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Dick B. Janssen – One of the best experts on this subject based on the ideXlab platform.

  • construction characterization and use of small insert gene banks of dna isolated from soil and enrichment cultures for the recovery of novel Amidases
    Environmental Microbiology, 2004
    Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. Janssen

    Abstract:

    Summary To obtain new Amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting Amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli , and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an Amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin Amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of b -lactam antibiotics.

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  • construction characterization and use of small insert gene banks of dna isolated from soil and enrichment cultures for the recovery of novel Amidases
    Environmental Microbiology, 2004
    Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. Janssen

    Abstract:

    Summary To obtain new Amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting Amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli , and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an Amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin Amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of b -lactam antibiotics.

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Sakayu Shimizu – One of the best experts on this subject based on the ideXlab platform.

  • A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides
    Applied and Environmental Microbiology, 2000
    Co-Authors: Chee-leong Soong, Jun Ogawa, Sakayu Shimizu

    Abstract:

    A variety of cyclic amide-metabolizing systems occur in nature and play important roles in pyrimidine and purine metabolism, amino acid metabolism (histidine degradation), antibiotic metabolism (β-lactam decomposition), creatinine degradation, etc.

    Cyclic imide is a kind of cyclic amide, and the metabolism of cyclic imides has been studied in relation to the detoxification of the antiepileptic agents ethotoin and phensuximide in mammals (3, 36). During the course of a study on cyclic amide transformation for hydantoin from the practical viewpoint of industrial d-amino acid production, we recently found that microorganisms also transform cyclic imides (21, 24–27). Microbial transformation of cyclic imides found in the bacterium Blastobacter sp. strain A17p-4 (22) involves ring opening of cyclic imide to monoamidated dicarboxylate (half-amide) catalyzed by imidase (23), half-amide hydrolysis to dicarboxylate catalyzed by Amidase, and subsequent trichloroacetic acid (TCA) cycle-like reactions (Fig. ​(Fig.1).1). The reactions and enzymes (imidase and Amidase) involved in the metabolism have practical potential for production of organic acids from cyclic imides or their metabolites and for fine enzymatic synthesis of useful compounds. For example, pyruvate, an effective precursor in the synthesis of various drugs and agrochemicals, was produced from succinimide or its metabolites (especially fumarate, a cheap material) through cyclic imide-transforming pathway (Fig. ​(Fig.1A1A and B) (28). Imidase was applied for the regiospecific synthesis of useful half-amide (3-carbamoyl-α-picolinic acid, an intermediate for herbicide) from a cyclic imide (2,3-pyridinedicarboximide) (J. Ogawa, M. Ito, T. Segawa, C.-L. Soong, and S. Shimizu, