A thermotolerant Endo-1,4-β-mannanase from Trichoderma virens UKM1: Cloning, recombinant expression and characterization

Abstract A gene encoding a thermotolerant endo-1,4-β-mannanase belonging to glycosyl hydrolase family 5 (GH5) was isolated from the fungal strain Trichoderma virens UKM1 ( manTV ). The aim of this work was to heterologously express and characterize manTV for subsequent applications. The 1329 bp β-mannanase gene was cloned and expressed in Pichia pastoris X33 yeast cells, and the recombinant mannanase (rMANTV) was expressed as a His 6 -tagged glycoprotein of approximately 65–70 kDa. The purified rMANTV showed a specific activity of 415.49 U mg −1 for 0.5% locust bean gum (LBG). This enzyme had a high optimum temperature, 70 °C, with stability from 20 °C to 65 °C. The rMANTV had its highest activity at pH 5, with a wide pH stability range of pH 3–9. It was relatively stable in the presence of several metal ions and chemical substances. In addition, rMANTV had K m values of 2.61 mg mL −1 and 1.49 mg mL −1 for LBG and Konjac glucomannan, respectively. Its catalytic efficiency ( K cat / K m ) was 225.41 ± 20.14 mL mg −1  s −1 for LBG and 336.67 ± 27.39 mL mg −1  s −1 for Konjac glucomannan. The high temperature tolerance of this endo-1,4-β-mannanase makes it a good potential candidate for industrial applications.