%D 2012 %0 MEDLINE %T Rice BGlu1 glycosynthase and wild type transglycosylation activities distinguished by cyclophellitol inhibition . %J Carbohydr Res %V 352 %P 51-9 %A Pengthaisong S %A Chen CF %A Withers SG %A Kuaprasert B %A Ketudat Cairns %M pub22418094 %X The rice BGlu1 beta-D-glucosidase nucleophile mutant E386G is a glycosynthase that catalyzes the synthesis of cellooligosaccharides from alpha-d-glucopyranosyl fluoride ( GlcF ) donor and p-nitrophenyl ( pNP ) cellobioside ( Glc2-pNP ) or cello-oligosaccharide acceptors . When activity with other donors and acceptors was tested , the initial enzyme preparation cleaved pNP-beta-D-glucopyranoside ( Glc-pNP ) and pNP-beta-D-fucopyranoside ( Fuc-pNP ) to pNP and glucose and fucose , suggesting contamination with wild type BGlu1 beta-glucosidase . The products from reaction of GlcF and Fuc-pNP included Fuc-beta- ( 1-->3 ) -Fuc-pNP , Glc-beta- ( 1-->3 ) -Fuc-pNP , and Fuc-beta- ( 1-->4 ) -Glc-beta- ( 1-->3 ) -Fuc-pNP , suggesting the presence of both wild type BGlu1 and its glycosynthase . Inhibition of the BGlu1 beta-glucosidase activity within this preparation by cyclophellitol confirmed that the E386G glycosynthase preparation was contaminated with wild type BGlu1 . Rice BGlu1 E386G-2 , generated from a new construct designed to minimize back-mutation , showed glycosynthase activity without wild type hydrolytic or transglycosylation activity . E386G-2 catalyzed transfer of glycosyl residues from GlcF , alpha-L-arabinosyl fluoride , alpha-D-fucosyl fluoride , alpha-D-galactosyl fluoride , alpha-D-mannosyl fluoride , and alpha-D-xylosyl fluoride donors to Glc2-pNP acceptor . The synthetic products from the reactions of alpha-fucosyl fluoride and alpha-mannosyl fluoride donors were confirmed to result from addition of a beta- ( 1-->4 ) -linked glycosyl residue . Moreover , the E386G glycosynthase transferred glucose from GlcF donor to glucose , cellobiose , Glc-pNP , Fuc-pNP , pNP-beta-D-galactopyranoside , and pNP-beta-D-xylopyranoside acceptors , but little to pNP-beta-D-mannopyranoside . Production of longer oligosaccharides occurred most readily on acceptors with an equatorial 4-OH . Elimination of wild type contamination thereby allowed a clear assessment of BGlu1 E386G glycosynthase catalytic abilities .