si e ve f o r n o l ess t h a n 72 h a t r o o m t e m2 p e r a t u r e . 1 . 2 A n a l yt ic a l P r oce d u re T h e a m m o n ol ysis r e a c t i o n w as m o ni t o r e d b y G C4890 gas c h r o m a t o g r a p h y w i t h a f l a m e i o niz a 2 ( ( ) t i o n de t e c t o r F I D a n d a c hi r a l c ol u m n 20 % p e r2 β) m e t h yl a t e d - c ycl o de xi n 30 T h e m ×0 . 25 m . μ c ol u m n t e m p e r a t u r e a n d p r ess u r e w e r e 160 ? a n d 1 . 02 Pa r es p e c t i vel y . N i t r o ge n w as us e d as t h e C o mp a ris o n of dif f e r e n t lip as es i n a m m o n ol ysis Fi g . 1 c a r ri e r gas w i t h a f l o w r a t e of 0 . 3 mL / mi n . T h e of ( ) ( ) R- a n d S- p h e n yl gl yci ne me t h yl es t e r i nj e c t i o n a n d t h e de t e c t o r t e m p e r a t u r e w e r e R e a c ti o n ti me : 23 h ; E1 : N ov oz y m435 ; E2 : L ip oz y me ; E3 : L ip as e M Y ; 280 ? a n d 300 ? r esp e c t i vel y . E4 : L ip as e O F ; E5 : L- 1754 ; E6 : N ov oz y m871 1 . 3 General Procedure f or Enz ymatic Am monol ysis 2 . 2 Eff ect of A m m oni u m Conce nt r a t i on on t he ( ) L ip as e N o v oz y m 435 50 m g w as a d de d t o ) ( ) ( A m m onol ys is of R - a n d S - P he n yl2 ( ) t e r t- b ut a n ol 10 mL c o n t a i ni n g a c e r t a i n a m o u n t gl yci ne Met h yl Es t e r ( ) ( ) of R S p h e n yl gl yci n e m e t h yl es t e r , a m2 - o r - Ef f e c t of a m m o ni u m c o nc e n t r a t i o n w as s t u d 2 ( m o ni u m c a r b a m a t e a n d n- t e t r a de c a n e as 20 L μAs c a n be s e e n i n Fi g . 2 a n d Fi g . 3 , t h e e nz y2 i e d . ) t h e i n t e r n a l s t a n d a r d . T h e mi xt u r e w as i nc u b a t 2 ( ) ( ) m a t ic a m m o n ol ysis of b o t h R- a n d S- p h e n yl2 e d w i t hi n a c o n t a i n e r c a p p e d w i t h a s e p t u m a n d w hil e t h e e n a n 2 gl yci n e m e t h yl es t e r a cc el e r a t e d ( s h a k e n i n a w a t e r- b a t h s h a k e r 150 r / mi n , i nc r e as e of a m 2 t i os el e c t i vi t y de c r e as e d w i t h t h e ) 30 ?.20 LA t r e gul a r t i m e i n t e r va ls s a m p l es μm o ni u m w i t hi n t h e sc o p e s t u di e d . Ta ki n g t h e r e2 w e r e t a k e n w i t h a s y ri n ge f o r G C a n a l ysis . T h e a c t i o n r a t e a n d t h e e n a n t i os el e c t i vi t y i n t o a c2 c o n ve rsi o n r a t i o of p h e n yl gl yci n e m e t h yl es t e r r e 2 c o u nt , 1 . 56 g/ L a m m o ni u m c a r b a m a t e w as a n d t h e r a t i o of t h e r e a c t i o n r a t e of t h e t w o e n a n 2 [ 4 ] ga r de d as t h e o p t i m u m . ( ) t i o m e rs v / vw e r e c a l c ul a t e d . R S B l a n k e xp e ri m e n ts s h o w e d t h a t n o a p p r e ci a2 bl e a m m o n ol ysis t o o k p l a c e i n t h e a bs e nc e of t h e e nz y m e . 1 . 4 Cont r ol of I ni t i a l W a t e r Act i vi t y T h e i ni t i a l w a t e r a c t i vi t y of s ol ve n ts a n d e n2 z y m e w as c o n t r oll e d b y gas e o us e q uili b ri u m w i t h [ 5 ] dif f e r e n t s a t u r a t e d s a l t s ol ut i o ns . Fi g . 