Sialyl Lewis x and derivatives have been synthesized using £-1,4-galactosyltransferase and recombinant a-2,3sialyltransferase and a-l,3-fucosyltransferase. The enzymatic glycosylations have been achieved on preparative scales with in situ regeneration of UDP-galactose, CMP-7V-acetylneuraminic acid, and GDP-fucose. Additionally, galactosyltransferase and fucosyltransferases have been studied with respect to their substrate specificity and inhibition. The enzymatic procedures have also been used in the synthesis of 2'-deoxy-LacNAc, 2'-amino-2'-deoxy-LacNAc, 2-azido-Lac, Lewis x, the Lewis x analog with GlcNAc replaced with 5-thioglucose, [Gal-l-13C]-LacNAc, [Gal-1 -13C]-sialyl Lewis x, and the corresponding terminal glycal. The synthesized 13C-labeled sialyl Lewis x and intermediates (including Lewis x and sialyl LacNAc) were used for conformational study using NMR techniques combined with calculations based on GESA and MM2 programs. GESA calculation of sialyl Lewis x gave four minimum-energy conformers, and the two (A and B) consistent with NMR results were further refined with MM2 calculation. The one (A') with lower energy was picked as the preferred conformer which had all intemuclear distances and glycosidic torsional angles consistent with the NMR analysis. The glycosidic torsional angle \p of Gal-GlcNAc, for example, was determined to be 18°on the basis of the coupling between Gal-l-13C and GlcNAc, while the predicted value was 15°. The tetrasaccharide appears to form a well-defined hydrophilic surface along NeuAc-Gal-Fuc, and a hydrophobic face underneath NeuAc-Gal-GlcNAc. Comparing the conformation of sialyl Lewis x to sialyl Lewis a indicates that the recognition domain of sialyl Lewis x mainly comes from the sialic acid-galactose-fucose residues.

The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H2 (2 atm [1 atm = 101.29 kPaJ) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alchohol-adapted strain. In the wild-type strain, ethanol (4%) and H2 (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H2 and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H2 significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which were present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.Thermophilic anaerobic bacteria have potential uses as new biocatalysts for the production of industrial ethanol because they can directly ferment inexpensive substrates such as cellulose, hemicellulose, and starch (18, 20, 28-30, 34, 36). Thermophiles lack industrial utility because they produce low concentrations of ethanol (<2.0% wt/vol). Herrero and co-workers (8-11) have studied the problem of ethanol tolerance in Clostridium thermocellum and concluded that low ethanol tolerance (<3% [wt/vol] ethanol) was a combined result of general solvent effects on membrane fluidity and a specific inhibition of enzymes involved in sugar phosphate metabolism.We previously demonstrated (21) that the wild-type strain 39E of C. thermohydrosulfuricum, which ferments starch, had low ethanol tolerance (i.e., no growth was achieved at 2% [wt/vol] ethanol); however, an ethanol-tolerant strain, 39EA, was selected which was tolerant of >4% (wt/vol) ethanol at 60°C and produced ethanol under these conditions. These studies concluded that direct enzymatic modifications could account for the higher ethanol tolerance of C. thermohydrosulfuricum at 60°C rather than those indirectly caused by membrane disruption in C. thermocellum. In addition to displaying low ethanol tolerance, wild-type strain 39E of C. thermohydrosulfuricum also differs from C. thermocellum (18) by being very sensitive to growth inhibition by hydrogen, another end product of saccharide fermentation (1, 31).The purpose of the present paper is to describe the biochemical basis for both hydrogen tolerance and ethanol tolerance in C. thermohydr...

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To exploit the enzymatic method for the synthesis of β-hydroxy-α-amino acids, the genes coding for the Escherichia coli l-threonine aldolase (LTA; EC 2.1.2.1) and Xanthomonus oryzae d-threonine aldolase (DTA) were cloned and overexpressed in E. coli through primer-directed polymerase chain reactions. The purified recombinant enzymes were studied with respect to kinetics, specificity, stability, additive requirement, temperature profile, and pH dependency. DTA requires magnesium ion as a cofactor, while LTA needs no metal ions. These enzymes work well in the presence of DMSO with concentration up to 40%, and DMSO-induced rate acceleration of LTA-catalyzed reaction was observed. Both enzymes use pyridoxal phosphate coenzyme to activate glycine to react with a wide range of aldehydes. LTA gave erythro-β-hydroxy-α-l-amino acids with aliphatic aldehydes and the threo isomer with aromatic aldehydes as kinetically controlled products. On the other hand, DTA formed threo-β-hydroxy-α-d-amino acids as kinetically controlled products with aliphatic and aromatic aldehydes but the diastereoselectivity was lower than that of LTA. Under optimal conditions, several β-hydroxy-α-amino acid derivatives (3-hydroxyleucines, γ-benzyloxythreonines, γ-benzyloxymethylthreonines, and polyoxamic acids) have been stereoselectively synthesized on preparative scales using these enzymes. Also, the tandem use of DTA and phosphatases has made possible the synthesis and separation of d-allo-threonine phosphate and d-threonine.

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