1184-46-9Relevant articles and documents
Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum
Nakai, Hiroyuki,Hachem, Maher Abou,Petersen, Bent O.,Westphal, Yvonne,Mannerstedt, Karin,Baumann, Martin J.,Dilokpimol, Adiphol,Schols, Henk A.,Duus, Jens ?.,Svensson, Birte
experimental part, p. 1818 - 1826 (2011/08/21)
Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-d-glucopyranosyl-[(1→4)- β-d-glucopyranosyl]n-(1→2)-d-glucopyranose, and β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]n- (1→3)-d-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.
Subsite Structure of Chalara paradoxa Glucoamylase and Interaction of the Glucoamylase with Cyclodextrins
Monma, Mitsuru,Yamamoto, Yoshihiro,Kainuma, Keiji
, p. 1503 - 1508 (2007/10/02)
The action of Chalara paradoxa glucoamylase (raw-starch-digesting enzyme) was studied with linear and cyclic maltodextrins.Subsite affinities (Ai) of the amylase were evaluated by the subsite theory.The active site was considered to be made up of seven subsites: A1 = 0.05 kcal/mol, A2 = 4.99 kcal/mol, A3 = 1.30 kcal/mol, A4 = 0.77 kcal/mol, A5 = 0.33 kcal/mol, A6 = 0.21 kcal/mol and A7 = 0.21 kcal/mol.Inhibitions by alpha-, beta-, and gamma-cyclodextrins were competitive for starch digestion by C. paradoxa glucoamylase.The inhibitor constants (Ki) of α-, β-, and γ-cyclodextrin for the amylase were 8.9, 1.4, and 3.9 mM, respectively.The Michaelis constant (Km) of 6-O-α-maltosyl-α-cyclodextrin digestion was 0.79 mM for the amylase.
