Expression, Purification And Enzymatic Properties Of Bacillus Cereus Colr75E Collagenase
Keywords
Type I Collagen, Collagenase, Enzymatic Properties, Bacillus Cereus
Abstract
Purpose: In order to further understand the characteristics of Bacillus cereus R75E collagenase colR75E, colR75E collagenase was expressed in the Escherichia coli prokaryotic expression system, and the expressed recombinant collagenase was purified and its enzymatic properties were studied. Methods: The collagenase colR75E gene was obtained by PCR using Bacillus cereus R75E genomic DNA as a template, and the pET28a/colR75E recombinant plasmid was constructed, induced and expressed in Escherichia coli BL21 (DE3), and purified by Ni-NTA to obtain high For the purity of ColR75E collagenase, the enzymatic properties of ColR75E collagenase were analyzed using collagenase spectrum, collagenase activity assay, electrophoresis detection of type I collagen degradation products, etc. Results: The protein purified by Ni-NTA showed the ability to degrade collagen when detected by collagen zymography and type I collagen degradation products. Under standard conditions, its specific activity was measured to be 3.62 U/mg, Km was 25.55 ¦Ìmol/L (2.93 mg/ml), and Vmax was 5.71 ¦Ìmol/(mg¡¤min). The optimal reaction temperature of ColR75E collagenase is 45¡æ, and the optimal reaction pH is 8.0. In addition, ColR75E collagenase has good stability to temperatures not higher than 50¡ãC and a pH range of 6.0 to 8.0. ColR75E collagenase can be activated by Ca2+, but is inhibited by metal ions such as Zn2+, Pb2+, Fe2+, Mn2+, etc. The inhibitory ability is in the order of Pb2+> Zn2+> Fe2+> Mn2+. The high sensitivity of ColR75E collagenase to EDTA and EGTA further confirmed that this collagenase is a metalloprotease. Conclusion: It is feasible to obtain high-purity and high-activity Bacillus cereus collagenase using the Escherichia coli prokaryotic expression system, which lays a theoretical foundation for the collagenase to be widely used in medical, food and other industrial fields.
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Original research was done by Zhang Zhen, Zhang Xixuan, Li Ye, Du Kanglong, Wang Yahang, Ruan Haihua
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