Folia Microbiol.37 (4), 256-260 (1992)
Immobilized fl-Glucosidase from Curvularia lunata U.C. BANERJEE Institute of Microbial Technology, Chandigarh - 160 014, India ReceivedAugust 14, 1991 ABSTRACT. fl-Glucosidasefrom Curvularia lunata was immobilizedin pellets of polyacrylamide,sodium alginate and agar.
The activityof the enzymewas estimatedat differenttimes by measuringthe absorbance of a solutioninto which 2-nitrophenol was released by the enzyme.The effectof pH and temperaturewas studied to select the optimumconditions.Thermostability of the fl-glucosidasein each of the carriers was assessed over a period of 12-26 d. The immobilizedenzymeon all the three carriers retained its activitylonger than free enzymedid. Polyacrylamidewas the best carrier both in terms of thermostability and of reusabilityof the immobilizedenzymepreparations. The Michaelisconstant (Km) for each of the immobilizedenzyme preparation was calculated. Microbial cellulolytic enzymes are of immense practical interest in the saccharification of cellulose and hemicellulose. Trichoderma reesei, the best known producer of cellulase has a low fl-glucosidase activity (Ryu and Mandles 1980). fl-Glucosidase (cellobiase, EC 3.2.1.21), an essential component of the cellulase complex, is responsible for the hydrolysis of cellobiose and higher oligosaccharides. It is known that the rate of the enzymic hydrolysis of cellulose is governed by the fl-glucosidase activity of the enzyme complex (Bisaria and Ghose 1981). Deficiency of this enzyme results in a build-up of inhibitory cellobiose in the saccharification medium, causing a decreased rate of cellulose hydrolysis. In view of the potential uses of this enzyme, it is desirable to study this enzyme from different microbial sources. Since the fl-glucosidase activity is inhibited by glucose but nevertheless large concentrations (about 20 %) of glucose are required for efficient fermentation to ethanol, there have been efforts to immobilize this enzyme in solid supports (Fadda et al. 1984; Simons et al. 1990). Curvularia lunata has been found to produce a number of extracellular enzymes (Banerjee 1991; Vohra et al. 1989; Banerjee and Vohra 1991) including fl-glucosidase, endoglucanase, rifamycin oxidase and laccase. The work presented in this study was done to find an effective immobilized enzyme preparation with reusable potential using different types of carriers for immobilization. Immobilized fl-glucosidase enzyme from C. lunata exhibited a fairly stable and reusable activity, which could increase the rate of cellulose hydrolysis.
MATERIALS
AND
METHODS
Chemicals. 2-Nitrophenyl fl-D-glucopyranoside (ONPG), acrylamide, N,N'-methylene-bisacrylamide, N,N,N',N'-tetramethylenediamine and ammonium persulfate were purchased from Sigma (USA). Sodium salt of alginic acid was purchased from Fluka (Germany). All other chemicals were of
analytical purity. Culture and enzyme preparation. C. lunata was isolated from soil and maintained on potato, dextrose and agar (PDA) plates at 28 ~ The organism was grown on yeast extract, peptone and dextrose medium ( 1 % each, W / V ) at 28-30 ~ for 4d, until the fl-glucosidase activity reached a maximum. The fungal culture was filtered through Whatman filter paper (No. 1) at 4 ~ This filtrate was designated as crude extract of the enzyme. Enzyme assay, fl-Glucosidase activity was measured with ONPG as substrate according to the method of Evans (1985). Fifty mg of immobilized enzyme preparation was added to 25/zL of an aliquot of substrate consisting of ONPG (40 mmol/L) and 0.5 mL citrate buffer (50 mmol/L, pH 4.4) was added to it. The reaction mixture was incubated for 1 h at 70 ~ After incubation, the amount of 2-nitrophenol (ONP) released was determined by absorbance measuremen.t at 410 nm. One unit of activity is defined as the amount of enzyme liberating one micromole of 2-nitrophenol per mg of immobilized enzyme preparation under the assay conditions. Estimation o f protein in crude enzyme extract. Protein in the enzyme extract was estimated according to Lowry. Bovine serum albumin was used as standard.
