蜡样芽孢杆菌ColR75E胶原酶的表达、纯化及酶学性质研究

doi:10.13523/j.cb.20151007

中国生物工程杂志

2015, Vol. 35

Issue (10): 44-52 DOI: 10.13523/j.cb.20151007

研究报告

蜡样芽孢杆菌ColR75E胶原酶的表达、纯化及酶学性质研究

张西轩, 李晔, 王亚航, 杜康龙, 张真, 阮海华

天津商业大学生物技术与食品科学学院天津市食品生物技术重点实验室天津 300134

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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摘要：

目的:为了进一步了解蜡样芽孢杆菌R75E胶原酶colR75E的特性,在大肠杆菌原核表达系统中表达colR75E胶原酶,并对表达的重组胶原酶进行纯化以及酶学性质研究。方法:以蜡样芽孢杆菌R75E基因组DNA为模板采用PCR法获得胶原酶colR75E基因,构建pET28a/colR75E重组质粒,并在大肠杆菌BL21(DE3)中进行诱导表达,利用Ni-NTA纯化的方式获得高纯度ColR75E胶原酶,利用胶原酶谱、胶原酶活力测定、Ⅰ型胶原蛋白降解产物电泳检测等方式对ColR75E胶原酶的酶学特性进行分析。结果:通过Ni-NTA纯化获得的蛋白经胶原酶谱、Ⅰ型胶原蛋白降解产物的检测时均表现出胶原蛋白的降解能力。在标准条件下测得其比活力为3.62 U/mg,K_m为25.55μmol/L (2.93 mg/ml),V_max为5.71μmol/(mg·min)。ColR75E胶原酶的最适反应温度为45℃,最佳反应pH为8.0。此外,ColR75E胶原酶对于不高于50℃的温度以及pH6.0~8.0的酸碱度范围具有良好的稳定性。ColR75E胶原酶可被Ca²⁺激活、但被Zn²⁺、Pb²⁺、Fe²⁺、Mn²⁺等金属离子抑制,抑制能力依次为Pb²⁺> Zn²⁺> Fe²⁺> Mn²⁺。ColR75E胶原酶对EDTA和EGTA的高敏感性进一步证实了该胶原酶为一种金属蛋白酶。结论:利用大肠杆菌原核表达系统获得高纯度高活性的蜡样芽孢杆菌胶原酶是可行的,为该胶原酶广泛应用于医疗、食品等工业领域中奠定了理论基础。

关键词： Ⅰ型胶原蛋白; 胶原酶; 酶学性质; 蜡样芽孢杆菌

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, K_m and V_max 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 K_m and V_max 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 words: Bacillus cereus Type I collagen Collagenase Enzymatic characteristics

收稿日期: 2015-07-02 出版日期: 2015-10-25

ZTFLH:

Q819

基金资助:

国家自然科学基金(81101220)、天津市应用基础与前沿研究计划(12JCQNJC08100)、"十二五"天津市中青年骨干创新人才支持计划,天津市创新团队建设(TD12-5049)资助项目

通讯作者: 阮海华 E-mail: ruanhaihua@tjcu.edu.cn

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引用本文:

张西轩, 李晔, 王亚航, 杜康龙, 张真, 阮海华. 蜡样芽孢杆菌ColR75E胶原酶的表达、纯化及酶学性质研究[J]. 中国生物工程杂志, 2015, 35(10): 44-52.

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.

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

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