Expression and characterization of thermo-activated β-glucosidase TaBgl3 from Thermofilum adornatum

Abstract:

[Objective] β-glucosidase, also known as β-D-glucosidase hydrolase, is a key rate-limiting enzyme for cellulose degradation, which is a cellulase. The β-glucosidases from thermophilic archaea have been verified to have tolerance to acid and high temperature, which have become one of the research hotspots of thermostable enzymes. We studied the prokaryotic expression and enzymatic properties of a GH3 family glucosidase from Thermofilum adornatum, which has not been reported, aiming to mine superior β-glucosidase. [Methods] We obtained the GH3 amino acid sequence of T.adornatum from NCBI database and constructed the recombinant plasmid pET-30a(+)-TaBgl3. The recombinant protein TaBgl3 was expressed in Escherichia coli BL21(DE3) competent cells under induction. The properties of the enzyme were studied after purification with magnetic beads. [Results] TaBgl3 had a molecular weight of 77.0 kDa and the best performance at pH 5.0 and 80℃. The treatment at 70℃ for 1-4 h improved the enzyme activity, and that at the optimum temperature of 80℃ for 2 h showed the most significant effect, which increased the enzyme activity by more than 40%. The enzyme still had the relative activity of above 60% after being treated 37℃ and pH 5.0-8.0 for 1 h. When the substrate was 4-p-nitrophenyl-β-D-glucopyranoside (pNPG), the enzyme had the specific activity of 144.23 U/mg, the K_m value of 1.81 mmol/L, the maximum reaction rate of 268.10 μmol/(mg·min), and the catalytic efficiency of 115.47/s. Cu²⁺, Li⁺ and EDTA at the final concentration of 5 mmol/L all increased the enzyme activity, which had the maximum increase of 39%. Fe³⁺ (5 mmol/L) and 5% β-mercaptoethanol had inhibitory effect on the enzyme activity. In addition, SDS, ethanol and glucose greatly inhibited the enzyme activity. [Conclusion] TaBgl3 is an acidic thermostable enzyme with good thermal stability and thermal activation characteristics, which can shed light on the future theoretical research and industrial production.