1992
IMMOBILIZED fl-GLUCOSIDASEFROM C. lunata
257
Preparation of immobilized enzyme in different carriers Polyacrylamide immobilization./~-Glucosidase was immobilized on polyacrylamide according to the method of Johansson and Mosbach (1974). 2.375 g acrylamide and 0.125 g methylene bisacrylamide were dissolved in 15 mL of EDTA (0.1 mmol/L), Tris-HCl buffer (0.1 mol/L, pH 6.5) and 9.50 mL crude enzyme suspension (protein content 1.1 g/L) was added to it and stirred for 10 min. Twenty/zL N,N,N',N'-tetramethylenediamine and 0.5 mL ammonium persulfate (20 g/L) were added. After casting, polymerization and repeated washing with the buffer, the gel was cut into pieces and suspended in 30 mL of EDTA (0.1 mmol/L), Tris-HCl buffer (0.1 mol/L, pH 6.5) at 4 ~ Finally it.was filtered and washed with buffer and then used as the enzyme source. Alginate immobilization. Fifteen mL of sodium alginate solution (5 %, W/V) was mixed with 10 mL of crude enzyme suspension (protein content 1.1 g/L) and mixed thoroughly. The beads were formed in CaC12 (0.2 mol/L) solution by extruding the alginate-enzyme mixture through a needle using a 10-mL syringe. These heads were kept in Tris-HCl buffer (0.1tool/L, pH 6.5) with CaCI2 (10 mmol/L). The procedure used was essentially the one described by Eikmeier and Rehm (1987). Agar immobilization, fl-Glucosidase was immobilized in agar gel according to the method of Matsunaga et al. (1980). 0.375 g agar was dissolved in 15 mL of 0.9 % (W/V) NaCI by heating at 100 ~ and then cooled to 50 *C. Ten mL of crude enzyme suspension (protein content 1.1 g/L) was added to the agar solution, which was kept at 50 ~ with constant stirring. After casting, polymerization and washing, the gel was cut into pieces and washed several times with phosphate buffer (0.1 mol/L, pH 6.5). The immobilized beads were used. Effect ofpH and temperature. The effect of pH and temperature on the activity of the immobilized fl-glucosidase was determined as follows. Fifty mg of the immobilized enzyme preparation was added to 25/zL aliquot of substrate consisting of ONPG (40 mmol/L) and 0.5 mL of citrate buffer (50 mmol/L) whose pH was varied from 3.6 to 6.0. After incubation at 70 ~ for 1 h, the amount of 2-nitrophenol released was determined by absorbance measurement at 410 nm. To study the effect of temperature, twelve identical sets of enzyme assays were set up and incubated at different temperatures from 30 to 80 ~ at intervals of 10 ~ for 1 h. Then the enzyme activity was assayed as described earlier. Thermostability studies. To find out the thermostability of the immobilized fl-glucosidase, the beads were put in phosphate or Tris-HC1 buffer (0.1 mol/L, pH 6.5) and kept in temperature-controlled incubators (37 and 50 ~ At different time intervals a few beads were taken out, washed with the respective buffers and their activities toward ONPG were measured. Reusability of immobilized enzyme preparations. The operational stability of the fl-glucosidase immobilized on different carriers was checked by incubating the immobilized enzyme preparations in the respective buffers with substrate (400 mmol/L) for 24h. The suspension was filtered and fl-glucosidase activity was determined by measuring the amount of 2-nitrophenol (ONP) released. The immobilized beads were washed twice with fresh buffer and used again with the substrate. The process was repeated each time. RESULTS A N D DISCUSSION
pH and temperature optima It is evident from Fig. 1 that all the immobilized enzyme preparations have the same pH optimum (4.4) of fl-glucosidase activity. The activity of the free enzyme is highest at pH 4.0 (Banerjee 1991). The type of carrier did not influence the pH optima for/~-glucosidase activity but the optimum pH was shifted from more acidic to less acidic region compared with the free enzyme. At pH 4.0, agarimmobilized fl-glucosidase retained 90 % of its activity, while at this pH, alginate- and polyacrylamideimmobilized fl-glucosidase retained 69 and 53 % of the maximum activity, respectively. Up to pH 4.8, all the three enzyme preparations retained 80 to 87 % of their maximum activity. At higher pH (4.8 to 6.0) the/~-glucosidase activity was found to be low. Fig. 2 shows the optimum temperature for fl-glucosidase activity immobilized on different carriers. The optimum temperature for/3-glucosidase activity was found to be same (70 ~ for all the immobilized enzyme preparations. The optimum temperature for the free ff-glucosidase activity is also 70 ~ activity was drastically reduced at a temperature higher than 70 ~ At 60 ~ most of the immobilized enzyme preparations retained 70-75 % of their activity.
