Molecular Modifications And Recombinant Overexpression In Pichia Pastoris Of Wheat Endoplasmic Reticulum Oxidoreductin 1

Wheat endoplasmic reticulum oxidoreductin 1?wEro1?can significantly improve the flour processing quality and is expected to be a new bio-improver.However,there are certain limitations in its industrial production applications.Due to the limitations of catalytic efficiency,expression and stability,the production cost is relatively high.In this study,wEro1 was used as a target.Based on the determination of the basic properties of wild-type wEro1,the molecular design was carried out by rational design,and finally the positive mutant with improved activity was obtained.At the same time,wild-type wEro1 was expressed by P.pastoris expression system,and the heterologous high-efficiency expression of wEro1 was achieved after optimization.The main research contents are as follows:?1?Determination of the basic properties of wEro1.The specific activity of recombinant E.coli recombinant wEro1 was 201.35±14.37 U/mg.The optimum reaction pH of wEro1 was 7.5,the optimum reaction temperature was 35?.wEro1 has a good stability under low temperature and neutral conditions.The metal ions such as Cu²⁺showed obvious inhibitory effects on the activity of wEro1,and SDS,Tween-20 and Triton X-100 showed some inhibition on the activity of wEro1.?2?Molecular modifications of wEro1.Guided by sequence homology analysis structure and homology modeling structure,9 cysteines at active sites or related to activity regulation were selected for site-directed mutagenesis through rational design.The site-directed mutagenesis of 9 cysteines was achieved by overlapping extension PCR technology,and the prokaryotic expression vector of recombinant mutant wero1 gene was further constructed.?3?Screening of positive mutants.8 molecularly engineered mutants were successfully soluble expressed in E.coli expression system.And 5 positive mutants with significantly improved activity and no significant decrease in stability were screened:C105A?Modification of Cysteine 105 of wEro1 to Alanine?,C124A,C149A,C220A and C229A.The activity of C220A and C105A increased by 119.64% and 101.74% respectively.?4?Overexpression of wEro1 in P.pastoris.The eukaryotic expression vector pPICZ?A-wero1 of wEro1 was successfully constructed and its expression in P.pastoris GS115 was achieved.The expression conditions were optimized by single factor experiment and orthogonal experiment.After optimization,the expression level could reach 53.24±2.11 U/mL.Under the same volume of fermentation broth,the expression level of P.pastoris expression system was 14.7 times that of E.coli expression system,and the high expression of wEro1 was achieved.