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Cloning Of Endoglucanase Gene And Expression In Pichia Pastoris GS1115

Posted on:2011-12-06Degree:MasterType:Thesis
Country:ChinaCandidate:Z Q WangFull Text:PDF
GTID:2120330332458146Subject:Biochemistry and Molecular Biology
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Lignocellulose is a renewable organic material and is the major structural component of all plant in living nature, which is the linnar molecular linked byβ-1, 4-glycosidic bond. Cellulases are called by a joint name belonging to glycosyl hydrolase family. They can be divided into three types according to their function, endo-β-1,4-glucanases,exo-β-1,4-glucanases andβ-1,4-Glucosidase. The cellulase act in synergistic manner in biomass-degrading microbes. The last hydrolyses of lignocellulose is cellulose. It needs to construct genetically manipulated bacteria to produce more cellulase due to the less cellulase and complitical culture conditions in nature microbes. Our works contain:1,Verify endoglucanase gene eg I existence in genomic DNA. First extracted DNA, using PCR, cloned into eg I gene, then eg I was connected to PMD18-T vector, the products were introduced into E. coli strain DH5α, positive clones were picked and sequencing. The sequencing results showed endoglucanase gene (EGⅠ-DNA) size of 833bp, the latter than in the NCBI using the software Vector NTI sequence analysis revealed that the gene compared with two introns and three exons three base mutation. Extract Aspergillus terreus M11 RNA and, clone egⅠ-cDNA sequence by RT-PCR method. The sequence length was 705bp after the removal of intron exon length exactly.3,Construct the recombinant expression vector.Frist,the eg I and the Pichia pastoris expression vector pPIC9K were digested by EcoRI and NotI. The eg I encoding the mature peptide was inserted into the Pichia pastoris expression vector pPIC9K,which resulted pPIC9K-egⅠ. Then the product was introduce into E. coli strain DH5a.4,pPIC9K-eg I was introduced into Pichia pastoris GS115 by electroporation. After the induction of methanol, eg I was expressed in the supernatant of the recombiantant Pichia pastoris GS115.5,The study about Optimizing fermentation conditions:such as re-engineering Pichia pastoris GS115 culture temperature,pH,methanol concentration, shaking speed, culturing time and so on.6,Preliminary study of the recombinant enzyme:the optimal reaction temperature, the optimum pH, thermal stability, pH stability, metal ion effects on enzyme activity.
Keywords/Search Tags:Aspergillus terreus M11, exo-β-1,4-glucanases, cloning and expressing, re-engineering Pichia pastoris GS115
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