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Influence of additives on the storage stability of cellulases from Aspergillus niger
Zentralbl. Mikrobiol. 146 (1991),391-392 Gustav Fischer Verlag lena
[Department of Biochemistry, N. D. University of Agriculture and Technology, Faizabad, India]
Influence of Additives on the Storage Stability of Cellulases from Aspergillus niger AJAY SINGH, A.
K. AGRAWAL, N. S. DARMWAL and A. B. ARIDI
Key words: Enzyme stability. cellulase, Aspergillus niger
Summary The storage stability of cellulases viz. exoglucanases, endoglucanases and cellobiases has been considerably improved by the use of additives like glycerol, BSA or ex-glycerophosphate. Glycerol was found to exhibit maximum protection of the purified enzymes.
Zusammenfassung Die Lagerstabilitat von Cellulasen, wie Exoglucanasen, Endoglucanasen und Cellobiasen laBt sich durch Verwendung von Zusatzen wie Glycerol, BSA oder ex-Glycerophosphat erheblich verbessem. Glycerol bedingt eine maximale Stabilitat der gereinigten Enzyme.
The instability of enzymes continues to be a barrier to their more widespread technological applications. Lot of studies have been done on the stabilization of enzymes either for their more practical application in solution or immobilized (BROUGHAM and JOHNSON 1981). Various methods have been studied which have the potential to overcome this problem. These include the use of soluble additives and chemical modification of soluble enzymes. The use of soluble additives has the advantage in view of avoiding the modification of enzymes with the
loss of activity which often take place. While carrying out the studies on cellulase purification and characterization (SINGH 1988), we obtained some interesting results on the storage stability of cellulases, which are presented in this communication.
Materials and Methods Aspergillus niger AS-IOI was isolated in our laboratory from decomposing substrates (SINGH et al. 1988a). The procedure for culturing this mould, preparation of crude enzyme extract and purification of cellulases have been described elsewhere (SINGH 1988; SINGH et al. 1988b). The purified enzyme showing only one protein band on polyacrylamide gel electrophoresis were used for the present investigation. However, the exoglucanase and endoglucanase preparations showed little activities towards carboxymethyl cellulose and filter paper, respectively also. Exoglucanase, endoglucanase and cellobiase enzyme activities were determined using filter paper, carboxymethyl cellulose and cellobiose as substrates, respectively (GHOSE 1987). The soluble enzymes in 50 mM citrate buffer, pH 5.5 with 0.02% sodium azide were incubated for 20 days at ambient temperature (25 ± 2 DC) with given concentration of various additives and the residual activities were determined at different time intervals.
27'"
392
A. SINGH et al.
Results and Discussion Table 1 presents the changes in activities of the cellulases during storage at 25 ± 2 "C. The enzymes are highly labile in the solution without any additive. The activities of exo-, Table l. Storage stability of cellulases (% activity remained) from Aspergillus niger AS-101 at 25
± 2°e l ) .
Days after storage Addition
5
10
20
100 100 100 100
32 70 80 65
10 54 63 48
0 32 40 25
100 100 100 100
25 75 85 65
12 55 70 38
0 38 45 18
100 100 100 100
23 69 81 64
6 58 66 46
0 39 41 29
Exoglucanase None BSA (10 rng/rnl) Glycerol (50%, v/v) o-Giycerophosphate (I mM) Endoglucanase None BSA (10 mg/rnl) Glycerol (50%, v/v) o-Glycerophosphate (I mM) Cellobiase None BSA (10 mg/ml) Glycerol (50%, v/v) o-Glycerophosphate (I mM) I) Mean of three determinations
endoglucanase and cellobiase decreased continously as the storage time prolonged. No activity was observed after 20 days of storage. However, addition of bovine serum albumin (BSA), glycerol or o-glycerophosphate resulted in much protection of enzymes. Among these, glycerol was found to exhibit maximum protection of the purified enzymes followed by BSA. Thus 40% of exoglucanase, 45% of endoglucanase and 41 % of cellobiase were retained with the addition of glycerol (50'/'0, v/v) when stored at ambient temperature for 20 days. BSA and glycerol have earlier been reported to afford protection to dehydrogenases (BROUGHAM and JOHNSON 1981; AGRAWAL and MOHAN RAO 1984). Further studies are needed to understand the mechanism of cellulase stabilization.
References AGRAWAL, A. K., MOHAN RAO, Y. Y.: Kinetic studies on glutamate dehydrogenase of Aspergillus ochraceus. Ind. J. Biochem. Biophys. 21 (1984), 386-388. BROUGHAM, M. J., JOHNSON, D. B.: Glycerol, o-glyccrophosphatc and other compounds as stabilizers of alcohol dehydrogenase from yeast. Enz. Microb. Technol. 3 (1981),225-228. GHOSE, T. K.: Measurement of cellulase activities. Pure Appl. Chern. 59 (1987),257-268. SINGH, A.: Biochemical studies on utilization of agricultural wastes using cellulolytic fungi. Ph. D. Thesis, N. D. University of Agriculture and Technology, Faizabad 1988. SINGH, A., ABIDl, A. B., AGRAWAL, A. K., DARMWAL, N. S., SRIVASTAVA, S.: Utilization of cellulosic substrates for the production of single cell protein and cellulase enzyme. Ind. J. Bio. Res. 6 (1988a), 1-6. SINGH, A., ABIDI, A. B., DARMWAL, N. S., AGRAWAL, A. K.: MIRCEN J. Appl. Microbiol. Biotechnol. 4 (l988b),473-479. Authors' address: Dr. AJAY SINGH, Institut fur Technische Chemie , CallinstraBe 3, 3000 Hannover I (BRD); Drs. A. K. AGRAWAL, N. S. DARMWAL and A. B. ABIDl, Department of Biochemistry, N. D. University of Agriculture and Technology, Faizabad-224229, India.
