不同信号肽对脂肪酶A在枯草芽孢杆菌中分泌表达的影响
首发时间:2009-08-12
摘要:本文以B. subtilis 168来源的脂肪酶A为报告基因,构建一系列枯草芽孢杆菌分泌表达载体,研究不同信号肽对脂肪酶分泌效率的影响。从B. subtilis 168基因组PCR扩增带自身信号肽的lipA基因,克隆入载体pUCX08,经定点突变在其信号肽与结构基因之间引入酶切位点EcoR I,利用突变后的lipA序列(含信号肽)克隆入穿梭质粒pMA5△MCS,构建分泌载体pMA5△MCS-lipA。PCR扩增AprE, LipB, WapA, WprA信号肽序列,替换pMA5△MCS-lipA中lipA基因信号肽序列部分,构建一系列分泌载体,均转化枯草杆菌WB600,得到重组菌株WB600(pMA5△MCS-lipA),WB600(pMAaprE-lipA),WB600(pMAlipB-lipA),WB600(pMAwapA-lipA),WB600(pMAwprA-lipA)。各重组菌株脂肪酶分泌表达情况有显著差异,具有自身信号肽的重组菌株WB600(pMA5△MCS-lipA)脂肪酶分泌表达最好,摇瓶发酵20 h,最高酶活可达7.64 U/mL,更换信号肽的重组菌株脂肪酶分泌表达均不如原始信号肽,但仍显示不同的结果,各自最高酶活分别为0.55 U/mL,1.38 U/mL,0.18 U/mL和0.77 U/mL。不同信号肽对脂肪酶A在枯草芽孢杆菌中的分泌表达有显著影响。
关键词: 信号肽 脂肪酶 lipA基因 枯草芽孢杆菌 重组菌株 分泌表达
For information in English, please click here
Effects of different signal peptides on the secretion of lipase A in Bacillus subtilis
Abstract:In this study, we constructed a series of expression-secretion vectors for Bacillus subtilis in which the lipase A gene origining from B.subtilis 168 was used as the reporter. Then, the effects of different signal peptides on the secretion efficiency of lipase were investigated. lipA with its signal peptide was amplified through PCR from B.subtilis 168 genome, and cloned into pUCX08. Then we made site-direct mutation between the signal peptide and structural gene of lipA creating a EcoR I site. The mutated lipA gene was cloned into the shuttle plasmid pMA5△MCS generating secretion vector pMA5△MCS-lipA. AprE, LipB, WapA, WprA signal peptides were amplified, then the signal peptide of lipA was replaced by these four in order to construct a series of secretion vectors. Finally, these vectors were transformed into WB600, getting WB600(pMA5△MCS-lipA),WB600(pMAaprE-lipA),WB600(pMAlipB-lipA),WB600(pMAwapA-lipA),WB600(pMAwprA-lipA).There were obvious lipase secretion efficiency differences from these recombinants. The recombinant WB600∕pMA5△MCS-lipA with its own signal peptide secreted best. After culturing for 20h, the highest enzyme activity reached 7.64 U/mL, and that of WB600(pMAaprE-lipA),WB600(pMAlipB-lipA),WB600(pMAwapA-lipA),WB600(pMAwprA-lipA) showed different results, reaching 0.55 U/mL,1.38 U/mL,0.18 U/mL and 0.77 U/mL separately, no matter they could not compare with the original signal peptide.There were notable effects of different signal peptide on the secretion of lipase in Bacillus subtilis.
Keywords: signal peptide lipase lipA gene Bacillus subtilis recombinant strain expression and secretion
基金:
论文图表:
引用
No.3442648784112500****
同行评议
共计0人参与
勘误表
不同信号肽对脂肪酶A在枯草芽孢杆菌中分泌表达的影响
评论
全部评论0/1000