Genome Mining Of A Thermotolerant　β-glucosidase And Its Catalytic Performance In Octyl Glucoside Synthesis

Alkylβ-D-glucosides, a group of green and non-ionic surfactants, are widely used in pharmaceuticals, detergents, and food ingredients. The enzymatic synthesis of alkyl glucosides has many attractive features:the reaction can avoid tedious protection/deprotection steps and meet the requirements of environmental safety and cleanliness. Reversed hydrolysis can accept unprotected glucose as substrate and the purification of the product is easier as it contains water as by-product.A newβ-glucosidase (DtGH) representing 40% identity with an apple seed glycosidase (ASG) was cloned from Dictyoglomus thermophilum. DtGH was overexpressed in Escherichia coli and purified by heat treatment and His-Trap affinity chromatography. The optimal temperature and pH were found to be 90℃and 7.0, respectively. The half-lives of the enzyme at 70,80, and 90℃were 533,44, and 5.0 h, respectively. The Km and kcatat values of the recombinant DtGH towards pNPG were 1.15 mM and 238 s-1. Besides the substrate of pNPG, DtGH also had a significant activity on pNPGal. Among alkyl glucosides, when the alkyl chain was changed from methyl to octyl, the activity of DtGH increased first and then decreased. DtGH could also hydrolyze arylalkyl glucosides such as salidroside.DtGH exhibited excellent stability under the reaction environment, retaining 70-80% of its initial activity after 7 d of incubation at 70℃in either glucose solution or octanol-aqueous (85:15, v/v) system. It could retain part of synthetic activity even in boiling water. By using this enzyme, when the temperature for direct glycosylation of n-octanol increased from 50℃to 70℃, the glucose based conversion was increased by 27%, but the time spent to reach equilibrium was decreased from 7 d to 3 d. Owing to the strong glucose-tolerance and extremely high thermostability, DtGH should be promising for various glucosides synthesis.