Abstract
β-Glucosidase (BG) is widely applied in the biofuel’s industry, as part of a cellulase cocktail to catalyze the hydrolysis of the β-1,4 linkages that join two glucose molecules in a cellulose polymer. The hydrolysis step is generally recognized as the major limiting step in the development of efficient enzyme-based technologies for the conversion of lignocellulosic biomass to sugars and the production of biofuels due to the accumulation of the reaction product, glucose. Relieving this glucose inhibition of BG is therefore a major challenge. In this study, O08324, a putative BG gene encoded in the hyperthermophilic archaeon Thermococcus sp., was cloned and overexpressed in Escherichia coli. O08324 showed maximum activity between pH 5–6.8 and at 78 °C and was thermostable with a half-life of 860 min at 78 °C in the presence of 1.5 M glucose. O08324 was not inhibited by glucose up to the highest assayable concentration of 4 M and also shows no decrease in activity in the presence of up to 4 M of sodium chloride or potassium chloride. O08324 supplementation of Trichoderma viride cellulase enhanced glucose production by more than 50 % compared to a commercially available BG, when Avicel (10 %, w/v) was used as a substrate at 37 °C. Multiple sequence alignments across previously reported glucose-tolerant BGs shows that many conserved residues previously implicated in glucose tolerance are not conserved in this BG suggesting a need for a relook at understanding the molecular basis of glucose tolerance.
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Acknowledgments
This work was supported in part by Rapid Grant for Young Investigators, Department of Biotechnology, Government of India, BT/PR6511/GBD/27/424/2012 (S.D.); Energy Bioscience Overseas Fellowship, Department of Biotechnology, Government of India, BT/NBDB/22/06/2011 (S.D); and IISER Kolkata Academic Research Fund. S.K.S is supported by a Junior Research Fellowship from CSIR, Govt. of India. We thank Ms. Baishali Roy for her help in the cloning and expression studies.
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S.D. and S.K.S designed the research. S.K.S performed the experiments. S.D. wrote the manuscript.
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Sinha, S.K., Datta, S. β-Glucosidase from the hyperthermophilic archaeon Thermococcus sp. is a salt-tolerant enzyme that is stabilized by its reaction product glucose. Appl Microbiol Biotechnol 100, 8399–8409 (2016). https://doi.org/10.1007/s00253-016-7601-x
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DOI: https://doi.org/10.1007/s00253-016-7601-x