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Acceleration of immobilized α-chymotrypsin activity with ultrasonic irradiation
JorrzaaEofMokczAr
Gdalysis.
Ql%evierSequoi2S.R,kmsann
12 (1981) 253 - 259 e~RintedinT%eNetherIands
ACCELERATION OF lMMOl3lLIZED WITH U.LTR,LSQNTC lRRADIATIOK
(r-CHyMOTRyPS~
253
ACTWm
YOSHXO.TSHIMORI,ESAOKA~~EsndSHL~C~S~UIU: Research Laboratory of Resources L’tilizafiorr, Tokyo insfifute of Techrmlagy, Nagutsufacko. -Midorifzu, Yokohama 227 (Japan}
aChymotrypsi.n was immobilized on agarose gel and the immobihzed enzyme was empIoyed for the proteclytic reaction under uhrasonics. C?sein was used as a substrate of crchymotrypsin. The activity of the immobtied ~chymotrypsin was accelerated with rrltrasonicirradiation (20 mlz, 10 - . 15 Wt. The a&vi@ was 2.0 - 2.2 times that of normaI conditions at the optimum conditions (pH 8.0,35 “C). Some inactivation of the immobibzed cxchymotrypsin under uItrasonics (20 Kl-iz, 15 W) was observed after four repeated uses. On the other hand, wken acetyltyrosine ethyI ester (ATEE) was u=d as a substrati, no acc&ration of activity was observed with uhrasonic irradiation. Casein was not degraded with 10 mm of uI&isonic irmd-iation. The ecceIeration of the enzyme activity under rrltrasomcs might be caused by the promotion of the penetration of casein into gels with uItrasonic irradiation.
U1trasordc.shave been used for many anaIyses and diagnoses during the last decade, ez+.eciaIIyin the medical fieId_ There-are many appkations for uItra.sonicssuck as cleaning, met&-welding, .soIderingand so forth, but there are few reports on the applications of uhrasonics to biochemistry such as enzyme reactions [II. RecentIy many enzymes have been immobikzed on various carriers and the immobiIized enzymes have been apphed to bioreactors to produce useful compounds.~thesesystems,thecontrol ofenzy7neaditi~i.s avery important factor. The.authors, therefore, have reported photo-, eIectric- and magneticconfzoI of enzyme activities [2.- 41. For .exampIe, the micro: environment around immobibzed enzymes can be changed witk a photochromic compound suckas spi.ropyr%nor E.hquid crystal. As a resuh,
254
activities are changed with tight irradiation or u+er e&tric fieid. The interadion between an enzyme and a substrate is very impotitt for the enzyme reaLon, especially in the case of immobilized enzymes, because of the steric hindrance betwthe immobilized enzyme and a sub&rate_ That is, the diffusion of a substrate is often a limiting factor for immobtied eilzyme reactions. Ultrasonics are used to bring about many types of oscillations. If ultrasonics are applied during an enzyme reaction, the adivrty of immobilized enzyme may be acc&rated since substrate diffusion into an immobilized carrier should be improved. In this paper, we describe the acceleration of ccbymotrypsin activity immobilized on agarose gel under ultrasonics. The mechanism of this phenomenon is also discussed. enzyme
Materials
ad
methods
crChymo*psin (EC. No. 3.4.21.1, from bovine pancreas) was purchased from Sigma Chemical Co. (Type II). Sepharose 4B was bought from Phrmacia Fine Chemicals. Milk casein was obtair,ed from Merck Co. Other reagents were commercially available & laboratory grade. Deionized water was supplied for all procedW. Appamtus The ultrasonic&or used was made by Ohtake Seisakujo Co., Tokyo.
Immobilizafion
of ~cfrymotrypsi~
on agarvse beads
Immobilized Q+ZhymOthJrpti was prepared as fol.lows: fifty grams (wet) of thoroughly washed Sepharose 4B was reacted with five grams of CNRr ($I was adjusted from 10.5 to 11.5 with 4 M NaOH) below 25 “C for 30 min. After being washed with 35 ml of cold 0.1 N N&C&, the gel was resuspended in oxhymotrypsin soIution (0.5 g/100 ml of 0.1 N NaJ3C03) and stirred slowly overnight at 4 “C. The gel suspended in wati was stored in a refrigerator after treatment with 1 M gIycine and washed with 0.1 h’ N&IC03 md then thoroughly with water. One gram gel (wet) contained two mil@ams of cr-ChymOtrypsir.according to spectrophotometric assay [53. The activity yield of the immobtied achymotrypsin was about 30%. a-Chymotrypsin. activity &tetimhtin aChymotrsFpsin activity was determined with 1% casein jO.1 M; pH 8.0 borate buff& solution) as a substrate [S] _ The enzyme reaction was
stopped by tie addition of 3 ml trichloPoacetic acid (5%). One unit of the enzyme was defined as one increase of O.D. 280 mn per minute.
