Blastobacter Sp Research Articles

Gene Cloning, Expression, and Substrate Specificity of an Imidase from the Strain Pseudomonas putida YZ-26

A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni(2+)-NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.

Cyclic ureide and imide metabolism in microorganisms producing a d-hydantoinase useful for d-amino acid production

The microbial transformation of dl-5-monosubstituted hydantoins has been applied to industrial scale production of optically active amino acids. Hydantoinase and N-carbamoyl amino acid amidohydrolase, which are the key enzymes in this transformation, from various microorganisms have been studied extensively. Blastobacter sp. A17p-4, which was isolated for d-amino acid production through hydantoin transformation, shows not only diverse cyclic ureide-metabolizing activities including those of d-hydantoinase and N-carbamoyl- d-amino acid amidohydrolase, but also cyclic imide-metabolizing activities. A recent study revealed the participation of d-hydantoinase in the metabolism of cyclic imides and the existence of novel enzymes, imidase and half-amidase, in this bacterium. d-hydantoinase functions in the metabolism of bulky cyclic imides, while imidase functions in that of simple cyclic imides in combination with half-amidase, which functions in the hydrolysis of the imidase reaction products, half-amides. Imidase and half-amidase are different from reported cyclic-amide-metabolizing enzymes, and are widely found in bacteria, yeasts and molds.

ChemInform Abstract: B‐90063, a Novel Endothelin Converting Enzyme Inhibitor Isolated from a New Marine Bacterium, Blastobacter sp. SANK 71894.

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B-90063, a novel endothelin converting enzyme inhibitor isolated from a new marine bacterium, Blastobacter sp. SANK 71894.

A novel endothelin-converting enzyme (ECE) inhibitor, B-90063, was isolated from the culture supernatant of the newly discovered marine bacterium Blastobacter sp. SANK 71894. Based on spectral analyses and chemical reactions, the structure of B-90063 was determined to be bis[6-formyl-4-hydroxy-2-(2'-n-pentyloxazol-4'-yl)-4-pyridon -3-yl]-disulfide (1a). Human and rat ECEs were inhibited more potently by B-90063, with respective IC50 values of 1.0 and 3.2 microM, than were other neutral endopeptidases such as NEP and type-I and -IV collagenases. B-90063 also inhibited the binding of ET-1 to rat ET(A) and bovine ET(B) receptors, though its antagonistic activities were weak. B-90063, thus, may abolish the physiological actions of endothelins through the ECE inhibitory and receptor antagonistic mechanisms.

Enzymatic preparation of d - p -trimethylsilylphenylalanine

In this paper we report on the enzymatic preparation of d-p-trimethylsilylphenylalanine (d-TMS-Phe). First, dl-5-(p-trimethylsilylphenylmethyl)hydantoin␣(dl-TMS-Phe-Hyd) was synthesized chemically and subjected to bacterial hydrolysis to obtain N-carbamoyl-d-p-trimethylsilylphenylalanine (C-d-TMS-Phe), but no strains examined showed sufficient hydantoinase activity on this compound. However, Blastobacter sp. A17p-4, which is known to produce N-carbamoyl-d-amino acid amidohydrolase (DCase), was found to be able to hydrolyze C-dl-TMS-Phe prepared chemically from the hydantoin. When C-dl-TMS-Phe was hydrolyzed with cells of Blastobacter sp. A17p-4, its optical purity was low because N-carbamoyl-l-amino acid amidohydrolase (LCase) coexisted in the cells. DCase and LCase in the cell-free extract of Blastobacter sp. A17p-4 could be separated by DEAE-Sephacel column chromatography. The optimum pH for the hydrolysis of C-dl-TMS-Phe by the partially purified DCase was 8.0 and addition of 2.5 % N,N-dimethylformamide was effective in raising the substrate concentration without inactivation of DCase. Under the optimized conditions, highly optically pure (98 % enantiomeric excess) d-TMS-Phe could be obtained from C-dl-TMS-Phe with partially purified DCase.

