Improved oligosaccharide synthesis by protein engineering of b-glucosidase from hyperthermophilic Pyrococcus furiosus

T. Hanson, T. Kaper, J. van der Oost, W.M. de Vos

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67 Citations (Scopus)

Abstract

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type -glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type -mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95°C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45 percent compared with the wild-type CelB (40 percent Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20 percent lactose concentrations compared to the wild-type. At a lactose concentration of 10 percent the oligosaccharide yield for the mutant was 40 percent compared to 18 percent for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10 percent lactose) compared to the wild-type (0.19, 70 percent lactose). At a lactose concentration as low as 10 percent only trisaccharides were synthesized by CelB wild-type. The -mannosidase BmnA from P. furiosus showed both -glucosidase and -galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44&Eth;The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating -glycosidases in oligosaccharide synthesis from lactose
Original languageEnglish
Pages (from-to)203-210
JournalBiotechnology and Bioengineering
Volume73
DOIs
Publication statusPublished - 2001

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