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Thermodynamic Properties of Free and Immobilized Subtilopeptidase from Bacillus subtilis PR-70
Zbl. Bakt. II. Abt. 135 (1980), 77-81 [Microbial Enzyme Unit, Microbiology Research Dept., Agric. Research Oentre of Egypt]
Thermodynamic Properties of Free and Immobilized Subtilopeptidase from Bacillus subtilis PR-70 RASMY M. ATTIA, RAWIA F. GAMAL, and A. M. DOKHAN With 5 Figures
Summary Thermodynamics of free and immobilized subtilopeptidase was studied over the temperature range from 30° to 80 °0 and from 30° to 90 °0, respectively. The apparent optimum temperature of the free enzyme was 50 °0 and of the immobilized cnzyme 60 °0. The hcat of the reaction (,1 E), accompanied by the process, was calculated from the slope of the Arrhenius equation to be 56770 cal/mole for free enzymc and 36640 cal/mole for immobilized enzyme. The standard free energy change (,1 G) and the standard entropy change (,1 S) were found to be 241 cal/mole and 186.6 cal/mole/dogree at 30 °0 for free enzyme and 142 cal/mole and 120.5 cal/molo/degree at 30 °0 for immobilized enzyme. The values of these thermodynamic quantities at other temperatures were also summarized. Studying the reaction order of hcat inactivation showed that free and immobilized enzymc followed a first order reaction. The energy of inaetivation was found to be 18900 cal/mole and 24400 cal/mole for free and immobilized enzyme, respectively.
+
+
+
Zusammenfassung Die Thermodynamik freier und gebundener Subtilopeptidase wurde im Temperaturbereich von 30-80 °0 bzw. 30-90 °C untersucht. Die scheinbare Optimaltemperatur des freien Enzyms war 50 °0 und die des gebundenon 60 °0. Die Reaktionswarme (,1 E) wurde aus del' ArrheniusGleiohung errechnot. Sie betrug 56770 cal/Mol fUr das freie und 36640 cal/Mol fUr das gebundene Enzym. ,1 G und ,1 S betrugen bei 30 °0 241 bzw. 186 cal/Mol/Grad fUr das freie und 142 bzw. 120,5 cal/Mol/Grad fUr das gebundene Enzym. Die Inaktivierung durch Ritze folgte einer Reaktion 1. Ordnung. Die Inaktivierungsenergie betrug 18900 cal/Mol fur das freie und 24400 call Mol fUr das gebundene Enzym.
+
+
+
During the past decade, the immobilization of enzymes on insoluble carrier has become increasingly important, as evidenced by the number of reports of success in this area (GOLDSTEIN and KATCHALSKI 1968 and GOLDSTEIN 1969), as well as the appearance of commercial insoluble enzymes and insolubilizing agents. The major difficulty that hindered progress in the understanding of the properties of insolubilized enzymes has been the lack of precise information, concerning the complicated changes in the physical and chemical properties of the enzyme after in solubilization (WEIBL and BRIGHT 1971). This paper deals with the thermodynamic properties of free and immobilized sub· tilopeptidase from Bacillus subtilis PR-70.
Material and Methods Free enzyme Subtilopeptidase (EO 3.4.4.16) was obtained from a locally isolated strain of B. subtilj:s PR-70 (ATTIA and SAMIA 1974), purified as previously mentioned by ATTIA and RAWIA (1977a, b).
78
R. M.
et al.
ATTIA
Immobilized enzyme A volume of enzyme solution (10 ml), containing 18000 LV unit, was added to 2 gram of Amb. CG-50 in a 25 ml Erlenmeyer fla sk. The vessel was placed in a water-bath shaker at 30°C and allowed to react for two hours. The vessel was then r emoved fr om the bath and the adsorption and diffusion was allowed to continue overnight at 5 °C with magnetic stirring. The water-insoluble subtilo-peptidaee was separated by filtration, washed with 1 M potassium chloride (100 ml), then with distilled water (50 ml), and dried at room temperature.
