Characterization of a Cu2+, SDS, alcohol and glucose tolerant GH1 β-glucosidase from Bacillus sp. CGMCC 1.16541

Abstract

A β-glucosidase gene (bsbgl1a) from Bacillus sp. CGMCC 1.16541 was expressed in Escherichia coli BL21 and subsequently characterized. The amino acid sequence shared 83.64% identity with β-glucosidase (WP_066390903.1) from Fictibacillus phosphorivorans. The recombinant β-glucosidase (BsBgl1A) had a molecular weight of 52.2 kDa and could hydrolyze cellobiose, cellotriose, cellotetrose, p-nitrophenyl-β-d-glucopyranoside (pNPG), and p-nitrophenyl-β-d-xylopyranoside (pNPX). Optimal activity for BsBgl1A was recorded at 45 °C with a pH between 5.6 and 7.6, and 100% of its activity was maintained after a 24 h incubation between pH 4 and 9. Kinetic characterization revealed an enzymatic turnover (Kcat) of 616 ± 2 s⁻¹ (with cellobiose) and 3.5 ± 0.1 s⁻¹ (with p-nitrophenyl-β-d-glucopyranoside). Interestingly, the recombinant enzyme showed cupric ion (Cu²⁺), sodium dodecyl sulfate (SDS) and alcohol tolerance at 10 mM for Cu²⁺ and 10% for both SDS and alcohol. Additionally, BsBgl1A had high tolerance for glucose (Ki = 2095 mM), which is an extremely desirable feature for industrial applications. Following the addition of BsBgl1A (0.05 mg/ml) to a commercial cellulase reaction system, glucose yields from sugarcane bagasse increased 100% after 1 day at 45 °C. This work identifies a Cu²⁺, SDS, alcohol, and glucose tolerant GH1 β-glucosidase with potential applications in the hydrolysis of cellulose for the bioenergy industry.