2 Ef f ec t of a m m o ni u m c o nce n t r a t i o n o n t h e 2 Res ul t s a n d D is c us s i on ( ) ( ) a m m o n ol ysis of R- a n d S- e n a n t i o me r 2 . 1 S el ect i on of Enz y me Wi t hi n t h e s e ve r a l li p as es e xa mi n e d , N o v o- z y m 871 e xp r ess e d e nz y m a t ic a c t i vi t y b ut n o e n a n2 ( ) t i os el e c t i vi t y t o w a r d t h e a m m o n ol ysis of R- a n d ( ) S - p h e n yl gl yci n e m e t h yl es t e r . N o v oz y m 435 s h o w e d hi g h a c t i vi t y a n d e n a n t i os el e c t i vi t y w i t h ( ) )( R- e n a n t i o m e r bei n g t h e p r ef e r r e d o n e Fi g . 1. Fi g . 3 Ef f ec t of a m m o ni u m c o nce n t r a t i o n o n t h e e n a n t i os elec t i vi t y of t h e a m m o n ol ysis ( )J o u r n al of S o ut h C hi n a U ni ve rsi t y of Tec h n ol ogy N a t u r al Scie nce Edi t i o n 16 Vol . 29 2 . 3 Eff ect of Re a ct i on Te m p e r a t u re on t he A m2 r a t e a n d e n a n 2 t i vi t y s t ri ki n gl y , a n d t h e r e a c t i o n ( ) ( ) t i os el e c t i vi t y c o ul d be w ell c o r r el a t e d w i t h l o g P m onol ys i s of R -S - P he n yl gl yci nea n d va l ues of o r ga ni c s ol ve n ts . T h e bi gge r t h e l o g P Met h yl Es t e r is , t h e hi g h e r t h e r e a c t i o n r a t e is a n d t h e l o w e r Te mp e r a t u r e h as g r e a t ef f e c t o n t h e e nz y m a t2 ic a c t i vi t y a n d e n a n t i os el e c t i vi t y . W h e n t e m p e r a2 t h e e n a n t i os el e c t i vi t y is . C o nsi de ri n g b o t h t h e e n2 z y m a t ic a c t i vi t y a n d e n a n t i os el e c t i vi t y , t e r t- b u2 t u r e w as bel o w 30 ?, i nc r e as e of t e m p e r a t u r e r e2 ) ( s ul t e d i n hi g h e r a m m o n ol ysis r a t e of b o t h R - t a n ol w as t h o u g h t t o be t h e m os t s ui t a bl e r e a c t i o n ) ( a n d S - e n a n t i o m e r , a n d t h e e nz y m a t ic r a t e of m e di u m f o r t h e r e a c t i o n . I n t e r es t i n gl y , e nz y m e ( ) R- e n a n t i o m e r w as e n h a nc e d m uc h g r e a t e r t h a n s h o w e d m uc h hi g h e r e n a n t i os el e c t i vi t y i n t e r t- b u2 ( ) ) ( t h a t of S - e n a n t i o m e r Fi g . 4 . H o w e ve r , t h e t a n ol t h a n i n be nz e n e a l t h o u g h t h e l o g P of t h e n e ga t i ve i nf l ue nc e of t e m p e r a t u r e o n t h e a m 2 f o r m e r is cl os e t o t h a t of t h e l a t t e r w hic h m a y be m o n ol ysis r a t e of t h e t w o e n a n t i o m e rs w as o b 2 d ue t o t h e bi g ge r s t e ric hi n d r a nc e of t e r t- b ut a n ol . Ta ble 1 Ef f ec t of o r ga nic s ol ve n ts o n t h e a m m o n ol ysis s e r ve d w h e n t e m p e r a t u r e e xc e e de d 30 ?, w hic h of t h e t w o e n a n t i o me rs m a y be t h e c o ns e q ue nc e of l ess a m m o ni a s ol u bi 2 - 1 - 1 - 1 m m ol?L m? g ?h li z e d i n t h e r e a c t i o n m e di a a t hi g h e r t e m p e r a t u r e . v v / v v O r ga nic s ol ve nts l og P RSRS 1 , 2- Dic hl o ri de e t h a ne 0 . 71 . 012 9 . 000 000 — 0 0 t e r t- B ut a n ol 1 . 