Det ermination of enzyme activity The enzyme activity was determined at pH 7.0 by the casein digestion method (KUNITZ 1947) as m.odified by HAGIHARA et 801. (1958) . The reaction mixture contained 5 ml of 1 % casein in 0.02 M phosphate buffer (pH 7.0) and 1 ml of the enzyme preparation. Digestion was allowed to proceed for 10 minutes at 30 °C. After the addition of 5 ml of TCA solution (TCA, 0.1 M; NaOAc, 0.22 M; HOAc, 0.33 M) and vigorous shaking, the mixture was allowed to stand at room temperature for 30 minutes and was then filtered. One ml of 3-times di luted Folin-Ciocalteau r eagent and 2,5 ml of 0.55 M sodium carbonate were added to 1 ml of the filtrate. After the solution had b een k ept at 30°C for 20 minutes, the absorbance at 660 nm was measured.
Results and Discussion Eff ec t of temperature on reaction rate The t emperature activity curve of an enzyme, like the pH curve, can be resolved into two components: the effect of increase in t emperature on the stability of the enzyme and on the rate of the catalyzed reaction. The former depends on the experimental conditions, while the latter is usually independent of them, except for reagents that alter the catalyst (SIZER 1943). Figures 1 and 2 are a plot of the observed reaction rate of free and immobilized subtilopeptidase at various temperatures under the standard conditions of the test (pH 7.0, 1 % casein). As expected, the apparent optimum temperature of free enzyme was 50 °C (ATTIA et a1. 1974),55 °C (FuKE 1963), and 57°C in the presence of calcium ions (MCCONN et a1. 1964). On the other hand, the apparent temperature of immobilized subtilopeptidase was higher than free en-
'tJ.
~ ~e/'/1]0'/I t) <... c:::
0~
I
"'Q-~o
'?
c:::
""Q-~o
~
;1)0/(1.
~
Q"~"
Q-/,;.,
.()
Thermal de.
~
.()
..:::'->
"~~/
t>
<1>
Q:
Q:
700
100
Opt/mum temp t a xbl
80
80 1----+---;1
61}
~o
ItO 1----
ItO
20
20
O~~~~~~~~~~~~~
o
~~.1
20
'to
~O
0
0
20
'to
60
Fig. 1. Effect of temperature on recti on rate of free enzyme catalyzed reaction. Fig . 2. Effect of t emp erature on r eaction rate of immobilized enzyme catalyzed reaction.
Thermodynamic Properties of Free and Immobilized Subtilopeptidase
79
2.5 2
~ 1
~
'-l
Immobll/zed
- -----:--- - -+--,
0.5
o -o.S
29.5"
- 1
JO.Y
30
31
32
33
Fig. 3. Plots of log k versus lIT t emp erature range 30 ", 60°C. Optimum temperatures are 50 and 60 °C for free and immobilized enzyme, respectively (activation en ergy).
zyme, being 60°C. This was expected because the bound enzyme is more stable to heat than the free enzyme. In a recent paper by MELROSE (1971), fifty immobilized enzyme systems were compared with their soluble counterparts with respect to their stability. Thirty of them were more stable. It indicates that in general increased stability will result from attachment to the solid phase. The reasons for increased stability of subtilopeptidase, resulting from attachment to Amb. CG-50, originated from the prevention of autodigestion, thus leading to greater stability. Another possible mechanism, leading to increased stability, is the prevention of conformation inactivation. Stabilization may also occur because active groups on the enzyme are sterically shielded from attack by reactive groups in the solution. Figure 3 shows the plot of JogK versus lIT for free and immobilized enzyme, aCcording to the Arrhenius equation: LogK
=
(-E/lnR) ·l/T
+ constant.
