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Cation-induced thermal stability of an alkaline protease from a Bacillus sp.
Bioresource Technology 50 (1994) 209-211 ElsevierScienceLimited Printed in Great Britain 0260-8774/94/$7.00
I~Jl~_. !',i ELSEVIER
0960-8524(94)00079-4
CATION-INDUCED T H E R M A L STABILITY OF A N ALKALINE PROTEASE FROM A BACILLUS SE Nisha Paliwal, a S. E
S i n g h b*
• S. K. Garga:~
"Department of Microbiology, G. B. Pant University of Agriculture and Technology, Pantnagar-263145, India hMicrobiology Unit, Department of Biosciences, Saurashtra University, Rajkot-360005, India
(Received 30 July 1994; accepted 10 August 1994)
Cations are known to protect enzymes in general against thermal denaturation by maintaining the necessary conformation of the molecule (Pan & Lin, 1991; Singh & Rogers, 1991; Steele et al., 1992; Donaghy & McKay, 1993). Therefore, in an attempt to enhance the thermal stability of alkaline protease from a Bacillus sp. the effect of various cations on the enzyme activity as a function of increasing temperature was examined.
Abstract A n alkafine protease produced from a Bacillus sp. was stimulated by the metal ions Ca 2÷, Mg 2+ and Mn2+; with Ca 2+ having the m a x i m u m effect. The thermal stability of the enzyme was also enhanced to varying degrees in the presence of these ions. Key words: Alkaline protease, Bacillus sp., thermostability, alkalophiles.
METHODS Growth of the organism The organism was earlier isolated in our laboratory and identified as a Bacillus sp. (Paliwal, 1993; Sinha, 1993; Sinha et al., 1994). The isolate was grown at 37°C in a growth medium that contained (g/litre): glucose, 10; peptone, 5; yeast extract, 5; KH2PO4, 10; MgSO4.7H:O, 0-2. For adjusting the pH to 10"5, separately sterilized Na2CO3 (20% w/v) was added to the sterilized medium at 10% (v/v). Cultures grown to an absorbance of 0"6 at 665 nm were used as inocula. The sterile medium (100 ml, pH 10.5) was inoculated with 0.50% (v/v) inoculum and incubated at 37°C for 12 h under shake-flask conditions (100 rev/min). After growth, the cultures were centrifuged at 10 000g for 25 min and supernatants were used for the enzyme assay.
INTRODUCTION Proteases are among the most important groups of industrial enzymes and constitute nearly 60% of the total world-wide enzyme sales (Ng & Kenealy, 1986). Alkaline proteases are of particular interest because of their potential to catalyse reactions under alkaline conditions, which makes them ideal for use in many industries such as detergent (Durham, 1987; Phadatare et al., 1993), leather (Grebeskova et al., 1988) and food manufacturers (Wilson et al., 1992). Of late, some newer applications, for instance silver recovery from photographic plates (Fujiwara et al., 1989, 1991) and peptide synthesis (Cerovsky, 1992) have also been explored. In view of the intensive commercial interest, microbes from varied habitats have been examined for industrially suitable alkaline proteases (Jones et al., 1988; Griffin et al., 1992; Snowden et al., 1992; Steele et al., 1992). The thermal stability of enzymes in general is considered as one of the most vital parameters for catalytic versatility in industrial applications. Some of the major commercial uses of alkaline proteases necessitate high temperatures, and thus improving the thermal stability of the enzyme would be distinctly advantageous. Thermostability can be enhanced either by adding certain stabilizers to the reaction mixture or by manipulating the tertiary structures of the enzymes through molecular approaches such as amino-acid substitution (Imanaka et al., 1986; Eijsink et al., 1991; Hardy et al., 1993).
