1. High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris
- Author
-
Rao Ben, Wang YaPing, Huang Can, Liu Xiaoyan, Yan Hong, Min Yong, Ma Lixin, and Wang Ying
- Subjects
0106 biological sciences ,Gene Expression ,01 natural sciences ,Pichia pastoris ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,law ,010608 biotechnology ,mental disorders ,Hydrolase ,Parathion methyl ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,biology.organism_classification ,Enzymes, Immobilized ,Phosphoric Monoester Hydrolases ,Recombinant Proteins ,Enzyme ,chemistry ,Biochemistry ,Saccharomycetales ,Recombinant DNA ,Plesiomonas ,Fermentation ,Specific activity ,Target protein ,human activities ,Biotechnology - Abstract
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.
- Published
- 2020