255 E&UIt.S
Some chemical reactions are accelerated by ultrasonic irradiation because of the generatid heat and strong agitation [71_ There have been, however, few reports on the effects of uhrasonics on enzyme activity. The acceIeration of &ee crtchymotrypsin activity using uitrasonics was examined. The enzyme reaction was performed as follows: one ml of 1% casein solution (0.1 M; pE%8.0 borate buEfer) and one ml of the buffer solution were mixed and F.re-irradiated with ukrasonics at appropriate intensity for 4 min. Five fiEof the enzyme solution (7.0 mg/ml of 1 mM HCI) was added and the reaction mixture was incubated for 5 mm under ultrasonic irradiation. The temperature of the reaction mixture was kept at 35 f 0.5 “C by cooling with cold water. In the case of normal cchymotrypsin reaction, the reaction mixture was stirred at 400 rpm with a magnetic stirrer. The results obtained are shown in Fig. I. The abscissa corresponds to the power or intensity of ultrasonic irradiation. The protease activity increased with increasing uXm~onic intensity up to 10 W and thereafter decreased gradu&y . At 20 W, the activity was lower than that for normal conditions_ This might he caused by the partial denaturation of the enzyme with cavjtation. The appropriate ultrasonic irradiation (Xl KHz and 10 W in this case) cotid accelerate the enzyme activiQ about ZC%. Agarose beads are a very popular support matetial for immobilized enzymes. Therefore, orchymotrypsin was immobilized on agarose beads. Irr determining the reaction rate it is important that a substrate, espectiy a large molecti weight one, penetrates into the inner gel. The activity of the immobilized achymotrypsin prepared increased gradually with increasing stirring speed and reached a plateau above a stirring speed of 8L70rpm, It is well-known that ultrasonics help the penetration or dispersion of reactits. Therefore, we examined tne effect of ultrasonic inter&s on the activity of immobilized crchymotrypsin. The crchymo+sypsm-agsrose gel (4 - LO mg wet) was suspended in one ml of i;he buffer sohrtion and pre-irradiated for
”
5
15
10 POnPr
[n]
20
_
Fig. I. Effect of ubasmic power OP the actirity of free achymotrypsiu. were performed under conditions des&bed iu the text.
The reactions
256
4 m~n. O n e m l o f 1% c a s e i n w a s a d d e d t o t h e r e a c t i o n m i x t u r e a n d t h e m i x t u r e s t o o d f o r 5 m i n u n d e r u l t r a s o n i c i r r a d i a t i o n . T h r e e m I o f 5% t r i c h l o r o a e e t i c acid (TCA) was a d d e d t o t h e r e a c t i o n m i x t u r e t o s t o p t h e
r e a c t i o n . T h e results are s h o w n in Fig. 2. T h e a c t i v i t y o f i m m o b i l i z e d ~chymotrypsin increased with increasing ultrasonic intensity. The activity was a l m o s t c o n s t a n t i n t h e 5 t o 1 5 W r a n g e a n d a b o u t 2 . 2 t i m e s t h a t o f normal conditions, but the rapid decrease of the activity observed over 20 W was d u e t o d e n a t u r a t i o n o f t h e e n z y m e w i t h s t r o n g cavitation. F i f t e e n W was t h e r e f o r e e m p l o y e d f o r s u b s e q u e n t e x p e r i m e n t s . S o u n d eneL,g y is p a r t l y c o n v e r t e d t o h e a t e n e r g y in l i q u i d . H o w e v e r , t h e t e m p e r a t u z ~ o f t h e r e a c t i o n m l x t u r e w a s ~ l m o s t c o n s t a n t ( a t m o s t 1 cC i n c r e a s e ) d u r i n g t h e r e a c t i o n . Since. t h e r e a c ~ o n t e m p e r a t u r e a f f e c t s t h e r e a c t i o n r~.te o f t h e e n z y m e s , t h e e f f e c t o f t h e r e a c t i o n t e m p e r a t u r e o n t h e a c t i v i t y 04 i m m o b i l i z e d a - c h y m o t r y p s i n w a s e ~ s m ~ n e d u n d e r u l t r a s o n i c s . T h e resul.