Characterization of a glucose-stimulated ß-glucosidase from Microbulbifer sp. ALW1.

Glucose-tolerant and/or glucose-stimulated ß-glucosidase is of great interest for its industrial utilization in enzymatic digestion of lignocellulosic biomass for biofuel production. In this study, a new gene of ß-glucosidase MaGlu1A was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene of MaGlu1A encoded a 472-amino acid protein classified into the glycosyl hydrolase family 1 (GH1). The recombinant ß-glucosidase was overexpressed and purified from Escherichia coli with a molecular mass of 65.0 kDa. Structure analysis illustrated the catalytic acid/base residue Glu186 and nucleophilic residue Glu370 in the enzyme. MaGlu1A displayed optimal activity at 40 °C and pH 4.5, respectively. It had substrate preference to the aryl-ß-glycosidic bonds with glucose, fucose, and galactose moieties, in addition to cellobiose. MaGlu1A demonstrated strong stimulation to the supplemental glucose. Site-directed mutagenesis suggested an essential role of Asn242 in glucose stimulation. The enzymatic characterization of MaGlu1A provides general information about its catalytic properties facilitating its practical applications.

A mutant of Pseudoalteromonas carrageenovora arylsulfatase with enhanced enzyme activity and its potential application in improvement of the agar quality.

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.