title: Isolation, purification and characterisation of an organic solvent-tolerant Ca2+-dependent protease from Bacillus megaterium AU02.
abstract: A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl2, and it retained 90 % activity at 50 °C for 3 h. The Km and Vmax values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH2) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.
authors.forename: J Deepa Arul
authors.initials: JD
authors.lastname: Priya
authors.forename: K
authors.initials: K
authors.lastname: Divakar
authors.forename: M Suriya
authors.initials: MS
authors.lastname: Prabha
authors.forename: G Panneer
authors.initials: GP
authors.lastname: Selvam
authors.forename: Pennathur
authors.initials: P
authors.lastname: Gautam
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects*; D000704:Analysis of Variance; D001218:Aspartame / Q000378:metabolism; D001410:Bacillus megaterium / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D019149:Bioreactors / Q000382:microbiology; D002118:Calcium / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009930:Organic Chemicals / Q000633:toxicity*; D010084:Oxidation-Reduction / Q000187:drug effects; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D010802:Phylogeny; D015203:Reproducibility of Results; D012997:Solvents / Q000633:toxicity*; D013379:Substrate Specificity / Q000187:drug effects; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
chemical_list: D007477:Ions; D008670:Metals; D009930:Organic Chemicals; D012997:Solvents; D013501:Surface-Active Agents; D010447:Peptide Hydrolases; D002118:Calcium; D001218:Aspartame