中文摘要：

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cozR75E基因，构建pET28a／colR75E重组质粒，并在大肠杆菌B121（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3．62U／mg，Km为25．55μmol／L（2．93mg／ml），Vmax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＋、Ph2＋、Fe、Mn2＋等金属离子抑制，抑制能力依次为Pbn〉Znn〉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。

英文摘要：

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 pErlE8a/colR75E plasmid. Then, the pET28a/colR75E plasmid was introduced into the E. coli BL21 （DE3）. After IFFG 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 5.71p, mol/（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