Collagenase Producing Bacteria Identification And Production Of Collagenase Enzymatic Properties

Objective: To isolate, purify and identify strains in human colostrum samples with proteolytic capacity to further determine the strain producing the enzyme collagenase coding genes and molecular structure characteristics, end-use prokaryotic expression systems in prokaryotic expression, and expression of the recombinant enzyme purification and Characterization Study. METHODS: In milk plate culture method and purified enzyme production strains using 16 Sr DNA sequence analysis and API CH50 media analysis of genetic characteristics and biochemical characteristics of the strain, the final identification of their type enzyme production strains. Secondly, the use of tandem mass spectrometry analysis of enzyme production strain type of enzyme produced, combined with mass spectrometry results in strain genomic DNA as a template using the PCR method to obtain colR75 E collagenase gene construct pET28 a / colR75 E recombinant plasmid in E. coli BL21(DE3) the expression was induced using Ni-NTA purified collagenase ColR75 E obtained high purity, using collagenase spectrum, collagenase activity assay, Type Ⅰ collagen degradation products electrophoresis, etc. on the enzymatic characteristics were analyzed ColR75 E collagenase. Results: 16 Sr DNA sequence analysis and API CH50 media analysis shows that human colostrum samples enzyme production strain is a Bacillus cereus, based on tandem mass spectrometry combined with the results of bioinformatics analysis shows that the strain produced by the enzyme is a protease when species can be secreted into the extracellular collagenase, the enzyme gene cloning and prokaryotic expression, the protein was purified by Ni-NTA of collagenase spectrum, Type Ⅰ collagen degradation products were detected showed degradation of collagen ability. Measured under standard conditions obtaining specific activity was 3.62 U / mg, Km is 25.55μmol / L(2.93 mg / mL), Vmax is 5.71μmol /(mg ? min). The optimum temperature ColR75 E collagenase was 45 ℃, the optimal reaction pH of 8.0. In addition, ColR75 E collagenase for not more than 50 ℃ temperature and pH range pH6.0-8.0 has good stability. ColR75 E collagenase can be activated Ca2+, but was Zn2 +, Pb2 +, Fe2 +, Mn2 + and other metal ions inhibited by capacity were Pb2 +> Zn2 +> Fe2 +> Mn2 +. ColR75 E collagenase high sensitivity EDTA and EGTA further confirmed that collagenase as a metalloprotease. Conclusion: This study identified a separate secrete collagenase Bacillus cereus, prokaryotic expression system appropriate collagenase, ultimately proves prokaryotic expression system to obtain a large number of high purity and high activity of Bacillus cereus collagenase is feasible and for collagenase widely used in industrial fields of medical, food and other laid a theoretical foundation.