~s a r e s h o w n i n Fi g. 3. T h e a c t i v i t y o f t h e e n z y m e u n d e r n o r m a l c o n d i t i o n s ( s tint i ng o n l y ) a t 3 5 °C w as d e f i n e d as 1 0 0 % . T h e a c t i v i t y was a l m o s t c o n s t a n t f r o m 3 5 t o 4 5 0(3. T h e r e f o r e , t h e r e a c t i o n w a s p e r f o r m e d a t 3 5 °C f o r t h e f u r t h e r e x p e r i m e n t s . A s h i f t o f t h e o p t i m u m p H a f t e r i m m o b i l i z a t i o n o f e n z y m e s has b e e n o ~ e n o b s e r v e d . T h e p H proEfle o f i m m o b i l i z e d c z ~ h y m o l ~ T p s i n w a s invest i g a t e d u n d e r ul~.rasonics. T h e r e s u l t s a r e s h o w n in Fi g. ~_. T h e o p t i m , , m p H ( a r o u n d 8 . 0 ) o f t h e immobfl_~zed e n z y m e w a s s i m i l a r t o t h a t o f n a t i v e ~chymotrypsin. However, the activity was higher than that of the native e n z y m e in a c i d i c c o n d i t i o n s u n d e r u l t r a s o n i c s . T h e s u b s e q u e n t e x p e r i m e n t s were performed at pH 8.0 (0.1 M borate buffer). T h e reusability o f the i m m o b i l i z e d ~ c h y m o t r y p s i n was investigated u n d e r u l t r a s o n i c s . T h e e x p e r i m e n t s w e r e p e r f o r m e d as f o l l o w s : t h e r e a c t i o n m i x t u r e ( a b o u t 2 0 m g w e t gel) w a s s u s p e n d e d i n 1 . 5 m l o f 0 . 1 M b o r a t e
!~0
200
>
/
/
> I~0
°I !oo
EO
0
f
i
r
5
i0
IS
F~e r
[W]
0 T
20
,
,
,
,
30
35
~0
~5
Te~-..e_r u t u r e
['r]
Fig. 2. E f f e c t o f u l t r a s o n i c p o w e r 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 a - c b y m o t w y p s i n . T h e e x p e r i m e a t s were p e r f o r m e d u n d e r standard condition_~. FiE. 3. P-,~fectof the ~ c t i o n temper~tu_~ o n the actlvi~y of ~mr-ohillzed r,-chymoL~yv~in. T h e experiments w e r e p e r f o r m e d u n d e r ~tandard conditior,~.
257
buffer, pH 8.0, and then pre-incubated under ukasonics (20 KHz, 15 W) :for 4 min. Next, 0.5 ml of 1% casein solution was added to the mixture which was then exposed to ukrasonics for 5 min. After this, the reaction gel was collected by filtration, md the same procedures were repeated four times. The results are iUus&&ed in Fig. 5. The specific activity of the gel hardLy decreased during four repeated uses. The enzyme on the gel was not denaturated under ukrasonics. On the other hand, the activity of kee achymotrypsin Fvasaffected by ultrasonic irradiation. The activity of the native enzyme pre-incubated under ukrasonics for 4 min was xound 75% of that under norm21 conditions. This shcws that the free.enzyme was partly denaturated under ultrasonics.
b.5
7.3
7.5
8.0
8.5
9.0
DH
Fig. 4. pH profile of immobilized crchpmotrypsin were performed under standad conditions. Fig. 5. FLe-ss=bXty
of immobilized achymotrypdn in the text.
1 Times
2
3
4
recycled
under uItrasonics. The experiment; under ultr~~nics.
