immobilized lipase to regio-specific interesterification

8/18/2019 Immobilized Lipase to Regio-Specific Interesterification

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European J App l Microbiol Biotechnol (1982) 14:1 -5

pplied

Microbiologyand

Biotechnology

9 S p f i n g e r - V e f l a g - 1 9 8 2

Application of Immobilized Lipase to Regio-Specif ic Interesterif ication

of Triglyceride in Organic Solvent

K e n z o

Yok ozek i I , Sh ige ru Y amanak a 1 Koich i Tak inam i I , Yosh io Hi rose 1 At suo Tanaka 2 ,

K e n j i S o n o m o t o

a n d

S a b u r o F u k u i 2

1 Centra lResearch Laboratoriesof A jinomoto Co., Inc., Suzuki-cho,Kawasaki-ku,Kawasaki210, Japan

2 Labora toryof Industrial Biochemistry,Departm ent of Ind ustrial Chem istry,Faculty of Engineering,Kyo to University,Yoshida,

Sakyo-ku, Kyo to 606 , Japan

S u m m a r y . L i p as e f r o m

R h i z o p u s d e l e m a r

was immobi -

l iz ed by en t rapm ent wi th pho to -c ross l i nkab le r e s in p re-

p o l y m e r s o r u r e t h a n e p r e p o l y m e r s o r b y b i n d in g t o

va r ious t ypes o f porous s il ic a beads . The immo bi l i z ed

l ipa se p repa ra t i ons t hu s ob t a ined w e re examined fo r

the i r ac t ivi ty in conver t ing ol ive off to an interester i f ied

fa t (cacao but te r- l ike fa t ) , who se ole ic ac id moie t ies a t

1- and 3-posi t ions were replaced wi th stear ic ac id

moie t i e s , i n t he r eac t i on so lven t n -hexane . Al thou gh a l l

o f t he immo bi l i zed p repa ra t ions exh ib i t ed some ac t i v i t y ,

l ipa se adsorbed on Ce l i te and t hen en t rapped wi th a hy-

d rophob ic pho to -c ross l i nkab le r e s in p repo lym er showed

the h ighes t a c t i v it y , abou t 75% o f t ha t o f li pa se s imp ly

a d s o r be d o n t o C e li te . E n t r a p m e n t m a r k e d l y e n h a n c e d

the opera t ion al s tabi l i ty o f lipase .

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

The p rodu c t ion o f f a t wi th de s i r ed phys i ca l and chemica l

p rope r t ie s b y r ep lac ing t he f a t t y ac id m oie t i e s o f t r ig ly -

ce r ide w i th o the r f a t t y acid( s) i s o f g rea t impor t ance and

in t e re s t f rom an i ndus t r i a l v i ewpoin t . A l though t ri gly-

ce r ide can be r e fo rme d chem ica l ly by h ydrog ena t i on o r

in t e re s t e r if i c a t i on , bo th r eac t ions occur a t r an dom posi -

t i ons and some o f t he cis u n s a t u r a te d f a t t y a c y l m o i e t ie s

are conver ted to the i r

trans

form s in the f i rs t reac t ion.

The use o f enzymes , such a s li pa se , wh ich p roduce n ew

types o f t r ig lyce r ide depend ing on t he i r subs t r a t e an d

posi t ion speci f ic i t ies , i s very important for food indust r ies .

Interes ter i f ica t ion w i th hog pancreas l ipase has been re-

* D e d i c a t e d t o P r o f e s s o r H . H o l z er , F r e i b u r g U n i v er s i t y , o n

h i s 6 0 t h b i r t h d a y J u n e 1 3 , 1 9 8 1 )

Offprint reque sts to

S. Fukui

por t ed i n an aqueous sys t em (S t evenson e t a l. 1979).

How eve r , t he p rodu c t y i e ld was no t good i n such aqueous

systems.

