Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

The Mutational Effect of Ile58 at Subsite C in Hen Egg-White Lysozyme on Substrate Binding,Enzymatic Activity,and Protein Stability

Partial acid hydrolysis of Saccharomyces cerevisiae mannan gave 2-O-α-d-Manp-d-Man (1), 3-O-α-d-Manp-d-Man (2), 6-O-α-d-Manp-d-Man (3), O-α-d Manp-(1→2)O-α-d-Manp-(1→2)-d-Man (4), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-d-Man (5), O-α-d Manp-(1→6)-6-O-α-d-Manp-(1→6)-d-Man (6), O-α-d Manp-(1→2)-O-α-d-Manp-(1→2)-6-O-α-d-Manp-(1→6)-d-Man (7), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-O-α-d-Manp-(1→6)-d-Man (8), and O-α-d-Manp-(1→6)-O-[α-d-Manp-(1→2)]-O-α-d-Manp-(1→6)-d-Man (9).     

Two novel pyrrolooxazole pigments formed by the Maillard reaction between glucose and threonine or serine

Pyrrolothiazolate formed by the Maillard reaction between l-cysteine and d-glucose has a pyrrolothiazole skeleton as a chromophore. We searched for a Maillard pigment having a pyrrolooxazole skeleton formed from l-threonine or l-serine instead of l-cysteine in the presence of d-glucose. As a result, two novel yellow pigments, named pyrrolooxazolates A and B, were isolated from model solutions of the Maillard reaction containing l-threonine and d-glucose, and l-serine and d-glucose, respectively, and identified as (2R,3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-2,5,7a-trimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid and (3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-5,7a-dimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid by instrumental analyses. These compounds were pyrrolooxazole derivatives carrying a carboxy group, and showed the absorption maxima at 300–360 nm under acidic and neutral conditions and at 320–390 nm under alkaline conditions.     

Distribution of 7-Keto-8-aminopelargonic Acid Synthetase in Bacteria and the Control Mechanism of the Enzyme Activity

The 7-keto-8-aminopelargonic acid (KAPA) synthetase activities of cell-free extracts from various bacteria were investigated. The experiments on the substrate specificity of KAPA synthetase, using crude cell-free extracts from bacteria having high enzyme activity, showed that l-serine and pyruvic acid could replace l-alanine, but that, when the enzyme was partially purified, these compounds were not effective. Many kinds of amino acids such as l-cysteine, l-serine, d-alanine, glycine, d-histidine, and l-histidine, inhibited the enzyme activity. This inhibition was found to be competitive with l-alanine. Pyridoxal 5′-phosphate, which is a cofactor of the enzyme, also inhibited the enzyme activity at high concentrations. The repression of KAPA synthetase by biotin occurred in Bacillus subtilis and B. sphaericus but not in Micrococcus roseus and Pseudomonas fluorescens, even at a concentration of 1000 mµg per ml of biotin.     

The Acceptor Specificity of Amylomaltase from Escherichia coli IFO 3806

The acceptor specificity of amylomaltase from Escherichia coli IFO 3806 was investigated using various sugars and sugar alcohols. d-Mannose, d-glucosamine, N-acetyl- d-glucosamine, d-xylose, d- allose, isomaltose, and cellobiose were efficient acceptors in the transglycosylation reaction of this enzyme. It was shown by chemical and enzymic methods that this enzyme could transfer glycosyl residues only to the C4-hydroxyl groups of d-mannose, iY-acetyl- d-glucosamine, d-allose, and d-xylose, producing oligosaccharides terminated by 4–0-α-d-glucopyranosyl-d-mannose, 4–0-α-d-glucopyranosyl-yV-acetyl-d-glucosamine, 4-O-α-d-glucopyranosyl-d-allose, and 4–0-α-d-gluco- pyranosyl-d-xylose at the reducing ends, respectively.     

Structure of Carbohydrate Moiety of Asterosaponin A

Partial acid hydrolysis of asterosaponin A, a steroidal saponin, afforded two new disaccharides in addition to O-(6-deoxy-α-d-glucopyranosyl)-(l→4)-6-deoxy-d-glucose which has been characterized in the preceding paper. The formers were demonstrated as O-(6-deoxy-α-d-galactopyranosyl)-(1→4)-6-deoxy-d-glucose and O-(6-deoxy-α-d-galactopyranosyl)-(l→4)-6-deoxy-d-galactose, respectively.

Accordingly, the structure of carbohydrate moiety being composed of two moles each of 6-deoxy-d-galactose and 6-deoxy-d-glucose, was established as O-(6-deoxy-α-d-galactopyranosyl)-(l→4)-O-(6-deoxy-α-d-galactopyranosyl)-(l→4)-O-(6-deoxy-α-d-glucopyranosyl)-(l→4)-6-deoxy-d-glucose, which is attached to the steroidal aglycone through an O-acetal glycosidic linkage.     

