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Cloning And High Efficiency Expression Of Glucose Oxidase From Aspergillus Niger

Posted on:2014-10-10Degree:MasterType:Thesis
Country:ChinaCandidate:Y LiuFull Text:PDF
GTID:2250330425452189Subject:Fermentation engineering
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Glucose oxidase (GOD) is the redox enzyme which can catalyze the oxidation ofβ-D-glucose to D-glucono-β-lactone and hydrogen peroxide using molecular oxygen asthe electron acceptor. GOD has been used for several industrial purposes includingmanufacture of glucose sensor, processing of flour and removing of extra glucose infood, production of gluconic acid and its salts and fuel cell making. However, due to thecost of the production, the application of glucose oxidase is seriously restricted. Toreduce the cost of its production, the gene of glucose oxidase was cloned formAspergillus niger preserved in our lab and planned to be expressed in Pichia pastoris.A high-efficiency glucose-gluconate conversion Aspergillus niger strain QYW3221,which can transform glucose into sodium gluconate at the highest molar ratio, wasisolated through primary screening depending on the halo size and followed secondaryscreening through fermentation test in shake flasks depending on the glucose-gluconatetransformation rate. This strain was used as original strain in the following molecularcloning experiment of glucose oxidase.To determine the activity of GOD in further study, continuous spectrophotometricrate determination (CSRD) and titrimetric analysis (TA) for GOD activity were studiedand compared. Several conclusions had been made form the research: good linearitywas obtained between activity range0.044-0.351U/mL and1.125-5.321U/mL for CSRDand TA, respectively; for CSRD, standard CSRD could be replaced by two-point assaywithin this range; the value of TA was lager than CSRD and the conversion formula ofthese two methods was: value of TA=(value of CSRD+0.1454)×1.2868+0.3793.CSRD was more convenient in large scale determination.In the gene cloning experiment, an1852bp-sequence with a CDS of1818bp wascloned through PCR using primers designed according to the result of multiplesequences alignment. The sequence were confirmed as A. niger glucose oxidase genethrough BLAST search. A16-amino-acid signal sequence was discovered using SignalP4.0as predictor.When P. pastoris was utilized as expression host, the CDS without signal peptidesequence was subcloned into secretive expression vector pPIC9K, thereby expressionvector pPIC9KGOD was generated. After that, P. pastoris SMD1168was electroporatedwith ethanol precipitated and condensed pPIC9KGOD DNA molecular after linearized with restriction enzyme Mss Ⅰ (Pme Ⅰ) and then spreaded on auxotrophiccomplementation plate for primary screening. Several colonies appeared on1mg/mLG418YPD plates in the second round screening. The positive recombinants of PCR testwhich could tolerate1mg/mL G418were inoculated in shake flasks. After a7-day-induction with methanol, the enzyme activity in the culture broth could be as highas14.9U/mL.
Keywords/Search Tags:Aspergillus niger, glucose oxidase, gene cloning, Pichia pastoris
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