Dataset Information

Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable ?-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 K m of 19.83?mM and a V max of 0.12??mol/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.

SUBMITTER: Ahmed SS

PROVIDER: S-EPMC6885295 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Ahmed Syed Shoaib SS Akhter Mohsina M Sajjad Muhammad M Gul Roquyya R Khurshid Sana S

BioMed research international 20191107

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, namel ...[more]

PMID: 31828148

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Project description:BackgroundEconomically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains.ResultsHere, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural), organic acids (acetate, itaconic acid, levulinic acid, succinic acid), amino acids (serine, threonine) and membrane-intercalating chemicals (decanoic acid, geraniol). The transcriptional response towards these chemicals revealed large overlaps of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, RpoS, OmpR, ArcA, Fur and GadX. These regulators, the genes within their regulons and the above mentioned cellular functions therefore constitute potential targets for increasing E. coli chemical tolerance. Fitness determination of genome-wide transposon mutants (Tn-seq) subjected to the same chemical stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene functions.ConclusionsThe combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals.

Dataset Information

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

Publications

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

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