Effects of Glycine and d-Amino Acids on Growth of Various Microorganisms

Growth of various microorganisms in media containing high concentrations of glycine or d-amino acids was examined. Susceptibilities to glycine or d-amino acids differed among microorganisms, and the differences in susceptibility have no direct relation with Gram staining, morphological forms, and aerobic or anaerobic nature of the organisms. Certain glycine-resistant bacteria tested, which included Bacillus cereus, Staphylococcus aureus and Serratia marcescens, exhibited relatively high oxidative activities towards glycine. The inhibition of the growth of Escherichia coli by either glycine or d-amino acids, which included d-threonine, d-alanine and d-lysine, was reversed by l-alanine, partialy by l-serine, and not by l-lysine or l-threonine. These results suggest that the growth inhibition of microorganisms by d-amino acids was similar to that by glycine. The incorporation of l-alanine into E. coli cells which were preincubated with glycine was less than those of preincubated without glycine. Particularly, the incorporation into the cell wall fraction was most susceptible to glycine. An additive effect of penicillin and glycine was observed in the inhibition of cell wall biosynthesis as determined by the intracellular accumulation of N-acetylamino sugar compounds.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

Synthesis of Certain Disaccharides Containing a α-or β- Rhamnopyranosidic Group and the Substrate Specificity of α-l-Rhamnosidase from Aspergillus niger

To investigate the substrate specificity of α-l-rhamnosidase from Aspergillus niger, the following seven substrates were synthesized: methyl 3-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (1), methyl 3-O-α-l-rhamnopyranosyl-α-l-xylopyranoside (2), methyl 3-0-α-l-rhamnopyranosyl-α-l-rhamnopyranoside (3), methyl 4-0-α-l-rhamnopyranosyl-α-d-galactopyranoside (4), methyl 4-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (5), methyl 4-0-α-l-rhamnopyra-nosyl-α-d-xylopyranoside (6), and 6-0-β-l-rhamnopyranosyl-d-mannopyranose (7). Compounds 1~6 were well-hydrolyzed by the crude enzyme, but 7 was unaffected.     

Pectin Isolated from the Midrib of Leaves of Nicotiana tabacum

A pectin isolated from tobacco midrib contained residues of d-galacturonic acid (83.7%), L-rhamnose (2.2%), l-arabinose (2.4%) and d-galactose (11.2%) and small amounts of d-xylose and d-glucose. Methylation analysis of the pectin gave 2, 3, 5-tri- and 2, 3-di-O-methyl-l-arabinose, 3, 4-di- and 3-O-methyl-l-rhamnose and 2, 3, 6-tri-O-methyl-d-galactose. Reduction with lithium aluminum hydride of the permethylated pectin gave mainly 2, 3-di-O-methyl-d-galactose and the above methylated sugars. Partial acid hydrolysis gave homologous series of β-(1 → 4)-linked oligosaccharides up to pentaose of d-galactopyranosyl residues, and 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, and di- and tri-saccharides of α-(1 → 4)-linked d-galactopyranosyluronic acid residues.

These results suggest that the tobacco pectin has a backbone consisting of α-(1 → 4)-linked d-galactopyranosyluronic acid residues which is interspersed with 2-linked l-rhamnopyranosyl residues. Some of the l-rhamnopyranosyl residues carry substituents on C-4. The pectin has long chain moieties of β-(1 → 4)-linked d-galactopyranosy] residues.     

Sugar Composition of Cell Wall Polysaccharides of Suspension-cultured Tobacco Cells

Neutral sugar composition of cell walls of suspension-cultured tobacco cells was examined with the advance of culture age by an anion-exchange chromatography. Isolated cell walls gave on hydrolysis the following sugars: 2% of l-rhamnose, 6% of d-mannose, 26% of l-arabinose, 13% of d-galactose, 8% of d-xylose and 47% of d-glucose as neutral sugars. Little changes in composition of cell wall polysaccharides were recognized with the advance of culture age. Sugar composition of the extra-cellular polysaccharides was similar to that of hemicellulose fraction from cell walls. Pectinic acid gave on hydrolysis 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, d-galacturonic acid and its oligosaccharides.     

A Simple Transductional Method for Construction of Adenylate- cyclase or cAMP Receptor Protein-deficient Mutants of Escherichia coli K-12

The substrate specificity of α-d-xylosidase from Bacillus sp. No. 693–1 was further investigated. The enzyme hydrolyzed α-1,2-, α-1,3-, and α-1,4-xylobioses. It also acted on some heterooligosaccharides such as O-α-d-xylopyranosyl-(1→6)-d-glucopyranose, O-α-d-xylopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→4)-d-glucopyranose, O-α- d-xylopyranosyl-(1→6)-O-d-glucopyranosyl-(1→4)-O-[α-d-xylopyranosyl-(1→6)]-d-glucopyranose, and O-α-d-xylopyranosyl-(1→3)-l-arabinopyranose. The enzyme was unable to hydrolyze tamarinde polysaccharides although it could hydrolyze low molecular weight substrates with similar linkages.     

