Endo-β-1,4-glucanase(EC3.2.1.4),as an important industrial enzyme,has been widely used in the many fields such as brewing and animal feed industry.With the developing of production,the need for it is increasing day and day.The purpose of this research is to clone thermostable endo-β-1,4-glucanase from a strain of Aspergillus niger, which effectively secreted EGâ… ,to improve the expression level with the methods of molecular biology and gene engineering and meet the need in industrial production.The gene encoding endo-β-1,4-glucanase from Aspergillus niger L3 was isolated with RT-PCR method.egâ… was eliminated its native signal peptide by PCR and inserted into the pPIC9K vector of Pichia pastoris in reading frame withα-factor secreting signal peptide sequence to construct the recombinant plasmid pPIC9K-egâ… .The recombinant plasmid pPIC9K-egâ… was transformed into P.pastoris GS115 with electroporation.Two of recombinant P.pastoris stains 1-1# and 1-5# were obtained by screening with MM,MD, CMC-Na and G418 plates and the activity of recombinant EGâ… could reach to 1456 U/mL and 1928U/mL respectively.The optimal temperature for the recombinant EGâ… was 70℃and the optimal pH was 5.0.To improve expression efficiency in recombinantβ-1,4-glucanase in P.pastoris,the egâ… gene from A.niger L3 was modified with codon optimization.A total of 193 nucleotides are changed,the G+C ratio was decreased from 54%to 44.22%.14 pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egâ… gene was synthesized using PCR-basic three-step DNA synthesis method.The modified syn-egâ… gene was inserted into the pPIC9K vector to construct the recombinant plasmid pPIC9K-syn-egâ… .The recombinant plasmid pPIC9K-syn-egâ… was transformed into P. pastoris GS115 with electroporation.A recombinant P.pastoris stains 2-7# was obtained by screening with MM,MD,CMC-Na and G418 plates.The activity of recombinant EGâ… at shaking flask level could reach to 3658 U/mL and 591.9 U/mL with 0.5%barleyβ-glucan and 1%CMC-Na as substrate,respectively.At 50 L fermentor level,theβ-1, 4-glucanase activity was 65649.6 and 39728.32 U/mL with 0.5%barleyβ-glucan and 1% CMC-Na as substrate.
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