Stability and Detergent Compatibility of a Predominantly β-Sheet Serine Protease from Halotolerant B. aquimaris VITP4 Strain

Abstract

The present study deals with the characterization of halotolerant protease produced by Bacillus aquimaris VITP4 strain isolated from Kumta coast, Karnataka, India. The studies were performed at 40°C and pH 8 in Tris buffer. Metal ions such as Mn(2+) and Ca(2+) increased the proteolytic activity of the enzyme by 34 and 30%, respectively, at 10mM concentration. Cu(2+) at 1mM concentration was found to enhance the enzyme activity by 16%, whereas inhibition was observed at higher concentration (>5mM). Slight inhibition was observed even with lower (>1mM) concentrations of Zn(2+), Hg(2+), Fe(3+), Ni(2+), and Co(2+).The activity of protease was completely inhibited by phenylmethylsulfonyl fluoride, indicating that the VITP4 protease is a serine protease. The presence of ethylenediaminetetraacetic acid and 1,10-phenanthroline (>5mM) moderately inhibited the activity, suggesting that the enzyme is activated by metal ions. The protease was purified to homogeneity with a purification fold of 15.7 with ammonium sulfate precipitation and 46.65 with gel filtration chromatography using Sephadex G-100, resulting in a specific activity of 424 ± 2.6Umg(-1). The VITP4 protease consists of a single polypeptide chain with a molecular mass of 34.7kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight. Among the different substrates used (casein, egg albumin, gelatin, and bovine serum albumin), the activity was higher with casein with V max, K m, and k cat values of 0.817mgmlmin(-1), 0.472mgml(-1), and 2.31s(-1), respectively. Circular dichroism studies revealed that the VITP4 protease has a predominantly β-sheet structure (51.6%) with a temperature for half denaturation of 85.8°C in the presence of 1mM CaCl2. Additionally, the VITP4 protease was found to retain more than 70% activity in the presence of 10mM concentration of different detergents (CTAB, urea, and sodium dodecyl sulfate) and surfactants (Triton X-100, Tween-20, and Tween-80), and the results of wash performance test with various commercial detergents confirmed that it can be used in detergent formulations.