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Expression Of Human Acetaldehyde Dehydrogenase2in Pichia Pastoris

Posted on:2011-10-05Degree:MasterType:Thesis
Country:ChinaCandidate:J ZhaoFull Text:PDF
GTID:2250330401479917Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
Aldehyde dehydrogenase (ALDH) is one of the key enzymes in thehuman metabolic pathway of ethanol. Alcohol dehydrogenase (ADH)converts alcohol to acetaldehyde, while ALDH converts acetaldehyde toacetic acid which is transformed into Acetyl-CoA and involves the othermetabolism pathway. The quantity and enzymatic activity of ADH is at thesimilar level among Chinese, while the activity of ALDH is very low in manypeople, which is related to drunkenness and other alcohol-related diseases.The high-level expression of human ALDH2in Pichia pastoris was studied inthis project.The plasmid pPIC9K-07ALDH2which contains the codon-optimizedhuman ALDH2was constructed by our lab. In this research, the new plasmidpPIC9K-ALDH2-delsign was constructed by removing the signal peptideα-factor. The plasmid pPIC9K-ALDH2-design was transformed into P.pastoris SMD1168by electroporation. A recombinant strain SMD1168(pPIC9K-ALDH2-delsign) was screened for the production of ALDH2. Theprocesses of fermentation using shaking flask and high-density fermentationin bioreactor of the recombinant strain were studied.The optimized ALDH2expression conditions using shaking flask are asfollows: optimal temperature was28℃, pH was7.0, initial OD600was15, theinduction period was72h,1.2%(v/v) of methanol was added every24hours.After48hours of induction, the activity of human ALDH2was approximately0.315U/mL of culture supernatant.The process of high-density fermentation for ALDH2using5Lbioreactor are as follows: during the growth stage using glycerol as carbonsource, the optimal conditions were with pH of5.5, temperature of30℃, wasair flow of3L/min, DO of30%, and stirring speed of300-750r/min. DOoption was set up to be related with stirring speed. During the glycerol fed-batch stage, pH was5.5, temperature was30℃, air flow was3L/min, DOwas30%, stirring speed was300-1050r/min, and DO was set up to be relatedwith stirring speed. Carbon-resource starvation continued for1h. In this stage,pH was5.5, temperature was30℃, air flow was3L/min, DO wasuncontrolled, and stirring speed was800r/min. In the methanol fed-batchstage, pH was7.0, temperature was25℃, air flow was4L/min, DO was20%,and stirring speed was800r/min. DO was set up to be related with methanolflow rate..Under this fermentation condition, we can get some conclusions. Duringthe growth stage using glycerol as carbon source, the yeast biomass was4.26g/L (OD600=14.17) after cultivated for18.9h when the glycerol in thesalt-based medium was consumed. During the glycerol fed-batch stage,500mL of50%(v/v) glycerol was flowed at the speed of25mL/h. After that,the yeast biomass achieved96.19g/L (OD600=319.18). In the methanolfed-batch stage, the biomass during induction phase changed very little, andachieved the maximum of120.42g/L (OD600=399.58) after70h offermentation. The whole fermentation process was100h The activity ofhuman ALDH2in culture supernatant achieved the maximum of0.944U/mLafter85.9h of fermentation.
Keywords/Search Tags:Aldehyde dehydrogenase, Pichia pastoris, shaking flaskfermentation, high-density fermentation
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