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

Abstract

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.

Enzymatic hydrolysis of lignocellulosic biomass using a novel, thermotolerant recombinant xylosidase enzyme from Clostridium clariflavum: a potential addition for biofuel industry

“…C. clariavum has got the potential to utilize xylose and xylan as sole sources of carbon. 33 In this study cloning and expression of a novel xylosidase gene from C. clariavum was performed into E. coli BL21 (DE3).…”

Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry

“…The cell contents were heated at 70 °C for 30 min to denature host proteins. 37 Host cell proteins were separated by centrifugation at 8000 rpm for 10 min.…”

Controlling the Adsorption of β-Glucosidase onto Wrinkled SiO2 Nanoparticles To Boost the Yield of Immobilization of an Efficient Biocatalyst

“…The spectrum of the free enzyme showed two minima centered at 215 and 222 nm, suggesting the presence of comparable amounts of β-sheet and α-helix components. 50 − 53 The spectra of the BG/WSNs systems are similar but slightly different from that of the free protein. Indeed, the two minima are better resolved and fall at about 210 and 220 nm.…”