Gao Jinhui, Dou Wenfang, Li Hui, Zhang Xiaomei, Xu Hongyu, Xu Zhenghong
Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2012 Jun;28(6):696-704.
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 mmol/L IPTG at 16 degrees C 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 degrees C and the optimal reaction pH is 7.0.
高分子量（HLW）的K5多糖可被K5裂解酶分解为低分子量（LMW）K5多糖，该裂解酶可用作部分合成低分子量硫酸乙酰肝素（HS）的底物。制备重组K5裂解酶（Elma）并分析其生物学活性。通过PCR扩增克隆Elma基因，并将其与pET-28a连接。然后将重组表达载体pET-28a-Elma转化到大肠杆菌BL21（DE3）中。在16℃用0.2 mmol/L IPTG诱导5小时后，Elma成功表达，SDS-PAGE分析表明该酶占总细胞蛋白的30%以上。经过Ni(2+)-NTA亲和层析和G-75层析后，酶的纯度超过95%。根据每毫升样品在232 nm处吸光度的变化测定酶活性。多糖分子量的降低可通过PAGE电泳检测。Elma可部分裂解透明质酸（HA）和硫酸乙酰肝素（HS）。其最佳反应温度为37℃，最佳反应pH为7.0。
