Identification and Characterization of a Novel α-L-Fucosidase from Enterococcus gallinarum and Its Application for Production of 2'-Fucosyllactose.

2'-fucosyllactose (2'FL) is an important nutrient in human milk that stimulates beneficial microbiota and prevents infection. α-L-fucosidase is a promising component for 2'FL synthesis. In this study, a soil-oriented α-L-fucosidase-producing strain from Enterococcus gallinarum ZS1 was isolated. Escherichia coli was employed as a host for cloning and expressing the α-L-fucosidase gene (entfuc). The EntFuc was predicted as a member of the GH29 family with a molecular mass of 58 kDa. The optimal pH and temperature for the activity of EntFuc were pH 7.0 and 30 °C, respectively. The enzyme exhibited a strictly specific activity for 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and had a negligible effect on hydrolyzing 2'FL. EntFuc could catalyze the synthesis of 2'FL via transfucosylation action from pNP-Fuc and lactose. The yield of 2'FL reached 35% under optimal conditions. This study indicated that EntFuc with a high conversion rate is a promising enzyme source for the biosynthesis of 2'FL.

Coevolutionary analysis reveals a distal amino acid residue pair affecting the catalytic activity of GH5 processive endoglucanase from Bacillus subtilis BS-5.

EG5C-1, processive endoglucanase from Bacillus subtilis, is a typical bifunctional cellulase with endoglucanase and exoglucanase activities. The engineering of processive endoglucanase focuses on the catalytic pocket or carbohydrate-binding module tailoring based on sequence/structure information. Herein, a computational strategy was applied to identify the desired mutants in the enzyme molecule by evolutionary-coupling analysis; subsequently, four residue pairs were selected as evolutionary mutational hotspots. Based on iterative-saturation mutagenesis and subsequent enzymatic activity analysis, a superior mutant K51T/L93T has been identified away from the active center. This variant had increased specific activity from 4170 U/µmol of wild-type (WT) to 5678 U/µmol towards carboxymethyl cellulose-Na and an increase towards the substrate Avicel from 320 U/µmol in WT to 521 U/µmol. In addition, kinetic measurements suggested that superior mutant K51T/L93T had a high substrate affinity (Km ) and a remarkable improvement in catalytic efficiency (kcat /Km ). Furthermore, molecular dynamics simulations revealed that the K51T/L93T mutation altered the spatial conformation at the active site cleft, enhancing the interaction frequency between active site residues and substrate, and improving catalytic efficiency and substrate affinity. The current studies provided some perspectives on the effects of distal residue substitution, which might assist in the engineering of processive endoglucanase or other glycoside hydrolases.