New Resolving Agents

Seven optical active 2-benzylamino alcohols were synthesized by reduction of N-benzoyl derivatives of L-alanine, L-valine, L-leucine, L-phenylalanine, L-aspartic acid, L-glutamic acid and L-lysine and applied for the resolution of (±)-trans-chrysanthemic acid. d-trans-Chrys-anthemic acid was obtained by resolution via the salts of 2-benzylamino alcohols derived from L-valine and L-leucine, while (?)-trans-chrysanthemic acid was prepared through the salts of the amino alcohols derived from L-alanine and L-phenylalanine.     

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.     

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.     

Further Characterization of Ureido Ring Synthetase from Pseudomonas graveolens

Detailed enzymatic properties of the ureido ring synthetase purified from Pseudomonas graveolens were investigated. Nucleotide specificity studies indicated that CTP, UTP, GTP, and ITP were each tenth to one-fifth as active as ATP. The effect of substrate concentration was examined. The Km values for 7,8-diaminopelargonic acid, biotin diaminocarboxylic acid, NaHCO₃, ATP, and MgCl₂ were 1 × 10^?4 M, 4 × 10^?5 M, 1 × 10^?2 m, 5 × 10^?5 M, and 3 × 10^?3 M, respectively. It was elucidated that only ADP was produced from ATP in both the reaction of desthiobiotin synthesis from 7,8-diaminopelargonic acid and biotin synthesis from biotin diaminocarboxylic acid. The reaction was remarkably inhibited by Ni²⁺, Cd²⁺, Cu²⁺, Ag⁺, and As³⁺, while Mn²⁺ remarkably enhanced the enzyme reaction. The reaction was remarkably inhibited by metal-chelating reagents. It was elucidated that ADP had a competitively inhibiting effect on this enzyme reaction. 7,8-DiaminopeIargonic acid, which is the substrate for the desthiobiotin synthesis, competitively inhibited the biotin synthesis from biotin diaminocarboxylic acid. The stoichiometry of the desthiobiotin synthesis indicated that the formation ratio of desthiobiotin to ADP was 1 to 1.     

Effect of Feeding Bengal Gram (Cicer arietinum) at Different Protein Levels on Plasma Lipids of Rats

L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.     

Production of 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus

Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good producers of L-DOPA from L-tyrosine out of various bacteria. The condition for the formation of L-DOPA by Vibrio tyrosinaticus ATCC 19378 was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5-ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives.

In the selected condition about 4 mg/ml of L-DOPA was produced from 4.3 mg/ml of L-tyrosine.     

Biotransformation of L-Lysine to L-Pipecolic Acid Catalyzed by L-Lysine 6-Aminotransferase and Pyrroline-5-carboxylate Reductase

The enzyme involved in the reduction of Δ ¹-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding “P6C reductase” that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100%.     

Substrate Specificity of the Protease from Streptomyces cellulosae

The substrate specificity and the mode of action of the protease from Streptomyces cellulosae were investigated, using many kinds of peptides and proteins as substrates. The protease hydrolyzed peptides consisting of hydrophobic amino acids such as L-Phe-L-Leu-NH₂, L-Pro-L-Phe-NH₂, l-Leu-L-Met, L-Leu-L-Leu, Gly-L-Ile, L-Phe-L-Phe, L-Pro-L-Leu-Gly-NH₂, etc. The protease hydrolyzed zein best among the proteins tested, but weakly hydrolyzed gelatin, myoglobin, bovine serum albumin, γ-globulin, and collagen. The protease mainly hydrolyzed Ser¹²-Leu¹³, Leu¹³-Tyr¹⁴, and Tyr¹⁴-Gln¹⁵ bonds in the oxidized A-chain of insulin and at least the Leu¹⁵-Tyr¹⁶ bond in the oxidized B-chain of insulin.     

Studies on the the Isoleucine Fermentation

Accumulation of L-isoleucine and L-valine was studied on 14 genera, 47 species and 110 strains of aerobic bacteria using bacterial type cultures. A large amount of L-isoleucine and a small amount of L-valine accumulated when 1% of DL-α-aminobutyric acid was added to the culture medium. As a rule, facultative aerobes such as Aerobacter, Erwinia, Serratia and Bacillus showed good accumulation. In the absence of α-aminobutyric acid, powerful L-isoleucine accumulators produced a large amount of L-valine, although the accumulation of L- isoleucine was scarcely observed under that condition. In the presence of α-aminobutyric acid, the accumulation of L-valine was generally suppressed, but in several strains, on the contrary, the accumulation increased as well as that of L-isoleucine. When DL-threonine was used instead of α-aminobutyric acid, the amount of L-isoleucine accumulated was not as high as that with α-aminobutyric acid in almost all strains except Serratia marcescens. It was concluded that a distinct relationship between bacterial genera or species and accumulation of L-isoleucine did not exist, that is, powerful accumulators were limited to special strains, and that the addition of α-aminobutyric acid was necessary for the accumulation of a large amount of L-isoleucine.     