The curve of the Arrhenius plots is linear over the temperature range of 30° to 60°C. The heat of the reaction (Ll E) , accompanied by the process, has been calculated from the slope of the plot to be +56770 cal/mole and + 36640 cal/mole for free and immobilized subtilopeptidase, respectively. Results in Table 1 show that the heat of hydrolyzing casein is positive, whereas it is compensated by the positive entropy change. Table 1. Thermodynamic quantities of free and immobilized subtilopeptidase at various temperatures Temperature °C) 30 40 50 60 1) LlG
LIE (cal/mole)
LlG (cal/mole)1)
Free
Immobilized
Free
Immobilized
Free
Immobilized
+56 770 + 56770 +56770
+ 36640 + 36640 +36640 +36640
+ 241 -1240 -3401
+ 142 - 945 - 2233 -3506
+ 186.6 + 185.3 +186.3
+ 120.5 + 120. 1 +120.3 +120.6
= -RT oInK. 2) LIS = (E - G)/T.
LIS (cal/mole/degreej2)
80
R. M.
ATTIA
at al.
R e action order of heat inactivation The heat stability of an enzyme is best expressed in terms of the energy of inacti. vation under specified conditions. To calculate this, it was first necessary to determine the reaction order of the heat inactivation of soluble and immobilized enzymes. This was performed as follows: the enzyme in phosphate buffer at pH 7.0 was rapidly brought to 60 °0 in a wat er-bath regulated to 0.05 °0, being stirred continuously. Aliquots were removed at 5 minutes' intervals and immediately cooled in a freezing mixture to 10 °0. The residual activity was plotted against the time of heating at 60 °0. This did not yield a linear relationship and neither did a plot of the reciprocal of relative activity against time of heating. Hence, the heat inactivation was not 2
,
~
:~ v
t)
't;
~
Z;
Q)
~
~
"
0 0
70
'to SO 30 20 Time of healing (minutes)
(j'0
Fig. 4. H eat inactivation of subtilopeptidase as a first order reaction (abscissa, minutes of heating of the en zyme in the absence of substrate at 60 °0 and pH 7.0).
r-
/ l
Free Enzy-me
O.O r
0.8 -
o.t,.
i
Slope = - EI"-.')8
,
~
0.2 ~
! I
l
..L~
0
JO
~-0.2 ~
I
-o·t,. r
-0.0
-0.8
ImmobIlized Enzxme
27.S"
29.S 28.S liT x lOt,.
Fig. 5. Plots of logk versus IjT, t emp erature range 60 to 90 °0 (inactivation energy).
Thermodynamic Properties of Free and Immobilized Subtilopeptidase
81
a zero order or second order reaction. When the log of activity was plotted against the time of heating (Figure 4), however, a straight line relationship was obtained, indicating that the heat inactivation of soluble and insoluble subtilopeptidase followed a first order reaction. The finding of a heat inactivation of first order reaction for this enzyme was expected, as most enzymes follow a first order rate in heat inactivation (SIZER 1943). Aliquotes of enzyme at pH 7.0 were rapidly brought to various temperatures, ranging from 60° to 80 °0 for free enzyme and from 70° to 90 °0 for immobilized enzyme. The enzyme was kept at these temperatures for 10 minutes, then cooled and analysed in each case by the casein method. Figure 5 shows the plot of log K versus liT for free and immobilized enzymes. The curve is linear over the temperature range from 60 to 80 °0 and from 70 to 90 °0. The heat of inactivation (LIE), accompanied by the process and calculated from the slope of the curve, was shown to be +18900 cal/mole and +24400 cal/mole for free and immobilized subtilopeptidase, respectively. From the above-mentioned results, it is clear that the energy of inactivation of immobilized enzyme was higher compared with free enzyme. This is to be expected, of course, because the immobilized enzyme is more stable to heat than its soluble counterpart.
