Expression and Characteristics of an Endoglucanase from Trichoderma atroviride (TaEGII) in Saccharomyces cerevisiae
2017
Huang, Xiao-mei | Li, Qing-qing | Chen, Xiu-ling | Fan, Jin-xia | Xu, Xiu-hong | Sun, Xu-dong | Li, Dong-yu | Zhao, Hong-xiao
Endoglucanase secreted by the fungus Trichoderma atroviride is a kind of cellulase. An endoglucanase gene egII was cloned from T. atroviride AS3.3013 and expressed in Saccharomyces cerevisiae INVScI. The open reading frame of the egII gene was composed of 1257 bp, encoding 418 amino acids with a molecular weight of 44.23 kDa plus a signal peptide of 21 amino acids. Based on sequence similarity, TaEGII belonged to the glycosyl hydrolase family 5. Expression of the egII gene in T. atroviride AS3.3013 can be induced by microcrystalline cellulose (MCC), bran, carboxymethyl cellulose (CMC), rice straw, and corn stalk but is inhibited by glucose. A highly efficient integrated expression vector (pYPIGH-B includes a sequence of the α-mating factor signal peptide (MF-α)) was constructed. The enzymatic activity of the supernatant of recombinant yeast YPIGH-B3 was 1.29 times higher than that of YES2-egII, demonstrating that the MF-α can significantly improve the expression of the recombinant EGII in S. cerevisiae. The recombinant endoglucanase TaEGII produced by S. cerevisiae showed maximum activity at pH 5.0 and temperature 60 °C. Under these conditions, the Km and Kcat values for Avicel and raffinose hydrolysis were 1.22 × 10–² mg ml⁻¹, 9.09 × 10⁻² s⁻¹ and 1.06 × 10–² mg ml⁻¹ , 9.18 × 10–² s⁻¹, respectively. The enzymatic activity of recombinant TaEGII was stable when incubated from 40 to 60 °C for 1 h. It was stable in a wide range of pH (4.0–7.0) and sensitive to various metal ions. Transgenic yeast strain YPIGH-B3 might be applied to cellulosic ethanol production.
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