Structure of Oligosaccharides Obtained by Controlled Degradation of Mung Bean Xyloglucan with Acid and Aspergillus oryzae Enzyme Preparation

A xyloglucan (MBXG) from the cell walls of etiolated mung bean hypocotyls was characterized by analyzing the fragment oligosaccharides from controlled degradation products of the polymer with acid and enzyme.

Cellobiose, cellotriose and cellotetraose were isolated from the partial acid hydrolyzate of MBXG. Isoprimeverose (6-O-α-d-xylopyranosyl-d-glucopyranose) and a pentasaccharide, α-l-fucosyl-(1 → 2)-β-d-galactosyl-(1 → 2)-α-d-xylosyl-(1 → 6)-β-d-glucosyl-(1 → 4)-d-glucose, were isolated from the hydrolyzate of MBXG with an Asp. oryzae enzyme preparation.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a need for larger amounts of different carbohydrate structures. Leloir glycosyltransferases catalyze the synthesis of complex oligosaccharides. However they are difficult or expensive to obtain, and require expensive nucleotide activated sugars. In contrast non-Leloir pathway enzymes use sucrose, which is known to be a high energy donor of d-glucose for glucosyltransferases like dextransucrase, or a donor of d-fructose for fructosyltransferases like inulin- and levansucrases for the synthesis of polysaccharides. Here we present the synthesis and kinetic studies of oligosaccharides using non-Leloir glycosyltransferases and sucrose analogues as new substrates, like β-d-fructofuranosyl-α-d-galactopyranoside (Gal-Fru) by a fructosyltransferase (FTF) from B. subtilis NCIMB 11871. The sucrose analogues carry a high binding energy in the glycosidic bond similar to that of sucrose. Thus, β-d-Fructofuranosyl-α-d-galactopyranoside (Gal-Fru) and β-d-Fructofuranosyl-α-d-fucopyranoside (d-Fuc-Fru) have been shown to be substrates for fructosyltransferases, which produce oligo- or polysaccharides, also in the presence of acceptors.     

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.     

Production of d(—)-α-Aminobenzylpenicillin by Kluyvera citrophila KY 3641

Intact cells of Kluyvera citrophila KY 3641 produced enzymaticaily d(—)-α-aminobenzyl-penicillin from 6-arainopenicillanic acid and phenylglycine derivatives. The optimum pH of the acylase was 6.5. Among various phenylglycine derivatives examined as substrates, d-phenylglycine methylester HC1 was the best compound giving the yields of about 10.7 mg/ml of d(—)-α-aminobenzy]penicillin in the enzymic reaction mixture. The product was isolated in a crystalline form and identified as d(—)-α-aminobenzylpenicillin.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was formed from the various carbohydrates including lactose, maltose, sucrose, d-glucose, d-fructose, d-galactose, d-mannose, d-mannitol, l-rhamnose, d-xylose, l-arabinose and glycerin. The influence of pH of the reaction mixture were tested, and inorganic phosphate was observed to be indispensable for α-ketoglutarate-fermentation. A cell of E. coli grown statically on glucose was found to reveal an ability of producing α-ketoglutarate under aerobic conditions. Optically dextro lactic acid was potent in the formation of a-ketoglutaric acid. The following reagents revealed the inhibiting effect on α-ketoglutarate-fermentation; CuSO₄, AgNO₃, iodoacetate, 2, 4-dinitrophenol, NaN₃, 3-sulfanilamido-6-methoxypyridazine and arsenite, while, kanamycin and 8-azaguanine has no inhibiting effect. When E. coli was grown in a glucose-medium, a small supply of air increased the yield of acetate against decreasing α-ketoglutarate.     

Functions,structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)₆-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.     

Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

7-Keto-8-aminopelargonic acid synthetase (KAPA synthetase) which catalyzes the formation of KAPA from pimelyl CoA and l-alanine, and is involved in biotin biosynthesis, was partially purified from a cell-free extract of Bacillus sphaericus by a procedure involving ammonium sulfate fraction ation, protamine treatment, and DEAE-cellulose column chromatography. The reaction product was bioautographically confirmed to be KAPA. Some properties of the enzyme were also investigated. Among the amino acids, only l-alanine was active as a substrate, condensing with pimelyl CoA, The reaction required pyridoxal phosphate but the other vitamin B₆ compounds were inert. Typical inhibitors of pyridoxal phosphate enzymes showed marked inhibition to the reaction. Various amino acids such as l-cysteine, glycine, d-alanine, l-serine, l-histidine, and d-histidine were also found to be inhibitory.