2007•Journal of Pharmaceutical and Biomedical SciencesRequires access
Expression of β-mannanase Gene from Trichoderma reesei RUTC-30 in Pichia pastoris
Xiaobing Chen
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Abstract
The man1 gene encoding mannanase without the signal peptide was cloned by RT-PCR using RNA of the Trichoderma reesei as template. The secreted expression plasmid pPIC9K-man1 of Pichia pastoris was constructed and digested with SacⅠand transformed into Pichia pastoris GS115 by PEG (polyethylene glycol). PCR and phenotype analysis showed man1 has been integrated into P.pastoris genome. After screening , the recombinant P. pastoris strain RMAN23 was obtained and fermented in large scale 5 L fermenter for 120 h. The recombinant mannanase activity could reach to 470 IU/mL .The properties of the recombinant mannanase were characterized. Optimum pH and temperature for the recombinant enzyme were 4.5 and 60℃, respectively. The enzyme was stable below 75℃ between pH 4.0~6.0