fungal enzymes (1)

8/3/2019 Fungal Enzymes (1)

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E L S E V IE R P I I :

Process Biochemistry,Vol. 32 , No . 5 , pp . 441-449 , 1997Copy righ t 1997 E lsev ier Science L td

Prin ted in Great Br i ta in . Al l r igh ts reserved0032-9592/97 $17.00 + 0.00

S 0 0 3 2 - 9 5 9 2 ( 9 6 ) 0 0 1 0 4 - 5

E ff e c t o f i m m o b i l i z a t i o n o n t h e s t a b i l i t y o fbac t e r i a l and funga l f i -D-g lucos idase

M . D . B u s t o , N . Ortega and M. Perez-Mateos*

Dep artm ent o f Biotechnology and Foo d Science, Faculty of Food Science an d Techn ology, University o f Burgos, Plaza M isaelBanuelos, s/n, E-09001 Burgos, Spain

(Received 6 Sep temb er 1996; revised version received and accepted 17 Nov emb er 1996)

A b s t r a c t

Th e therm al and p roteo lytic stability of free and imm obilized fl-o-glucosidase, isolated fro mP s e u d o m o n a spicket t i iand Aspergi l lus n iger,were d etermin ed. Th e optimal temp eratures of soluble a nd entra~gped fl-gluco-sidase extracted fromP. picket t i iwere 40 an d 50C , respectively. In contrast , the optimal tem peratu re o fenzyme isolated fromA. n i ge rremained unaltered (60C). Free and immobil izedA. n igerfl-glucosidaseshowed an unusual discontinuity around 40C in the A rrhenius plot , suggest ing th at the enzym e could existin two (or mo re) interconvert ible forms with different act ivat ion energies.

Th e po lymeric network influenced the react ivi ty of bo th fungal and bacterial fl-glucosidases since the ir E~values chang ed with respect to their soluble co unterparts. Nevertheless, both the therm al stabili ty and theresistance to proteolysis were ap parently related to the origin (bacterial or fungal) and locat ion (intra-cellular or exocellular) of the enzyme. The half-lives of soluble and immobilized fl-glucosidases at sixdifferent temperatures were also calculated. The propert ies assayed were compared cri t ical ly with thoserepo rted by othe r authors. 1997 Elsevier Science Ltd

Keywords :enzy me stabilization, enzy me immo bilization, fl-glucosidase, calcium alginate, enzy me half-life,protease.

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

T h e i m m o b i l i z a ti o n o f e n z y m e s i n c r e a s e s t h e i r s t a b il it yin many cases [1 , 2 ] . I t i s d i ff icu l t to p red ic t , however,w h e t h e r a p a r t i c u l a r m e t h o d o f i m m o b i l i z a ti o n wi llresu l t in such an increase [3 , 4 ] . Obviously, thee n h a n c e m e n t o f s t a b i l i t y i s a d v a n t a g e o u s f o r t h e i n d u s -t r i a l ap p l i ca t i o n o f en zy me s [5, 6] an d i t is th u si m p o r t a n t i n d e t e r m i n i n g t h e f e a s ib i li t y o f a n e n z y m esy s t em fo r a p a r t i cu l a r ap p l i ca t i o n . No t wi t h s t an d i n g ,t h e l o ss o f e n z y m e a c t iv i ty w i t h t i m e is o n e o f t h e m o s ti m p o r t a n t f a c t o r s i n d e t e r m i n i n g o r i n d e e d l i m i t i n g t h es y s t e m p e r f o r m a n c e a n d e c o n o m i c s [ 7 ] . T h r e e b a s i ct y p e s o f e n z y m e a c t iv i ty lo s s c a n o c c u r : ( i ) t h e e n z y m ec a n b e l o s t f r o m t h e s y s t e m b e c a u s e o f d e s o r p t i o n ,s e v e r in g o f c h e m i c a l b o n d s , o r e r o s i o n o f t h e s u p p o r tma t e r i a l [8 ] ; ( i i ) i t c an b eco me i n ac t i v e d u e t o t h e rma ld e n a t u r a t i o n , i n h i b i t i o n , i n a c t i v a t i o n o r d e s t r u c t i o n b yp ro t e o l y t i c en zy m es [9] ; o r ( i i i) i t c an b e c o a t ed o ro t h e r w i s e b l o c k e d f r o m c o n t a c t w i th t h e s u b s t r a t e [ 10 ].

