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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (4): 56-67    DOI: 10.13523/j.cb.20170408
研究报告     
对SDS稳定的V8(V125T)蛋白酶突变体的高效表达及性质研究
程可利1, 刘晓2, 李素霞1
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
Study on High-level Expression and Characterization of a V125T V8 Protease Mutant with Tolerance to SDS
CHENG Ke-li1, LIU Xiao2, LI Su-xia1
1 East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering, Shanghai 200237, China;
2 Shanghai Yaxin Biotechnology Co., Ltd, Shanghai 201108, China
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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50 L发酵罐中发酵，融合蛋白为可溶性表达，可得菌湿重50 g/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0.998 mg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-pNA作为底物测得V8（V125T）蛋白酶的Km为0.339 mmol/L，Vmax为16.642 μmol/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 ℃，12 h内在4~35℃有很好的温度稳定性；25 ℃条件下1 mmol/L的金属离子对酶具有不同程度的影响，其中Fe3+的抑制作用最强；2 mol/L尿素及1 mmol/L EDTA对酶活性无影响，在0.1% SDS中保存12 h、在0.5% SDS中4 h和在1% SDS中1 h，活性均能维持90%以上，在0.5%，0.1% SDS保存12 h，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。
关键词： 重组V8蛋白酶;  纯化;  稳定性;  SDS耐受性;  酶学性质;  突变体    
Abstract: 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 BL21 (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 16.642 μmol/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.
Key words: Mutant    Stability    Enzyme properties    Recombinant V8 protease    Purification    Tolerance to SDS
收稿日期: 2016-11-17 出版日期: 2017-04-25
ZTFLH:  Q786  
通讯作者: 李素霞     E-mail: lisuxia@ecust.edu.cn
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引用本文:

程可利, 刘晓, 李素霞. 对SDS稳定的V8(V125T)蛋白酶突变体的高效表达及性质研究[J]. 中国生物工程杂志, 2017, 37(4): 56-67.

CHENG Ke-li, LIU Xiao, LI Su-xia. Study on High-level Expression and Characterization of a V125T V8 Protease Mutant with Tolerance to SDS. China Biotechnology, 2017, 37(4): 56-67.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170408    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I4/56

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