80 0 . 017 4 0 . 000 440 39 . 0 B e nz e ne 2 . 00 0 . 017 6 0 . 000 988 17 . 6 M IB K 2 . 20 0 . 018 2 0 . 001 100 16 . 5 ( )met hyl is obut yl ket one 3 . 20 0 . 021 9 0 . 001 320 16 . 7 Cycl o h e xa ne Hep t a n e 4 . 00 0 . 024 7 0 . 001 650 15 . 0 All or ga nic s olve nts a re de hydrat ed by molecula r sieve bef ore use . C o nsi de ri n g b o t h t h e e nz y m a t ic a c t i vi t y a n d e n a n t i os el e c t i vi t y , w e t h o u g h t t e r t- b ut a n ol w as Fi g . 4 Ef f ec t of t e mp e r a t u r e o n t h e a m m o n ol ysis t h e m os t s ui t a bl e m e di u m f o r t h e r e a c t i o n . ( ) ( ) of R- a n d S- e n a n t i o me rs Eff ect of I ni t i a l W a t e r Act i vi t y on t he A m2 2 . 5 E nz y m a t i c h y d r ol ysis m a y c o mp e t e w i t h t h e ( ) ( ) m onol ys is of R - a n d S - P he n yl gl yci ne 2 a m m o n ol ysis of t h e es t e rs , b ut n o a p p r e ci a bl e h yMet h yl Es t e r ( ( ) ) Sd r ol ysis of R- a n d - e n a n t i o m e rs c o ul d be de 2 W a t e r p l a ys a c r uci a l r ol e i n t h e e nz y m a t ic ( ) t e c t e d w i t hi n t h e s c o p e s t u di e d d a t a n o t s h o w n . m e di a . E nz y m e s h o ws n o r e a c t i o ns i n o r ga nic 2 . 4 Effect of Orga nic Sol vents on t he Am monoly sis of t o t a ll y a n h y d r o us e n vi r o n2 c a t a l yt i c a c t i vi t y i n ( ) ( ) R- a nd S- Phenylgl yci ne Met h yl Ester m e n t , b ut e xc essi ve w a t e r w ill l e a d t o t h e i nc r e as e As t h e r e a c t i o n m e di u m , o r ga ni c s ol ve n ts i n2 of m ass t r a nsf e r li mi t a t i o n . I n o u r s t u d y , t h e e x2 f l ue nc e n o t o nl y t h e e nz y m a t ic a c t i vi t y b ut t h e is t e nc e of w a t e r w ill a cc el e r a t e t h e h y d r ol ysis of e n a n t i os el e c t i vi t y m a r k e dl y . O w i n g t o t h e p o o r t h e es t e r , s o i t is of g r e a t si g nif i c a nc e t o c o n t r ol s ol u bili t y of p h e n yl gl yci n e m e t h yl es t e r i n m os t t h e a m o u n t of w a t e r i n t his s ys t e m . o r ga nic s ol ve n ts , w e s t u di e d t h e r el a t i ve ef f e c t of c a n be s e e n i n Fi g . 5 , t h e a m m o n ol ysis As dif f e r e n t s ol ve n ts o n t h e a m m o n ol ysis of t h e t w o ( )R - e n a n t i o m e r i nc r e as e d w i t h t h e i n 2 r a t e of )( e n a n t i o m e rs b y diss ol vi n g t h e m i n 5 mL of t e r t- c r e as e of t h e i ni t i a l w a t e r a c t i vi t y a u n t il t h e w b ut a n ol a n d t h e n a d di n g 5 mL of dif f e r e n t o r ga ni c w a t e r a c t i vi t y r e a c h e d 0 . 75 , a b o ve w hi c h o p p o2 ( ) ( si t e ef f e c t of a o n t h e r e a c t i o n r a t e of R- e n a n2 s ol ve n ts 1 , 2- di c hl o ri de e t h a n e , t e r t- b ut a n ol , w ) t i o m e r w as o bs e r ve d . T h o u g h t h e a m m o n ol ysis be nz e n e , M IB K , c ycl o h e xa n e a n d h e p t a n e . As ( ) r a t e of S- e n a n t i o m e r i nc r e as e d s t e a dil y w i t h t h e c a n be s e e n i n Ta bl e 1 , o r ga nic s ol ve n ts i nf l u2 a sc op e s t u di e d , i t c h a n ge d m uc h l ess t h a n t h a t e nc e d t h e e nz y m a t ic a c t i vi t y a n d e n a n t i os el e c 2 w Te rt - but a nol was t he best me di um f or t he reacti o n . ) ( of R- e n a n t i o m e r . S o 0 . 