Enzyme assay The assay was based on Anson-Hagihara's method (Hagihara et al., 1958) using casein as substrate. The enzyme solution (0"5 ml) was added to 3"0 ml casein solution (0"6% w/v casein solution prepared in 20 mM borax NaOH buffer of pH 10-5) and allowed to react for 10 min at 37°C. The reaction was terminated by the addition of 3.2 ml of a solution of 0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0"33 M acetic acid. The reaction mixture was left at 370C for 10 min and filtered through a Whatman No. 1 filter paper. The absorbance of the filtrate was measured at 275 nm. A unit of alkaline protease activity (U) was defined as the amount of enzyme liberating 1/~g of tyrosine per min under the conditions described above. The enzyme estimation was based on a calibration curve using tyrosine.
*To whom correspondence should be addressed. :~Present address: Avadh University, Faizabad, India. 209
210
Nisha Paliwal, S. P. Singh, S. K. Garg o ~
~
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~
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•~
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~
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=
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o:=. ~ ~~,~..~ "~.~ "~ ~ <
.~
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~~ , . ~
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~o
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>.-,0
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0 ;zL ¢~
I
I
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,O
Thermal stability of an alkaline protease Effect of cations on e n z y m e activities
The effects of increasing concentrations (5-20 mM) of Ca 2+, Mg 2+ and Mn 2+ on enzyme activity were investigated at 37°C. All cations were prepared in b o r a x - N a O H buffer (pH 10-5) and diluted appropriately in the same buffer to give the desired concentrations in the assay mixtures. However, for examining the thermal stability, the enzyme was preincubated with the cations (2"5-20 mM) for 10-60 min at 37, 50, 60 and 70°C before the enzyme assay. The cations were used in the form of CaCI 2, MgCI2 and MnCL. RESULTS AND DISCUSSION As shown in Fig. 1, all the three cations, Ca 2+, Mg 2÷, and Mn 2+, induced enzyme activity. At 5 mM concentration, all the cations had almost similar effects on the enzyme activity, but while Ca 2÷ exhibited an increasing activity up to 15 mM, with Mg 2+ and Mn 2+ no further enhancement was evident beyond 5 mM (Fig. 1 ). The thermal stability of the enzyme as a function of varying cation concentrations at 50, 60 and 70°C was monitored. The results are graphically depicted in Fig. 2. The enzyme activity showed a decrease at temperatures beyond 37°C when no cation was added. At 70°C, the activity dropped to nearly zero after 10 min of incubation. Cations Ca 2+, Mg :+ and Mn 2÷ apparently protected the enzyme against thermal denaturation. With Ca 2+, however, this effect was pronounced at concentrations above 5 mM. Whether these cations added to the thermal stability of the enzyme at 70°C is not clear because of the drastic reduction in the activity at this temperature. Nevertheless, the cations showed stimulation as compared to the control. This may, perhaps, be taken as indirect evidence of the enzyme protection by the cations at higher temperatures. On the whole, Ca 2+, Mg 2+ and Mn 2÷ substantially stimulated the enzyme catalysis, and the thermal denaturation of the enzyme was retarded to varying extents in the presence of the cations. The results indicate that the metal ions play a vital role in maintaining the active conformation of the enzyme at higher temperatures, as also recently suggested for two alkaline proteases from Kurthia spiroforme sp. nov. (Steele et al., 1992) and Aureobasdium pullulans (Donaghy & McKay, 1993). Other experiments (Singh & Pailwal, unpublished data) have indicated that these cations also stimulate enzyme production when added during bacterial growth.
REFERENCES
Cerovsky, V. (1992). Protease-catalysed peptide synthesis in solvent-free system. Biotechnol. Techniques, 6 (2), 155-60. Donaghy, J. A. & McKay, A. M. (1993). Production and properties of an alkaline protease by Aureobasidium pullulans. J. Appl. Bacteriol., 74,000-000. Durham, D. R. (1987). Utility of Subtilisin GX as a detergent additive. J. Appl. Bacteriol., 63 (5), 381-6.
