Expression Of Functional Enzymes In Inpichia Pastoris Expression System

Pichia pastoris expression system is a further study of gene engineering expression system in recent years. Because of its characteristics of high concentrations of culture, high expression of protein quantity, high enzyme activity, senior level of translation modification after machining system, low cultivate cost, hard to dye the mixed bacterium, it become the great choice of industrialized production of high quality functional enzymes.This topic systematic studied and explored the system through expressing different species and different types of functional enzyme in pichia. Had representative chosen the source of yeast:xylose reductase, Rhizomucor.sp, Mucor sp and Mammal Species chymosin.Acquired the genes through different approaches respectively and express in pichia system. Measure the enzymology properties and product. Obtained the experiment results as followed:1. Cloned XYL1from Candida tropicalis genome and constructed the engineering yeast pPICZaA-XYL1/GS115. Successful secretory expressed XR and measured the enzyme activity through the in vitro experiment.The highest enzyme activity can reach2.1U·mg-1,higher than the original strains of enzyme activity0.5U·mg-1, and coenzyme dependence ratio is NADPH/NADH=1/0.62.2. Construction engineered yeast pPIC3.5K-XYL1/GS115and expressed XR in intracellular with plasmid pPIC3.5K which can screened multi-copy. Worked under the coenzyme NADH and NADPH, xylose reduced into xylitol. Optimized the copies, dissolved oxygen condition, the initial level of methanol, the adding period and concentration of xylose, selected a multi-copy with high-yielding xylitol strain pPIC3.5k-XYL1-21/GS115. Its xylitol yield increased from2.45%to33.86%and the conversion rate grew from2.94%to34.24%. Adding60g·L-1xylose at Oh is better,the yield is33.86%and the highest conversion rate is34.24%.And in96h fermentation20.32g·L-1xylitol can be obtained.3. Aquired the RM-CHY and MP-CHY From the Rhizomucor miehei and Mucor pusillus genome, and syntheticed BOVINE source chymosin gene BOVINE-CHY. Secreted expressed with plasmid pPICZaA and optimized the induction time, reaction temperature, pH, concentration of Ca2+to screened highly enzyme activities expressed yeast pPICZaA-CHY/GS115.At the conditions of induced168h,40℃, pH6.5, the Ca2+concentration of40to60mM, obtained a higher enzyme activity:strains MP-CHY1, RM-CHY3and BOVINE-CHY3respective had enzyme activities of778SU·mL-1,240SU·mL-1and80SU·mL-14. In consideration of food safety, I try to replace the inducible promoter AOX1into constitutive promoter GAP, which does not need methanol induction and only glucose can be a carbon source. To rebuild the engineering yeast pGAPZaA-CHY/GS115contain a GAP promoter and three sources of CHY. Screened several strains which had higher enzyme activity, respectively at a condition of fermentation culture72h, strains RM-CHY4、MP-CHY9and BOVINE-CHY10got667SU·mL-1、158SU·mL-1、78SU·mL-1of enzyme activity.