The experimenti
were performed as described
Discus.sioon
The activity of lmnobilized uchymotrypsin increased under ultrasonics using casein as a substrate. Ultrasonics have many effects such as he&ing, circulation and diffusion based on cavitation. Reaction ternperatrrre is a very important factor determining the reaction rate. At first, it was supposed that the acceleration of the immobilized achymotrypsin reaction would be due to the increase of the reaction temperat-. Therefore, the temperature increase of the reaction misture under ukrasonics of various intensities was exakned (Fig. 6), However, the temperatxre increase of the reaction mixture was about one degree centigrade in zany cases. The increase of the reaction mixture temperature would not be the reason for the acceleration of the immobiked ~~chymotrypsin activity. Mechanochemicd a&ions of ~Itrasonics cause breakdown of polymers. Thergfore. the structure of casein might be degraded w-&h&rasonic irradkt-
256
Fig. 6. Temperature increase of the reaction mixture under ultrasonics. were pe;fo_xned under st~nckrd conditions except for ultrarJnic power. 3,lO TJ; 4, 5 w. Fig. 7. Gel chrom&ogram of casein. Sephadex G-100 (IX.. 100 ml) 5C m&I Lhosphate bufFer (pH 7.0) was packed in a column (65 2 K casein was charged on the gc: and eluted with the buffer described (flow rate: ca. 10 the elute was coUe&ed with a fract- 1z.1 cAlector were performed et 5 “L . i-): casein treated with ultrasonics (20 (- - - -): norm?: casein.
The experiments 1, 20 W; 2,15
W;
pre-equitibrated with 30 cm). Twr, ml OF 1% above. Each 5 ml of ml/h). All procedures KHz;
15 W; 5 min).
tion. The struc~:lre change of casein by ultrasonic irradiation was examined. Figure 7 shows the kel chromatogmms of both treated and untreated casein. Czsein solution was irradiated for 5 minutes under the conditions described above. No diffzretnce in chromatograms was obseervedexcept for the small peak of fraction numbers 18 to 20. Fuzthermore, the same sampks were employed to measure their intrinsic -viscosiw with an Ubbelohde viscometer, the values obtained showing good agreement with a relative error of 3 %. Therefore, casein was not degraded by ultrasonic irradiation. Ultrasonics may affect the properties of proti in. The hydrophobic&y change of casein after ultrasonic irradiation was examined fh~oros~ctrophotometrically wi;h ‘use of TNS C6-(p-toluidino)-2-naphthalene-srrIfonic acid] as a probe. The peak (440 nm) of untrea’r;edcasein decreased slightly (about 5%). Therefore, hydrophobi&y of casein was not changed by ultrasonic irradiation. The promotion of subs&ate diffusion into gel matrix might acceterate the activity of immobilized achy-motrypsin. ETgure 8 shows the LineweaverE3urk plot of immobilized crchymotrypsin under ultrasonics and normal conditions. Km’ and Ym’ are shown in Table I. Km’ under ultrasonic was about half of Km’ under normal conditions. This demonstites that the apparent &E&y between immobilized ~~&~ymotrfpsi.n and casein increases under ultrasonics. Therefore, the promotion of casein diffusion into gel under ultraso_nicsplays a main role in the acceieration of immobilized Qchymotrypsin. Actually, the atiivity of immobilized uchymotrypsin gradually inaeaed with imzreasing stirring speed. iTherr ATEE (N-acetyl-L-
::59
Fig. 8. Lineweaver-Burk plots of immobilized ar-chymotrypsin. The experiments were performed under standard coaditions except for the glucose concentration. l : under uItrz.sonics (20 K&s, 16 W); 0: norm& conditions. TABLE V&z”
1
of Kin’ aad Vm'
Under ukasonics Normal conditions
(ullhlug-Knin)
Km’
(W)
Vm’
4.24
x 1o-3
I.83 x 1O-2 1.0s x lo-2
9.57 x lo-”
ethyl ester) was useed as a substrate for immobilized crchymotrypsin, the ax&ration in activity was not observed. Therefore, the Gffusion of substrate into gel was not a r&edetermiGn g step in this case. En eondusion, Ultrasonics can be used for the acceleration of immobilized enzyme a&v-iQ where the &ffusion of a substrate into the gel is aratedefiemkhg step. tyro_fine
L. A C?wnbers, J. Bill Chem.. IL7 (1937) 639. L Karube, Y. Yugek and S. Suz&i, Biokclrnol. Bioeng., 19 (1977) 1493. I. Karube, S. Suzuki, Y. Nakamoto and M. Nishida,J. iKoZ. Catat.. 6 (1979) 51. I. &n&e, Y. N&amato. M. Nishida and S. Suzuki, BiotecFnzol. Bioeng.. !9 (1977) 1549. 0. H. Lowry, N. J. Roserough, A U. Fsrr and R. J. RendaII, J. Eiol. Chem.. 193 (1951) 263. M. Kunitz,J. Gem PizysioL, 30 (1947) 291. E. W. Barrett tmd C. W. Poter, J. Am. CFrem. Sot., 63 (1943) 3434.
