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 1329bp 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 65–70kDa. The purified rMANTV showed a specific activity of 415.49Umg−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 2.61mgmL−1 and 1.49mgmL−1 for LBG and Konjac glucomannan, respectively. Its catalytic efficiency (Kcat/Km) was 225.41±20.14mLmg−1s−1 for LBG and 336.67±27.39mLmg−1s−1 for Konjac glucomannan. The high temperature tolerance of this endo-1,4-β-mannanase makes it a good potential candidate for industrial applications.
Published Version
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