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

gene encoding a thermotolerant endo-1,4-beta-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 by beta-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 degrees C, with stability from 20 degrees C to 65 degrees 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 Km 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-beta-mannanase makes it a good potential candidate for industrial applications. (C) 2016 Elsevier B.V. All rights reserved.