I n a d d i t i o n , t h e e x a m i n a t i o n o f t h e c h a n g e s i n o p t i -m a l t e m p e r a t u r e a n d p H t h a t o c c u r a s a co n s e q u e n c e

*To whom correspondence should be addressed .

44 1

o f i m m o b i l i z a t i o n o f t h e e n z y m e p r o v i d e s u s e f u l i n f o r -m a t i o n a b o u t t h e r e l a t i o n s h i p b e t w e e n t h e e n z y m ep r o t e i n s t r u c t u r e a n d t h e e n z y m e a ct iv i ty a n d a b o u t t h ep h y s i c a l o r c h e m i c a l p r o p e r t i e s o f t h e c a r r i e r s u r f a c e[11].

O n t h e o t h e r h a n d , t h e b r e a k d o w n o f n a ti v e c e l-l u l o se t o so l u b l e su g a rs i s a p ro cess wh i ch i n v o l v es t h ea c t i o n o f a m u l t i - en z y m i c sy s t em . O n e c o m p o n e n t o fth i s sys tem is the f l -D-g lucosidase (ce l lob iase , f l -o -g lu-co s i d ase g l u co h y d ro l a se , EC 3 .2 .1 .2 1 ) wh i ch ca t a l y se st h e h y d ro l y s i s o f c e l l o b i o se t o D-g l u co se [1 2 ] . M u cha t t e n t i o n h a s b e e n f o c u s e d o n t h i s e n z y m e s i n c e c e l l o -b i o s e i s n o t o n l y t h e m a j o r p r o d u c t o f t h e c e l l u lo s eso l u b i l iz i n g en zy m es b u t i s a l so a p o t en t i n h i b i t o r o ft h e s e e n z y m e s a n d i f a l l o w e d t o a c c u m u l a t e d c a u s e s as i g n i f i can t d ec l i n e i n t h e ra t e , an d fo r p rac t i ca l p u r-p o ses a d ec re a se i n t h e o v e ra l l l ev e l, o f c e l l u l o seh y d ro l y s i s [1 3 ] . Th e e ff i c i en t h y d ro l y s i s o f c eUo b i o se t oD-g l u co se ca t a l y sed b y f l -o -g l u co s i d ase i s , t h e re fo re ,c r i t ic a l t o t h e o v e ra l l d i g e s t i o n o f ce ll u l o se .

I m m o b i l i z a t i o n o f f l - g lu c o s id a s e s h a s b e e n s u g g e s t e das a way t o ach i ev e t wo o b j ec t i v e s : f i r s t , t o i mp ro v een zy me s t ab i l i t y an d seco n d , t o fac i l i t a t e a d ec rea se i ne n z y m e c o n s u m p t i o n [ 14 , 1 6].


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442 M.D. Bustoet al.

Th e wo r k r e p o r t e d h e r e i s a n a t t e mp t t o g a i n k n o w-l e d g e o n t h e e f f e c t s o n e n z y me p r o p e r t i e s o fimmobi l i za t ion o f funga l and bac te r i a l f l -g lucos idasesin ca lc ium a lg ina te ge l s . The in f luence o f enzymeimmobi l i za t ion upon the k ine t i c p rope r t i e s was ve r i f i edin p rev ious work [17 ]. Th e re su l t s o f th i s p rev ious workfo rm the bas i s o f the p resen t s tudy, in wh ich we g ivethe r e su l t s o f expe r im en ts con ce rn ing the f l -g lucos idaseac t iv ity wi th r e la t ion to the the rm a l a nd p ro teo ly t i cs t ab i li ty. Op t im a l t e m pera tu re a s we l l a s the ac t iva t ionene rg ie s were a l so inves t iga ted .

