褐藻胶裂解酶基因的克隆、表达载体构建以及表达条件的研究  

Preliminary studies on cloning,vector construction and expression conditions of alginate lyase gene

作　　者：刘航[1,2] 曹海龙[1] 岳敏[1,2] 张建平[1,2] 李曙光[1] 杜昱光[1] 

机构地区：[1]中国科学院大连化学物理研究所生物技术部,辽宁大连116023 [2]中国科学院研究生院,北京100049

出　　处：《华中师范大学学报（自然科学版）》2012年第4期456-460,467,共6页Journal of Central China Normal University：Natural Sciences

基　　金：国家"863"计划项目(2011AA090704);辽宁省博士启动基金项目(20111150);公益性行业(农业)科研专项项目(200903052)

摘　　要：以产褐藻胶裂解酶的紫色链霉菌(Streptomyces violaceoruber)为出发菌株,应用基因工程技术,将褐藻胶裂解酶aly基因进行克隆,并构建了该基因的重组表达载体pET23b(+)-aly.通过表达条件的初步优化,实现了aly基因在大肠杆菌Escherichia coli BL21(DE3)中的成功表达.在IPTG诱导浓度为0.1mmol/L,诱导时间为8h,诱导温度为16℃的条件下,细胞破壁后上清中ALY粗酶液活力为6U/mg.根据褐藻胶裂解酶ALY的蛋白质结构预测结果推断该酶属于Algi-nate lyase-2家族,理论pI为8.85,理论分子量大小为28 500,化学式为C1252N1920H356O394S9.SDS-PAGE蛋白电泳检测发现,ALY蛋白的分子量约为28 000,与预测的蛋白分子量较为接近.The alginate lyase-eneoding gene aly of Streptomyces violaceoruber was cloned and inserted into the expression vector pET23b （＋） with genetic engineering technology. Recombinant vector pET23 b（＋）-aly was transformed into host Escherichia coli BL21 （DE3）. The expression parameters of pET23b （ ＋ ）-aly in the E. coli BL21 （DE3） were optimized for maximum alginate lyase activity with respect to IPTG concentration and incubation time. The cell free extract of E. coli BL21 （DE3） harboring pET23b（＋）-aly exhibited a alginate lyase activity of 6U/mg crude protein, when induced with 0.1 mmol/L IPTG and incubated at 16 ℃ for 8 hours. Furthermore, the ALY was deduced as a member of Alginate lyase-2 family by protein structure analysis. The theory pI of ALY is 8.85, theory molecular weight size of ALY is 28 500, and chemical formula of ALY is C1252 N1920 H356 0394 S9. The molecular weight size of ALY protein was detected on SDS-PAGE, which is closed to 28 500 of calculation.

关 键 词：褐藻胶裂解酶 aly基因 pET23b(+)克隆 ESCHERICHIA coli表达 

分 类 号：Q815[生物学—生物工程]