A Novel Glucose-Tolerant GH1 β-Glucosidase and Improvement of Its Glucose Tolerance Using Site-Directed Mutation.
2020
A novel GH1 β-glucosidase gene (bgla) from marine bacterium was sequenced and expressed in Escherichia coli. After purification by Ni2+ affinity chromatography, the recombinant protein was characterized. The purified recombinant enzyme showed maximum activity at 40 °C, pH 7.5 and was stable between temperatures that range from 4 to 30 °C and over the pH range of 6-10. The enzyme displayed a high tolerance to glucose and maximum stimulation at the presence of 100 mM glucose. To improve glucose tolerance of the enzyme, a site-directed mutation (f171w) was introduced into β-glucosidase. The recombinant F171W showed a higher glucose tolerance than the wild type and maintained more than 40% residual activity at the presence of 4 M glucose. Additionally, the recombinant enzymes showed notable tolerance to ethanol. These properties suggest the enzymes may have potential applications for the fermentation of lignocellulosic sugars and the production of biofuels.
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