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

Cell growth an d enzyme production

Th e b a c t e r i u m,Pseudomonas pickettii ,i so la ted in ou rlabora to ry f rom so i l [18 ] , was g rown in c losed cu l tu resby incuba t ion a t 30C in an o rb i t a l sha ke r a t 150 rpmo n t h e mi n e r a l s a l t s me d i u m d e s c r i b e d b y Wo l l u m I I[19 ] supp lemen ted wi th 0 .4% ce l lob iose a s a so le ca r-b o n a n d e n e rg y s o u r c e. To o b t a i n t h e b a c t e r i a l e n z y me ,the cu l tu re s were cen t r i fug ed a t 21 000g fo r 30 min a t4C. The re su l t ing pe l l e ts were then w ashed w i th 10 mld i s ti ll e d wa t e r, r e s u s p e n d e d i n 1 0 ml Tr i s - m a l e a t ebuffe r (pH 7 -0 ) and son ica ted f ive t imes fo r 60 s ( a t9 0 W a n d a t e mp e r a t u r e o f 4 C) i n a Br a n s o n $ 2 5 0son ica to r a t a f r eq uenc y o f 20 kHz . Th e so n ica te d sus-pens ion w as cen t r i fuged a t 30 000 g fo r 1 h a t 4 C andt h e r e s u l t i n g s u p e r n a t a n t wa s c o l l e c t e d a n d u s e d f o rassay o f the endo ce l lu la r f l -g lucos idase ac t iv i ty.

To o b t a i n t h e f u n g a l e n z y me , s p o r e s f r o mAspergillusniger( s tr a in o b t a i n e d f r o m t h e Sp a n i s h Cu l t u r e Co l l e c -t i o n ) we r e p r o d u c e d o n Cz a p e k - Do x a g a r s l a n t s f o r7 days a t 30C. A sp ore susp ens ion was then inocu la tedin 250 ml E r len me yer f l a sks con ta in ing 100 ml m ine ra lm ed ium (0.5 g NaN O3, 1 .0 g K2HP O4, 0 .5 gMgS O4"7H2 0 and 0 .01 g FeSO4"7H 20 pe r l i t re ) a t pH7 , 0 .2% pep tone and 0 .4% ce l lob iose a s a ca rbonsource . Th e f l a sks were incub a ted fo r 7 days a t 30Cwi thou t ag i t a t ion . The cu l tu res were f i l t e red th roughme mb rane s wi th a po re s i ze o f 0-45 /~m, h a rves t ing theexoce l lu la r f l -g lucos idase in the ex t ramyce l i a l supe r-n a t a n t .

Imm obilization o f fl-glucosidase

Ca l c i u m a l g i n a t e b e a d s we r e p r e p a r e d , a s d e s c r i b e dprev ious ly [17 ] , by d ropp ing a 2% sod ium a lg ina tes o l u t io n ( i n wh ic h a s u i t a b le a mo u n t o f e n z y m e h a dbeen d i lu ted ) in to a 0 .2 M ca lc ium ch lo r ide so lu t ionu n d e r c o n t i n u o u s s t i r r i n g . Th e b e a d s we r e s t i r r e d i nthe ca lc ium ch lo r ide so lu t ion fo r 4 h , washed seve ra lt imes wi th a 0 .03 M CaC12 so lu t ion (un t i l no ac t iv itywas obse rved in the f ina l wash ing) an d s to red in th isso lu t ion a t 4 C p r io r to use .

