Journal of Pure and Applied MicrobiologyVol. 11 No. 1

Purification and Characterization of Alkaline Metalloprotease from Bacillus sp. MTCC 9558 and its Application in Bioactive Peptide Production

Roopa Prasad1*, Ananthakrishnan Jayakumaran Nair2 and Theruvath Koshy Abraham1

1Department of Biotechnology and Biochemical Engineering, Mohandas College of Engineering and Technology, Anad, Nedumangadu, Trivandrum, India. 2Centre for Genomics and Gene Technology, Department of Biotechnology, University of Kerala, Trivandrum, India.

Received on 13 October 2016 and accepted on 19 December 2016

 

ABSTRACT

The present study describes the protease purification from halophilic Bacillus sp. MTCC 9558 by a single step hydrophobic interaction chromatographic using Phenyl-Sepharose CL - 4B column. After the final purification steps, protease was purified to 59.62-fold with a specific activity of 165.75 U/mg protein and overall recovery of 5.89%. The homogeneity of the enzyme preparation was confirmed by procedures such as SDS - PAGE, zymogram analysis and MALDI-TOF techniques. The molecular weight was determined to be 80.7 KDa and casein zymogram analysis of purified protease showed a single band indicating that the protease is active as a monomer. This paper also describes comparison of purified protease with crude in order to determine the effect of impurities or contaminating proteins on its characteristics. The purified protease exhibited improved thermostability and stability in presence of organic solvents in comparison with crude enzyme. The alkaline protease was determined to be halotolerant and Zn2+ activated metalloprotease. The purified protease was characterized to determine its kinetic constants such as Km, Vmax, kcat and kcat/Km values. The purified enzyme exhibited promising characteristics of potential biotechnological interest. In this paper, one of the applications of this protease in protein hydrolysis for bioactive peptide production with emphasis on antibacterial studies is discussed.

Keywords : MALDI-TOF, Thermostability, Metalloprotease, Kinetic constants, Bioactive peptides.