References ATTIA, R. M., and GAMAL, R. F.: Subtilopeptidase A produced by Bacillu8 8ubtili8 PR·70, I. Kinetic behaviour of solubilized enzyme. Zbl. Bakt. II 134 (1979), 275-281. - and EL-DEMERDASH, A. M.: Subtilopeptidase A produced by Bacillus 8ubtilis PR-70_ II. Kinetie behaviour of immobilized enzyme. Zbl. Bakt. II 134 (1979), 353 - 370. and ALI, S. A.: Isolation of Bacillu8 8ubtili8 producing subtilopeptidase A. Agrie. Res. Review 52 (1974). Ill. SHAWKI, H. A., EL-GAMAL, S. E., and SAMI, S. M.: Purification and physical properties of subtilopeptidase isolated from Bacillus 8ubtilis PR-70. Proc. XIII. Conf. Pharm. Sci., Cairo, Egypt (1974), 207. FUKE, I.: J. Biochom. (Tokyo) 53 (1963). In: "Methods in Enzymology", vol. 19 (G. E. Perlman and L. Lorand, ods.), p. 209, Academic Press, New York 1970. GOLDSTEIN, L.: In: "Fermentation Advances" (D. Perlman, ed.), p. 391. Academic Press, New York 1970. - and KATCHALSKI, E. G.: Z. Anal. Chern. 234 (1968), 375. In: "Methods in Enzymology", vol. 19 (G. E. Perlman and L. Lorand, eds.), p. 9:15. Academic Press. New York 1970. HAGIHARA, B., MATSUBARA, R., NAKAI, M., and OKUNUKI, K.: J. Biochem. (Japan) 45 (1958), 185. In: "Methods in Enzymology", vol. 19 (G. E. Perlman and L. Lorand, eds.), p.569. Academie Press, New York 1970. KUNITZ, M.: Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol. 30 (1947),291. MCCONN, J. D., TsuRlr, D., and YASUNOBU: Bacillu8 8ubtilis neutral proteinase. I. A zinc enzyme of high specific activity. J. BioI. Chern. 239 (1964), 3706. MELROSE, G. J. M.: Insolubilized enzymes. Biochemical applications of synthctic polymers. Rev. Pure Appl. Chern. 21 (1971), 83. SIZER, I. W.: In: "Advances in Enzymology", vol. 3 (F. F. Nord, ed.), p. 35. Interscience Publishers, Inc., New York 1943. WEIBL, M. K., and BRIGHT, H. J.: The glucose oxidase mechanism. Interpretation of the pH dependence. J. BioI. Chern. 246 (1971),2734. Authors' addresses: Dr. RASMY M. ATTIA. Dept. of Microbiology, Agricultural Research Centre of Egypt, Giza, Cairo, Egypt, and Mrs. Dr. RAWIA F. GANIAL, Dept. of Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. 6 ZbI. Bakt. II. Abt., Bd. 135
Thermodynamic Properties of Free and Immobilized Subtilopeptidase from Bacillus subtilis PR-70 RASMY M. ATTIA, RAWIA F. GAMAL, and A. M. DOKHAN With 5 Figures
Summary Thermodynamics of free and immobilized subtilopeptidase was studied over the temperature range from 30° to 80 °0 and from 30° to 90 °0, respectively. The apparent optimum temperature of the free enzyme was 50 °0 and of the immobilized cnzyme 60 °0. The hcat of the reaction (,1 E), accompanied by the process, was calculated from the slope of the Arrhenius equation to be 56770 cal/mole for free enzymc and 36640 cal/mole for immobilized enzyme. The standard free energy change (,1 G) and the standard entropy change (,1 S) were found to be 241 cal/mole and 186.6 cal/mole/dogree at 30 °0 for free enzyme and 142 cal/mole and 120.5 cal/molo/degree at 30 °0 for immobilized enzyme. The values of these thermodynamic quantities at other temperatures were also summarized. Studying the reaction order of hcat inactivation showed that free and immobilized enzymc followed a first order reaction. The energy of inaetivation was found to be 18900 cal/mole and 24400 cal/mole for free and immobilized enzyme, respectively.
+
+
+
Zusammenfassung Die Thermodynamik freier und gebundener Subtilopeptidase wurde im Temperaturbereich von 30-80 °0 bzw. 30-90 °C untersucht. Die scheinbare Optimaltemperatur des freien Enzyms war 50 °0 und die des gebundenon 60 °0. Die Reaktionswarme (,1 E) wurde aus del' ArrheniusGleiohung errechnot. Sie betrug 56770 cal/Mol fUr das freie und 36640 cal/Mol fUr das gebundene Enzym. ,1 G und ,1 S betrugen bei 30 °0 241 bzw. 186 cal/Mol/Grad fUr das freie und 142 bzw. 120,5 cal/Mol/Grad fUr das gebundene Enzym. Die Inaktivierung durch Ritze folgte einer Reaktion 1. Ordnung. Die Inaktivierungsenergie betrug 18900 cal/Mol fur das freie und 24400 call Mol fUr das gebundene Enzym.
