title: 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 10 mM concentration. Cu(2+) at 1 mM concentration was found to enhance the enzyme activity by 16 %, whereas inhibition was observed at higher concentration (>5 mM). Slight inhibition was observed even with lower (>1 mM) 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 (>5 mM) 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 ± 2.6 U mg(-1). The VITP4 protease consists of a single polypeptide chain with a molecular mass of 34.7 kDa 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 0.817 mg ml min(-1), 0.472 mg ml(-1), and 2.31 s(-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 1 mM CaCl2. Additionally, the VITP4 protease was found to retain more than 70 % activity in the presence of 10 mM 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.
authors.affiliation: VIT University, Vellore, Tamil Nadu, India.
authors.forename: Chittoor Jabeena
authors.initials: CJ
authors.lastname: Thaz
authors.forename: Gurunathan
authors.initials: G
authors.lastname: Jayaraman
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects*; D000645:Ammonium Sulfate / Q000737:chemistry; D001407:Bacillus / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology*; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D017433:Protein Structure, Secondary; D059748:Proteolysis / Q000187:drug effects; D057057:Serine Proteases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013501:Surface-Active Agents / Q000494:pharmacology
chemical_list: D003902:Detergents; D007477:Ions; D008670:Metals; D011480:Protease Inhibitors; D013501:Surface-Active Agents; D057057:Serine Proteases; D000645:Ammonium Sulfate