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

• An endo-β-mannanase from T. virens UKM1 was successfully cloned and expressed as functionally active enzyme in P. pastoris.
• Expressed recombinant mannanase (rMANTV) showed high optimum temperature at 70 °C.
• It showed 70% of its activity at 80 °C and good stability along 20–65 °C.
• rMANTV showed good thermotolerance with more than 60% of retaining activity at 60 °C up to one hour.
• rMANTV has a half-life of 26 min at 70 °C.

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 His6-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 Km values of 2.61 mg mL−1 and 1.49 mg mL−1 for LBG and Konjac glucomannan, respectively. Its catalytic efficiency (Kcat/Km) 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.

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