Gdalysis.
Ql%evierSequoi2S.R,kmsann
12 (1981) 253 - 259 e~RintedinT%eNetherIands
ACCELERATION OF lMMOl3lLIZED WITH U.LTR,LSQNTC lRRADIATIOK
(r-CHyMOTRyPS~
253
ACTWm
YOSHXO.TSHIMORI,ESAOKA~~EsndSHL~C~S~UIU: Research Laboratory of Resources L’tilizafiorr, Tokyo insfifute of Techrmlagy, Nagutsufacko. -Midorifzu, Yokohama 227 (Japan}
aChymotrypsi.n was immobilized on agarose gel and the immobihzed enzyme was empIoyed for the proteclytic reaction under uhrasonics. C?sein was used as a substrate of crchymotrypsin. The activity of the immobtied ~chymotrypsin was accelerated with rrltrasonicirradiation (20 mlz, 10 - . 15 Wt. The a&vi@ was 2.0 - 2.2 times that of normaI conditions at the optimum conditions (pH 8.0,35 “C). Some inactivation of the immobibzed cxchymotrypsin under uItrasonics (20 Kl-iz, 15 W) was observed after four repeated uses. On the other hand, wken acetyltyrosine ethyI ester (ATEE) was u=d as a substrati, no acc&ration of activity was observed with uhrasonic irradiation. Casein was not degraded with 10 mm of uI&isonic irmd-iation. The ecceIeration of the enzyme activity under rrltrasomcs might be caused by the promotion of the penetration of casein into gels with uItrasonic irradiation.
U1trasordc.shave been used for many anaIyses and diagnoses during the last decade, ez+.eciaIIyin the medical fieId_ There-are many appkations for uItra.sonicssuck as cleaning, met&-welding, .soIderingand so forth, but there are few reports on the applications of uhrasonics to biochemistry such as enzyme reactions [II. RecentIy many enzymes have been immobikzed on various carriers and the immobiIized enzymes have been apphed to bioreactors to produce useful compounds.~thesesystems,thecontrol ofenzy7neaditi~i.s avery important factor. The.authors, therefore, have reported photo-, eIectric- and magneticconfzoI of enzyme activities [2.- 41. For .exampIe, the micro: environment around immobibzed enzymes can be changed witk a photochromic compound suckas spi.ropyr%nor E.hquid crystal. As a resuh,
254
activities are changed with tight irradiation or u+er e&tric fieid. The interadion between an enzyme and a substrate is very impotitt for the enzyme reaLon, especially in the case of immobilized enzymes, because of the steric hindrance betwthe immobilized enzyme and a sub&rate_ That is, the diffusion of a substrate is often a limiting factor for immobtied eilzyme reactions. Ultrasonics are used to bring about many types of oscillations. If ultrasonics are applied during an enzyme reaction, the adivrty of immobilized enzyme may be acc&rated since substrate diffusion into an immobilized carrier should be improved. In this paper, we describe the acceleration of ccbymotrypsin activity immobilized on agarose gel under ultrasonics. The mechanism of this phenomenon is also discussed. enzyme
Materials
ad
methods
crChymo*psin (EC. No. 3.4.21.1, from bovine pancreas) was purchased from Sigma Chemical Co. (Type II). Sepharose 4B was bought from Phrmacia Fine Chemicals. Milk casein was obtair,ed from Merck Co. Other reagents were commercially available & laboratory grade. Deionized water was supplied for all procedW. Appamtus The ultrasonic&or used was made by Ohtake Seisakujo Co., Tokyo.
Immobilizafion
of ~cfrymotrypsi~
on agarvse beads
Immobilized Q+ZhymOthJrpti was prepared as fol.lows: fifty grams (wet) of thoroughly washed Sepharose 4B was reacted with five grams of CNRr ($I was adjusted from 10.5 to 11.5 with 4 M NaOH) below 25 “C for 30 min. After being washed with 35 ml of cold 0.1 N N&C&, the gel was resuspended in oxhymotrypsin soIution (0.5 g/100 ml of 0.1 N NaJ3C03) and stirred slowly overnight at 4 “C. The gel suspended in wati was stored in a refrigerator after treatment with 1 M gIycine and washed with 0.1 h’ N&IC03 md then thoroughly with water. One gram gel (wet) contained two mil@ams of cr-ChymOtrypsir.according to spectrophotometric assay [53. The activity yield of the immobtied achymotrypsin was about 30%. a-Chymotrypsin. activity &tetimhtin aChymotrsFpsin activity was determined with 1% casein jO.1 M; pH 8.0 borate buff& solution) as a substrate [S] _ The enzyme reaction was
stopped by tie addition of 3 ml trichloPoacetic acid (5%). One unit of the enzyme was defined as one increase of O.D. 280 mn per minute.