Ass ay of fl-glucosidase activity

f i -Glucos idase ac t iv i ty was de te rm ined by incuba t ing a t37C in a ro ta ry sha ke r (150 rpm) a r eac t ion mix tu rec o n t a i n i n g 3 ml o f 0 "1 M Tr i s - m a l e a t e b u f f e r ( p H 5 .4 ),1 m l o f a 1 0 mM 4-n i t ropheny l f l -D-g lucopyranos ides o l u t io n ( p N PG) a n d 1 ml o f t h e n a t iv e o r i mmo b i l i z e de n z y me s o l u t i o n . Th e e n z y mi c r e a c t i o n wa s s t o p p e da f te r 1 h o f inc uba t ion by ad d ing 8 ml o f 0 -1 M Tr i s -h y d r o x y m e t i l a m i n o m e t h a n e ( T H A M ) a t p H 1 2 .0 . T h ea b s o r b a n c e o f t h e r e s u l ti n g c o l o u r d u e t o t h e r e l e a s e dp - n i t r o p h e n o l wa s m e a s u r e d a t 4 1 0 n m a f t e r fi l tr a ti o n .Con t ro l s , in wh ich the subs t ra te so lu t ions were addeda f t e r i n c u b a t i o n , we r e i n c l u d e d t o d i s c o u n t a n y n o n -enzym ic ac t ivi ty. Va lues show n in the f igu res and t ab le srep resen t the ave rage o f a t l ea s t th ree r ep l i ca tedsamples .

Effect o f temperature

T h e o p t i m u m t e m p e r a t u r e f o r h yd r ol y si s o f p N P G w a sd e t e r m i n e d f r o m t h e e n z y me a c t iv i ty me a s u r e m e n t sma d e o v e r a t e m p e r a t u r e r a n g e o f 3 0 - 9 0 C . Th e a c t i -v a t i o n e n e rg y(Ea) v a l u e s we r e d e t e r mi n e d f r o m t h e s ed a t a , f o r t e mp e r a t u r e s r a n g i n g b e t we e n 3 0 a n d 6 0 C ,b y u s in g t h e A r r h e n i u s e q u a t i o n p l o t.

Re s i s t a n c e t o s t o r a g e a t e l e v a t e d t e mp e r a t u r e s o ff ree and en t rapp ed f l -g lucos idase was examin ed . Al lprepara t ions were incubated a t 30 , 40 , 50 , 60 , 70 or90C fo r 60 min be fo re the r e s idua l enz ym e ac t iv i ty wasme a s u r e d . Th e t h e r ma l s t a b i li ty o f t h e e n z y me a ts e l e c t e d t e mp e r a t u r e s wa s a l s o s t u d i e d b y i n c u b a t i n gt h e e n z y me s o l u t i o n s i s o l a t e d f r o m P.pickettii a n d A .nigera t d i f f e r e n t t e mp e r a t u r e s r a n g i n g f ro m 4 0 t o 5 0 Cand f rom 60 to 70C, r e spec t ive ly, and wi thd rawingsamples fo r a s says a t f ixed in te rva l s ove r an incuba t ionpe r iod o f 7 h .

Effect of protease

To e x a mi n e t h e e f f e c t o f p r o t e a s e i s o la t e d f r o mStrep-tomyces griseus( Bo e r h i n g e r Ma n n h e i m) o n t h e a c t i v i t yo f so lub le and immobi l i zed f l -o -g lucos idase , p repa ra -t ions o f th i s enzym e were a l lowed to r eac t wi th thep r o t e a s e d i s so l v e d i n 0 -2 M Tr i s - m a l e a t e b u f f e r ( p H7.5) to g ive d i ffe ren t co nc en tra t i on s (0.01, 0 .05, 0 .10,0.50, 1.00 an d 3"00 g l i tre ~) at 37C fo r 1 h. Th e reac -t i o n wa s t e r mi n a t e d b y r a p i d l y r e d u c i n g t h et e mp e r a t u r e o f s a mp l e s i n a n i ce b u c k e t. Th e p H o ft h e r e a c t i o n mi x t u r e wa s r e d u c e d t o p H 5 . 4 b y a d d i n gHC1 and the r e s idua l f l -g lucos idase ac t iv i ty me asu red .

