文献类型： 外文期刊

作者： Wang Ying ¹ ; Wang YaPing ² ; Huang Can ² ; Ma Lixin ² ; Yan Hong ² ; Min Yong ¹ ; Liu Xiaoyan ¹ ; Rao Ben ¹ ;

作者机构： 1.Hubei Acad Agr Sci, Natl Biopesticide Engn Technol Res Ctr, Hubei Biopesticide Engn Res Ctr, Biopesticide Branch,Hubei Innovat Ctr Agr Sci & T, Wuhan, Peoples R China

2.Hubei Univ, Hubei Key Lab Ind Biotechnol, Engn Hubei Collaborat Innovat Ctr Green Transform, State Key Lab Biocatalysis & Enzyme,Biol Fac, Wuhan 430062, Hubei, Peoples R China

关键词： Multi-copy strains; High-density fermentation; Enzyme immobilisation; Methyl parathion hydrolase; Pichia pastoris

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：1.513； 五年影响因子：1.381 ）

ISSN： 1046-5928

年卷期： 2021 年 183 卷

页码：

收录情况： SCI

摘要： Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.