255 E&UIt.S
Some chemical reactions are accelerated by ultrasonic irradiation because of the generatid heat and strong agitation [71_ There have been, however, few reports on the effects of uhrasonics on enzyme activity. The acceIeration of &ee crtchymotrypsin activity using uitrasonics was examined. The enzyme reaction was performed as follows: one ml of 1% casein solution (0.1 M; pE%8.0 borate buEfer) and one ml of the buffer solution were mixed and F.re-irradiated with ukrasonics at appropriate intensity for 4 min. Five fiEof the enzyme solution (7.0 mg/ml of 1 mM HCI) was added and the reaction mixture was incubated for 5 mm under ultrasonic irradiation. The temperature of the reaction mixture was kept at 35 f 0.5 “C by cooling with cold water. In the case of normal cchymotrypsin reaction, the reaction mixture was stirred at 400 rpm with a magnetic stirrer. The results obtained are shown in Fig. I. The abscissa corresponds to the power or intensity of ultrasonic irradiation. The protease activity increased with increasing uXm~onic intensity up to 10 W and thereafter decreased gradu&y . At 20 W, the activity was lower than that for normal conditions_ This might he caused by the partial denaturation of the enzyme with cavjtation. The appropriate ultrasonic irradiation (Xl KHz and 10 W in this case) cotid accelerate the enzyme activiQ about ZC%. Agarose beads are a very popular support matetial for immobilized enzymes. Therefore, orchymotrypsin was immobilized on agarose beads. Irr determining the reaction rate it is important that a substrate, espectiy a large molecti weight one, penetrates into the inner gel. The activity of the immobilized achymotrypsin prepared increased gradually with increasing stirring speed and reached a plateau above a stirring speed of 8L70rpm, It is well-known that ultrasonics help the penetration or dispersion of reactits. Therefore, we examined tne effect of ultrasonic inter&s on the activity of immobilized crchymotrypsin. The crchymo+sypsm-agsrose gel (4 - LO mg wet) was suspended in one ml of i;he buffer sohrtion and pre-irradiated for
”
5
15
10 POnPr
[n]
20
_
Fig. I. Effect of ubasmic power OP the actirity of free achymotrypsiu. were performed under conditions des&bed iu the text.
The reactions
256
4 m~n. O n e m l o f 1% c a s e i n w a s a d d e d t o t h e r e a c t i o n m i x t u r e a n d t h e m i x t u r e s t o o d f o r 5 m i n u n d e r u l t r a s o n i c i r r a d i a t i o n . T h r e e m I o f 5% t r i c h l o r o a e e t i c acid (TCA) was a d d e d t o t h e r e a c t i o n m i x t u r e t o s t o p t h e
r e a c t i o n . T h e results are s h o w n in Fig. 2. T h e a c t i v i t y o f i m m o b i l i z e d ~chymotrypsin increased with increasing ultrasonic intensity. The activity was a l m o s t c o n s t a n t i n t h e 5 t o 1 5 W r a n g e a n d a b o u t 2 . 2 t i m e s t h a t o f normal conditions, but the rapid decrease of the activity observed over 20 W was d u e t o d e n a t u r a t i o n o f t h e e n z y m e w i t h s t r o n g cavitation. F i f t e e n W was t h e r e f o r e e m p l o y e d f o r s u b s e q u e n t e x p e r i m e n t s . S o u n d eneL,g y is p a r t l y c o n v e r t e d t o h e a t e n e r g y in l i q u i d . H o w e v e r , t h e t e m p e r a t u z ~ o f t h e r e a c t i o n m l x t u r e w a s ~ l m o s t c o n s t a n t ( a t m o s t 1 cC i n c r e a s e ) d u r i n g t h e r e a c t i o n . Since. t h e r e a c ~ o n t e m p e r a t u r e a f f e c t s t h e r e a c t i o n r~.te o f t h e e n z y m e s , t h e e f f e c t o f t h e r e a c t i o n t e m p e r a t u r e o n t h e a c t i v i t y 04 i m m o b i l i z e d a - c h y m o t r y p s i n w a s e ~ s m ~ n e d u n d e r u l t r a s o n i c s . T h e resul.