黄单胞杆菌纤维素酶的原核表达及固定化研究

Prokaryotic expression of the cellulase of Xanthomonas campestris pv.Campestris and its immobilization

从野油菜黄单胞杆菌(Xanthomonas campestris pv.Campestris)中PCR克隆得到删除信号肽的纤维素酶基因engXCAΔSP,并成功使用CPEC(circular polymerase extension cloning)方法构建了原核表达载体pET28a-engXCAΔSP;将该载体转入大肠杆菌rosetta(DE3)中,用IPTG诱导蛋白质的表达;通过Ni-NTA树脂非变性亲和纯化到了较纯的蛋白。SDS-PAGE电泳结果表明:engXCAΔSP基因编码出约50 kD的蛋白质ENGXCAΔSP。然后对该酶成功进行了固定化。该酶比活为60 U.mg-1,固定前后最适温度为53℃与62℃,最适pH为5.4与5.8,动力学常数分别为Vmax:411μmol.mL-1.h-1与383μmol.mL-1.h-1,Km:0.2500%与0.3125%。

We cloned the cellulase gene of Xanthomonas campestris pv.Campestris with deleted signal peptide,using PCR（polymerase chain reaction） technique.The gene was ligated into the expression vector pET28a applying CPEC（circular polymerase extension cloning）method to construct a recombinated plasmid pET28a-engXCAΔSP,and then was transformed into the E.Coli rosetta（DE 3）.The protein was successfully expressed by 0.4 mmol·L-1 IPTG（isopropyl-β-D-1-thiogalactopyranoside） induction,then purified by the affinity Ni-NTA.Its molecular weight was about 50 kD,which was determined by SDS-PAGE.The activity of recombinant enzyme was 60 U·mg-1.We immobilized the enzyme as well.The optimum reaction temperature and pH value for soluble enzyme and insoluble enzyme were 53℃,62℃ and 5.4,5.8,respectively.The results of their enzymatic properties using sodium carboxymethylcellulose（CMC） as the substrate showed that their Vmax were 411 μmol·mL-1·h-1 and 383 μmol·mL-1·h-1,while Km were 0.2500% and 0.3125%,respectively.