258
U.C. BANERJEE
120
Vol. 37
J
i
I
%
100
80
1
2O
0 3
120
I
I
I
t.
5
6
I
I
Fig. 1. Effect of pH on immobilized fl-glucosidase activity (%) from C. lunata; 1 - polyacrylamide, 2 - alginate, 3 - agar. pH
7
I
%
100
80
60
40
Fig. 2. Effect of temperature (~ on immobilized fl-glucosidase activity (%) from C. lunata; 1 - polyacrylamide, 2 - alginate, 3 agar.
20
20
t.O
80
60
'C
Km values of immobilized enzyme preparations
The Michaelis constants (Kin) were calculated from the initial reaction rate data plotted as a Lineweaver-Burk plot: the plots deviated from linearity at low substrate concentrations, in most of the cases. The immobilized enzyme preparations showed substantially higher Km values than the free enzyme (Table I). T h e increase of Kin values is possibly caused by diffusional limitations. This is further shown by the lower Km value obtained with alginate-immobilized enzyme preparation, where diffusional limitations are lower c o m p a r e d with polyacrylamide- or agar-immobilized enzyme preparations.
1992
I M M O B I L I Z E D f l - G L U C O S I D A S E F R O M C. lunata
Table I, Km of the immobilized fl-glueosidase on different carriers
259
Thermostability of the immobilized enzyme preparations
Thermostability of the immobilized enzyme preparations was determined at 37 and 50 ~ It is evident Enzyme preparation Km, tool O N P G / L from Table II that polyacrylamide is the best of the three carriers. The other two supports permit a faster Free 28 decay. The agar-immobilized enzyme preparation Polyacrylamide 326 showed a similar type of behavior at 37 and 50 ~ but Alginate 312 the percentage of retained enzyme activity was very Agar 383 low compared to the other two supports. The loss in activity may be due to the temperature inactivation of the enzyme or to the leaching of the enzyme from the beads and diffusional limitations in the case of polyacrylamide- and agar-immobilized enzyme preparations. Thermostability of the immobilized /~-glucosidase also did not improve at 70 ~ (data not shown), but at 37 and 50 ~ the thermostability was found to be improved compared to the free enzyme (Banerjee 1991).
Table II. Thermostability of immobilized fl-glucosidase activity from Curvularia l u n a t a a
Polyacrylamide
Ca-alginate
Agar
Days 37 ~ 0 2 5 8 11 14 17 20 23 26
7.21 7.11 7.53 7.46 6.93 7.21 6.23 6.45 7.00 6.42
50 ~
37 ~
50 ~
37 ~
50 ~
6.38 7.21 7.01 6.25 6.51 5.62 5.21 4.26 4.50 4.10
7.01 6.56 6.21 6.01 5.22 5.00 4.21
6.53 5.32 5.16 4.85 3.96 3.00 2.11
6.42 6.00 5.32 5.01 3.21
6.11 5.26 5.12 4.51 3.76
-
-
aActivity,/~mol O N P per h per g immobilized enzyme preparation.
Table III.
