Heterologous expression and characterization of glycoside hydrolase with its potential applications in hyperthermic environment

Abstract

With the progressive focus on renewable energy via biofuels production from lignocellulosic biomass, cellulases are the key enzymes that play a fundamental role in this regard. This study aims to unravel the characteristics of Thermotoga maritima MSB8 (Tma) (a hyperthermophile from hot springs) thermostable glycoside hydrolase enzyme. Here, a glycoside hydrolase gene of Thermotoga maritima (Tma) was heterologously expressed and characterized. The gene was placed in the pQE-30 expression vector under the T5 promotor, and the construct pQE-30-Gh was then successfully integrated into Escherichia coli BL21 (DH5α) genome by transformation. Sequence of the glycoside hydrolase contained an open reading frame of 2.124 kbp, encoded a polypeptide of 721 amino acid residues. The molecular weight of the recombinant protein estimated was 79 kDa. The glycoside hydrolase was purified by Ni⁺²-NTA affinity chromatography and its enzymatic activity was investigated. The recombinant enzyme is highly stable within an extreme pH range (2.0-7.0) and highly thermostable at 80 °C for 72 h indicating its viability in hyperthermic environment and acidic nature. Moreover, the Ca²⁺ and Mn²⁺ introduction stimulated the residual activity of recombinant enzyme. Conclusively, the thermostable glycoside hydrolase possesses potential to be exploited for industrial applications at hyperthermic environment.

Keywords: Escherichia coli; Heterologous expression; Hyperthermic environment; Thermotoga maritime.