- Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum
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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.
- Nakai, Hiroyuki,Hachem, Maher Abou,Petersen, Bent O.,Westphal, Yvonne,Mannerstedt, Karin,Baumann, Martin J.,Dilokpimol, Adiphol,Schols, Henk A.,Duus, Jens ?.,Svensson, Birte
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experimental part
p. 1818 - 1826
(2011/08/21)
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- Study of the action of human salivary alpha-amylase on 2-chloro-4-nitrophenyl α-maltotrioside in the presence of potassium thiocyanate
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The degradation mechanism of a synthetic substrate, 2-chloro-4-nitrophenyl α-maltotrioside (CNP-G3), by human salivary alpha-amylase (HSA) was investigated by kinetic and product analyses. It was observed that the enzyme attacked the various CNP-maltooligosaccharides (CNP-G3, to CNP-G6) releasing free CNP. Addition of 500 mM potassium thiocyanate (KSCN) was also found to greatly increase the rates of CNP-release. It was the fastest with CNP-G3, and, in the presence of KSCN, was almost comparable to that of degradation of maltopentaose (G5). On the other hand, addition of KSCN decreased the rate of cleavage between glucan-glucan bonds in maltopentaose. Product analysis showed that KSCN addition altered the cleavage distribution which occurred 100% at the bond between CNP and G3, and that product distribution of free CNP was largely dependent on substrate concentration. Formation of CNP-G6, a larger product than the original substrate CNP-G3, was found to be present in the digest at high concentrations of substrate and in the presence of KSCN. Based on these results, a degradation pathway for CNP-G3 involving transglycosylation besides direct hydrolysis is proposed. The increase of the CNP-release by the addition of KSCN would result from a corresponding increase in the interaction between the CNP moiety and the corresponding subsite near the catalytic site, as well as the enhancement of the catalytic efficiency.
- Suganuma, Toshihiko,Maeda, Yoshiaki,Kitahara, Kanefumi,Nagahama, Tomonori
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p. 219 - 227
(2007/10/03)
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- Subsite Structure of Chalara paradoxa Glucoamylase and Interaction of the Glucoamylase with Cyclodextrins
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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.
- Monma, Mitsuru,Yamamoto, Yoshihiro,Kainuma, Keiji
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p. 1503 - 1508
(2007/10/02)
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