Stereoselective Incorporation of Isoleucine into Cypridina Luciferin in Cypridina hilgendorfii (Vargula hilgendorfii)

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four ²H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.     

Enzymatic Synthesis of L-Pipecolic Acid by Δ1-Piperideine-2-carboxylate Reductase from Pseudomonas putida

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine α-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Δ¹-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.     

Bacterial Degradation of N-Lauroyl-L-valine

Studies were conducted on the degradation of N-lauroyl-L-valine by type cultured bacteria. Many strains could utilize sodium N-lauroyl-L-valinate as carbon and nitrogen sources for their growth. Metabolism of N-lauroyl-L-valine was investigated in detail using Ps. aeruginosa AJ2116. Laurie acid was identified by gas chromatography suggesting cleavage of N-acyl linkage in N-lauroyl-L-valine.

Laurie acid might be metabolized to capric acid (C₁₀) and caprylic acid (C₈) becuase the accumulated substances gave nearly identical peaks with those of authentic fatty acids on gas chromatograms. The experiment using N-lauroyl-L-valine (¹⁴C) indicated that ¹⁴CO₂ was produced as a final product. Valine was not detected because it might be metabolized very rapidly immediately after its release.

It was supposed that the enzymes or enzyme systems degrading N-lauroyl-L-valine might be constitutive from the experiment using two kinds of cells grown in the medium containing N-lauroyl-L-valine or nutrient broth.     

The Contribution of Arabidopsis Homologs of L-Gulono-1,4-lactone Oxidase to the Biosynthesis of Ascorbic Acid

To clarify the involvement of seven Arabidopsis homologs of rat L-gulono-1,4-lactone (L-GulL) oxidase, AtGulLOs, in the biosynthesis of L-ascorbic acid (AsA), transgenic tobacco cells overexpressing the various AtGulLOs were generated. Under treatment with L-GulL, the levels of total AsA in three transgenic tobacco cell lines, overexpressing AtGulLO2, 3, or 5, were significantly increased as compared with those in control cells.     

Preparation of Optically Active Allothreonine by Separating from a Diastereoisomeric Mixture with Threonine

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH₃) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH₃ by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.     

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.     

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO₂ (SC-CO₂). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO₂ exposure by up to 15%. L-AAO was more stable in supercritical CO₂ than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO₂, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO₂. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3?h per change of enzyme activity after 3?h was the highest in SC-CO₂ (1.474?mmol?U^?1), where catalase was present, and the lowest in the EMR (0.457?mmol?U^?1).     

Properties of Revertants Appearing in l-Leucine Fermentation Culture Broth

The l-leucine productivity of an l-leucine producing strain, H-1204, of Corynebacterium glutamicum substantially decreased during a large-scale culture or repetitive subculturing. This instability was found to be due to the appearance of revertants with lower or no l-leucine productivity. Strains in the culture broth could be roughly classified into three types on the basis of their phenotypes: l-type, original l-leucine producing strain, Val^L Leu⁺ (valine leaky); M-type, Val⁺ Leu⁺ (prototroph); V-type, Val⁺ Leu^- (leucine auxotroph). The appearance of these revertants was determined to be caused by the distribution imbalance of α-ketoisovaleric acid, the common precursor for l-leucine and l-valine biosynthesis.     

Gene Cloning of α-Methylserine Aldolase from Variovorax paradoxus and Purification and Characterization of the Recombinant Enzyme

The α-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from α-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The V _max and K _m of the enzyme for the formaldehyde release reaction from α-methyl-L-serine were 1.89 μmol min^?1 mg^?1 and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of α-methyl-L-serine and α-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5′-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.     

New L-Amino Acid Ligases Catalyzing Oligopeptide Synthesis from Various Microorganisms

L-Amino acid ligase synthesizes various peptides from unprotected L-amino acids in an ATP-dependent manner. Known L-amino acid ligases catalyze only dipeptide synthesis, but recently we found that RizB of Bacillus subtilis NBRC 3134 catalyzes oligopeptide synthesis. In the present study, we searched for new members of the L-amino acid ligase group that catalyze oligopeptide synthesis. Several hypothetical proteins possessing the ATP-grasp motif were selected by in silico analysis. These recombinant proteins were assayed for L-amino acid ligase activity. We obtained five L-amino acid ligases showing oligopeptide synthesis activities. These proteins showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacterium adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones. We also examined some of their characteristics.