Targeted screening, recombinant expression and enzymatic properties of β-glucosidase Bgl747

Abstract:

[Background] β-glucosidase (EC3.2.1.21) is one of the important components of the three cellulase enzymes. At present, most of the industrial cellulase are derived from fungi such as Trichoderma, and less derived from bacteria, and there are still problems in application such as narrow application range of reaction conditions (such as temperature, pH), low enzyme activity, and high acquisition cost. These greatly limit the application of β-glucosidase. Screening β-glucosidase from soil bacteria has a great possibility to screen out enzymes with better enzymatic properties, thus solving existing industrial problems. [Objective] Use functional screening method to screen β-glucosidase from soil, obtain a new type of β-glucosidase through gene recombination, expression optimization and protein purification, explore its enzymatic properties, and its industrial application lay the foundation. [Methods] The β-glucosidase was screened from the soil by the functional screening method. Because its full length is 747 bp, it was named Bgl747, and the recombinant expression plasmid pET-28a-Bgl747 was constructed with Escherichia coli BL21(DE3). Induced by IPTG to achieve soluble expression and optimize expression conditions, the purified enzyme was purified by His-tag protein purification kit, and its enzymatic properties were explored. [Results] β-glucosidase Bgl747 as part of the BglB superfamily, its molecular weight is 27.23 kD, and an optimal pH of 4.0, an optimal reaction temperature of 45℃; The optimal induction conditions are:when OD₆₀₀ 1.0, add the final concentration IPTG 0.6 mmol/L, the highest expression level of β-glucosidase Bgl747 protein was 1.82 mg/mL after being induced at 37℃ and 220 r/min for 10 h. The specific enzyme activity when the substrate is p-nitrophenyl-β-D- galactopyranoside (pNPG) is 225.07 U/mg, the Michaelis constant K_m value and the maximum reaction rate are respectively 0.268 mmol/L, 547.23 μmol/(L·min); 1 mmol/L K⁺, 1 mmol/L and 10 mmol/L Fe²⁺, 30% methanol, 30% ethanol, 1 mmol/L and 10 mmol/L guanidine hydrochloride all have a promoting effect on enzyme activity, 30% TritonX-100 and 10 mmol/L SDS inhibits enzyme activity more obviously; The enzyme is feedback inhibition by the product glucose, the greater the glucose concentration, the more obvious the inhibitory effect, but when the glucose concentration is 1 mol/L, the enzyme activity remains above 50%. [Conclusion] Bgl747 has a wide and stable reaction temperature range and excellent enzymatic properties, and lays the foundation for its industrial applications such as cellulose degradation.