75 w as c o nsi de r e d t o be Wit hi n t he scop e st udie d , t he op ti mal i nitial wat e r t h e op t i m a l i ni t i a l w a t e r a c t i vi t y . acti vit y was f oun d t o be 0. 75. Ref e re nces : Z oe t e M C , Koc k- V a n D A C , R a n t wij k V F , e t al . [ 1 ] L ip as e- c a t al yze d a m m o ni ol ysis of lip i ds : a f acile s y n2 t h esis of f a t t y aci d a mi des J . J o u r n al of M olec ul a r ( ) Ca t al ysis B : Enz y m a t ic , 1996 , 2 2: 141 - 145. 2 ] L i t je ns M J J , S h a M , St r a t h of A J J , e t al . C o mp e t i2 [ t i ve lip as e- c a t al yze d es t e r h y d r ol ysis a n d a m m o n ol ysis i n o r ga nic s ol ve n ts ; e q uili b ri u m m o del of a s oli d- li q2 Fi g . 5 Ef f ec t of i ni t i al w a t e r ac t i vi t y o n t h e a m m o n ol ysis ui d- vap o r s ys t e m J . B i ot ec h n ol B i oe n g , 1999 , 65 ( ) ( ) of R- a n d S- e n a n t i o me rs ( ) 3: 347 - 356. T h e h y d r ol ysis r e a c t i o n w as a ls o m o ni t o r e d [ 3 ] W e g m a n M A , Hac ki n g M A P J , R op s J , e t al . D y2 u n de r t h e a b o ve c o n di t i o ns , s h o w i n g t h a t h y d r ol2 n a mic ki ne t ic r es ol ut i o n of p h e n yl gl yci ne es t e rs vi a li2 ( af t e r 30 h , ysis of t h e es t e r c o ul d be n e gl e c t e d p as e- c a t al ys e d a m m o n ol ysis J . Te t r a h e d r o n : As y m2 )t h e c o n ve rsi o n r a t i o w as bel o w 5 % me t r y , 1999 , 10 : 1 739 - 1 750. [ 4 ] C h e n C S , Fuji m ot o Y , Gi r da u k as G . Q ua n t i t a t i ve 3 Concl us i on a n al ys es of 2 bi oc h e mic al ki ne t ic r es ol ut i o ns of e n a n t i o me rs J . J A m C h e m S oc , 1982 , 104 : 7 294 - N ovoz ym435 was select e d f r o m seve ral li p ases 7 299 . beca use of its hi gh activit y a n d e na ntiselectivit y t o2 [ 5 ] A mléie D , Tra ni M , R obe rt L . L ip ase- cat alyze d e na n2 ( ( ))wa r d t he a m mo nol ysis of R- a n d S- p he nylgl yci ne ti oselective est e rif icati on of i bup r of e n i n orga nic s olve nts met h yl est e r . The co nce nt rati o n of a m mo ni um ha d unde r cont r olle d wat e r activit y J . Enz yme a nd Micr o2 great i nf lue nce o n t he a m mo nol ysis ; 30 ? was co n- bial Tec h nol ogy , 1998 , 22 : 212 - 216. si de re d t o be t he op ti mum reacti o n t e mp e rat ure . ( ) 有机介质中酶促 R- 及 3( ) S- 苯甘氨酸甲酯的氨解反应 杜 伟 李慧青 宗敏华 郭 勇 ( )华南理工大学 生物工程系 , 广东 广州 510640 摘 要 : 酶促氨解反应是近年来发现的一类新反应. 脂肪酶 Novozym435 在催化苯甘氨酸甲酯的氨解反应中 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 现 出较高的酶活性和对映体选择性. 本文系统地研究了氨基甲酸铵浓度 、温度 、溶剂及初始水活度对酶促氨解反应的 影响 ,结果表明酯的酶促氨解反应不仅是酰胺合成的有效方法,也是手性 酸 、手性醇及手性酯拆分的有效途径. 关键词 : 脂肪酶 ; 氨解反应 ; 苯甘氨酸甲酯 文献标识码 : A 中图分类号 : Q 556 ( ) 文章编号 : 1000 - 565 X 200110 - 0014 - 04 收稿日期 : 2000 - 03 - 12 ( ) 3 基金项目 : 广东省自然科学基金资助项目 980543 ( ) 作者简介 : 杜 伟 1975 - , 女 , 博士生 , 主要从事非水相酶催化方面的研究.