Electronic Journal of Biotechnology (Jul 2023)
Biochemical and in silico evaluation of a recombinant, glucose tolerant, and highly thermostable β-glucosidase from Thermococcus radiotolerans DSM-15228
Abstract
Background: β-glucosidase (EC 3.2.1.21) catalyzing the β-glycosidic linkages in polysaccharides is a ubiquitous enzyme with great importance in biofuel and other industries. Enzyme inhibition by glucose has been considered as the major hurdle in the practical applications of this enzyme. Therefore, there has been a continuous search for novel β-glucosidase with high glucose tolerance and stability at industrial temperature. In the present study, recombinant of β-glucosidase from Thermococcus radiotolerans has been produced and characterized. Results: The enzyme was overexpressed in Escherichia coli strain BL21 (DE3) codon plus RIPL and purified by selective heat denaturation, ethanol precipitation and anion exchange chromatography. Purified enzyme displayed a band on SDS-PAGE with a molecular weight of 50 kDa. Optimum enzyme activity was found at pH 5, and 85°C, it retained more than 46% activity when incubated at 100°C for 5 min and exhibited 80% activity in the presence of 800 mM glucose. Km and Vmax values of the purified enzyme were found as 16.3 mM of pNPG and 25.8 µ moles per min. Molecular docking studies have shown a strong binding affinity of pNPG with the enzyme active site consisting of Glu365, Asn266, and Trp295 as major the active site amino acids. MD simulation analysis has shown a significantly high stability of enzyme active site and high potential of enzyme substrate complex formation. Conclusions: The novel characteristics such as relatively low Km value, extremely high-temperature stability and tolerance of high glucose concentration advocate the enzyme as a potential candidate for the industrial applications.How to cite: Albalawi H, Altayeb HN, Iftikhar S, et al. Biochemical and in silico evaluation of a recombinant, glucose tolerant, and highly thermostable β-glucosidase from T. radiotolerans DSM-15228. Electron J Biotechnol 2023; 64. https://doi.org/10.1016/j.ejbt.2023.03.002.