211
Eijsink, V. G. H., Zee, J. R., Burg, B., Vriend, G. & Venema, G. (1991). Improving the thermostability of the neutral portease of Bacillus stearothermophilus by replacing a buried asparagine by leucine. FEBS L ett., 282 ( 1), 13-16. Fujiwara, N., Tsumiya, T., Katada, T., Hosobuchi, T. & Yamamoto, K. (1989). Continuous recovery of silver from used X-ray films using a proteolytic enzyme. Proc. Biochem., 24, 155-6. Fujiwara, N., Yamamoto, K. & Masui, A. (1991). Utilization of a thermostable alkaline protease from an alkalophilic thermophile for the recovery of silver from used X-ray film. J. Ferment. Bioengng, 72 (4), 306-8. Grebeskova, R. N., Ryshlava, J. M., Fedorova, L. G., Kochetova, S. P., Babloyan, O. O. & Vinogredona, G. L. (1988). Using alkaline protease to intensify the processing of leather raw material. Biotechnolgica, 4 (6), 788-91. Griffin, H. L., Greene, R. V. & Cotta, M. A. (1992). Isolation and characterization of an alkaline protease from the marine shipworm bacterium. Curr. Microbiol., 24, 111-17. Hagihara, B., Matsubara, H., Nakai, M. & Okunuki, K. ( 1958). Crystalline bacterial proteinase of Bacillus subtilis. J. Biochem., 45, 185-94. Hardy, F. G., Vriend, O. R., Veltnan, B., Vinne, V. D., Venema, G. & Eijsink, V. G. H. (1993). Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines. FEBS Lett., 317 (1-2), 89-92. Imanaka, T., Shibazaki, M. & Takagi, M. (1986). A new way of enhancing the thermostability of proteases. Nature, 324,695-7. Jones, C. W., Morgan, H. W. & Daniel, R. M. (1988). Aspects of protease production by Thermus Stain OK6 and other New Zealand isolates. J. Gen. Microbiol., 134,191-8. Ng, T. K. & Kenealy, W. R. (1986). Industrial applications of thermostable enzymes. In Thermophiles. General, Molecular and Applied Microbiology, ed. T. D. Brock. John Wiley, New York, pp. 197-205. Paliwal, N. (1993). Production of alkaline protease from Bacillus sp. MSc thesis submitted to G. B. Pant University of Agriculture and Technology, Pantnagar, India. Pan, T. & Lin, S. (1991). Fermentative production of alkaline protease as detergent additive. J. Chinese Biochem. Soc., 20 (1), 49-60. Phadatare, S. V., Deshpande, V. U. & Srinivasan, M. C. (1993). High activity alkaline protease from Conidiobolus coronatus: enzyme production and compatibility with commercial detergents. Enzyme Microbiol. Technol., 15 (1), 72-5. Singh, S. P. & Rogers, P. J. (1991). Isolation and kinetic properties of pyruvate kinase from Brochothrix thermosphaeta. Biochem. Int., 25 (1), 35-45. Sinha, R. (1993). Purification and characterization of a Bacil/us alkaline protease. MSc thesis submitted to G. B. Pant University of Agriculture and Technology, Pantnagar, India. Sinha, R., Singh, S. P., Garg, S. K. & Ahmad, S. (1994). Aqueous two phase system: partitioning and purification of a bacterial alkaline protease. Proc. Ann. Conf. Assoc. Microbiologists, Ludhiana, India, p. 70. Snowden, J., Bluenentals, I. I. & Kelly, R. M. (1992). Proteolytic activity in the hyperthermophile Pyrococcus furiosus. Appl. Environ. Microbiol., 58, 1134-41. Steele, D. B., Fiske, M. J., Steele, B. P. & Kelly, V. C. (1992). Production of a low molecular weight alkaline active, thermostable protease by a novel, spiral shaped bacterium, Kurthia spireforme sp. nov. Enzyme Microbiol. Technol., 14,358-60. Wilson, S. A., Young, O. A., Coolbear, T. & Daniel, R. M. (1992). The use of proteases from extreme thermophiles for meat tenderization. Meat Sci., 32, 93-103.