There are severa l problems in establ i shing such reac-

t i on sys t em. O ne i s the ve ry l ow so lub i l i t y o f t he subs t r a t e s

and p roduc t s i n an aqueous sys t em. A two-phase sys t em

c o m p o s e d o f a n a q u e o u s m e d i u m a n d a n o i l w o u l d l o w e r

the r ac t i on r a t e and be i nconven ien t fo r ope ra t i ng a con-

t i nuous sys t em. Fur the rmo re , an excess o f wa t e r i n t he r e -

ac t i on mix tu re t ends t o f avour hydro lys i s o f t r ig lyce r ide s

ra the r t ha n i n t e re s t e r i f ic a t i on . An app ropr i a te o rgan ic

so lven t is r equ i red a s a r e ac ti on med ium to se t up a ho-

mogen eous r eac ti on sys t em and t o sh i f t t he r eac t i on

equi l ibr ium to the desi red di rec t ion.

The p rodu c t ion o f c acao bu t t e r -l i ke f a t f rom o li ve o i l

and s t ea r i c a c id o r pa lmi t i c a c id b y enzym a t i c i n t ere s t er -

i f ica t ion in an organic solvent system has been successful ly

a t tem pted wi th C el i te -adsorbed 1- and 3-po si t ional spec i f ic

l ipase f rom

R h i z o p u s d e l e m a r

and t h i s sys t em has p roved

to be supe r io r t o t he aqu eous sys t em (Tanaka e t a l. 1981 ;

F ig . 1 ) . Long- t e rm repea t ed use o f t he enzy me w ould be

a use fu l improvem ent o f t h i s sys tem. Imm obi l i z a t i on o f

l ipase in or on a n adequ ate sup port i s expected to stabi-

l iz e t he enz yme in an o rgan i c so lven t and t o pe rm i t i t s

r e u s e .

In t he mean t ime , b ioconve rs ions o f s t e ro ids and t e r -

pen oid in water-immiscible organic solvents have bee n

successful ly carr ied ou t using microbia l ce l ls ent rapp ed

in gel s o f an appropr i a t e hydro pho b ic cha rac t e r (Fuku i

e t a l . 1980; Omata e t a l . 1979a and b, 1980 and 1981;

Yam ane e t a l . 1979) . These system s seemed to be appli -

cable to the biocon versions of a var ie ty of water- insol-

ub l e o r h igh ly l ipoph i l ic com poun ds such a s o il s .

This paper describes regio-specific interesterification

of t r ig lyce f ide i n n -hexane w i th l ipa se immob i l i z ed by

v a ri o us m e t h o d s b u t p r e d o m i n a n t l y w i t h t h e e n z y m e

en t rapped by pho to -c ross f i nkab le r e sin p repo lymers .

0 1 7 1 - 1 7 4 1 / 8 2 / 0 0 1 4 / 0 0 0 1 / 0 1 . 0 0


2/5

2 K . Yok oz e k i e t a l . : In te re s te r i f i c a t ion of Tr ig lyc e r ide by Im m obi l i z e d L ipa se

H2~-O-CO-R H2~-O-CO-X

HC-O-CO-R + 2 X-COOH . HC-O-CO-R

g

HZC-O-C0-R HzC-O-C0-X

R - C00H R - C00H

Fig . 1 . 1- a nd 3-Pos i t iona l spe c i f ic in te re s te r i f ic a t ion of t r ig ly-

c e r ide by l ipa se f rom Rhizopus delemar

T h e e f f e c t o f th e h y d r o p h o b i c i t y o f t h e g el s o n t h e

a c t i v i t y a n d t h e s t a b i l i t y o f t h e i m m o b i l i z e d l ip a s e is

a l s o d i s c u s s e d .

M a t e r ia l s a n d M e t h o d s

Enzyme.. Lipa se f rom Rhizopus delemar (6 ,000 uni t s pe r r a g) wa s

purc ha se d f rom Se ika ga ku Kogyo Co. , L td . , J a pa n .

Chemicals. Four k inds of hydrophi l i c photo-c ros s l inka ble r e s in

p r e p o l y m e r s , E N T - 1 0 0 0 , 2 0 0 0 , 4 0 0 0 a n d 6 0 0 0 ( e a c h n u m b e r

i n d i c at e s t h e a p p r o x i m a t e m o l e c u l ar w e i g h t o f p o l y ( e t h y l e n e g l y -

c ol ) ske le ton) (Ta na ka e t a l . 1978) , a nd a hydrophobic photo-

c ros s l inka ble r e s in pre polym e r , ENTP-2000 (Sonom oto e t a l .

