褐藻胶裂解酶的基因克隆及表达条件优化

Conditions optimization of gene cloning and expression of alginate lyase

采用大肠杆菌(Escherichia coli)表达海洋弧菌(Vibrio sp.)X511的褐藻胶裂解酶基因,以实现对该酶的大量制备及酶学性质研究。通过序列分析,预测该酶的前20个氨基酸为信号肽,去除信号肽后的蛋白质分子质量约为30 254.28 Da,等电点8.75,分子式为C1368H2100N364O401S6,对应的基因序列命名为alg1987。按如下方案构建重组菌:设计特异性核酸引物,PCR扩增得到alg1987;将重组质粒p ET-30(a)-alg1987导入大肠杆菌Trans5α进行测序;提取阳性p ET-30(a)-alg1987质粒导入大肠杆菌BL21,利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测诱导后的重组菌胞液上清,发现异源表达的褐藻胶裂解酶分子质量大小与预测值相近。对重组菌中的褐藻胶裂解酶进行表达条件优化,结果显示,最适参数为诱导温度16℃,诱导时间16 h,诱导剂IPTG浓度0.8 mmol/L。

The alginate lyase gene in marine Vibrio sp. X511 was expressed by Escherichia coli to realize the large-scale preparation of alginate lyase and research enzymatic properties. By analyzing the sequence, the first 20 amino acids of the enzyme were predicted as the signal peptide, the protein molecular mass without the signal peptide was 30 254.28 Da, the isoelectric point was 8.75, the molecular formula was C1368H2100N364O401S6 , and the corresponding gene sequence was named as alg1987. The construction program of bacteria recombination was as follows： the specific nucleic acid primers were designed and the alg1987 was obtained by PCR; the recombinant plasmid pET-30（a）-alg1987 was introduced into E. coli Trans5α for sequencing; the positive plasmid pET-30（a）-alg1987 was extracted and introduced into E. coli BL21, then expressed by the induce of IPTG. The supernatant of recombinant bacteria cytosol was induced and detected by SDS-PAGE, it was found that the molecular mass of the alginate lyase by heterologous expression was similar to the predicted value. The expression conditions of alginate lyase in recombinant bacteria were optimized. The results showed that optimum parameters were as follows： induction temperature 16 ℃, time 16 h, induction agent IPTG concentration 0.8 mmol/L.