I~Jl~_. !',i ELSEVIER
0960-8524(94)00079-4
CATION-INDUCED T H E R M A L STABILITY OF A N ALKALINE PROTEASE FROM A BACILLUS SE Nisha Paliwal, a S. E
S i n g h b*
• S. K. Garga:~
"Department of Microbiology, G. B. Pant University of Agriculture and Technology, Pantnagar-263145, India hMicrobiology Unit, Department of Biosciences, Saurashtra University, Rajkot-360005, India
(Received 30 July 1994; accepted 10 August 1994)
Cations are known to protect enzymes in general against thermal denaturation by maintaining the necessary conformation of the molecule (Pan & Lin, 1991; Singh & Rogers, 1991; Steele et al., 1992; Donaghy & McKay, 1993). Therefore, in an attempt to enhance the thermal stability of alkaline protease from a Bacillus sp. the effect of various cations on the enzyme activity as a function of increasing temperature was examined.
Abstract A n alkafine protease produced from a Bacillus sp. was stimulated by the metal ions Ca 2÷, Mg 2+ and Mn2+; with Ca 2+ having the m a x i m u m effect. The thermal stability of the enzyme was also enhanced to varying degrees in the presence of these ions. Key words: Alkaline protease, Bacillus sp., thermostability, alkalophiles.
METHODS Growth of the organism The organism was earlier isolated in our laboratory and identified as a Bacillus sp. (Paliwal, 1993; Sinha, 1993; Sinha et al., 1994). The isolate was grown at 37°C in a growth medium that contained (g/litre): glucose, 10; peptone, 5; yeast extract, 5; KH2PO4, 10; MgSO4.7H:O, 0-2. For adjusting the pH to 10"5, separately sterilized Na2CO3 (20% w/v) was added to the sterilized medium at 10% (v/v). Cultures grown to an absorbance of 0"6 at 665 nm were used as inocula. The sterile medium (100 ml, pH 10.5) was inoculated with 0.50% (v/v) inoculum and incubated at 37°C for 12 h under shake-flask conditions (100 rev/min). After growth, the cultures were centrifuged at 10 000g for 25 min and supernatants were used for the enzyme assay.
INTRODUCTION Proteases are among the most important groups of industrial enzymes and constitute nearly 60% of the total world-wide enzyme sales (Ng & Kenealy, 1986). Alkaline proteases are of particular interest because of their potential to catalyse reactions under alkaline conditions, which makes them ideal for use in many industries such as detergent (Durham, 1987; Phadatare et al., 1993), leather (Grebeskova et al., 1988) and food manufacturers (Wilson et al., 1992). Of late, some newer applications, for instance silver recovery from photographic plates (Fujiwara et al., 1989, 1991) and peptide synthesis (Cerovsky, 1992) have also been explored. In view of the intensive commercial interest, microbes from varied habitats have been examined for industrially suitable alkaline proteases (Jones et al., 1988; Griffin et al., 1992; Snowden et al., 1992; Steele et al., 1992). The thermal stability of enzymes in general is considered as one of the most vital parameters for catalytic versatility in industrial applications. Some of the major commercial uses of alkaline proteases necessitate high temperatures, and thus improving the thermal stability of the enzyme would be distinctly advantageous. Thermostability can be enhanced either by adding certain stabilizers to the reaction mixture or by manipulating the tertiary structures of the enzymes through molecular approaches such as amino-acid substitution (Imanaka et al., 1986; Eijsink et al., 1991; Hardy et al., 1993).
Enzyme assay The assay was based on Anson-Hagihara's method (Hagihara et al., 1958) using casein as substrate. The enzyme solution (0"5 ml) was added to 3"0 ml casein solution (0"6% w/v casein solution prepared in 20 mM borax NaOH buffer of pH 10-5) and allowed to react for 10 min at 37°C. The reaction was terminated by the addition of 3.2 ml of a solution of 0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0"33 M acetic acid. The reaction mixture was left at 370C for 10 min and filtered through a Whatman No. 1 filter paper. The absorbance of the filtrate was measured at 275 nm. A unit of alkaline protease activity (U) was defined as the amount of enzyme liberating 1/~g of tyrosine per min under the conditions described above. The enzyme estimation was based on a calibration curve using tyrosine.