Operational stability of immobilized fl-glucosidase from
Curvularia l u n a t a a
Reusability of hnmobilized enzyme preparations
In order to determine the operational stability of the immobilized Polyacrylamide Ca-alginate Agar enzyme preparations, the once used enzyme complexes were used re0 8.83 7.65 5.27 peatedly under identical conditions, 2 8.61 7.29 5.21 4 8.72 7.46 5.02 to assess the enzyme activity. The re6 8.26 7.21 4.93 sults showed (Table III) that immo8 8.42 7.52 5.21 bilized enzyme preparations retained 10 8.60 6.93 4.63 the original activity of fl-glucosidase 12 8.21 6.06 3.81 enzyme up to 16-18 d in the case of 14 8.36 5.00 2.52 polyacrylamide immobilized enzyme, 16 8.00 4.01 1.33 8-10 d in the case of alginate immo18 7.92 2.26 20 6.93 1.92 bilized enzyme and 6 - 8 d in the case of agar immobilized enzyme. aActivity,/xmol per h per g immobilized enzyme preparation. Reusability of the polyacrylamideimmobilized enzyme system is much better compared to that of alginate- or agar-immobilized enzyme preparation. Diffusional limitations may be one of the reasons for the lower reusability of the agar-immobilized enzyme preparation. Leaching of the enzyme from the immobilized enzyme preparations was also checked by measuring the protein content in the supernatant. No leaching was found in the case of polyacrylamide- and agarN u m b e r of times of reuse
260
U.C. BANERJEE
Vol. 37
immobilized system but a considerable amount of leaching was noticed in the case of alginateimmobilized enzyme preparations. The technical assistance of Mr. J.P. Srivastava and Miss B. Saxena is greatly appreciated.
REFERENCES
BANEILIEEU.C.: Production of/~-glucosidase (cellobiase) by Curvularia sp. Lett,4ppLMicrobioL 10, 197-199 (1990). BAI~ERJEEU.C., VOHRA R.M.: Production of iaccase by Curvulatia lunata. Folia Microbiol. 36, 343- 346 (1991). BmARIAV.S., GHOSE T.K.: Biodegradation of cellulosic materials: substrates, microorganisms, enzymes and products. Enz. Microb.Technol. 3, 90-104 (1981). E~gMEIER H., REHM HJ.: Stability of calcium alginate during citric acid production of immobilised Aspergillus niger. Appl. Microbiol.BiotechnoL 26, 105-111 (1987). EVANS C.S.: Properties of the fl-glucosidase (cellobiase) from the wood-rotting fungus Coriolus versicolor. Appl.MicrobioL BiotechnoL 22, 128-131 (1985). FADDAM.B., DESS!M.R., MAURlClR., RINALD!A., SA'ITAG.: Highly efficient solubilization of natural lignocellulosic materials by a commercial cellulose immobilised on various solid supports. Appl.Microbiol.BiotechnoL 19, 306-311 (1984). JOHANSSONA.C., MOSBACHK.: Acrylic copolymers as matrices for the immobilisation of enzymes: 1. Covalent binding or entrapping of various enzymes to bead-formed acrylic copolymers. Biochim.Biophys,4cta 370, 339-347 (1974). MAISUNAGAT., KARUBEI., SUZUKI S.: Some observations on immobilised hydrogen-producing bacteria: Behaviour of hydrogen in gel membranes. Biotechnol.Bioeng. 22, 2607-2615 (1980). Rvu D.D., MANDLES M.: Cellulase: biosynthesis and applications. Enz.Microb.Technol. 2, 91 - 102 (1980). SIMONS G., GEORGATSOSJ.G.: Immobilization of barley fl-glucosidase on solid supports - yields and properties. Appl. Microbiol.Bioteclmol. 33, 51-53 (1990). VOHRA R.M., BANERJEEU.C., DAS S., DUBE S.: Microbial transformation of rifamycin B: A new extracellular oxidase from Curvularia lunata. Biotectmol.Lett. 11, 851-854 (1989).