Efficient preparation of optically active p-trimethylsilylphenylalanine by using cell-free extract of Blastobacter sp. A17p-4

Optically active p-trimethylsilylphenylalanine (TMS-Phe) was prepared by enantioselective hydrolysis of N-carbamoyl- dl- p-trimethylsilylphenylalanine (C- dl-TMS-Phe) with the cell-free extract of Blastobacter sp. A17p-4, which is known to produce N-carbamoyl- d-amino acid amidohydrolase (DCase). Although this bacterium also produced N-carbamoyl- l-amino acid amidohydrolase (LCase), heat treatment of the cell-free extract for 40 min at 50°C and pH 7.0 was found to be effective in completely inactivating the LCase without loss of DCase activity, which provided a far simpler and more convenient method of preparing optically pure d-TMS-Phe (99% enantiomeric excess, ee). Furthermore, optically pure l-TMS-Phe (99% ee) could be obtained by LCase-catalyzed hydrolysis of the residual substrate with the non-treated cell-free extract as the enzyme source. The optimum pH for the hydrolysis of C- l-TMS-Phe was 7.0, and addition of 2 mM Mn 2+ and 5% N,N-dimethylformamide were effective in accelerating the activity of LCase and raising the substrate concentration, respectively.

Imidase, a dihydropyrimidinase-like enzyme involved in the metabolism of cyclic imides.

Imidase, which preferably hydrolyzed cyclic imides to monoamidated dicarboxylates, was purified to homogeneity from a cell-free extract of Blastobacter sp. A17p-4. Cyclic imides are known to be hydrolyzed by mammalian dihydropyrimidinases. However, imidase was quite different from known dihydropyrimidinases in structure and substrate specificity. The enzyme has a relative molecular mass of 105 000 and consists of three identical subunits. The purified enzyme showed higher activity and affinity toward cyclic imides, such as succinimide (Km = 0.94 mM; Vmax = 910 micromol x min(-1) x mg(-1)), glutarimide (Km = 4.5 mM; Vmax = 1000 micromol min (-1) x mg (-1) and maleimide (Km = 0.34 mM; Vmax = 5800 micromol x min(-1)x mg(-1)), than toward cyclic ureides, which are the substrates of dihydropyrimidinases, such as dihydrouracil and hydantoin. Sulfur-containing cyclic imides, such as 2,4-thiazolidinedione and rhodanine, were also hydrolyzed. The enzyme catalyzed the reverse reaction, cyclization, but with much lower activity and affinity. The enzyme was non-competitively inhibited by succinate, which was found to be a key compound in cyclic-imide transformation in relation with the tricarboxylic acid cycle in this bacterium, suggesting that the role of imidase is to catalyze the initial step of cyclic-imide degradation.

Thermostable [formula omitted] acid amidohydrolase: screening, purification and characterization

A thermostable N- carbamoyl- d-amino acid amidohydrolase was found in the cells of newly isolated bacterium, Blastobacter sp. A17p-4. The bacterium also showed d-specific hydantoinase activity. The N- carbamoyl- d-amino acid amidohydrolase activity of the cells exhibited a temperature optimum at 50–55°C, and was stable up to 50°C. The N- carbamoyl- d-amino acid amidohydrolase of Blastobacter sp. A17p-4 was purified to homogeneity and characterized. It has a relative molecular weight of about 120 000 and consists of three identical subunits with a relative molecular weight of about 40 000. N- Carbamoyl- d-amino acids having hydrophobic groups served as good substrates for the enzyme. It has been suggested that d-amino acid production from dl-5-substituted hydantoin involves the action of a series of enzymes involved in pyrimidine degradation, namely amide-ring opening enzyme, dihydropyrimidinase, and N-carbamoylamide hydrolyzing enzyme, β-ureidopropionase. However, the purified enzyme did not hydrolyze β-ureidopropionate; suggesting that the N- carbamoyl- d-amino acid amidohydrolase coexisting with d-specific hydantoinase, probably dihydropyrimidinase, in Blastobacter sp. A17p-4 is different from β-ureidopropionase.

Evaluation of pyrimidine- and hydantoin-degrading enzyme activities in aerobic bacteria

Abstract The enzyme activities responsible for the reductive pyrimidine base degradation by aerobic bacteria, which produce hydantoin-degrading enzymes, were investigated. Pseudomonas putida IFO 12996, which is a D -stereospecific hydantoinase producer, has dihydropyrimidinase activity, and Comamonas sp. E222c and Blastobacter sp. A17p-4, which are N- carbamoyl- D -amino acid amidohydrolase producers, have β-ureidopropionase activity. Blastobacter sp. also possesses both D -stereospecific hydantoinase and dihydropyrimidinase activities. Thus, two amide ring-opening activities and/or two N -carbamoyl amino acid-hydrolyzing activities coexist in these bacteria. However, the differences of the induction levels of each enzyme activities for the several pyrimidine- and hydantoin-related compounds suggest that these corresponding amide ring-opening or N -carbamoyl amino acid-hydrolyzing activities are not always catalyzed by the same enzymes.