Practical Preparation of D-Galactosyl-β1→4-L-rhamnose Employing the Combined Action of Phosphorylases

D-Galactosyl-β1→4-L-rhamnose (GalRha) was produced enzymatically from 1.1 M sucrose and 1.0 M L-rhamnose by the concomitant actions of four enzymes (sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, and D-galactosyl-β1→4-L-rhamnose phosphorylase) in the presence of 1.0 mM UDP-glucose and 30 mM inorganic phosphate. The accumulation of GalRha in 1 liter of the reaction mixture reached 230 g (the reaction yield was 71% from L-rhamnose). Sucrose and fructose in the reaction mixture were removed by yeast treatment, but isolation of GalRha by crystallization after yeast treatment was unsuccessful. Finally, 49 g of GalRha was isolated from part of the reaction mixture with yeast treatment by gel-filtration chromatography.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Homogeneous Enzymatic Assay for L-Cysteine with βC-S Lyase

We have developed a new enzymatic assay for determining L-cysteine concentration. The method involves the use of βC-S lyase from Streptococcus anginosus, which catalyzes the α,β-elimination of L-cysteine to hydrogen sulfide, pyruvate, and ammonia. The production of pyruvate is measured by D-lactate dehydrogenase and NADH. The decrease in NADH was proportional to the L-cysteine concentration up to 1.0 mM. When serum samples were used, within-day and day-to-day coefficient variations were below 4%. This method is simple, and can easily and reliably be used for accurate determination of L-cysteine concentration in serum or other samples.     

Characterization of Mesorhizobium loti L-Rhamnose Isomerase and Its Application to L-Talose Production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.     

Isolation and Biological Activity of Aspermutarubrol,a Self-growth- inhibitor from Aspergillus sydowi

Isolated hepatocytes are known to maintain their physiological functions for over a week when cultured on Matrigel, artificially reconstituted from basement membrane components. Although this culture technique has been frequently used in research on hepatocyte functions, there has been a limitation on its application for small scale experiments due to some technical problems. By using micro-culture plates with 96 round-bottom wells, we succeeded in coating the wells uniformly with Matrigel. When the cultured hepatocytes were treated with either 10 mM, 15 mM, or 20 mM of acetaminophen or 1 mM, 10 mM, or 20 mM of D-galactosamine, the viability of the hepatocytes became 91.1%, 75.3%, 64.7%, and 79.0%, 43.8%, 26.2% of the non-treated control at 48 hours, respectively. Fractionated extracts of Glycyrrhiza glabra L. and Schisandra chinensis Baillon inhibited the action of acetaminophen or D-galactosamine in this model. From these results, we concluded that the microculture system presented here is capable of maintaining the in vivo characteristics of hepatocytes and is suitable for the screening of hepatoprotective substances.     

Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.     

Main Polyol Dehydrogenase of Gluconobacter suboxydans IFO 3255, Membrane-bound D-Sorbitol Dehydrogenase,That Needs Product of Upstream Gene,sldB, for Activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

Antioxidative Effect of an Isolate from a Culture Filtrate of Aspergillus niger

D-Ins(1,3,4,5)P₄ and unnatural L-Ins(1,3,4,5)P₄ were prepared in gram-quantities from D- and L-2,6-di-O-benzyl-myo-inositol by a chemical phosphorylation and deprotection step in high yield and purity without extensive purification. The optically pure benzyl derivatives were obtained by enzyme-catalyzed resolution of racemic 2,6-di-O-benzyl-myo-inositol under acyl-transfer conditions in vinyl acetate as the acyl donor. The lipase of Candida antarctica only acetylated regio- and enantio-selectively the L-enantiomer, providing exclusively L-5-acetyl-2,6-di-O-benzyl-myo-inositol, whereas the D-enantiomer remained unchanged.     

Substrate Specificity of Thermostable D-Alanine-D-alanine ligase from Thermotoga maritima ATCC 43589

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 °C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

New Polar Constituents in the Pupae of the Silkworm Bombyx mori L. (II): Developmental Changes of the Constituents

A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. ¹H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.     

Structural Analysis of the Fundamental Polymer of the Sheath Constructed by Sphaerotilus natans

The sheath of Sphaerotilus natans is composed of cysteine-rich peptide and polysaccharide moieties. The polysaccharide was prepared by treating the sheath with hydrazine, and was determined to be a mucopolysaccharide containing β-D-GlcA, β-D-Glc, α-D-GalN, and β-D-GalN. To elucidate the structure of the peptide, the sheath was labeled with a thiol-selective fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Enantiomeric determination of the S-derivatized Cys in the fluorescent sheath suggested that it contained L-Cys mainly. Fluorescent cysteinylglycine was detected in the partial acid hydrolysate of the fluorescent sheath. The sheath-degrading enzyme secreted by Paenibacillus koleovorans produced a fluorescent disaccharide-dipeptide composed of GalN, Gly, and N-acetylated Cys from the fluorescent sheath. The disaccharide and dipeptide moieties were found to be connected by an amide bond. Based on these results, the sheath was deduced to be formed by association of a mucopolysaccharide modified with N-acetyl-L-cysteinylglycine.     

Conversion of L-Galactono-1,4-lactone to L-Ascorbate Is Regulated by the Photosynthetic Electron Transport Chain in Arabidopsis

In this study we focused on the effects of light irradiation and the addition of L-galactono-1,4-lactone (L-GalL) on the conversion of exogenous L-GalL to L-ascorbate (AsA) and the total AsA pool size in detached leaves of Arabidopsis plants and transgenic plants expressing the rat L-gulono-1,4-lactone oxidase gene. Increases in the total AsA level in L-GalL-treated leaves depended entirely on light irradiation. Treatment with an inhibitor of photosynthetic electron transport together with L-GalL reduced the increase in total AsA under light. Light, particularly the redox state of photosynthetic electron transport, appeared to play an important role in the regulation of the conversion of L-GalL to AsA in the mitochondria, reflecting the cellular level of AsA in plants.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Cloning,Expression, and Identification of Genes Involved in the Conversion of DL-2-Amino-Δ2-thiazoline-4-carboxylic Acid to L-Cysteine via S-Carbamyl-L-cysteine Pathway in Pseudomonas sp. TS1138

Two novel genes (tsB, tsC) involved in the conversion of DL-2-amino-Δ²-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine through S-carbamyl-L-cysteine (L-SCC) pathway were cloned from the genomic DNA library of Pseudomonas sp. TS1138. The recombinant proteins of these two genes were expressed in Escherichia coli BL21, and their enzymatic activity assays were performed in vitro. It was found that the tsB gene encoded an L-ATC hydrolase, which catalyzed the conversion of L-ATC to L-SCC, while the tsC gene encoded an L-SCC amidohydrolase, which showed the catalytic ability to convert L-SCC to L-cysteine. These results suggest that tsB and tsC play important roles in the L-SCC pathway and L-cysteine biosynthesis in Pseudomonas sp. TS1138, and that they have potential applications in the industrial production of L-cysteine.     

Regioselectivity in β-Galactosidase-catalyzed Transglycosylation for the Enzymatic Assembly of D-Galactosyl-D-mannose

The regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.     

Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.     

Genetic Engineering of Candida utilis Yeast for Efficient Production of L-Lactic Acid

Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.     

Effects of a Feedback-Resistant Aspartokinase III Gene on L-Isoleucine Production in Escherichia coli K-12

An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.