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

Du e to the f ac t tha t bo th the ac t iv i ty a s we l l a s thes tab i l i ty o f an enzyme va r ie s wi th t empera tu re , a c losec o n t r o l o f t h i s p a r a me t e r d u r i n g t h e i mmo b i l i z a t i o np r o c e s s wa s n e c e s s a r y t o e n s u r e t h a t t h e o p e r a t i o n


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Bacterial and funga l fl-o-glucosidase 443

e lapsed under op t imum condi tions . The e ffec t o f tem-perature on the activi ty of f l-glucosidase isolated fromP pickettii an d A. n igeris shown in Fig. 1. Under theassayed condit ions, the catalytic capacity of the freeand immobi l ized enzymes changed no tab ly wi th theI:emperature. Th e m axim um a ctivity of the solub leJ~acterial and fungal fl-glucosidase occurred at 50 and60C, respectively. Below these temperatures enzymeac t iv i t ies decreased , bu t above these tempera tu res theac t iv i t ies decreased rap id ly (a t 50-70C about 48% ofthe enz ym e activi ty was lost) .

The mode of act ion and characteris t ics of fungal andoacte rial cellulases diffe red significantly. In fact, som ecellulases, particula rly of bac terial origin, are kno wn tobe strongly associated with microbial cel ls in contrastto the extracellular fungal cel lulases [20-22]. Further-more , the ce l lu lase components f rom a lmost anyorgan ism (i.e. /3-glucosidases) occ ur as a num be r ofmult iple forms differing in their composit ion and cata-lyt ic characteris t ics [23]. Neve rtheless , the optim umtemp era tu re o f the f l-g lucos idase ex t rac ted f rom A.~iger was similar and/or c oincident to those o f othe rfungal origins. The fl-glucosidases isolated fromTricho-~lerma virid eand Aspergillus wentiihave been repor tedto be optimal at 65 [24] and 60C [12], respectively. Onthe other hand, the activi ty of purif ied f l-glucosidaseisolated fromA. nigerhas been repor ted to be maximalat 55C [25]. Although the p rodu ction of cel lulolyticenzymes using fungi has been extensively s tudied [26],much less is known about bacterial cel lulases and fewstud ies have been pub l i shed on the p roduc t ion andproper t ies o f these enzym es [2 7 , 28] . For exam ple ,Thermomonosphora and Thermoactinomycesspp. are

wor th s tudy ing s ince they p roduce thermostab le (op t i -mum tempera tu re 65-70C) ce l lu lases . However, theseorganisms form an intracellular/ /-glucosidase [21].

F igure 1 shows tha t whi le the op t imum temp era tu re(60C) of soluble and immobil ized fungal f l-glucosidasewas the same, the f ree and en t rapped enzyme f rombacterial origin exhibited a difference of 10C in theirop t imal tempera tu res . How ever, a decrease in the op t i-mum tempera tu re o f the immobi l ized enzyme does no tnecessarily imply that its stability will be lower.Al though the tempera tu re op t imum may decrease by10-20C, the immobi l ized enzyme may be very s tab lea t the lower tem pera tu re [3 ] . Th is d isp lacement o f thet e mp e ra tu r e o p t imu m ma y b e c a u s e d b y th e mic ro-environm ental effects of the calcium alginate m atrix[29]. In contrast , the temperature-activi ty profi le of thesoluble and immobil ized f l-glucosidase fromA. nigerremained a lmos t una l te red . T he resu l t s o f theimm obil ization of enzym es are unpredictable . In som ecases , bo th the op t imal tempera tu re and the tempera-ture-a ctivi ty profi le change [30, 31 ] uponimmobi l iza t ion , whereas in o ther cases they do no t[11].

In genera l , the e ffec ts o f changes in tempera tu re onthe ra tes o f enzyme-ca ta lysed reac t ions do no t p rov idemuch in format ion on the mechan ism of cata lys is . How -ever, these effects can be impo rtant in indicatingstructural chang es in enzyme s [32]. Fro m the transit ionstate theory of chemical reactions, an expression forthe variat ion of the rate constant , k , with temperaturecan be derived. This is of the form of the followingequation, which is sometimes referred to as the Arrhe-nius expression:

120

Fig. 1.

