Cloning of endo-β-glucanase I gene and expression in Pichia pastoris
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Total RNA of Thermoascus aurantiacus was isolated from its mycelium and acted as template for RT-PCR. The full-length cDNA encoding an endo-β-glucanase I was cloned via RACE-PCR method and the cDNA contained an ORF of 1005 bp encoding 305 amino acids. A recombinant plasmid, pPIC9k-egI, was constructed by inserting the ORF sequence of endo-β-glucanase I gene (egI) into the yeast expression vector pPIC9k and transformed to Pichia pastoris GS115. The results showed that the recombinant endo-β-glucanase I was excreted into the fermentation medium. The highest activity of endo-β-glucanase I and the protein content were up to 45.42 U/mL and 788.26 μg/mL at incubation time of 144 h. The optimal temperature and pH for the recombinant endo-β-glucanase I were found to be 70°C and 3.5, respectively.
KeywordsThermoascus aurantiacus endo-1 4-β-glucanase RACE Pichia pastoris gene expression
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