1979; O m a ta e t a l . 1979b) we re the produc t s of Ka nsa i Pa in t Co. ,

L td . Thre e k inds o f wa te r -m isc ib le ure tha ne pre po lym e rs (PU-3 ,

PU-6 a nd PU-9) (Fukushim a e t a l . 1978; Om a ta e t a l . 1979a ;

S o n o m o t o e t a i. 1 9 8 0 ) w e r e o b t a i n e d f r o m T o y o R u b b e r I n d u s t r y

C o . , J ap a n . P o r o u s s il ic a be a d s, S p h e r o s i l (X O B 0 1 5 ; - O - S i O 2 -

O H : Q M A ; - [ ( C H 3 ) 3N ] + C I - : a n d g l u t a r a ld e h y d e - a c ti v a t ed X O B

0 1 5 ; - N = C H - ( C H 2 ) 3 - C H O ) w e r e t h e p ro d u c ts o f R h d n e-

Poule nc , Fra nc e . Ce l i t e (No. 535) wa s the produc t of Johns -Ma r -

v i ll e C o ., U S A . n - H e x a n e a n d T E S ( N - T r i s ( h y d r o x y m e t h y l ) m e t h y t -

2-a m inoe tha ne sul fonic a c id) buf fe r we re obta ine d f rom Wa ko

Pure Che m ic a l s, a nd s te a r ic a c id f rom N a ka ra i Che m ic a l s L td . ,

J a pa n . O l ive o i l wa s the prod uc t of Yoshida Pha rm a c e u t ic a l s L td . ,

J a pa n .

Adsorption of Lipase on C eli te. Usua l ly , 5 m g of l ipa se d i s so lve d

in 200/~1 of 0 .3 M TES buf fe r , pH 6 .5 , wa s m ix e d thoro ughly w i th

0 .25 g o f Ce l i t e . Ce l i t e -a dsorbe d l ipa se thus o bta ine d wa s de s igna te d

as C-lipase.

Imm obilization o f Lipase with Hyclrophilic Photo.Crosslinkable

Resin Prepolymer. E N T - 1 0 0 0 , 2 0 0 0 , 4 0 0 0 o r 6 0 0 0 ( 0 .5 g ) w as

m i x e d w i t h 5 m g o f a p h o t o s e n s i ti z e r , b e n z o i n e t h y l e t h e r a n d

100 Izl of 0 . 3 M TES b uf fe r , pH 6 .5 . The m ix ture wa s m e l te d by

w a r m i n g a t 6 0 ~ c o o l e d t o ro o m t e m p e r a t u r e an d t h e n 5 m g o f

f r e e l ipa se in 100 t zl o f the buf fe r wa s a dde d to the m ol te n m ixtu re .

The m ix ture wa s ge l le d by a shor t i l lum ina t ion w i th ne a r -UV

l ight a nd the ge l form e d wa s c u t in to sm a l l p ie c e s (3 x 3 r a m ) , a s

de sc r ibe d pre vious ly (Fuk ui e t a l . 1976; Ta na ka e t a l . 1978) . In

som e c a se s, C- lipase c or re spond ing to 5 m g o f l ipa se wa s im m o -

bi l i z e d by th e s a m e proc e dure .

Immobilization of Lipase with Hydrophobic Photo Crosslinkable

Resin PrepoIymer. E N T P - 2 0 0 0 ( 0 .5 g ) a n d 5 m g o f b e n z o i n e t h y l

e the r we re d i s so lve d in 400 1 of wa te r - sa tura te d n-he xa ne . 5 m g

of f r e e l ipa se in 200/~ 1 of 0 .3 M T ES buf fe r , pH 6 .5 , c onta in ing

40 m g o f Twe e n 80 or C- l ipa se (5 m g o f l ipa se ) was a dde d to the

m ixtu re a nd the im m o bi l i z a t ion wa s c a r r i e d out a s de sc ribe d

a bove .

Immobilization o f Lipase with Urethane Prepolymer. PU-3, PU-6

or PU-9 (0 .5 g) m e l te d a t 60 ~ i f ne c e s sa ry a nd c oole d to roo m

t e m p e r a t u r e , w a s m i x e d q u i c k l y w i t h i 0 0 ; zl o f 0 . 3 M T E S b u f f e r ,

pH 6 .5 , c onta in ing 5 m g of l ipase a nd the n the m ix ture wa s ke pt

a t 4 ~ for 1 h to c om p le te ge la t ion . The gel thus form e d wa s

c ut in to sm a l l p ie c e s (3 x 3 x 3 r a m ) a nd u se d for the r e a c t ion .