Immobilized fl-Glucosidase from Curvularia lunata U.C. BANERJEE Institute of Microbial Technology, Chandigarh - 160 014, India ReceivedAugust 14, 1991 ABSTRACT. fl-Glucosidasefrom Curvularia lunata was immobilizedin pellets of polyacrylamide,sodium alginate and agar.
The activityof the enzymewas estimatedat differenttimes by measuringthe absorbance of a solutioninto which 2-nitrophenol was released by the enzyme.The effectof pH and temperaturewas studied to select the optimumconditions.Thermostability of the fl-glucosidasein each of the carriers was assessed over a period of 12-26 d. The immobilizedenzymeon all the three carriers retained its activitylonger than free enzymedid. Polyacrylamidewas the best carrier both in terms of thermostability and of reusabilityof the immobilizedenzymepreparations. The Michaelisconstant (Km) for each of the immobilizedenzyme preparation was calculated. Microbial cellulolytic enzymes are of immense practical interest in the saccharification of cellulose and hemicellulose. Trichoderma reesei, the best known producer of cellulase has a low fl-glucosidase activity (Ryu and Mandles 1980). fl-Glucosidase (cellobiase, EC 3.2.1.21), an essential component of the cellulase complex, is responsible for the hydrolysis of cellobiose and higher oligosaccharides. It is known that the rate of the enzymic hydrolysis of cellulose is governed by the fl-glucosidase activity of the enzyme complex (Bisaria and Ghose 1981). Deficiency of this enzyme results in a build-up of inhibitory cellobiose in the saccharification medium, causing a decreased rate of cellulose hydrolysis. In view of the potential uses of this enzyme, it is desirable to study this enzyme from different microbial sources. Since the fl-glucosidase activity is inhibited by glucose but nevertheless large concentrations (about 20 %) of glucose are required for efficient fermentation to ethanol, there have been efforts to immobilize this enzyme in solid supports (Fadda et al. 1984; Simons et al. 1990). Curvularia lunata has been found to produce a number of extracellular enzymes (Banerjee 1991; Vohra et al. 1989; Banerjee and Vohra 1991) including fl-glucosidase, endoglucanase, rifamycin oxidase and laccase. The work presented in this study was done to find an effective immobilized enzyme preparation with reusable potential using different types of carriers for immobilization. Immobilized fl-glucosidase enzyme from C. lunata exhibited a fairly stable and reusable activity, which could increase the rate of cellulose hydrolysis.
MATERIALS
AND
METHODS
Chemicals. 2-Nitrophenyl fl-D-glucopyranoside (ONPG), acrylamide, N,N'-methylene-bisacrylamide, N,N,N',N'-tetramethylenediamine and ammonium persulfate were purchased from Sigma (USA). Sodium salt of alginic acid was purchased from Fluka (Germany). All other chemicals were of
analytical purity. Culture and enzyme preparation. C. lunata was isolated from soil and maintained on potato, dextrose and agar (PDA) plates at 28 ~ The organism was grown on yeast extract, peptone and dextrose medium ( 1 % each, W / V ) at 28-30 ~ for 4d, until the fl-glucosidase activity reached a maximum. The fungal culture was filtered through Whatman filter paper (No. 1) at 4 ~ This filtrate was designated as crude extract of the enzyme. Enzyme assay, fl-Glucosidase activity was measured with ONPG as substrate according to the method of Evans (1985). Fifty mg of immobilized enzyme preparation was added to 25/zL of an aliquot of substrate consisting of ONPG (40 mmol/L) and 0.5 mL citrate buffer (50 mmol/L, pH 4.4) was added to it. The reaction mixture was incubated for 1 h at 70 ~ After incubation, the amount of 2-nitrophenol (ONP) released was determined by absorbance measuremen.t at 410 nm. One unit of activity is defined as the amount of enzyme liberating one micromole of 2-nitrophenol per mg of immobilized enzyme preparation under the assay conditions. Estimation o f protein in crude enzyme extract. Protein in the enzyme extract was estimated according to Lowry. Bovine serum albumin was used as standard.
