Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli

Abstract EN

This study aims to achieve high-level soluble expression and characterization of a thermostable industrially important enzyme, i.e., beta-glucosidase (BglA; EC: 3.2.1.21), from Clostridium thermocellum (C.

thermocellum) by cloning in an Escherichia coli (E.

coli) expression system.

BglA was expressed as a partially soluble component of total cellular protein (TCP) having a molecular weight of ∼53 kDa with 50% of it as soluble fraction.

Purification in two steps, namely, heat inactivation and Ni-chromatography, yielded approximately 30% and 15% of BglA, respectively.

The purified (∼98%) BglA enzyme showed promising activity against the salicin substrate having a Km of 19.83 mM and a Vmax of 0.12 μmol/min.

The enzyme had an optimal temperature and pH of 50°C and 7.0, respectively, while retaining its catalytic activity up till 60°C and at pH 7.

The optimized maximum expression level was attained in M9NG medium with lactose as an inducer.

Circular dichroism revealed presence of alpha helix (43.50%) and small percentage of beta sheets (10.60%).

Factors like high-end cellulolytic activity, fair thermal stability, stability against low pH, and ease of purification make BglA from C.

thermocellum a potential candidate in industrial applications.