中文摘要：

【目的】利用毕赤酵母真核表达系统表达蜡样芽孢杆菌胶原酶Col R75E,寻找一种安全、稳定的方式体外制备具有高活性的胶原酶。【方法】以蜡样芽孢杆菌R75E基因组DNA为模板,采用PCR法扩增胶原酶col R75E基因,构建p PICZαA/col R75E重组质粒,将该质粒线性化后电转化至毕赤酵母X-33菌株,诱导其表达并对表达条件进行优化。将表达后的酵母发酵液上清通过硫酸铵沉淀、脱盐处理及亲和层析纯化步骤获得高纯度重组Col R75E胶原酶。利用胶原酶活力测定、SDS-PAGE电泳、胶原酶谱、I型胶原蛋白及不同底物蛋白降解产物电泳等方法对重组胶原酶Col R75E的活性及底物特异性进行分析。【结果】毕赤酵母中最佳表达重组胶原酶Col R75E的条件为p H 6.0,甲醇终浓度为2.5%,诱导时间72 h,诱导后的蛋白经SDS-PAGE、胶原酶谱以及I型胶原蛋白降解产物电泳分析发现,毕赤酵母中表达的重组胶原酶分子量符合预期,蛋白纯度超过95%,具有较好的胶原蛋白水解活性并测得其比活力为4.977 U/mg。该酶对I型胶原蛋白表现出较好的专一性,但是对牛血清白蛋白、酪蛋白及溶菌酶蛋白没有水解活性。【结论】利用毕赤酵母真核表达系统能够获得高活性的蜡样芽孢杆菌胶原酶Col R75E,为该胶原酶广泛应用于医疗、食品等工业领域奠定了理论和方法基础。

英文摘要：

[Objective] In order to find a safe and stable method to produce collagenase in vitro, we expressed the Bacillus cereus collagenase col R75 E in Pichia pastoris. [Methods] With the Bacillus cereus genomic DNA as template, we successfully amplified the collagenase col R75 E DNA fragment by PCR and cloned it into p PICZαA plasmid. The p PICZαA/col R75 E recombinant plasmid was lineared with Sac I, and then the lineared plasmid was transformed into Pichia pastoris X-33 competent cell in order to integrate the inducible AOX1 promoter controlled col R75 E fragment into Pichia pastoris X-33 genomic DNA. The successfully integrated Pichia pastoris X-33 strains was cultured and induced by methanol addition. To acquire the highest production, the optimized conditions for Col R75 E collagenase expression in Pichia pastoris X-33 were investigated here. After induction, we purified recombinant Col R75 E collagenase in supernatant sequentially by ammonium sulfate precipitation, desalting and affinity capture. Finally, the recombinant collagenase Col R75 E was analyzed by catalytic activity assay, SDS-PAGE, zymography, type I collagen proteolysis and substrate specificity assay. [Results] The highest level of collagenase Col R75 E induction was gained under p H 6.0 for 72 hours incubation by 2.5% methanol. As expected, the molecular weight of the recombinant collagenase is nearly 110 k D to Col R75 E. The results of zymography and type I collagen degradation analysis uncovered that the recombinant collagenase Col R75 E had an excellent collagen proteolysis activity. Its specific activity after purification reached to nearly 4.977 U/mg under standard conditions. The recombinant collagenase Col R75 E exhibited specific proteolysis to type I collagen, but not to BSA, Casein or Lysozyme. [Conclusion] Pichia eukaryotic expression system is suitable for the expression of Bacillus cereus collagenase Col R75 E, which supplied a good basement both for its subsequent theoretic research and industrial exploitation.