[Objective] In order to find a safe and stable method to produce collagenase in vitro,we expressed the Bacillus cereus collagenase colR75E in Pichia pastoris.[Methods] With the Bacillus cereus genomic DNA as template,we successfully amplified the collagenase colR75E DNA fragment by PCR and cloned it into pPICZαA plasmid.The pPICZαA/colR75E 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 colR75E 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 ColR75E collagenase expression in Pichia pastoris X-33 were investigated here.After induction,we purified recombinant ColR75E collagenase in supernatant sequentially by ammonium sulfate precipitation,desalting and affinity capture.Finally,the recombinant collagenase ColR75E was analyzed by catalytic activity assay,SDS-PAGE,zymography,type Ⅰ collagen proteolysis and substrate specificity assay.[Results] The highest level of collagenase ColR75E induction was gained under pH 6.0 for 72 hours incubation by 2.5％ methanol.As expected,the molecular weight of the recombinant collagenase is nearly 110 kD to ColR75E.The results of zymography and type Ⅰ collagen degradation analysis uncovered that the recombinant collagenase ColR75E had an excellent collagen proteolysis activity.Its specific activity after purification reached to nearly 4.977 U/mg under standard conditions.The recombinant collagenase ColR75E 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 ColR75E,which supplied a good basement both for its subsequent theoretic research and industrial exploitation.%[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ColR75E,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colR75E基因,构建pPICZαA/colR75E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ColR75E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ColR75E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ColR75E的条件为pH 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l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ColR75E,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.
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