K5裂解酶在大肠杆菌中的表达、纯化及酶学性质分析

Expression, purification and characterization of K5 lyase in Escherichia coli

K5多糖裂解酶(Elma)能够裂解半合成肝素的底物-K5多糖,裂解产物是半合成法生产低分子量肝素的底物。利用PCR方法扩增elma,构建表达载体pET-28a-Elma,将构建好的质粒转化至大肠杆菌BL21中,以0.2 mmol/L的IPTG在16℃诱导5 h实现了高效表达,SDS-PAGE分析表明Elma表达量可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法和G-75分子筛层析纯化目的蛋白,其纯度大于95%。通过PAGE多糖电泳发现裂解前后的K5多糖分子量有明显的减小。根据Elma裂解产物产生双键从而在232 nm处有吸光度的变化来测Elma的酶活。其最适反应温度为37℃,反应的最适pH值为7.0。底物特异性分析发现Elma除K5多糖外对肝素和透明质酸也有降解作用。

K5 polysaccharide of high molecular weight （HLW） can be splitted into low molecular weight （LMW） K5 polysaccharide by K5 lyase which can be used as the substrate of partial synthesis low molecular heparin sulfate （HS）. To prepare recombinant K5 lyase （Elma） and analyze its biological activity, The gene of Elma was cloned by PCR amplification and was ligated with pET-28a. Then the recombinant expression vector pET-28a-Elma was transformed into Escherichia coli BL21 （DE3）. After induction with 0.2 mrnol/L IPTG at 16 ℃ for 5 h, Elma was successfully expressed,SDS-PAGE analysis demonstrated that the enzyme constituted more than 30% of the total cell proteins. After Ni^2＋-NTA affinity and G-75 chromatography, the purity of enzyme was more than 95%. Enzymatic activity was determined according to the change of absorbance at 232 nm per ml of the sample. The reduction of the polysaccharide molecular weight could be detected by PAGE electrophoresis. Elma can partially split HA and HS. Its optimal reatcion temperature is 37 ℃ and the optimal reaction pH is 7.0.