Klac PNP基因密码子优化及在枯草芽胞杆菌中的高效表达  

Codon optimization and efficient expression of Klac PNPgene in Bacillus subtilis

作　　者：赵越[1] 王新秀 吴思 陈作慧 张会[1] 惠觅宙 李杰[1] 双宝[1] ZHAO Yue;WANG Xinxiu;WU Si;CHEN Zuohui;ZHANG Hui;HUI Miuzhou;LI Jie;SHUANG Bao(College of Life Sciences,Northeast Agricultural University,Harbin 150006,China)

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150006

出　　处：《食品与发酵工业》2023年第19期53-59,共7页Food and Fermentation Industries

基　　金：中国博士后科学基金项目(2019M651149)。

摘　　要：嘌呤核苷磷酸化酶(purine nucleoside phosphorylase,PNP)是嘌呤补救合成途径中的关键酶,嘌呤降解途径涉及氧化嘌呤环裂解,在人体中形成尿酸作为最终产物,增加患痛风的风险。为提高PNP的发酵水平,将来源于乳酸克鲁维酵母的PNP基因进行密码子优化,分别将优化前后的目的PNP基因连接至分泌表达载体pBSA43,并在枯草芽胞杆菌WB600中重组表达PNP-YP-pBSA43、PNP-YO-pBSA43。结果显示,密码子优化前的酶活力为333.69 U/mL,优化后的酶活力达到351.61 U/mL。为进一步提高重组蛋白表达量,通过响应面分析得出优化后重组菌株产PNP的最佳发酵条件为初始pH为6.67、发酵时间55 h、发酵温度28.7℃。在该条件下,重组PNP活力为372.79 U/mL,相比未优化前提高了12%。来源于乳酸克鲁维酵母的PNP基因经密码子优化后重组表达,酶活力显著提高,对PNP在枯草芽胞杆菌中高效表达奠定了基础。The purine nucleoside phosphorylase(PNPase)is a key enzyme in the purine remediation synthesis pathway.The purine degradation pathway involves oxidative purine ring cleavage,which forms uric acid as an end product in body and increases the risk of gout.To improve the fermentation level of purine nucleoside phosphorylase,in this study,the purine nucleoside phosphorylase gene derived from Kluyveromyces lactis was codon optimized,and the target PNP gene before and after optimization was ligated to the secretory expression vector pBSA43,respectively,and PNP-YP-pBSA43 and PNP-YO-pBSA43 were recombinantly expressed in Bacillus subtilis WB600.The results showed that the enzyme activity was 333.69 U/mL before codon optimization and 351.61 U/mL after optimization.To further improve the recombinant protein expression,the optimal fermentation conditions for PNPase production by the optimized recombinant strain were derived from response surface analysis:initial pH 6.67,55 h,and 28.7℃.Under these conditions,the recombinant purine nucleoside phosphorylase activity was 372.79 U/mL,which was 12%higher than that before optimization.The recombinant expression of purine nucleoside phosphorylase gene from K.lactis with codon optimization significantly improved the enzyme activity and laid the foundation for the efficient expression of purine nucleoside phosphorylase in B.subtilis.

关 键 词：嘌呤核苷磷酸化酶 枯草芽胞杆菌 密码子优化 异源表达 响应面分析 

分 类 号：TS201.3[轻工技术与工程—食品科学]