• Ashwini Nilesh Puntambekar Protein Biochemistry Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune - 411 033, Maharashtra, India
  • Manjusha Sudhakar Dake Protein Biochemistry Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune - 411 033, Maharashtra, India



 Objective: The main objective of this study is to investigate the industrial applications of a thermophillic alkaline protease from a hot water spring bacterial isolate “A†and to study its production, optimization, and purification.

Methods: The alkaline protease was produced using shake flask studies maintaining a pH of 9.0 and a temperature of 50°C. Optimization studies of the enzyme were carried out using variable pH, temperature, organic carbon, and nitrogen sources followed by purification of the enzyme using DEAE-cellulose ion exchange chromatography technique. Stability of the enzyme was analyzed in the presence of organic solvents and surfactants. The efficiency of the enzyme in the removal of proteinaceous stains in the presence of strong detergents under extreme conditions was assessed. The fibrinolytic activity of the enzyme in dissolving the blood clot was confirmed.

Results: The isolated alkaline protease was purified to homogeneity with a 16-fold increase. Media optimization studies revealed that 1% glucose and 1 % casein-induced the production of alkaline protease. The purified enzyme retained stability in the presence of ethanol, methanol, and acetone and surfactants such as 0.5% (w/v) sodium dodecyl sulfate (SDS) and 0.5% (v/v) Triton-X-100. The isolated alkaline protease successfully removed the proteinaceous stains and showed significant results in the dissolution of blood clot.

Conclusion: The above experimental results confirm that the isolated enzyme has both thermophilic and alkaliphilic protease properties. Thereby the enzyme finds promising industrial applications even in extreme conditions.

Keywords: Protease, Thermophillic, Alkaliphillic, Purification, Applications.

Author Biographies

Ashwini Nilesh Puntambekar, Protein Biochemistry Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune - 411 033, Maharashtra, India
Manjusha Sudhakar Dake, Protein Biochemistry Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune - 411 033, Maharashtra, India


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How to Cite
Puntambekar, A. N., and M. S. Dake. “ISOLATION, PURIFICATION, AND OPTIMIZATION OF THERMOPHILIC AND ALKALIPHILC PROTEASE ORIGINATING FROM HOT WATER SPRING BACTERIA”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 10, no. 9, Sept. 2017, pp. 284-91, doi:10.22159/ajpcr.2017.v10i9.19717.
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