~s a r e s h o w n i n Fi g. 3. T h e a c t i v i t y o f t h e e n z y m e u n d e r n o r m a l c o n d i t i o n s ( s tint i ng o n l y ) a t 3 5 °C w as d e f i n e d as 1 0 0 % . T h e a c t i v i t y was a l m o s t c o n s t a n t f r o m 3 5 t o 4 5 0(3. T h e r e f o r e , t h e r e a c t i o n w a s p e r f o r m e d a t 3 5 °C f o r t h e f u r t h e r e x p e r i m e n t s . A s h i f t o f t h e o p t i m u m p H a f t e r i m m o b i l i z a t i o n o f e n z y m e s has b e e n o ~ e n o b s e r v e d . T h e p H proEfle o f i m m o b i l i z e d c z ~ h y m o l ~ T p s i n w a s invest i g a t e d u n d e r ul~.rasonics. T h e r e s u l t s a r e s h o w n in Fi g. ~_. T h e o p t i m , , m p H ( a r o u n d 8 . 0 ) o f t h e immobfl_~zed e n z y m e w a s s i m i l a r t o t h a t o f n a t i v e ~chymotrypsin. However, the activity was higher than that of the native e n z y m e in a c i d i c c o n d i t i o n s u n d e r u l t r a s o n i c s . T h e s u b s e q u e n t e x p e r i m e n t s were performed at pH 8.0 (0.1 M borate buffer). T h e reusability o f the i m m o b i l i z e d ~ c h y m o t r y p s i n was investigated u n d e r u l t r a s o n i c s . T h e e x p e r i m e n t s w e r e p e r f o r m e d as f o l l o w s : t h e r e a c t i o n m i x t u r e ( a b o u t 2 0 m g w e t gel) w a s s u s p e n d e d i n 1 . 5 m l o f 0 . 1 M b o r a t e
!~0
200
>
/
/
> I~0
°I !oo
EO
0
f
i
r
5
i0
IS
F~e r
[W]
0 T
20
,
,
,
,
30
35
~0
~5
Te~-..e_r u t u r e
['r]
Fig. 2. E f f e c t o f u l t r a s o n i c p o w e r 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 a - c b y m o t w y p s i n . T h e e x p e r i m e a t s were p e r f o r m e d u n d e r standard condition_~. FiE. 3. P-,~fectof the ~ c t i o n temper~tu_~ o n the actlvi~y of ~mr-ohillzed r,-chymoL~yv~in. T h e experiments w e r e p e r f o r m e d u n d e r ~tandard conditior,~.
257
buffer, pH 8.0, and then pre-incubated under ukasonics (20 KHz, 15 W) :for 4 min. Next, 0.5 ml of 1% casein solution was added to the mixture which was then exposed to ukrasonics for 5 min. After this, the reaction gel was collected by filtration, md the same procedures were repeated four times. The results are iUus&&ed in Fig. 5. The specific activity of the gel hardLy decreased during four repeated uses. The enzyme on the gel was not denaturated under ukrasonics. On the other hand, the activity of kee achymotrypsin Fvasaffected by ultrasonic irradiation. The activity of the native enzyme pre-incubated under ukrasonics for 4 min was xound 75% of that under norm21 conditions. This shcws that the free.enzyme was partly denaturated under ultrasonics.
b.5
7.3
7.5
8.0
8.5
9.0
DH
Fig. 4. pH profile of immobilized crchpmotrypsin were performed under standad conditions. Fig. 5. FLe-ss=bXty
of immobilized achymotrypdn in the text.
1 Times
2
3
4
recycled
under uItrasonics. The experiment; under ultr~~nics.