Immobilization o f Lipase with Porous S~ica Bead~

O n e g r a m o f

porous s i li c a be a ds , Sphe ros i l XOB 015, QMA or g lu ta ra lde hyd e -

-a c t iva ted XOB 015 we re m ixe d w i th 10 m g of lipa se d i s so lve d in

3 m l o f 0 . 0 3 M T E S b u f fe r , p H 6 . 5 , a n d k e p t at 4 ~ f o r 1 h w i t h

m o de ra te s t i rr ing . The be a ds we re c o l l e c te d on a g la ss f i l t e r a nd

r inse d w i th the buf fe r . Th e im m o bi l i z e d l ipa se on th e poro us s i li -

c a be a ds wa s lyophi l i z e d a nd use d for the r e a c t ion .

Enzyme Reaction. The r e a c t ion wa s c a r r i e d ou t a t 40 ~ w i th

shaking (120 s t roke s pe r m in) . The r e a c t ion m ixture wa s c om -

pose d of the im m obi l i z e d l ipase or C- lipase ( c or re sponding to

5 m g of l ipa se ) , 0 . 25 g of o l ive o i l a nd 0 .25 g o f s t e a r ic a c id in

10 m l of wa te r - sa tura te d n-he xa ne .

Analysis.

To i so la te t r ig lyc e r ide from th e r e a c t ion m ix ture , 1 m l

of the r e a c t ion m ixture w a s loa de d on a th in- la ye r p la te (S i l ic a

ge l -60 , No. 574 7, M e rc k , Ge rm a ny ) a nd de ve lop e d wi th a so lve nt

sys te m o f he xa ne -e thy l e the r -a c e t i c a c id (80: 20 : 1 by v olum e ) .

Tr ig lyc e r ide on the p la te w a s v i sua l i z e d unde r UV l igh t by spra y-

ing 0 .1% 2 ' , 7 ' -d ic h lorof luore sc e in a nd e xt r a c te d w i th a bout

10 m l of e thy l e the r f rom the s i l i c a ge l s c ra pe d of f f rom the p la te .

The e thyl e the r wa s e va pora te d to dryne ss a nd the t r ig lyc e r ide

t h u s e x t r a c te d ( 1 0 - 1 0 0 m g ) w a s h y d r o l y z e d w i t h 1 .5 m l o f

0 .5 N Na OH in m e th a no l at 80 ~ for 7 ra in . The f a t ty a c ids

form e d we re e s te r i f i e d w i th 1 .5 m l of 7% BF3 in m e tha nol for

2 r a in a t 80 ~ a nd the r e a c t ion m ixture wa s ke pt fo r a fur the r

1 r a in at 80 ~ a f t e r the a ddi t ion o f i m l of he xa ne . A f te r c ool ing

to room te m pe ra tu re , 5 m l of s atura te d Na C1 a que ou s so lu t ion

wa s a dde d to the m ixture a nd the he xa ne l a ye r obta ine d wa s de -

h y d r a t e d o n a n h y d r o u s N a 2 S O 4 . T h e s a m p l e t h u s p r e p a r e d w a s

a n a l y z ed b y g a s c h r o m a t o g r a p h y w i t h a J E O L J C G - 2 0 K F L G a s

C h r o m a t o g r a p h e q u i p p e d w i t h a h y d r o g e n f l a m e i o n i z a ti o n d e -

te c tor . A s ta in les s s t e el c o lum n (2 m x 3 m m ID ) was pa c ke d wi th

15% DEGS Dia sol id L (Ga sukuro K ogyo , J a pa n) . The t e m pe ra ture s

of the in je c t ion a nd c olum n we re 220 ~ a nd 175 ~ r e spe c tiv-

e ly . F low ra te of he l ium , the c a r r ie r gas , wa s 40 m l p e r r a in . Pro-

por t io n of s t e a r ic a c id to the to ta l f a t ty a c ids in t r ig lyc e r ide

w a s c a l cu l a t e d f r o m t h e g as c h r o m a t o g r a p h i c p a t t e r n o f f a t t y

a c ids a nd the r a t io of s t e a r ic a c id inc orpora te d wa s obta ine d by

subt ra c t ing the e ndoge nous propor t ion of s t e a r ic a c id in o l ive

oi l (2.9% by we ight ) .