1992
IMMOBILIZED fl-GLUCOSIDASEFROM C. lunata
257
Preparation of immobilized enzyme in different carriers Polyacrylamide immobilization./~-Glucosidase was immobilized on polyacrylamide according to the method of Johansson and Mosbach (1974). 2.375 g acrylamide and 0.125 g methylene bisacrylamide were dissolved in 15 mL of EDTA (0.1 mmol/L), Tris-HCl buffer (0.1 mol/L, pH 6.5) and 9.50 mL crude enzyme suspension (protein content 1.1 g/L) was added to it and stirred for 10 min. Twenty/zL N,N,N',N'-tetramethylenediamine and 0.5 mL ammonium persulfate (20 g/L) were added. After casting, polymerization and repeated washing with the buffer, the gel was cut into pieces and suspended in 30 mL of EDTA (0.1 mmol/L), Tris-HCl buffer (0.1 mol/L, pH 6.5) at 4 ~ Finally it.was filtered and washed with buffer and then used as the enzyme source. Alginate immobilization. Fifteen mL of sodium alginate solution (5 %, W/V) was mixed with 10 mL of crude enzyme suspension (protein content 1.1 g/L) and mixed thoroughly. The beads were formed in CaC12 (0.2 mol/L) solution by extruding the alginate-enzyme mixture through a needle using a 10-mL syringe. These heads were kept in Tris-HCl buffer (0.1tool/L, pH 6.5) with CaCI2 (10 mmol/L). The procedure used was essentially the one described by Eikmeier and Rehm (1987). Agar immobilization, fl-Glucosidase was immobilized in agar gel according to the method of Matsunaga et al. (1980). 0.375 g agar was dissolved in 15 mL of 0.9 % (W/V) NaCI by heating at 100 ~ and then cooled to 50 *C. Ten mL of crude enzyme suspension (protein content 1.1 g/L) was added to the agar solution, which was kept at 50 ~ with constant stirring. After casting, polymerization and washing, the gel was cut into pieces and washed several times with phosphate buffer (0.1 mol/L, pH 6.5). The immobilized beads were used. Effect ofpH and temperature. The effect of pH and temperature on the activity of the immobilized fl-glucosidase was determined as follows. Fifty mg of the immobilized enzyme preparation was added to 25/zL aliquot of substrate consisting of ONPG (40 mmol/L) and 0.5 mL of citrate buffer (50 mmol/L) whose pH was varied from 3.6 to 6.0. After incubation at 70 ~ for 1 h, the amount of 2-nitrophenol released was determined by absorbance measurement at 410 nm. To study the effect of temperature, twelve identical sets of enzyme assays were set up and incubated at different temperatures from 30 to 80 ~ at intervals of 10 ~ for 1 h. Then the enzyme activity was assayed as described earlier. Thermostability studies. To find out the thermostability of the immobilized fl-glucosidase, the beads were put in phosphate or Tris-HC1 buffer (0.1 mol/L, pH 6.5) and kept in temperature-controlled incubators (37 and 50 ~ At different time intervals a few beads were taken out, washed with the respective buffers and their activities toward ONPG were measured. Reusability of immobilized enzyme preparations. The operational stability of the fl-glucosidase immobilized on different carriers was checked by incubating the immobilized enzyme preparations in the respective buffers with substrate (400 mmol/L) for 24h. The suspension was filtered and fl-glucosidase activity was determined by measuring the amount of 2-nitrophenol (ONP) released. The immobilized beads were washed twice with fresh buffer and used again with the substrate. The process was repeated each time. RESULTS A N D DISCUSSION
pH and temperature optima It is evident from Fig. 1 that all the immobilized enzyme preparations have the same pH optimum (4.4) of fl-glucosidase activity. The activity of the free enzyme is highest at pH 4.0 (Banerjee 1991). The type of carrier did not influence the pH optima for/~-glucosidase activity but the optimum pH was shifted from more acidic to less acidic region compared with the free enzyme. At pH 4.0, agarimmobilized fl-glucosidase retained 90 % of its activity, while at this pH, alginate- and polyacrylamideimmobilized fl-glucosidase retained 69 and 53 % of the maximum activity, respectively. Up to pH 4.8, all the three enzyme preparations retained 80 to 87 % of their maximum activity. At higher pH (4.8 to 6.0) the/~-glucosidase activity was found to be low. Fig. 2 shows the optimum temperature for fl-glucosidase activity immobilized on different carriers. The optimum temperature for/3-glucosidase activity was found to be same (70 ~ for all the immobilized enzyme preparations. The optimum temperature for the free ff-glucosidase activity is also 70 ~ activity was drastically reduced at a temperature higher than 70 ~ At 60 ~ most of the immobilized enzyme preparations retained 70-75 % of their activity.
