肝素黄杆菌硫酸软骨素酶AC的高效重组表达体系构建及其酶学性质研究  

Gene Cloning and Recombinant Expression of Chondroitinase AC from Pedobacter heparinus and Characterization of Recombinant Fusion Enzyme

作　　者：吴敬君[1] 李晔[1,2] 张翀[1] 李梅[1] 邢新会[1] 

机构地区：[1]清华大学化学工程系工业生物催化教育部重点实验室,生物化工研究所,北京100084 [2]北京电子科技职业学院生物技术系,北京100029

出　　处：《食品科学》2013年第9期127-134,共8页Food Science

基　　金：国家“863”计划项目(2012AA022201);北京市属高等学校人才强教计划项目(PHR201107151);北京电子科技职业学院院内重点课题(YZKB2011003);北京市属高等学校人才强教深化计划资助项目

摘　　要：为实现硫酸软骨素酶AC(chondroitinase AC,ChonAC)在大肠杆菌中高效表达,通过PCR方法从肝素黄杆菌(Pedobacter heparinus)中扩增得到ChonAC基因cslA,采用融合蛋白技术构建麦芽糖结合蛋白(maltose-bindingprotein,MBP)与ChonAC的融合表达载体(MBP-ChonAC),并在低温(15℃)条件下实现MBP-ChonAC的可溶活性表达,通过MBPTrap HP可实现一步纯化,使纯度可达95%以上,酶比活力可达94.1IU/mg融合蛋白(即相当于143.8IU/mgChonAC蛋白当量)。对MBP-ChonAC的酶学研究发现,其最佳催化pH值为7.5～8.0,最佳Ca2+浓度为20mmol/L,最适NaCl浓度为50mmol/L,最佳作用温度为20～35℃。MBP-ChonAC具有较好的热稳定性,在30℃时其半衰期可达8.3h。同时对其以硫酸软骨素A为底物的动力学常数测定发现,MBP-ChonAC相比ChonAC其Km稍有增大而催化常数kcat稍有降低,但是差别不大,表明MBP的融合没有对ChonAC的催化能力造成影响。通过宿主优化、诱导浓度优化及培养基优化进一步提高了MBP-ChonAC的表达量,其摇瓶培养酶活力就可达10800.5IU/L。Chondroitinase AC（ChonAC） is an important enzyme to reveal the biological function,structure and mechanism of chondroitin sulfate,and it is important for low molecular weight chondroitin sulfate preparation and diseases treatment.To achieve the efficient expression of ChonAC with high activity in recombinant Escherichia coli（E.coli）,the ChonAC gene（cslA） from Pedobacter heparinus was amplified by polymerase chain reaction（PCR）,and the fusion expression vector for expressing maltose-binding protein（MBP） fused ChonAC（MBP-ChonAC） was constructed.The results showed that the fusion strategy using MBP was effective to enhance the solubility of the MBP-ChonAC when induced at low temperature of 15 ℃,the purity and the specific activity of MBP-ChonAC could reach 95% and 94.1 IU/mg-fusion protein（equivalent to 143.8 IU/mg ChonAC） by one-step MBPTrap HP purification,respectively.The enzyme characteristic study showed that the optimum pH value,Ca2＋concentration,NaCl concentration,and reaction temperature was 7.5—8.0,20 mmol/L,50 mmol/L,and 20—35 ℃,respectively.The half-life of MBP-ChonAC could reach 8.3 h at 30 ℃.The kinetic characterization showed that the Kmwas higher while the kcat was a little bit lower than the reported native ChonAC for chondroitin sulfate A,while the biocatalysis experiment for MBP-ChonAC indicated that the fusion of ChonAC with MBP did not affect the enzyme function.Furthermore,the total activity of MBP-ChonAC by shake flask cultivation could reach 10800.5 IU/L,the highest value reported so far,through the optimizations of host cells,IPTG induction concentration,and M9-based culture medium.

关 键 词：亲和纯化 硫酸软骨素酶AC 酶学性质 表达量优化 麦芽糖结合蛋白 重组表达 

分 类 号：Q814.1[生物学—生物工程]