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

E f f e c t o f S u p p o r ts o n t h e A c t i v i t y o f I m m o b i l i z e d L i p as e

L i p a s e f r o m R . d e l e m a r w a s i m m o b i l i z e d b y a d s o r p t i o n ,

i o n i c b in d i n g , c o v a l e n t b i n d i n g o r e n t r a p m e n t u s i ng v a r-

i o u s t y p e s o f s u p p o r t s . T h e r e l a t i v e a c ti v i t ie s o f i m m o -

b i l i z e d l i p as e i n i n t e r e s t e r i f i c a t i o n o f t r i g l y c e r i d e i s s u m -

m a r i z e d i n T a b l e 1 . F r e e l ip a s e s h o w e d a l m o s t n o a c t i v i t y

i n w a t e r - s a t u r a t e d h e x a n e , w h i l e C e l i t e - a d s o r b e d l i p a s e

( d e s i g n a t e d a s C - li p a s e) c a t a l y z e d t h e f a c t i o n w e l l t h r o u g h

t h e e f f e c t iv e d i s p e rs i o n o f t h e e n z y m e i n t o t h e r e a c t io n


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K. Yok ozek i et al . : Interesterification of Triglyceride by Im mobilized Lipase

Table 1. Interesterification activity o f lipase immobilized by different m ethod s

Suppo r t Adsorpt ion Immobi lizat ion metho d Prote in Act iv i ty

on Celite imm obilized (%) yield (%)

None + - 100 100

None - - - 0

Spherosil XOB 15 - Adsorption 74 16

QMA Ionic binding 80 28

XOB 15 (glutaraldehyde-activated) - Covalent binding 70 9

ENT-1000 - Ent rapm ent 100 13




ENTP-2000 - En t r a pme n t 100 29 - 82

ENTP-2000 + Ent rapm ent 100 75

PU-3 - Ent rapm ent 100 19



C-Lipase (l ipase adsorbed on Celite) was prepared as described in Materials and M ethods . The enzymatic reac tion was carried

f o r l h

ou t

A 1 5

O A f

I I I

1

0

o

c_

x : ~ 5 -

'Z

oo

0 100 200 300

Vo lum e of buf fer p . l )

/ s

I I

400 500

Fig. 2. Effect of buffer volume on interesterification of tr igly-

ceride. Lipase (5 mg) dissolved in the indicated volume of 0.3 M

TES buffer, pH 6.5, was mixed with 0.25 g of Celite. The enzym -

atic reaction was carried ou t for I h. (a), C-Lipase; (o), ENTP-

2000-entrapped C-lipase

m i x t u r e . A l t h o u g h li p as e e n t r a p p e d w i t h a h y d r o p h o b i c

p h o t o - c r o s s li n k a b l e r e s in p r e p o l y m e r , E N T P - 2 0 0 0 , e x -

h i b i t e d t h e b e s t a c t i v it y o f th e i m m o b i l i z e d p r e p a r a t i o n s

e x a m i n e d , e n t r a p m e n t o f f r e e li p as e d i d n o t g iv e re p r o -

d u c i b le r e s u lt s. T h i s m a y b e d u e t o t h e d i f f i c u l t y o f h o -

m o g e n e o u s d i s p e r s i o n o f li p a se i n t h e g e l d u r i n g t h e i m -

m o b i l i z a t i o n p r o ce s s . O n t h e c o n t r a r y , E N T P - 2 0 0 0 - e n -

t r a p p e d C - l i p as e s h o w e d a h i g h a n d r e p r o d u c i b l e a c t i v i t y .