258
U.C. BANERJEE
120
Vol. 37
J
i
I
%
100
80
1
2O
0 3
120
I
I
I
t.
5
6
I
I
Fig. 1. Effect of pH on immobilized fl-glucosidase activity (%) from C. lunata; 1 - polyacrylamide, 2 - alginate, 3 - agar. pH
7
I
%
100
80
60
40
Fig. 2. Effect of temperature (~ on immobilized fl-glucosidase activity (%) from C. lunata; 1 - polyacrylamide, 2 - alginate, 3 agar.
20
20
t.O
80
60
'C
Km values of immobilized enzyme preparations
The Michaelis constants (Kin) were calculated from the initial reaction rate data plotted as a Lineweaver-Burk plot: the plots deviated from linearity at low substrate concentrations, in most of the cases. The immobilized enzyme preparations showed substantially higher Km values than the free enzyme (Table I). T h e increase of Kin values is possibly caused by diffusional limitations. This is further shown by the lower Km value obtained with alginate-immobilized enzyme preparation, where diffusional limitations are lower c o m p a r e d with polyacrylamide- or agar-immobilized enzyme preparations.
1992
I M M O B I L I Z E D f l - G L U C O S I D A S E F R O M C. lunata
Table I, Km of the immobilized fl-glueosidase on different carriers
259
Thermostability of the immobilized enzyme preparations
Thermostability of the immobilized enzyme preparations was determined at 37 and 50 ~ It is evident Enzyme preparation Km, tool O N P G / L from Table II that polyacrylamide is the best of the three carriers. The other two supports permit a faster Free 28 decay. The agar-immobilized enzyme preparation Polyacrylamide 326 showed a similar type of behavior at 37 and 50 ~ but Alginate 312 the percentage of retained enzyme activity was very Agar 383 low compared to the other two supports. The loss in activity may be due to the temperature inactivation of the enzyme or to the leaching of the enzyme from the beads and diffusional limitations in the case of polyacrylamide- and agar-immobilized enzyme preparations. Thermostability of the immobilized /~-glucosidase also did not improve at 70 ~ (data not shown), but at 37 and 50 ~ the thermostability was found to be improved compared to the free enzyme (Banerjee 1991).
Table II. Thermostability of immobilized fl-glucosidase activity from Curvularia l u n a t a a
Polyacrylamide
Ca-alginate
Agar
Days 37 ~ 0 2 5 8 11 14 17 20 23 26
7.21 7.11 7.53 7.46 6.93 7.21 6.23 6.45 7.00 6.42
50 ~
37 ~
50 ~
37 ~
50 ~
6.38 7.21 7.01 6.25 6.51 5.62 5.21 4.26 4.50 4.10
7.01 6.56 6.21 6.01 5.22 5.00 4.21
6.53 5.32 5.16 4.85 3.96 3.00 2.11
6.42 6.00 5.32 5.01 3.21
6.11 5.26 5.12 4.51 3.76
-
-
aActivity,/~mol O N P per h per g immobilized enzyme preparation.
Table III.
