Expression Of Soybean Peroxidase From Soy Bean In Pichia Pastoris

Soybean peroxidase SBP326gene and C-truncated20amino acids SBP306gene were cloned in vector pARS2,electroporation and induce express in P.pichia X-33. Secretion express by alpha factor peptide from Saccharomyces cerevisiae,Detection enzyme activity of fermentation broth showed that whole sequence SBP expressed in pichia are inactive,only the C-truncated SBP get activity of23.5U/mL.By analysis amino acid sequence of SBP, found that seven structure of Asn-X-Thr (Ser) in amino acid sequence, which easily to glycosylation in yeast after translation. Design primers to mutation these glycosylation sites asparagine become glutamine, study the effect of glycosylation sites mutation on enzyme activity. After deglycosylation showed that:144,185,197are very important for enzyme activity,cannot be mutation,211and216sites had little effect on activity of SBP. SBP is compose of a single peptide chain and Heme group.The Heme group is the center of enzyme. Reconstruction Heme synthesis way in P.pichia by over-express four key genes (ALAS, PBGD, UroD, Fer)on Heme synthesis metabolic pathway. Indicate that over-expression of ALAS, UroD, Fer three gene in Heme synthesis metabolic pathway increase SBP activity obviously. Optimized the induced condition by flask culture, established the optimum culture conditions for fermentation:growth in YPCS medium to OD600≈8.Induction under the anhydrous methanol by final concentration of1%、 add Heme by final concentration of50μM and0.1%Tween80at the starting of induction. Get the highest activity of465U/mL in shake flask.With the strain of X-33-SBP306-4(After high copy screening by G418+resistant) and Heme synthesis metabolism transformation strain(over-expressed ALAS、Fe、 UroD) in5L fermentor.The highest activity of SBP was respectively510U/mL and330.6U/mL in fermentation broth, which is the highest level of the reported.