L i p a s e g a v e a l o w a c t i v i ty w h e n e n t r a p p e d w i t h p h o t o -

c r o s sl i nk a b l e r e s in p r e p o l y m e r s o f h y d r o p h i l i c c h a r a c t e r

( E N T - 1 0 0 0 , E N T - 2 0 0 0 , E N T - 4 0 0 0 a n d E N T - 6 0 0 0 ) . P re -

t r e a t m e n t o f li p a s e w i t h C e l it e h a d l i tt l e e f f e c t o n t h e

e n z y m e a c t i v i t y . T h e s e r e s u l t s s u g g e s t e d t h a t t h e h y d r o -

p h o b i c i t y o f th e g e ls e n tr a p p i n g l i pa s e w o u l d m a r k e d l y

a f f e c t t h e d i f f u si o n o f h y d r o p h o b i c s u b s tr a te s , s u c h a s

o i ls , i n t o t h e g e ls , a s i s t h e c a s e w i t h s t e r o i d b i o c o n v e r -

s i o n in o r g a n i c s o l v e n t s ( F u k u i e t a l . 1 9 8 0 ; O m a t a e t a l .

1 9 7 9 a a n d 1 9 8 0 ) . L i p a se e n t r a p p e d w i t h u r e t h a n e p r e -

p o l y m e r s , P U - 3 , P U - 6 a n d P U - 9 , s h o w e d a r a th e r l o w

a c t i v i t y . H o w e v e r , e v e n i n t h is c a s e , t h e g e l h y d r o p h o -

b i c i t y a f f e c t e d t h e e n z y m e a c t i v i t y . T h a t i s, l i p a s e e n -

t r a p p e d i n t h e h y d r o p h o b i c g e l, P U - 3 , e x h i b i t e d a h i g h e r

a c t iv i t y th a n t h a t e n t r a p p e d i n h y d r o p h i l i c P U - 6 a n d

P U - 9 . T h e e n z y m e b o u n d o n t o p o r o u s s i li ca b e a d s ( S p h e -

r o s i l ) s h o w e d a m o d e s t a c t i v i t y .

Optimization of Reaction Conditions

I n t h i s s t u d y , n - h e x a n e w a s s e l e c t e d a s t h e r e a c t i o n s o l v e n t

b e c a u s e t h e s u b s t r a t e s a n d t h e p r o d u c t s a r e h i g h l y s o l u b l e

i n it a n d b e c a u s e i t h a s a l es s d e n a t u r a t i n g e f f e c t o n t h e

e n z y m e . H e x a n e o b t a i n e d f r o m c o m m e r c i a l s o u r c e s o f t e n

g a v e fl u c t u a t i n g r e su l ts p r o b a b l y d u e t o d i f f e r e n t w a t e r

c o n t e n t s . F u r t h e r m o r e , w a t e r - u n s a t u r a t e d h e x a n e a p p e a r s

t o e x t r a c t w a t e r , w h i c h i s e s s e n t i a l f o r t h e c a t a l y t i c a c t i o n

o f t h e e n z y m e , f r o m t h e g e l s a n d c o n s e q u e n t l y , f r o m t h e

m i c r o e n v i r o n m e n t o f th e e n z y m e . T o a v o i d t h e se u n d e -

s i ra b l e e f f e c t s o f t h e o r g a n i c s o l v e n t , h e x a n e s a t u r a t e d w i t h

w a t e r w a s e m p l o y e d a s t h e s o l v e n t t h r o u g h o u t t h e e x p e r i-

m e n t s .


4/5

4 K . Yok oz e k i e t a l .: In te re s te r i f i c a t ion of Tr ig lyc e r ide by Im m obi l i z e d L ipa se

100

o

~ 75

>

50

ID

>

g 2~.

0

5.5 6.0 6.5

pH

7.0 7.5

~ 40

3

_

~ - 2 0 -

.x2

._u 10 -

o

\

\

\

O0 3 6 9 12

Incuba t ion per iod [day )

Fig . 3 . Ef fe c t o f buf fe r pH on in te re s te r i f i c a t ion o f t rig lyc e r ide .

C-Lipa se wa s pre pa re d a s de sc r ibe d in Ma te r ia l s a nd M e thods

e x c e p t t h a t t h e p H o f t h e b u f f e r w a s v a r ie d . T h e e n z y m a t i c r ea c -

t ion wa s c a r r i e d ou t fo r 1 h . ( zx), C-Lipa se ; (o) , ENTP-200 0-e n-

trapped C-l ipase

Fig . 5 . Re pe a te d use of l ipa se pre pa ra t ions for in te re s te r i f i c a t ion

of t r ig lyc e r ide . C-Lipa se wa s pre pa re d a s de sc r ibe d in Ma te r ia l s

a nd Me thods . Ea c h e nz ym a t ic r e a c t ion wa s c a r r i e d out for 24 h .

