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Secretory Expression Of The Aspergillus Niger Glucose Oxidase Gene In Pichia Pastoris And Aspergillus Niger

Posted on:2013-06-17Degree:MasterType:Thesis
Country:ChinaCandidate:G N ZhangFull Text:PDF
GTID:2230330377957626Subject:Genetics
Abstract/Summary:PDF Full Text Request
Glucose oxidase (glucose oxidase EC1.1.3.4.) Full name of the (3-D-pyranose glucose aerobic dehydrogenase, Referred to as GOD. β-D-glucose in the presence of oxygen it will be oxidized to gluconic acid to the oxygen removal. Because of its natural non-toxic it has been widely used in food processing, pharmaceutical and biological fields. The industrial production of glucose oxidase using Aspergillus niger or Penicillium fermentation, But in the fermentation process, the low expression level and the existence of the hybrid protein has caused great difficulties to the separation and purification, Caused the product to higher costs. Through gene engineering method to construct engineering bacteria in order to realize the rapid and efficient expression of glucose oxidase, is an economic way.In this study, glucose oxidase gene was cloned from a production of glucose oxidase Aspergillus niger, constructed Pichia pastoris expression vector and Aspergillus niger expression vectors, The recombinant vector was transformed into Pichia pastoris and high-yield production strains of Aspergillus niger glucoamylase, in order to achieve efficient secretory expression of the glucose oxidase, to solve the problem of low yield of glucose oxidase unit, to explore a new way for its industrial production.The main results are as follows:1. GOD gene expression in Pichia pastorisMaterials on a production of glucose oxidase of Aspergillus niger, GOD gene was amplified by PCR cloning of the genome as a template. The sequencing results showed that the GOD gene a total length of1818bp. Blast analysis found the GOD gene has similarity of100%with gene which accession number is EU532181.1. Connec GOD gene with laboratory pre-build the Pichia pastoris expression vector pGAPHaM, Construction recombinant plasmid pGAPHaM-GOD. Recombinant plasmid restriction endonuclease by BglⅡ linearized then electroporation transfected into Pichia pastoris GS115, Recombinant transformants identification by PCR, but did not detect enzyme activity in the fermentation supernatant.2. The establishment ofAgrobacterium mediated transformation system of Aspergillus nigerFrom the choice of medium, culture temperature and time, temperature and time of screening, cefotaxime the hip sodium inhibitory concentration of Agrobacterium, hygromycin screening of Aspergillus niger pressure fumbling, Ultimately determine the use of PDA medium, co-cultured72h at28℃,32℃screened more than a week co-culture acetosyringone (AS) the final concentration of200μmol/L, and screening cephalosporin and hygromycin B final concentration of400mg/L and200mg/L.3. The construction of Aspergillus niger expression vector and expression in Aspergillus nigerBy overlap extension PCR technology, to connect5’Gla、GOD and GOD、3’Gla, obtain the overlap extension product5’Gla-GOD and GOD-3’Gla, digested the two overlap extension products and connect digestion products, get splicing product5’Gla-GOD-3’Gla. Connection this product with laboratory pre-built Aspergillus niger expression vectors pSZH, Construction recombinant expression vector pSZH-GOD.The plasmid pSZH-GOD through the freeze-thaw method transforming the Agrobacterium AGLI, Pick Agrobacterium-mediated transformation identified by PCR then use the correct transformant co-cultured with Aspergillus niger under screen conditions which have established. Extract genom and Identification by PCR to resistant Aspergillus niger. Show that all transformants were homologous recombination transformants, but mixed with non-homologous recombinant transformants and hard to separate. Then determination of enzyme activity, enzyme activity data showed that GOD gene in recombinant Aspergillus niger has been secreted expression. Secretory expression in different media is different, pH5.5the starch medium added in0.01%Triton enable the activity to achieve the highest for13.208U/mL.
Keywords/Search Tags:glucose oxidase, Aspergillus niger, conversion, secretion expression
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