[Department of Biochemistry, N. D. University of Agriculture and Technology, Faizabad, India]
Influence of Additives on the Storage Stability of Cellulases from Aspergillus niger AJAY SINGH, A.
K. AGRAWAL, N. S. DARMWAL and A. B. ARIDI
Key words: Enzyme stability. cellulase, Aspergillus niger
Summary The storage stability of cellulases viz. exoglucanases, endoglucanases and cellobiases has been considerably improved by the use of additives like glycerol, BSA or ex-glycerophosphate. Glycerol was found to exhibit maximum protection of the purified enzymes.
Zusammenfassung Die Lagerstabilitat von Cellulasen, wie Exoglucanasen, Endoglucanasen und Cellobiasen laBt sich durch Verwendung von Zusatzen wie Glycerol, BSA oder ex-Glycerophosphat erheblich verbessem. Glycerol bedingt eine maximale Stabilitat der gereinigten Enzyme.
The instability of enzymes continues to be a barrier to their more widespread technological applications. Lot of studies have been done on the stabilization of enzymes either for their more practical application in solution or immobilized (BROUGHAM and JOHNSON 1981). Various methods have been studied which have the potential to overcome this problem. These include the use of soluble additives and chemical modification of soluble enzymes. The use of soluble additives has the advantage in view of avoiding the modification of enzymes with the
loss of activity which often take place. While carrying out the studies on cellulase purification and characterization (SINGH 1988), we obtained some interesting results on the storage stability of cellulases, which are presented in this communication.
Materials and Methods Aspergillus niger AS-IOI was isolated in our laboratory from decomposing substrates (SINGH et al. 1988a). The procedure for culturing this mould, preparation of crude enzyme extract and purification of cellulases have been described elsewhere (SINGH 1988; SINGH et al. 1988b). The purified enzyme showing only one protein band on polyacrylamide gel electrophoresis were used for the present investigation. However, the exoglucanase and endoglucanase preparations showed little activities towards carboxymethyl cellulose and filter paper, respectively also. Exoglucanase, endoglucanase and cellobiase enzyme activities were determined using filter paper, carboxymethyl cellulose and cellobiose as substrates, respectively (GHOSE 1987). The soluble enzymes in 50 mM citrate buffer, pH 5.5 with 0.02% sodium azide were incubated for 20 days at ambient temperature (25 ± 2 DC) with given concentration of various additives and the residual activities were determined at different time intervals.
27'"
392
A. SINGH et al.
Results and Discussion Table 1 presents the changes in activities of the cellulases during storage at 25 ± 2 "C. The enzymes are highly labile in the solution without any additive. The activities of exo-, Table l. Storage stability of cellulases (% activity remained) from Aspergillus niger AS-101 at 25
± 2°e l ) .
Days after storage Addition
5
10
20
100 100 100 100
32 70 80 65
10 54 63 48
0 32 40 25
100 100 100 100
25 75 85 65
12 55 70 38
0 38 45 18
100 100 100 100
23 69 81 64
6 58 66 46
0 39 41 29
Exoglucanase None BSA (10 rng/rnl) Glycerol (50%, v/v) o-Giycerophosphate (I mM) Endoglucanase None BSA (10 mg/rnl) Glycerol (50%, v/v) o-Glycerophosphate (I mM) Cellobiase None BSA (10 mg/ml) Glycerol (50%, v/v) o-Glycerophosphate (I mM) I) Mean of three determinations
endoglucanase and cellobiase decreased continously as the storage time prolonged. No activity was observed after 20 days of storage. However, addition of bovine serum albumin (BSA), glycerol or o-glycerophosphate resulted in much protection of enzymes. Among these, glycerol was found to exhibit maximum protection of the purified enzymes followed by BSA. Thus 40% of exoglucanase, 45% of endoglucanase and 41 % of cellobiase were retained with the addition of glycerol (50'/'0, v/v) when stored at ambient temperature for 20 days. BSA and glycerol have earlier been reported to afford protection to dehydrogenases (BROUGHAM and JOHNSON 1981; AGRAWAL and MOHAN RAO 1984). Further studies are needed to understand the mechanism of cellulase stabilization.
References AGRAWAL, A. K., MOHAN RAO, Y. Y.: Kinetic studies on glutamate dehydrogenase of Aspergillus ochraceus. Ind. J. Biochem. Biophys. 21 (1984), 386-388. BROUGHAM, M. J., JOHNSON, D. B.: Glycerol, o-glyccrophosphatc and other compounds as stabilizers of alcohol dehydrogenase from yeast. Enz. Microb. Technol. 3 (1981),225-228. GHOSE, T. K.: Measurement of cellulase activities. Pure Appl. Chern. 59 (1987),257-268. SINGH, A.: Biochemical studies on utilization of agricultural wastes using cellulolytic fungi. Ph. D. Thesis, N. D. University of Agriculture and Technology, Faizabad 1988. SINGH, A., ABIDl, A. B., AGRAWAL, A. K., DARMWAL, N. S., SRIVASTAVA, S.: Utilization of cellulosic substrates for the production of single cell protein and cellulase enzyme. Ind. J. Bio. Res. 6 (1988a), 1-6. SINGH, A., ABIDI, A. B., DARMWAL, N. S., AGRAWAL, A. K.: MIRCEN J. Appl. Microbiol. Biotechnol. 4 (l988b),473-479. Authors' address: Dr. AJAY SINGH, Institut fur Technische Chemie , CallinstraBe 3, 3000 Hannover I (BRD); Drs. A. K. AGRAWAL, N. S. DARMWAL and A. B. ABIDl, Department of Biochemistry, N. D. University of Agriculture and Technology, Faizabad-224229, India.