(A), C-Lipase; (o) , ENTP-2000-entrapped C-l ipase

50

30

-~ 20

10

o

O~

0

m

5 10 15 20

Incuba t ion t ime h r )

Fig . 4 . T im e -c ourse o f in te re s te r i f i c a t ion o f t r ig lyc e r ide . C-Lipa se

wa s pre pa re d a s de sc r ibe d in Ma te r ia l s a nd Me th ods . ( z x), C-Li-

pa se ; (o) , ENT P-2000 -e nt r a ppe d C- lipase

A s m e n t i o n e d a b o v e , th e c o n c e n t r a t i o n o f w a t e r i n t h e

reac t i on mix tu re w i l l a f f ec t t he y i e ld o f r e fo rmed f a t ,

f avour ing e i ther i n t e res t e r if i ca t i on o r hyd ro lys i s . F igu re 2

s h o w s t h a t t h e o p t i m u m v o l u m e o f t he b u f f e r w a s f r o m

200 t o 300/11 wh en 5 mg o f f r ee l ipase w as adso rbed on

0.25 g of Ce l i te to prepare C-lipase. Ent ra pm en t of C-l i-

pase w i th ENTP-2000 d id no t sh i f t the op t imal r ange o f

t he bu f fe r vo lume.

The pH depe nden cy o f t he i n t e res t e r i fi ca t i on is il lu s -

r

t r a t ed i n F ig . 3 . Bo th C- li pase and EN TP-2000-en t r apped

C- li pase showed t he same pH op t imu m of abo u t pH 6 .5 .

Time Course o f Reaction

Time-cou r ses o f i n t e res t e r i f ica t i on wi th C- li pase o r ENTP-

2000-en t r apped C- li pase under t he op t imized con d i t i ons

me n t ioned above a re shown in F ig . 4 . Inco rpo ra t i on o f

stear ic acid in to o l ive oi l was l inear wi th t im e up to 4 h

and , t hereaf t e r , i ncreased g radual l y t o r each t he ma x imu m

of abou t 40 . Th i s va lue i s r a t her smal l e r t han t he t heo -

ret ical value of abo ut 65 , suggest ing the existence of an

equ i l ib r i um o r com pet i t i on be tween s t ear ic ac id added as

the subst rate and oleic acid released f rom ol ive oi l . From

the r eac t i on r a t es shown in F ig . 4 , t he ac t i v i t y o f en t r ap -

ped C-l ipase was foun d to be 75 of that of C-lipase.

Stability o f Lipase

The C- li pase had l o s t a bou t h a l f o f i t s ac t i v i t y a f t e r 5 ex -

per imen t s (ope ra t i on per i od , 5 da ys ) . En t r appe d C- l ipase

was f a r more s t ab l e , more t ha n 90 o f i ts ac t i v i t y r emain -

ing a f t e r 12 exper im en t s (opera t i on per i od , 12 days)

(F ig . 5 ) . Main t enance o f t he enzym e ac t i v i t y by im mo-

b i l iza t i on m ay be ascr i bed t o t he p ro t e c t i on o f t he enzym e

f r o m d e n a t u r a t i o n b y n - h e x a n e , p r e v e n t io n o f l e a k ag e o f

t he p ro t e in f rom Cel i te o r t o bo th .


5/5

K. Yokozeki et al.: Interesterification of Triglyceride by Immobilized Lipase 5

As described above, interest erificat ion of olive oil

with stearic acid to produce cacao butter-like fat could

be carried o ut successfully in an organic solvent by hy-

drophobi c gel-entrapped lipase. This technique appears

to be applicable to various reactions involving lipophilic

and water-insoluble reactants.

A c k n o w l e d g e m e n t s T h e authors thank to Mr. T. Tanaka, Ajino-

moto Co., Inc., for his useful discussion, and are indebted to

Mr. T. Yamamoto and Mr. T. Iida, Kansai Paint Co., to Mr. S.

Fukushima, Toyo Rubber Industry Co. , and to Dr. B. Mirabel,

Rhdne-Poulenc, for their generous supply of the supports used

in this study.

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Received August 17, 1981

immobilized lipase to regio-specific interesterification

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