+
+
+
During the past decade, the immobilization of enzymes on insoluble carrier has become increasingly important, as evidenced by the number of reports of success in this area (GOLDSTEIN and KATCHALSKI 1968 and GOLDSTEIN 1969), as well as the appearance of commercial insoluble enzymes and insolubilizing agents. The major difficulty that hindered progress in the understanding of the properties of insolubilized enzymes has been the lack of precise information, concerning the complicated changes in the physical and chemical properties of the enzyme after in solubilization (WEIBL and BRIGHT 1971). This paper deals with the thermodynamic properties of free and immobilized sub· tilopeptidase from Bacillus subtilis PR-70.
Material and Methods Free enzyme Subtilopeptidase (EO 3.4.4.16) was obtained from a locally isolated strain of B. subtilj:s PR-70 (ATTIA and SAMIA 1974), purified as previously mentioned by ATTIA and RAWIA (1977a, b).
78
R. M.
et al.
ATTIA
Immobilized enzyme A volume of enzyme solution (10 ml), containing 18000 LV unit, was added to 2 gram of Amb. CG-50 in a 25 ml Erlenmeyer fla sk. The vessel was placed in a water-bath shaker at 30°C and allowed to react for two hours. The vessel was then r emoved fr om the bath and the adsorption and diffusion was allowed to continue overnight at 5 °C with magnetic stirring. The water-insoluble subtilo-peptidaee was separated by filtration, washed with 1 M potassium chloride (100 ml), then with distilled water (50 ml), and dried at room temperature.
Det ermination of enzyme activity The enzyme activity was determined at pH 7.0 by the casein digestion method (KUNITZ 1947) as m.odified by HAGIHARA et 801. (1958) . The reaction mixture contained 5 ml of 1 % casein in 0.02 M phosphate buffer (pH 7.0) and 1 ml of the enzyme preparation. Digestion was allowed to proceed for 10 minutes at 30 °C. After the addition of 5 ml of TCA solution (TCA, 0.1 M; NaOAc, 0.22 M; HOAc, 0.33 M) and vigorous shaking, the mixture was allowed to stand at room temperature for 30 minutes and was then filtered. One ml of 3-times di luted Folin-Ciocalteau r eagent and 2,5 ml of 0.55 M sodium carbonate were added to 1 ml of the filtrate. After the solution had b een k ept at 30°C for 20 minutes, the absorbance at 660 nm was measured.
Results and Discussion Eff ec t of temperature on reaction rate The t emperature activity curve of an enzyme, like the pH curve, can be resolved into two components: the effect of increase in t emperature on the stability of the enzyme and on the rate of the catalyzed reaction. The former depends on the experimental conditions, while the latter is usually independent of them, except for reagents that alter the catalyst (SIZER 1943). Figures 1 and 2 are a plot of the observed reaction rate of free and immobilized subtilopeptidase at various temperatures under the standard conditions of the test (pH 7.0, 1 % casein). As expected, the apparent optimum temperature of free enzyme was 50 °C (ATTIA et a1. 1974),55 °C (FuKE 1963), and 57°C in the presence of calcium ions (MCCONN et a1. 1964). On the other hand, the apparent temperature of immobilized subtilopeptidase was higher than free en-
'tJ.
~ ~e/'/1]0'/I t) <... c:::
0~
I
"'Q-~o
'?
c:::
""Q-~o
~
;1)0/(1.
~
Q"~"
Q-/,;.,
.()
Thermal de.
~
.()
..:::'->
"~~/
t>
<1>
Q:
Q:
700
100
Opt/mum temp t a xbl
80
80 1----+---;1
61}
~o
ItO 1----
ItO
20
20
O~~~~~~~~~~~~~
o
~~.1
20
'to
~O
0
0
20
'to
60
Fig. 1. Effect of temperature on recti on rate of free enzyme catalyzed reaction. Fig . 2. Effect of t emp erature on r eaction rate of immobilized enzyme catalyzed reaction.