The experimenti
were performed as described
Discus.sioon
The activity of lmnobilized uchymotrypsin increased under ultrasonics using casein as a substrate. Ultrasonics have many effects such as he&ing, circulation and diffusion based on cavitation. Reaction ternperatrrre is a very important factor determining the reaction rate. At first, it was supposed that the acceleration of the immobilized achymotrypsin reaction would be due to the increase of the reaction temperat-. Therefore, the temperature increase of the reaction misture under ukrasonics of various intensities was exakned (Fig. 6), However, the temperatxre increase of the reaction mixture was about one degree centigrade in zany cases. The increase of the reaction mixture temperature would not be the reason for the acceleration of the immobiked ~~chymotrypsin activity. Mechanochemicd a&ions of ~Itrasonics cause breakdown of polymers. Thergfore. the structure of casein might be degraded w-&h&rasonic irradkt-
256
Fig. 6. Temperature increase of the reaction mixture under ultrasonics. were pe;fo_xned under st~nckrd conditions except for ultrarJnic power. 3,lO TJ; 4, 5 w. Fig. 7. Gel chrom&ogram of casein. Sephadex G-100 (IX.. 100 ml) 5C m&I Lhosphate bufFer (pH 7.0) was packed in a column (65 2 K casein was charged on the gc: and eluted with the buffer described (flow rate: ca. 10 the elute was coUe&ed with a fract- 1z.1 cAlector were performed et 5 “L . i-): casein treated with ultrasonics (20 (- - - -): norm?: casein.
The experiments 1, 20 W; 2,15
W;
pre-equitibrated with 30 cm). Twr, ml OF 1% above. Each 5 ml of ml/h). All procedures KHz;
15 W; 5 min).
tion. The struc~:lre change of casein by ultrasonic irradiation was examined. Figure 7 shows the kel chromatogmms of both treated and untreated casein. Czsein solution was irradiated for 5 minutes under the conditions described above. No diffzretnce in chromatograms was obseervedexcept for the small peak of fraction numbers 18 to 20. Fuzthermore, the same sampks were employed to measure their intrinsic -viscosiw with an Ubbelohde viscometer, the values obtained showing good agreement with a relative error of 3 %. Therefore, casein was not degraded by ultrasonic irradiation. Ultrasonics may affect the properties of proti in. The hydrophobic&y change of casein after ultrasonic irradiation was examined fh~oros~ctrophotometrically wi;h ‘use of TNS C6-(p-toluidino)-2-naphthalene-srrIfonic acid] as a probe. The peak (440 nm) of untrea’r;edcasein decreased slightly (about 5%). Therefore, hydrophobi&y of casein was not changed by ultrasonic irradiation. The promotion of subs&ate diffusion into gel matrix might acceterate the activity of immobilized achy-motrypsin. ETgure 8 shows the LineweaverE3urk plot of immobilized crchymotrypsin under ultrasonics and normal conditions. Km’ and Ym’ are shown in Table I. Km’ under ultrasonic was about half of Km’ under normal conditions. This demonstites that the apparent &E&y between immobilized ~~&~ymotrfpsi.n and casein increases under ultrasonics. Therefore, the promotion of casein diffusion into gel under ultraso_nicsplays a main role in the acceieration of immobilized Qchymotrypsin. Actually, the atiivity of immobilized uchymotrypsin gradually inaeaed with imzreasing stirring speed. iTherr ATEE (N-acetyl-L-
::59
Fig. 8. Lineweaver-Burk plots of immobilized ar-chymotrypsin. The experiments were performed under standard coaditions except for the glucose concentration. l : under uItrz.sonics (20 K&s, 16 W); 0: norm& conditions. TABLE V&z”
1
of Kin’ aad Vm'
Under ukasonics Normal conditions
(ullhlug-Knin)
Km’
(W)
Vm’
4.24
x 1o-3
I.83 x 1O-2 1.0s x lo-2
9.57 x lo-”
ethyl ester) was useed as a substrate for immobilized crchymotrypsin, the ax&ration in activity was not observed. Therefore, the Gffusion of substrate into gel was not a r&edetermiGn g step in this case. En eondusion, Ultrasonics can be used for the acceleration of immobilized enzyme a&v-iQ where the &ffusion of a substrate into the gel is aratedefiemkhg step. tyro_fine
L. A C?wnbers, J. Bill Chem.. IL7 (1937) 639. L Karube, Y. Yugek and S. Suz&i, Biokclrnol. Bioeng., 19 (1977) 1493. I. Karube, S. Suzuki, Y. Nakamoto and M. Nishida,J. iKoZ. Catat.. 6 (1979) 51. I. &n&e, Y. N&amato. M. Nishida and S. Suzuki, BiotecFnzol. Bioeng.. !9 (1977) 1549. 0. H. Lowry, N. J. Roserough, A U. Fsrr and R. J. RendaII, J. Eiol. Chem.. 193 (1951) 263. M. Kunitz,J. Gem PizysioL, 30 (1947) 291. E. W. Barrett tmd C. W. Poter, J. Am. CFrem. Sot., 63 (1943) 3434.