蜡样芽孢杆菌胶原蛋白酶基因的异源表达与活性分析

Heterologous Expression and Activity Analysis of Collagenase Gene from Bacillus cereus

研究采用降解骨胶原蛋白的蜡样芽孢杆菌MBL13-U作为原菌种,并对蜡样芽孢杆菌MBL13-U进行全基因组测序,克隆获得胶原蛋白酶(ColM13)基因。将目的基因与大肠杆菌的表达载体pET30a进行连接,转入大肠杆菌宿主菌株BL21,获得降解骨胶原蛋白的工程菌pET30a-ColM13/BL21。通过SDS-PAGE测定异源表达的重组蛋白酶ColM13的分子质量,并测定不同诱导时间和诱导浓度对ColM13酶活性的影响,最终确定重组蛋白酶ColM13的酶活最适条件:6‰异丙基硫代半乳糖苷(IPTG,100 mmol/L),37℃诱导6 h,优化后酶活力最高为64.99 U/mL,较优化前提高4.24倍。通过水解圈试验、紫外光谱和扫描电子显微镜等方式验证,表明工程菌构建成功,这为我国畜禽骨骼资源的深度开发和综合利用提供了新的研究思路。

In this study,Bacillus cereus MBL13-U,which degrades bone collagen,was used as the original strain.The whole genome of B.cereus MBL13-U was sequenced,and the collagenase(ColM13)gene was cloned.The target gene was ligated with the expression vector pET30a of Escherichia coli and transferred into E.coli host strain BL21 to obtain an engineering strain pET30a-ColM13/BL21 that degrades collagen.The molecular weight of the recombinant enzyme ColM13 expressed in heterologous was determined by SDS-PAGE,and the enzyme activity of the enzyme producing bacteria were studied under the different induction time and induced concentration.Finally,the best enzyme activity conditions of recombinant enzyme ColM13 were as follows:6‰IPTG(100 mmol/L),6 h at 37℃,the optimized enzyme activity was 64.99 U/mL,increased 4.25 times than the pre-optimization.And further through the hydrolysis ring test,full-wavelength ultraviolet scanning spectroscopy and scanning electron microscopy were validated experiments,indicating that the successful construction of engineering bacteria,which provided a new approach for the depth development and comprehensive utilization of animals’bone resources in China.