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0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

Inc uba t ion t e mpe ra tu re (oC ) Inc uba t ion t e mpe ra tu re (oC )

Effec t o f t empe ra tu re on ac t iv i ty o f so lub le an d immo bi l i zed f l-g lucos idase f rom (A)P. pick ettiia n d ( B )A. niger.


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444 M . D . B u s t oet al.

k = A e ( --Ea/RT)

In this equation, A is known as the pre-exponentialfactor, R is the gas constant , T is the absolute tempera-ture and E, is the activation energy for the reaction.According to the Arrhenius expression, a plot of In(ve-locity) against 1 /T gives a straight line of slope- E , / 2 . 3 0 3 R [33]. Reactions rates normally increasewith temperature as long as the enzyme activi ty loss isnot significant durin g activity dete rm inatio n.

Taking into account the loss of catalytic act ivi ty athigh temperatures, i t would be expected that theArrhen ius p lo t fo r an enzyme ca ta lysed reac t ion wouldresemble that shown in Fig. 2(A) for free andimmobilized /~-glucosidase fromP. p icket t i i . Compl ica -t ions appeared when the enzyme a rose in two (o rmore) interconvert ible forms with different act ivationenergies. In this case, a discontinuity in the Arrheniusp lo t a round the tempera tu re where the change overbetw een the two forms bec am e signif icant [32]. Anexample o f th is type o f behav iour i s p rov ided by thefree a nd imm obil ized /3-glucosidase isolated from A.n ige r (Fig. 2(B)) . A change in s lope was noted for bothso lub le and bound enzyme a t the same tempera tu rewhere the transit ion probably arose causing structuralchanges in the enzyme. These data suggest possiblethermal deactivation around 40C. Similar s lopes forboth cases imply a freedom from internal mass trans-port resis tance [14].

Fro m the slope of the s traight lines prod uce d in theArrhenius equations between 30 and 60C (Fig. 2) , anac t iva t ion energy of 15 .1 kc a lm ol- I (63"2 kJ mol l )(run a) , 5-8 kcal tool 1 (24-4 kJ m ol -I ) (run b) and2 .9 kca l mol i (12-3 kJ m ol - I ) were deduc ed fo r the

soluble f l-glucosidase isolated fro mA . n ige rand P. p ick -ettii, respectively. The activation energies in theimmobi l ized phases were 11"3 kca l m ol - 1(47.3 kJ tool ]) (run a) , 6"2 kcal m ol -i (26.1 kJ m o l- l)( ru n b) a n d l l . 5 k c a l m o l i ( 4 8 . 2 k J m o l - 1 ) f o r t h efungal an d bacterial f l-glucosidases, respectively.

The p resence o f the po lymer ic ne twork seemed toinfluence the reactivi ty of the f l-glucosidases isolatedfrom different sources, s ince the E~ values changedwith respect to the free counterparts . The activationenergy of an enzyme reac t ion may or may no t changeas a consequence o f the immobi l iza t ion p rocess . Forexam ple, the activation energ ies of imm obil izedenzymes were a lmos t the same as those o f the cor re -sponding native enzym es in the case of lactase [34] andglucoamylase [36] immobil ized by alkylat ion withchloro-s-tr iazinylcellulose or chloro-s-tr iazinyl D E A E -cellulose. On the other hand, the activation energy ofaminoacylases [36, 37] immobil ized by ionic bindingw i t h D E A E - c e l l u l o s e a n d D E A E - S e p h a d e x i n c r e a s e din comparison to that of the native enzymes, whiledecreases in the activation energy were observed inaminoacylases immobil ized by alkylat ion with iodoace-tylcellulose [38].

