枯草芽孢杆菌纤维素酶基因整合载体的构建

摘要

Aim:To obtain an engineered bacterial strain able to express cellulose-degrading enzymes through constructing an integration vector using Bacillus subtilis as the host.Methods:The homologous fragments M1 and M2 were cloned by polymerase chain reaction (PCR) from the genome of B.subtilis LN.The glucosidase gene CelKg,homologous fragments M1 and M2 and the strong promoter P43 were ligated to the pGEM-T vector by T4 DNA Ligase to construct the integrated vector pGEM-Kmpgrnt.The vector was then transformed to B.subtilis LN by double crossover homologous recombination method.Results:The integration vector was successfully constructed and integrated into the genome of B.subtilis LN,as verified by PCR.Congo red staining indicated that the recombinant B.subtilis could obviously degrade sodium carboxymethyl cellulose in the medium.The cellulase activity from the culture supematant harvested after 18 h culture of the recombinant strain in a modified medium at 37 ℃ with shaking was increased by 115％ as compared with the wild-type B.subtilis LN.%目的:以枯草芽孢杆菌(Bacillus subtilis)为宿主,构建纤维素酶基因整合表达载体,获得能够表达纤维素酶并且降解纤维素的工程菌.方法:通过聚合酶链式反应(polymerasc chain reaction,PCR)从B.subtilis LN 基因组中克隆同源片段M1、M2基因片段,以质粒pGEM-T为载体,将同源片段M1、M2、启动子P43和葡萄糖苷酶基因CelKg连接在一起构建整合载体pGEM-Kmpgmt,并采用双交换同源重组的方式将其转化进入B.subtilis LN 基因组中.结果:通过PCR和双酶切验证整合载体构建完成,并成功整合到野生型B.subtilis LN中,获得重组菌B.subalis Kpg.刚果红染色结果显示重组菌对羧甲基纤维素钠有降解作用.改良培养基37℃条件下摇瓶培养,重组菌B.subtilis Kpg生长至18h时上清液中纤维素酶活力比野生型B.subtilisLN提高了115％.