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Expression Of Endo-β-glucanase Gene

Posted on:2012-07-06Degree:MasterType:Thesis
Country:ChinaCandidate:N MengFull Text:PDF
GTID:2210330338972385Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
Endo-β-glucanase (CMCase) is one of the most important parts of cellulase, and has great value in cellulose degrading, food, feedstuff production and fabric washing fields. This paper focused on construction, screening and CMCase production of high endo-p-glucanase producing strains based on gene engineering technology.An endo-β-glucanase gene afl from anaerobic fungus was inserted into Escherichia coli expression vector pET-28a(+). Then the recombinant plasmid pET-28a(+)-af1 was transformed into Escherichia coli BL21(DE3) to obtain recombinant strain BL21(DE3)/pET-28a(+)-af1. The appropriate conditions to cultivate recombinant Escherichia coli were:added IPTG to 0.1 mM when the cell concentration reached OD600=0.8, then cultivated at 31℃. SDS-PAGE analysis of its expression product showed a clear protein band near 40 kDa, which accorded with that of AF1 protein theoretic size. The enzyme exhibited maximum CMCase activity at pH 6.0,45℃, which belonged to the neutral cellulase. It had been proved that gene afl had a complete sequence ready to express product.Furthermore, afl was inserted into Pichia pastoris expression vector pPIC9K, digested by enzyme Bglll, and then transformed into Pichia pastoris GS115 using Gene Delivery Instrument. Transformants were screened by selective plates, and 6 positive transformants with high G418 resistance were obtained. PCR analysis of their genome showed clear bands relative to the size of gene afl, which demonstrated that afl was successfully integrated into GS115 genome and contributed to stable inheritance.The expression product was analyzed by SDS-PAGE, and a clear protein band was found near 52 kDa. The 6 positive transformants selected were cultivated respectively and showed similar and stable enzyme producing performance.Moreover, in shaking flask experiments with methonal induction, the CMCase activity of the supernatant could reach 600 U/mL at 96 h. This result laid good foundation for neutral cellulase production using recombinant Pichia pastoris strains.Through secreening processes, T/afl-3 and T/egⅡ-5 with good endo-β-glucanase producing performance were respectively obtained from recombinant Trichoderma reesei T/af1 and T/egⅡ, which included about 300 strains respectively. Shaking flask experiments demonstrated that, T/af1-3 contained endo-β-glucanase gene af1 from anaerobic fungus, and the neutral CMCase activity in the supernatant could reach 1280 U/mL measured at pH 6.0,50℃, which was 3.9-fold compared with that of original strain; T/egⅡ-5 contained endo-β-glucanase gene egⅡfrom Trichoderma reesei, and the acid CMCase activity in the supernatant could reach 3500 U/mL measured at pH 4.8,50℃, which was 4.9 times that of original strain. As a genetic engineering host, Trichoderma reesei system has plentiful advantages, such as high expression level of foreign gene, simple procedures for product separation, etc. This paper is useful for cellulase directed evolution and construction of industrial strains with high cellulase activity.
Keywords/Search Tags:endo-β-glucanase, gene recombination, Escherichia coli, Pichia pastoris, Trichoderma reesei, gene expression, cellulase
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