The deactivation energies were also s tudied, result-ing in resu lt s o f 16 .2kca lm ol 1 (6 8 .0 kJm ol- 1) ( runa ' ) and 2.2 kcal mo1-1 (9.0 kJ mol -~) (run b ' ) for thesoluble fungal ~-glucosidase and 11.0 kcal mol - i(46.2 kJ m ol- 1) (run a ' ) and 0-1 kcal mol 1(0-5kJmo1-1) ( run b ' ) fo r the immobi l ized enzyme.The deactivation energies for the native andimmobi l ized enzyme f rom bac te r ia l o r ig in were7.05 kcal mo l i (29.5 kJ m o1- 1) and 5.90 kcal mo l -i(24.8 kJ mo l i), respectively.

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Arrhen ius e quation plot of fl-glucosidase from (A ) P.pickettiiand (B)A. niger.

2 .9 3 3.1 3.2 3.3

1 0 3 l I T ( o K - Q

3 . 4


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Bacterial and fungal fl-o-glucosidase 445

In a s tudy o f f l -D-g lucos idase p roduced byAsperg i l lu swen t t i iPt 2804 , t he ac t iva t ion and deac t iva t ion energyva lues fo r the hydro lys i s o f 4NP G we re 33-2 and111.3 kJ mo l ' , respect ively, an d for the hydrolysis of, :e l lobiose we re 43.6 an d 63.7 kJ tool i [12]. L i t t le i s,

._> 80

t~ 6O

O

4 O

20

F r e e

0 10 20 30 0 10 20 30 40 50

Temp era tu r e (oC)

The rma l stability of soluble and imm obilized/t-glucosidase from (A) P.pickettiiand (B)A. niger.

I m m o b i l i z e d

60 70 80 90

Temp era tu r e (oC)100


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4 4 6 M . D . Busto e t a l .

2 . 5

>

~ 2

~ 1.5Ot.)

O I

I

1t-

r-

E

0 . 5

0

2 .5

mco

" o

1 .50U

i

ca 1,,.

E

0 .5

0.. J

0

0

e i

0 1 2 3 4 5 6 7 8 8

T i m e ( h )

2 ~

4 0 o c

5 0 o 5 o c ~ e

i

1 2 3 4 5 6 7

T i m e ( h )

F i g . 4 . Semi-logarithmic plot of remaining (A ) soluble and (B) immo bil ized/3 -gluco sidase act ivi ty fromP p i c k e t t iia s a funct ionof t ime of incubation at different temperatures.

terms of their half- l ife were reported by Vallender andEriksson [15]. The range of half- l ives of/~-glucosidaseswas w ide, ranging fro m 5 to 198 h. The results pre-sented by these authors clearly showed that thestability of some /~-glucosidases drastically decreases

with temperature in the vicinity of 50C. Crude [:t-o-glucosidase isolated f rom A s p e rg i l l u s p h o e n i c i sbySternberg e t a l . [44] had a half-life of 6"5 h at 60C andless than 0.5 h at 70C.

In the enzyme reactions that use crude enzymepreparations, inactivation of the enzy me is ofte n accel-erated by contamination with proteolytic enzymes.However, in some cases the resistance to variousproteolytic enzymes is increased by immobilization [45]

and this is very advantageous. Pro nase was used to testthe resis tance of the f ree and immobi l ized enzymes toproteolysis (Tab le 2). f l-Glucosidases isolated from A.n ige rshow ed resistance to proteolysis, exhibiting, at the

2 .5

A>,,.

>

20

Q~0 )

m"i o

t~ 1 .50o

i

O~ 1C.-

E

~" 0 . 5

o-J

C

, 0 o c

;>

m

m" 0

m0o

2 . 5

1 .5

i

t,,,-.,.

E

0. 5

e l)0

. - I

7 0 o c

6 5 o c

6 0 o c

r , OI

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

T i m e ( h ) T i m e ( h )

Fig . 5 . S e m i - l o g a r i t h m i c p l o t o f r e m a i n i n g ( A ) s o l u b l e a n d ( B ) i m m o b i l i z e d / ~ - g lu c o s i d a s e a c ti v it y f r o mA. niger a s a f u n c t i o no f t im e o f i n c u b a t i o n a t d i f f e r e n t t e m p e r a t u r e s .


