华根霉脂肪酶在黑曲霉中的重组表达研究

摘要

将华根霉脂肪酶基因RCL在黑曲霉中进行了重组表达。通过PCR技术分别获得黑曲霉Pgpd启动子和RCL-TtrpC融合片段并克隆至质粒pCHAMBIA230，构建出RCL超表达载体pCHAMBIA2302∷Pgpd-RCL-TtrpC。将该载体通过冻融法转化农杆菌EHA105，进而通过农杆菌介导转化法转化黑曲霉，随机挑选7株转化子，进行PCR和Southern杂交鉴定，均为阳性。对这7株转化子进行发酵和脂肪酶活力测定，结果显示，所有转化子均能表达RCL，其中T6转化子的脂肪酶活力最高，达到71 U/mL。SDS-PAGE分析表明，重组表达的RCL的分子量约为37 kD。%This article carried out the recombinant expression ofRhizopus chinensis lipase(RCL)gene inAspergillus niger. Promoter of Pgpd fromA. niger and RCL-TtrpC fusing fragment were respectively obtained by PCR and further cloned to plasmid pCHAMBIA2302 to generate the RCL over-expression vector pCHAMBIA2302∷Pgpd-RCL-TtrpC. The resulting vector was introduced intoAgrobacterium tumefaciens EHA105, and then transformed intoA. niger through the mediation ofA. tumefaciens. Seven randomly selected transformants were analyzed by PCR and Southern blot. As a result, all the transformants were found to be positive. Then, the seven transformants were subjected to fermentation and lipase assay, and the results showed that all the transformants secreted recombinant RCL, among which transformant T6 produced the highest lipase specific activity, reaching up to approximately 71 U/mL. The fermentation broth was further analyzed by SDS-PAGE, which revealed the molecular weight of the recombinant RCL was 37 kD.