Main content area

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

Chai, Sin Yee, Abu Bakar, Farah Diba, Mahadi, Nor Muhammad, Murad, Abdul Munir Abdul
Journal of molecular catalysis 2016 v.125 pp. 49-57
Pichia pastoris, Trichoderma virens, beta-mannosidase, catalytic activity, genes, glycoproteins, heat tolerance, industrial applications, konjac mannan, locust bean gum, metal ions, molecular cloning, pH, temperature, yeasts
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.