Heterologous expression and biochemical characterization of a thermostable endo-β-1,4-glucanase fromColletotrichum orchidophilum

To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene fromColletotrichum orchidophilumwas synthesized and a recombinantPichia pastorisGS115/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 degrees C and high thermostability (about 85% residual activity) at the temperature of 55 degrees C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0-5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0-6.0) at 4 degrees C for 24 h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and beta-d-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parametersV(max),K-m, andK(cat)/K(m)values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against beta-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.

Automated summary

The newly developed endoglucanase CoCel5A shows strong adaptability to temperature and pH, with optimal activity at 55-75 degrees Celsius and in citric acid buffer with pH 4.0-5.0. CoCel5A exhibits high affinity and catalytic efficiency towards carboxymethyl cellulose sodium salt and beta-d-glucan, suggesting its potential applications in biotechnological and environmental fields.