Cloning And Expression Of An Endo-1, 4-β-glucanase Ⅰ Gene(eg Ⅰ) From Volvariella Volvacea

An endoglucanaseⅠ(EGⅠ) cDNA including signal peptide was isolated from V. volvacea V23 by RT-PCR. The full-length cDNA contained an ORF of 1167 bp which encoded a signal peptide of 23 amino-acids and a mature peptide of 366 amino-acids. Similarity analysis showed the EGⅠbelonged to family 5 of glycosyl hydrolases.The cDNA encoding the mature peptide was cloned into expression plasmid pET-28a, and expressed in Escherichia coli BL21(DE3) by IPTG induction, SDS-PAGE showed that EGⅠwas expressed successfully, the molecular weight of EGⅠwas about 39.5 kD but without enzymatic activity.The mature peptide cDNA was cloned into the secretive expression vector pPIC9K.The recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after linearized by SalⅠ.After screening, a high yielding recombinant strain P. pastoris-EGⅠ1 was obtained. SDS-PAGE showed the molecular weight of EGⅠwas about 42 kD,it was higher than the theoretic molecular weight which may be due to glycosylation after translation.The conditions of expressing EGⅠin Pichia pastoris were optimized, The optimum conditions for EGⅠaccumulation were as follows: induced time 96 h by 2.0% methanol; the initial induced pH7.5;the concentration of glycerin in BMGY 4.0%; glycerin addition quantity per 24 h in BMMY 0.75%; the concentration of the surface active agent tween 20 in BMMY 0.15%,the enzyme activity was up to 4612U/mL at these conditions.Part of the enzymatic properties of EGⅠwere determined, The results revealed that the optimal temperature and optimal pH for enzyme reaction were 55℃and 7.5;the enzyme had good stability when the temperature bellow 55℃while declined quickly above 55℃,but still holded 60% of the enzymatic activity when the temperature reached 65℃.the enzyme had good pH stability in pH6.5-9.0 and holded above 90%enzymatic activity.