Enzymatic synthesis of [beta]-glucosylglycerol using a continuous-flow microreactor containing thermostable [beta]-glycoside hydrolase CelB immobilized on coated microchannel walls

β-Glucosylglycerol (βGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable β-glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of βGG under conditions of continuous flow at 70...C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 ...L, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2-nitrophenyl-β-D-glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25-1.0 M), and the average residence time (0.2-90 s). Glycerol caused a concentration-dependent decrease in the conversion of the cdonor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of βGG were ≥80% and ...60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (≥80%), giving about 120 mM (30 g/L) of βGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1-O-β-D-glucopyranosyl-rac-glycerol (79%) and 2-O-β-D-glucopyranosyl-sn-glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme. (ProQuest: ... denotes formulae/symbols omitted.)