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中国生物工程杂志

China Biotechnology
China Biotechnology  2015, Vol. 35 Issue (10): 44-52    DOI: 10.13523/j.cb.20151007
    
Expression, Purification and Enzymatic Characterization of ColR75E Collagenase of Bacillus cereus R75E
ZHANG Xi-xuan, LI Ye, WANG Ya-hang, DU Kang-long, ZHANG Zhen, RUAN Hai-hua
College of Biotechnology & Food Science, Tianjin Key Laboratory of Food Science & Biotechnology, Tianjin University of Commerce, Tianjin 300134, China
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Abstract  

Objective: In order to investigate the enzymatic characteristics of ColR75E collagenase of Bacillus cereus R75E, the colR75E collagenase gene was expressed in E. coli prokaryotic expression system and acquired the recombinant ColR75E collagenase protein with high purity. Methods: The colR75E collagenase gene fragments were amplified using the Bacillus cereus R75E genomic DNA as template. The fragments were cloned into the pET28a vector, constructing recombinant pET28a/colR75E plasmid. Then, the pET28a/colR75E plasmid was introduced into the E.coli BL21(DE3). After IPTG induction, the recombinant ColR75E collagenase protein was purified with Ni-NTA affinity chromatography column. Subsequently, the characteristics of ColR75E collagenase of Bacillus cereus R75E were analyzed by collagen-zymography, collagen hydrolysis activity assay, Km and Vmax calculation and collagen degradative products SDS-PAGE detection. Results: The purified recombinant ColR75E collagenase of Bacillus cereus R75E exhibited the collagen hydrolysis activity, both showing with the obvious negative staining in collagen-zymography gel and progressive degradation of native type I collagen extracted from scales of grass carp with the prolong of treatment time by SDS-PAGE. The specific activity of recombinant ColR75E collagenase was 3.62 U/mg, its Km and Vmax were 25.55 μmol/L (2.93 mg/ml)and 5.71μmol/(mg·min), respectively. The optimum temperature to ColR75E collagenase was 45℃ and the optimum pH was 8.0 .In addition, the activity of recombinant ColR75E collagenase was stable both under the temperature no higher than 50℃ and the pH of buffer from 6.0 to 8.0. The recombinant ColR75E collagenase was activated by adding of Ca2+, but inhibited by adding of Zn2+, Pb2+, Fe2+, Mn2+, respectively. The inhibitory capability of these metal ions was Pb2+> Zn2+> Fe2+> Mn2+, subsequently. The high sensitivity of ColR75E collagenase to EDTA and EGTA further confirmed the ColR75E collagenase was a metallo-proteinase. Conclusion: It is feasible to acquire the recombinant ColR75E collagenase with both high purity and perfect hydrolysis activity in E. coli prokaryotic expression system, which supplies a foundation for the extensive application of ColR75E collagenase in field of medical and food industry.



Key wordsBacillus cereus      Type I collagen      Collagenase      Enzymatic characteristics     
Received: 02 July 2015      Published: 25 October 2015
ZTFLH:  Q819  
Cite this article:

ZHANG Xi-xuan, LI Ye, WANG Ya-hang, DU Kang-long, ZHANG Zhen, RUAN Hai-hua. Expression, Purification and Enzymatic Characterization of ColR75E Collagenase of Bacillus cereus R75E. China Biotechnology, 2015, 35(10): 44-52.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20151007     OR     https://manu60.magtech.com.cn/biotech/Y2015/V35/I10/44

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