Abstract
To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55-75°C and high thermostability (about 85% residual activity) at the temperature of 55°C after incubation for 3h. The highest activity of CoCel5A was detected when 100mM citric acid buffer (pH 4.0-5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100mM citric acid buffer (pH 3.0-6.0) at 4°C for 24h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and β-D-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters Vmax, Km, and Kcat/Km values against CMC were 290.70U/mg, 2.65mg/mL, and 75.67mL/mg/s, respectively; and 228.31U/mg, 2.06mg/mL, and 76.45mL/mg/s against β-D-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.
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