The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties,such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4M glucose/1.0M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-D-glucopyranoside as substrate the values were Vmax (7614 U/mg), and KM (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and KM 0.877mM (glucose), and 1.410mM (xylose).
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process no 2018/07522-6) and a scholarship to Tassio B. Oliveira (grant 2017/09000-4). MLTMP is a Research Fellow of Conselho de Desenvolvimento Científico e Tecnológico (CNPq, process 301963/2017-7). The project also received grants from National Institute of Science and Technology of Bioethanol,INCT, CNPq 465319/2014-9/FAPESP nº 2014/50884-5). PZA was a fellow from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) funding code 001 and Programa de Doutorado Sanduiche no Exterior, PDSE no 88881.135684/2016-01.