Thermodynamic Properties of Free and Immobilized Subtilopeptidase
79
2.5 2
~ 1
~
'-l
Immobll/zed
- -----:--- - -+--,
0.5
o -o.S
29.5"
- 1
JO.Y
30
31
32
33
Fig. 3. Plots of log k versus lIT t emp erature range 30 ", 60°C. Optimum temperatures are 50 and 60 °C for free and immobilized enzyme, respectively (activation en ergy).
zyme, being 60°C. This was expected because the bound enzyme is more stable to heat than the free enzyme. In a recent paper by MELROSE (1971), fifty immobilized enzyme systems were compared with their soluble counterparts with respect to their stability. Thirty of them were more stable. It indicates that in general increased stability will result from attachment to the solid phase. The reasons for increased stability of subtilopeptidase, resulting from attachment to Amb. CG-50, originated from the prevention of autodigestion, thus leading to greater stability. Another possible mechanism, leading to increased stability, is the prevention of conformation inactivation. Stabilization may also occur because active groups on the enzyme are sterically shielded from attack by reactive groups in the solution. Figure 3 shows the plot of JogK versus lIT for free and immobilized enzyme, aCcording to the Arrhenius equation: LogK
=
(-E/lnR) ·l/T
+ constant.
The curve of the Arrhenius plots is linear over the temperature range of 30° to 60°C. The heat of the reaction (Ll E) , accompanied by the process, has been calculated from the slope of the plot to be +56770 cal/mole and + 36640 cal/mole for free and immobilized subtilopeptidase, respectively. Results in Table 1 show that the heat of hydrolyzing casein is positive, whereas it is compensated by the positive entropy change. Table 1. Thermodynamic quantities of free and immobilized subtilopeptidase at various temperatures Temperature °C) 30 40 50 60 1) LlG
LIE (cal/mole)
LlG (cal/mole)1)
Free
Immobilized
Free
Immobilized
Free
Immobilized
+56 770 + 56770 +56770
+ 36640 + 36640 +36640 +36640
+ 241 -1240 -3401
+ 142 - 945 - 2233 -3506
+ 186.6 + 185.3 +186.3
+ 120.5 + 120. 1 +120.3 +120.6
= -RT oInK. 2) LIS = (E - G)/T.
LIS (cal/mole/degreej2)
80
R. M.
ATTIA
at al.
R e action order of heat inactivation The heat stability of an enzyme is best expressed in terms of the energy of inacti. vation under specified conditions. To calculate this, it was first necessary to determine the reaction order of the heat inactivation of soluble and immobilized enzymes. This was performed as follows: the enzyme in phosphate buffer at pH 7.0 was rapidly brought to 60 °0 in a wat er-bath regulated to 0.05 °0, being stirred continuously. Aliquots were removed at 5 minutes' intervals and immediately cooled in a freezing mixture to 10 °0. The residual activity was plotted against the time of heating at 60 °0. This did not yield a linear relationship and neither did a plot of the reciprocal of relative activity against time of heating. Hence, the heat inactivation was not 2
,
~
:~ v
t)
't;
~
Z;
Q)
~
~
"
0 0
70
'to SO 30 20 Time of healing (minutes)
(j'0
Fig. 4. H eat inactivation of subtilopeptidase as a first order reaction (abscissa, minutes of heating of the en zyme in the absence of substrate at 60 °0 and pH 7.0).
r-
/ l
Free Enzy-me
O.O r
0.8 -
o.t,.
i
Slope = - EI"-.')8
,
~
0.2 ~
! I
l
..L~
0
JO
~-0.2 ~
I
-o·t,. r
-0.0
-0.8
ImmobIlized Enzxme
27.S"
29.S 28.S liT x lOt,.
Fig. 5. Plots of logk versus IjT, t emp erature range 60 to 90 °0 (inactivation energy).