Operational stability of immobilized fl-glucosidase from
Curvularia l u n a t a a
Reusability of hnmobilized enzyme preparations
In order to determine the operational stability of the immobilized Polyacrylamide Ca-alginate Agar enzyme preparations, the once used enzyme complexes were used re0 8.83 7.65 5.27 peatedly under identical conditions, 2 8.61 7.29 5.21 4 8.72 7.46 5.02 to assess the enzyme activity. The re6 8.26 7.21 4.93 sults showed (Table III) that immo8 8.42 7.52 5.21 bilized enzyme preparations retained 10 8.60 6.93 4.63 the original activity of fl-glucosidase 12 8.21 6.06 3.81 enzyme up to 16-18 d in the case of 14 8.36 5.00 2.52 polyacrylamide immobilized enzyme, 16 8.00 4.01 1.33 8-10 d in the case of alginate immo18 7.92 2.26 20 6.93 1.92 bilized enzyme and 6 - 8 d in the case of agar immobilized enzyme. aActivity,/xmol per h per g immobilized enzyme preparation. Reusability of the polyacrylamideimmobilized enzyme system is much better compared to that of alginate- or agar-immobilized enzyme preparation. Diffusional limitations may be one of the reasons for the lower reusability of the agar-immobilized enzyme preparation. Leaching of the enzyme from the immobilized enzyme preparations was also checked by measuring the protein content in the supernatant. No leaching was found in the case of polyacrylamide- and agarN u m b e r of times of reuse
260
U.C. BANERJEE
Vol. 37
immobilized system but a considerable amount of leaching was noticed in the case of alginateimmobilized enzyme preparations. The technical assistance of Mr. J.P. Srivastava and Miss B. Saxena is greatly appreciated.
REFERENCES
BANEILIEEU.C.: Production of/~-glucosidase (cellobiase) by Curvularia sp. Lett,4ppLMicrobioL 10, 197-199 (1990). BAI~ERJEEU.C., VOHRA R.M.: Production of iaccase by Curvulatia lunata. Folia Microbiol. 36, 343- 346 (1991). BmARIAV.S., GHOSE T.K.: Biodegradation of cellulosic materials: substrates, microorganisms, enzymes and products. Enz. Microb.Technol. 3, 90-104 (1981). E~gMEIER H., REHM HJ.: Stability of calcium alginate during citric acid production of immobilised Aspergillus niger. Appl. Microbiol.BiotechnoL 26, 105-111 (1987). EVANS C.S.: Properties of the fl-glucosidase (cellobiase) from the wood-rotting fungus Coriolus versicolor. Appl.MicrobioL BiotechnoL 22, 128-131 (1985). FADDAM.B., DESS!M.R., MAURlClR., RINALD!A., SA'ITAG.: Highly efficient solubilization of natural lignocellulosic materials by a commercial cellulose immobilised on various solid supports. Appl.Microbiol.BiotechnoL 19, 306-311 (1984). JOHANSSONA.C., MOSBACHK.: Acrylic copolymers as matrices for the immobilisation of enzymes: 1. Covalent binding or entrapping of various enzymes to bead-formed acrylic copolymers. Biochim.Biophys,4cta 370, 339-347 (1974). MAISUNAGAT., KARUBEI., SUZUKI S.: Some observations on immobilised hydrogen-producing bacteria: Behaviour of hydrogen in gel membranes. Biotechnol.Bioeng. 22, 2607-2615 (1980). Rvu D.D., MANDLES M.: Cellulase: biosynthesis and applications. Enz.Microb.Technol. 2, 91 - 102 (1980). SIMONS G., GEORGATSOSJ.G.: Immobilization of barley fl-glucosidase on solid supports - yields and properties. Appl. Microbiol.Bioteclmol. 33, 51-53 (1990). VOHRA R.M., BANERJEEU.C., DAS S., DUBE S.: Microbial transformation of rifamycin B: A new extracellular oxidase from Curvularia lunata. Biotectmol.Lett. 11, 851-854 (1989).