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Bacterial and fungal [I-o-glucosidase 447

Fable 1. Half-lives of soluble a nd immobilized fl-glucosidases isolated from P.pickettiiand A. niger

Origin Soluble Imm obilized

Te m pe ratu re Half-life r" Half-life(of) (h ) (h )

P. pick ettii 40 16-83 0-9962 4.54 0-988245 5-95 0.9979 1.50 0.989550 3" 14 0.992 5 1"16 0-9897

4. niger 60 3.67 0-9968 5-12 1-000065 2' 17 0-9945 2.29 0'999170 1'12 0.9971 1.56 0'9984

'Regressio n co efficient of lg AflGR = fi t ) plot .

Tab le 2. E ffect of pron ase on the activity of free and immo bilized/~-glucosidases isolated fro mP. pick ettiiand A. niger

/3-Glucosidase activity (%)

P. pickettii A. niger

Pron ase Soluble Immobilized Soluble Imm obilized(g l i t re- t )

0.00 100.00 100.00 100.00 100.000.01 95.51 65.07 96.32 99-970.05 91-08 49.61 95.80 95.430.10 82-26 49.40 93.09 92.55

0.50 78.19 53.43 92.82 94.361-00 75.10 83.38 91.18 89.80

en d o f i n cu b a t i o n , r e s i d u a l a c t i v i t i e s ran g i n g f ro m 9 6 t o9 1 % ( f r e e ) o r f r o m 9 9 to 8 9 . 8 % ( i m m o b i l i z e d ) o f t h ei n i t i a l a c t i v i t i e s . Ho wev e r, t h e b ac t e r i a l / %g l u co s i d ase ssh o wed l e s s re s i s t an ce t o p ro t eo l y s i s , r e t a i n i n g 7 8( f ree ) an d 5 3 % ( i mm o b i l i z ed ) o f t h e i r i n i ti a l a c t i v it i e sa f t e r 1 h o f e x p o s u r e t o 0 .5 g l i t r e - ~ p r o n a s e . O b v i -o u s ly, t h e s t a b il i za t io n o f t h e b a c t e r i a l e n z y m e t o w a r d sp r o t e o l y t i c d e a c t i v a t io n d i d n o t s e e m t o b e p a r t i c u l a r lye f f e c ti v e , s i nc e t h e a c ti v it y o f t h e e n t r a p p e d e n z y m ew a s d e c r e a s e d w i t h r e s p e c t t o i t s s o l u b l e c o u n t e r p a r t .

B o t h t h e r e s i s t a n c e t o t h e r m a l d e n a t u r a t i o n a n d t op r o t e o l y s is w e r e a p p a r e n t l y r e l a t e d t o t h e o r i g i n a n dl o c a t i o n o f t h e e n z y m e . C o n c l u s i v e l y, t h e c h a n g e s o fe n z y m i c p r o p e r t i e s c a u s e d b y t h e i m m o b i l i z a t i o n p r o -ce ss a re co n s i d e red t o b e d u e a t l e a s t t o t wo fac t o rs :o n e i s t h e c h a n g e s p r o d u c e d i n th e e n z y m e s t r u c t u r ea n d t h e o t h e r 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 o ft h e c a r r i e r s u s e d f o r i m m o b i l i z a t i o n . T h e o b s e r v e dc h a n g e s i n e n z y m i c p r o p e r t i e s a f t e r i m m o b i l i z a t i o n a r et h e r e s u l t o f c o m p l i c a t e d i n t e r a c t i o n s o f t h e s e f a c t o r san d i t i s d i ff i cu l t t o d e t e rmi n e t h e ex ac t e ffec t o f ag i v e n f a c t o r in t e r m s o f c h a n g e s o f e n z y m i c p r o p e r t i e s[39].

A c k n o w l e d g e m e n t

F i n a n c i a l s u p p o r t f r o m t h e C I C Y T ( G r a n t N o . A L I 9 4 -0 9 5 6 . C O 2 . O 2 ) is g r a te f u l ly a c k n o w l e d g e d .

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fungal enzymes (1)

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