Thermodynamic Properties of Free and Immobilized Subtilopeptidase
81
a zero order or second order reaction. When the log of activity was plotted against the time of heating (Figure 4), however, a straight line relationship was obtained, indicating that the heat inactivation of soluble and insoluble subtilopeptidase followed a first order reaction. The finding of a heat inactivation of first order reaction for this enzyme was expected, as most enzymes follow a first order rate in heat inactivation (SIZER 1943). Aliquotes of enzyme at pH 7.0 were rapidly brought to various temperatures, ranging from 60° to 80 °0 for free enzyme and from 70° to 90 °0 for immobilized enzyme. The enzyme was kept at these temperatures for 10 minutes, then cooled and analysed in each case by the casein method. Figure 5 shows the plot of log K versus liT for free and immobilized enzymes. The curve is linear over the temperature range from 60 to 80 °0 and from 70 to 90 °0. The heat of inactivation (LIE), accompanied by the process and calculated from the slope of the curve, was shown to be +18900 cal/mole and +24400 cal/mole for free and immobilized subtilopeptidase, respectively. From the above-mentioned results, it is clear that the energy of inactivation of immobilized enzyme was higher compared with free enzyme. This is to be expected, of course, because the immobilized enzyme is more stable to heat than its soluble counterpart.
References ATTIA, R. M., and GAMAL, R. F.: Subtilopeptidase A produced by Bacillu8 8ubtili8 PR·70, I. Kinetic behaviour of solubilized enzyme. Zbl. Bakt. II 134 (1979), 275-281. - and EL-DEMERDASH, A. M.: Subtilopeptidase A produced by Bacillus 8ubtilis PR-70_ II. Kinetie behaviour of immobilized enzyme. Zbl. Bakt. II 134 (1979), 353 - 370. and ALI, S. A.: Isolation of Bacillu8 8ubtili8 producing subtilopeptidase A. Agrie. Res. Review 52 (1974). Ill. SHAWKI, H. A., EL-GAMAL, S. E., and SAMI, S. M.: Purification and physical properties of subtilopeptidase isolated from Bacillus 8ubtilis PR-70. Proc. XIII. Conf. Pharm. Sci., Cairo, Egypt (1974), 207. FUKE, I.: J. Biochom. (Tokyo) 53 (1963). In: "Methods in Enzymology", vol. 19 (G. E. Perlman and L. Lorand, ods.), p. 209, Academic Press, New York 1970. GOLDSTEIN, L.: In: "Fermentation Advances" (D. Perlman, ed.), p. 391. Academic Press, New York 1970. - and KATCHALSKI, E. G.: Z. Anal. Chern. 234 (1968), 375. In: "Methods in Enzymology", vol. 19 (G. E. Perlman and L. Lorand, eds.), p. 9:15. Academic Press. New York 1970. HAGIHARA, B., MATSUBARA, R., NAKAI, M., and OKUNUKI, K.: J. Biochem. (Japan) 45 (1958), 185. In: "Methods in Enzymology", vol. 19 (G. E. Perlman and L. Lorand, eds.), p.569. Academie Press, New York 1970. KUNITZ, M.: Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol. 30 (1947),291. MCCONN, J. D., TsuRlr, D., and YASUNOBU: Bacillu8 8ubtilis neutral proteinase. I. A zinc enzyme of high specific activity. J. BioI. Chern. 239 (1964), 3706. MELROSE, G. J. M.: Insolubilized enzymes. Biochemical applications of synthctic polymers. Rev. Pure Appl. Chern. 21 (1971), 83. SIZER, I. W.: In: "Advances in Enzymology", vol. 3 (F. F. Nord, ed.), p. 35. Interscience Publishers, Inc., New York 1943. WEIBL, M. K., and BRIGHT, H. J.: The glucose oxidase mechanism. Interpretation of the pH dependence. J. BioI. Chern. 246 (1971),2734. Authors' addresses: Dr. RASMY M. ATTIA. Dept. of Microbiology, Agricultural Research Centre of Egypt, Giza, Cairo, Egypt, and Mrs. Dr. RAWIA F. GANIAL, Dept. of Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. 6 ZbI. Bakt. II. Abt., Bd. 135