褐藻胶裂解酶基因在大肠杆菌中的表达及其酶学性质

作者从蜡样芽孢杆菌F1-5-10中提取DNA,通过PCR克隆得到褐藻胶裂解酶基因后,将其构建到pET-30a(+)上并在大肠杆菌BL21菌株中进行高效诱导表达,继而对纯化得到的褐藻胶裂解酶蛋白进行活性检测和酶学特性研究。实验结果表明:PCR扩增出1035bp大小的褐藻胶裂解酶基因algl(GenBank登录号:GU585575),SDS-PAGE电泳结果显示出相对分子质量约为45 000的特异性蛋白质条带。表达条件优化实验结果表明0.4mmol/L浓度的IPTG 32℃诱导4h重组蛋白的表达量最高,约占菌体总蛋白质质量的36%。测定褐藻胶裂解酶酶活性,酶活力为11.7U/mg。酶学性质研究表明:ALGL的酶活最适温度为35℃,热稳定性较差,最适pH值为6.6,Ca2+、Mg2对酶活有激活作用,ALGL催化褐藻胶的Km值为1.89mg/mL,Vmax为15.01U/mL。

Expression of Alginate lyase Gene in E.coli and Characterization of the Enzyme

The algl gene(GenBank accession number: GU585575) encoding alginate lyase(ALGL) from Bacillus cereus F1-5-10 was cloned into the vector pET-30a and expressed in Escherichia coli BL21 in this study.The optimum expression conditions of algl in E.coli was study and listed as follows: the concentration of IPTG 0.4 mmol/L,expression time 4 h,and expression temperature 32 ℃.Under the optimum conditions,the activity of recombinant enzyme achieved at 11.7 U/mg.Then,the recombinant enzyme was purified and the characteristics were determined.It was found that(1) the molecular weight was approximately 45 000;(2)the optimum pH and temperature were 6.6 and 35 ℃,respectively;(3)the activity was stimulated by 1.0 mmol/L Ca2+ and Mg2+;(4)the Km and Vmax values for alginate were 1.89 mg/mL and 15.01 U/mL,respectively.