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High-level expression and purification of a molluskan endoglucanase from Ampullaria crossean in Pichia pastoris.

EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the mollusk Ampullaria crossean. In this study, the mature EG27I peptide gene fused to the HFBII secretion signal of Trichoderma reesei was expressed under the GAP promoter of Pichia pastoris in SMD1163 strain. A bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into our culture medium. The respective final OD600 and hydrolytic activity were 333 and 1.28 U/mL when high-cell-density fermentation of the recombinant P. pastoris was performed in a 7.5 L fermenter through a fed-batch strategy for 132 h. The recombinant protein concentration of the fermentation supernatant was 47.7 mg/L. EG27I was consecutively purified from the fermentation supernatant through ultrafiltration, cation exchange, and hydrophobic interaction. The specific activity of the recombinant EG27I was 26.8 U/mg, and the optimal pH and temperature of the enzyme were 5 and 50 °C, respectively. The half-life of the enzyme activity at 100 °C could reach 40 min. The N-terminal amino acid sequence analysis of the purified recombinant protein confirmed that the amino terminal sequence was consistent with the natural structure. The high quantity and purity of the EG27I provide a basis for future structural and functional studies.

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