Cloning and characterization of thermo-alkalistable and surfactant stable endoglucanase from Puga hot spring metagenome of Ladakh (J&K)

Abstract

A thermo-alkalistable and surfactant stable endoglucanase (PHS) gene consisting of 554 amino acids was identified from metagenomic library of Puga hot spring using functional screening. PHS gene was overexpressed and purified to homogeneity using affinity chromatography The purified PHS protein presented a single band of 60kDa on the SDS-PAGE gel and zymogram. The recombinant PHS exhibited activity over a broad range of pH and temperature with optima at pH 8.0 and 65°C, respectively and having optimum stability at 60°C and pH 8.0, respectively. The recombinant PHS showed highest substrate specificity using CMC (218.4U/mg) as compared with Barley β-glucan (89.2U/mg) and Avicel (0.8U/mg). The K_m and V_max of recombinant PHS for CMC were 3.85mg/ml and 370.37μmolmin^-1mg^-1, respectively. The activity of the recombinant PHS was enhanced by treatment with 10mM non-ionic detergents such as Tween 20, Tween 40, Tween 80, Triton X- 100 and PEG and was inhibited by CTAB, SDS. Its functionality was stable in the presence of Fe³⁺ but inhibited by Cu²⁺, Hg²⁺, Mn²⁺ and Zn²⁺. These properties make PHS endoglucanase a potential candidate for use in laundry, textile,paper and pulp industries.

Keywords: Endoglucanase; Metagenomic library; Puga hot spring; Soil metagenome; Thermostable.