Glycosynthase Activity of Bacillus licheniformis 1,3-1,4-β-Glucanase Mutants:  Specificity, Kinetics, and Mechanism

Abstract

Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficiently catalyze transglycosylation reactions. The mechanism of the glycosynthase reaction is probed with the E134A mutant of Bacillus licheniformis 1,3-1,4-beta-glucanase. This endo-glycosynthase is regiospecific for formation of a beta-1,4-glycosidic bond with alpha-glycosyl fluoride donors (laminaribiosyl as the minimal donor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (aryl monosaccharides or oligosaccharides). The pH dependence of the glycosynthase activity reflects general base catalysis with a kinetic pK(a) of 5.2 +/- 0.1. Kinetics of enzyme inactivation by a water-soluble carbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pK(a) of 5.3 +/- 0.2. The general base is Glu138 (the residue acting as the general acid-base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A. It is devoid of glycosynthase activity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a beta-glycosyl azide product. The pK(a) of Glu138 (kinetic pK(a) on k(cat)/K(M) and pK(a) of EDC inactivation) for the E134A glycosynthase has dropped 1.8 pH units compared to the pK(a) values of the wild type, enabling the same residue to act as a general base in the glycosynthase enzyme. Kinetic parameters of the E134A glycosynthase-catalyzed condensation between Glcbeta4Glcbeta3GlcalphaF (2) as a donor and Glcbeta4Glcbeta-pNP (15) as an acceptor are as follows: k(cat) = 1.7 s(-)(1), K(M)(acceptor) = 11 mM, and K(M)(donor) < 0.3 mM. Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions for preparative use of the glycosynthase reaction in oligosaccharide synthesis. Yields are 70-90% with aryl monosaccharide and cellobioside acceptors, but 25-55% with laminaribiosides, the lower yields (and lower initial rates) due to competitive inhibition of the beta-1,3-linked disaccharide acceptor for the donor subsites of the enzyme.

Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

“…Half of the culture (0.5 ml) was transferred to 100 ml of fresh medium and incubated at 37 C with shaking. When the absorbance at 600 nm reached 0.6, protein expression was induced by the addition of isopropyl -D-1-thiogalactopyranoside to a final concentration of 1 mM, and cultivation was continued overnight at 30 C with shaking. The cells were harvested by centrifugation at 10;000 Â g for 10 min and resuspended in 2 ml of 10 mM phosphate buffer (pH 7).…”

“…The hydrolytic activities of all mutant xylanases were confirmed using xylohexaose (X 6 ) and 4-nitrophenyl -D-xylobioside (pNP-X 2 ). The mutant xylanases (0.5 mg/ml) were incubated with 40 mM X 6 in 10 mM MES buffer (pH 6.1) at 30 C for 5 h. The derivatives hydrolyzed by mutant xylanases from X 6 were analyzed by thin layer chromatography (TLC). In the case of the highly sensitive enzyme assay method, the mutant xylanases (2 mg/ml) were incubated with 1 mM pNP-X 2 in 50 mM MES (pH 6.1) at 30 C for 90 min.…”

“…The mutant xylanases (0.5 mg/ml) were incubated with 40 mM X 6 in 10 mM MES buffer (pH 6.1) at 30 C for 5 h. The derivatives hydrolyzed by mutant xylanases from X 6 were analyzed by thin layer chromatography (TLC). In the case of the highly sensitive enzyme assay method, the mutant xylanases (2 mg/ml) were incubated with 1 mM pNP-X 2 in 50 mM MES (pH 6.1) at 30 C for 90 min. The amount of 4-nitrophenol liberated from pNP-X 2 was quantified by measuring the absorbance at 400 nm after adding an equal volume of 1 M Na 2 CO 3 .…”

“…20) Subsequently, several glycosynthases derived from variousglycosidases have been reported from both exo- [21][22][23][24][25][26] and endo-hydrolases. [27][28][29][30][31][32][33] An -glycoside-forming glycosynthase was also demonstrated with an -glucosidase belonging to GH 31. 34) Recently, we reported the first glycosynthase derived from an inverting enzyme, reducing-end-xylose releasing exo-oligoxylanase, belonging to GH 8.…”

The Family 11 Carbohydrate-binding Module of Clostridium thermocellum Lic26A-Cel5E Accommodates β-1,4- and β-1,3–1,4-Mixed Linked Glucans at a Single Binding Site

“…Cello-oligosaccharides were from Seikagaku Corp. Avicel (PH101) was obtained from Serva, whereas acid-swollen cellulose was prepared as described previously (18). The mixed linked gluco-oligosaccharides Glc-␤1,4-Glc-␤1,4-Glc-␤1,3-Glc and Glc-␤1,3-Glc-␤1,4-Glc-␤1,3-Glc were synthesized by the method of Faijes et al (19).…”