Expression of a thermostable glucose-stimulated β-glucosidase from a hot-spring metagenome and its promising application to produce gardenia blue

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This study reports a thermostable glucose-stimulated β-glucosidase, BglY442, from hot-spring metagenomic data that was cloned and expressed in Escherichia coli BL21 (DE3). The molecular mass of recombinant BglY442 was 69.9kDa and was used in the production of gardenia blue. The recombinant BglY442 showed its maximum activity at pH 6.0 and 75°C, maintained 50% activity at 70°C for 36h, presented over 90% activity in a broad pH range and a wide range of pH stability. Moreover, BglY442 exhibited excellent tolerance toward methanol and ethanol. The specific activity of BglY442 was 235 U/mg at pH 6.0 and 75°C with 10mM pNPG as substrate. BglY442 activity increased by over fourfold with 2M glucose or xylose. Specifically, the enzyme kinetics of BglY442 seem to be non-Michaelis-Menten kinetics or atypical kinetics because the Michaelis-Menten saturation kinetics were not observed with pNPG, oNPG or geniposide as substrates. Under optimum conditions, geniposide was dehydrated by BglY442 and reacted with nine amino acids respectively by the one-pot method. Only the Arg or Met derived pigments showed bright blue, and these two pigments had similar ultraviolet absorption spectra. The OD590 nm of GB was detected to be 1.06 after 24h with the addition of Arg and 1.61 after 36h with the addition of Met. The intermediate was elucidated and identified as ginipin. Molecular docking analysis indicated that the enzyme had a similar catalytic mechanism to the reported GH1 Bgls. BglY442 exhibited potential for gardenia blue production by the one-pot method. With outstanding thermostability and glucose tolerance, BglY442 should be considered a potential β-glucosidase in biotechnology applications.