*To whom correspondence should be addressed. :~Present address: Avadh University, Faizabad, India. 209
210
Nisha Paliwal, S. P. Singh, S. K. Garg o ~
~
I1~ ~
I~l
~
~
o~ tt3 ~ ~
•~
~,~
ua .g
~
m~
<~
o "6
g=
,--.4
~..~ ~E=..
=
~.~
o:=. ~ ~~,~..~ "~.~ "~ ~ <
.~
o .~
°~ ~
(itu/f~) ,(I!A!~OV
~~ , . ~
D
~o
|
\\.
>.-,0
\.
0 ;zL ¢~
I
I
°~o
l
(ltUlTl)~!A.q~V ,,,,,,t
,O
Thermal stability of an alkaline protease Effect of cations on e n z y m e activities
The effects of increasing concentrations (5-20 mM) of Ca 2+, Mg 2+ and Mn 2+ on enzyme activity were investigated at 37°C. All cations were prepared in b o r a x - N a O H buffer (pH 10-5) and diluted appropriately in the same buffer to give the desired concentrations in the assay mixtures. However, for examining the thermal stability, the enzyme was preincubated with the cations (2"5-20 mM) for 10-60 min at 37, 50, 60 and 70°C before the enzyme assay. The cations were used in the form of CaCI 2, MgCI2 and MnCL. RESULTS AND DISCUSSION As shown in Fig. 1, all the three cations, Ca 2+, Mg 2÷, and Mn 2+, induced enzyme activity. At 5 mM concentration, all the cations had almost similar effects on the enzyme activity, but while Ca 2÷ exhibited an increasing activity up to 15 mM, with Mg 2+ and Mn 2+ no further enhancement was evident beyond 5 mM (Fig. 1 ). The thermal stability of the enzyme as a function of varying cation concentrations at 50, 60 and 70°C was monitored. The results are graphically depicted in Fig. 2. The enzyme activity showed a decrease at temperatures beyond 37°C when no cation was added. At 70°C, the activity dropped to nearly zero after 10 min of incubation. Cations Ca 2+, Mg :+ and Mn 2÷ apparently protected the enzyme against thermal denaturation. With Ca 2+, however, this effect was pronounced at concentrations above 5 mM. Whether these cations added to the thermal stability of the enzyme at 70°C is not clear because of the drastic reduction in the activity at this temperature. Nevertheless, the cations showed stimulation as compared to the control. This may, perhaps, be taken as indirect evidence of the enzyme protection by the cations at higher temperatures. On the whole, Ca 2+, Mg 2+ and Mn 2÷ substantially stimulated the enzyme catalysis, and the thermal denaturation of the enzyme was retarded to varying extents in the presence of the cations. The results indicate that the metal ions play a vital role in maintaining the active conformation of the enzyme at higher temperatures, as also recently suggested for two alkaline proteases from Kurthia spiroforme sp. nov. (Steele et al., 1992) and Aureobasdium pullulans (Donaghy & McKay, 1993). Other experiments (Singh & Pailwal, unpublished data) have indicated that these cations also stimulate enzyme production when added during bacterial growth.
REFERENCES
Cerovsky, V. (1992). Protease-catalysed peptide synthesis in solvent-free system. Biotechnol. Techniques, 6 (2), 155-60. Donaghy, J. A. & McKay, A. M. (1993). Production and properties of an alkaline protease by Aureobasidium pullulans. J. Appl. Bacteriol., 74,000-000. Durham, D. R. (1987). Utility of Subtilisin GX as a detergent additive. J. Appl. Bacteriol., 63 (5), 381-6.
