文献类型：期刊文献

中文题名：对SDS稳定的V8(V125T)蛋白酶突变体的高效表达及性质研究

英文题名：Study on High-level Expression and Characterization of a V125T V8 Protease Mutant with Tolerance to SDS

作者：程可利[1];刘晓[2];李素霞[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]上海雅心生物技术有限公司,上海201108

年份：2017

卷号：37

期号：4

起止页码：56

中文期刊名：中国生物工程杂志

外文期刊名：China Biotechnology

收录：CSTPCD;;北大核心:【北大核心2014】；CSCD:【CSCD2017_2018】；

基金：华东理工大学生物反应器工程国家重点实验室对本研究提供的基金支持

语种：中文

中文关键词：重组V8蛋白酶;突变体;纯化;酶学性质;稳定性;SDS耐受性

外文关键词：Recombinant VS protease Mutant Purification Enzyme properties Stability Tolerance to SDS

摘要：谷氨酰内切酶能特异性切割谷氨酸、天冬氨酸残基羧基端结合的肽键。将含有V8蛋白酶突变体(V125T)基因的表达质粒的重组大肠杆菌BL21(DE3),在50L发酵罐中发酵,融合蛋白为可溶性表达,可得菌湿重50g/L,相对蛋白表达量为33%。融合蛋白采用GST亲和纯化、肠激酶激活、DEAE-FF阴离子交换层析,得到纯的V8(V125T)突变体,经纯化后可获得0.998mg蛋白/g菌(湿重),比活为13.47 U/mg pro.纯化过程的酶活回收率达到了97.9%。对纯酶进行酶学性质分析,以Z-Phe-Leu-Glu-p NA作为底物测得V8(V125T)蛋白酶的Km为0.339mmol/L,Vmax为16.642μmol/min。其最适pH为8.0,在pH4.0~10.0之间较稳定;最适反应温度在45℃,12h内在4~35℃有很好的温度稳定性;25℃条件下1mmol/L的金属离子对酶具有不同程度的影响,其中Fe^(3+)的抑制作用最强;2mol/L尿素及1mmol/L EDTA对酶活性无影响,在0.1%SDS中保存12h、在0.5%SDS中4h和在1%SDS中1h,活性均能维持90%以上,在0.5%,0.1%SDS保存12h,仍能保持80%和64%的活性,与未突变的重组V8蛋白酶相比,该突变体对SDS的耐受性得到极大提高。
Glutamyl endopeptidase enzyme can cleave specifically the peptide bonds on the carboxyl- terminal side of aspartate and glutamate residues. The gene of V8 （V125T） protease mutant was cloned into plasmid pGEX-4T-3 and then the recombinant plasmid was transformed into E. coli BI21 （DE3）. After the fermentation in 50 L fermenter, 50 g/L wet cell was obtained, and the fusion protein was expressed as soluble one, the ratio of expressed aim protein reached to 33%. The fusion protein was purified with GST affinity column, activated by enterokinase, purified with anion-exchange chromatography DEAE-FF, 0. 998mg purified aim protein per gram wet cell was obtained, the specific activity was 13.47 U/mg pro. with Z-Phe-Leu-Glu-pNA as a substrate. The total activity recovery rate was 97.9%. The values of Km and Vmax of the recombinant V8 （V125T） mutant were 0.339 mmol/L and 16. 642μmol/min respectively. The optimum pH was pH8.0 and was stable from pH4.0 to pH 10.0. The optimum temperature was 45℃, and the protease was stable from 4℃ to 35℃ after incubated for 12 h. At 25℃, the protease activity was affected by some 1 mmol/L metal ions, especially by Fe3＋ metal ion. The enzyme activity was not affected by 2 moL/L urea and 1 mmol/L EDTA. More than 90% of total activity was kept when it was in 0.1% SDS for 12 h, in 0.5% SDS for 4 h or in 1% SDS for 1 h. The residual activity still was 80% in 0. 5% SDS for 12 h and 64% in 1% SDS for 12 h. The tolerance of recombinant V8 （V125T） mutant to SDS was vastly improved compared with the wild-type recombinant V8 protease.