211
Eijsink, V. G. H., Zee, J. R., Burg, B., Vriend, G. & Venema, G. (1991). Improving the thermostability of the neutral portease of Bacillus stearothermophilus by replacing a buried asparagine by leucine. FEBS L ett., 282 ( 1), 13-16. Fujiwara, N., Tsumiya, T., Katada, T., Hosobuchi, T. & Yamamoto, K. (1989). Continuous recovery of silver from used X-ray films using a proteolytic enzyme. Proc. Biochem., 24, 155-6. Fujiwara, N., Yamamoto, K. & Masui, A. (1991). Utilization of a thermostable alkaline protease from an alkalophilic thermophile for the recovery of silver from used X-ray film. J. Ferment. Bioengng, 72 (4), 306-8. Grebeskova, R. N., Ryshlava, J. M., Fedorova, L. G., Kochetova, S. P., Babloyan, O. O. & Vinogredona, G. L. (1988). Using alkaline protease to intensify the processing of leather raw material. Biotechnolgica, 4 (6), 788-91. Griffin, H. L., Greene, R. V. & Cotta, M. A. (1992). Isolation and characterization of an alkaline protease from the marine shipworm bacterium. Curr. Microbiol., 24, 111-17. Hagihara, B., Matsubara, H., Nakai, M. & Okunuki, K. ( 1958). Crystalline bacterial proteinase of Bacillus subtilis. J. Biochem., 45, 185-94. Hardy, F. G., Vriend, O. R., Veltnan, B., Vinne, V. D., Venema, G. & Eijsink, V. G. H. (1993). Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines. FEBS Lett., 317 (1-2), 89-92. Imanaka, T., Shibazaki, M. & Takagi, M. (1986). A new way of enhancing the thermostability of proteases. Nature, 324,695-7. Jones, C. W., Morgan, H. W. & Daniel, R. M. (1988). Aspects of protease production by Thermus Stain OK6 and other New Zealand isolates. J. Gen. Microbiol., 134,191-8. Ng, T. K. & Kenealy, W. R. (1986). Industrial applications of thermostable enzymes. In Thermophiles. General, Molecular and Applied Microbiology, ed. T. D. Brock. John Wiley, New York, pp. 197-205. Paliwal, N. (1993). Production of alkaline protease from Bacillus sp. MSc thesis submitted to G. B. Pant University of Agriculture and Technology, Pantnagar, India. Pan, T. & Lin, S. (1991). Fermentative production of alkaline protease as detergent additive. J. Chinese Biochem. Soc., 20 (1), 49-60. Phadatare, S. V., Deshpande, V. U. & Srinivasan, M. C. (1993). High activity alkaline protease from Conidiobolus coronatus: enzyme production and compatibility with commercial detergents. Enzyme Microbiol. Technol., 15 (1), 72-5. Singh, S. P. & Rogers, P. J. (1991). Isolation and kinetic properties of pyruvate kinase from Brochothrix thermosphaeta. Biochem. Int., 25 (1), 35-45. Sinha, R. (1993). Purification and characterization of a Bacil/us alkaline protease. MSc thesis submitted to G. B. Pant University of Agriculture and Technology, Pantnagar, India. Sinha, R., Singh, S. P., Garg, S. K. & Ahmad, S. (1994). Aqueous two phase system: partitioning and purification of a bacterial alkaline protease. Proc. Ann. Conf. Assoc. Microbiologists, Ludhiana, India, p. 70. Snowden, J., Bluenentals, I. I. & Kelly, R. M. (1992). Proteolytic activity in the hyperthermophile Pyrococcus furiosus. Appl. Environ. Microbiol., 58, 1134-41. Steele, D. B., Fiske, M. J., Steele, B. P. & Kelly, V. C. (1992). Production of a low molecular weight alkaline active, thermostable protease by a novel, spiral shaped bacterium, Kurthia spireforme sp. nov. Enzyme Microbiol. Technol., 14,358-60. Wilson, S. A., Young, O. A., Coolbear, T. & Daniel, R. M. (1992). The use of proteases from extreme thermophiles for meat tenderization. Meat Sci., 32, 93-103.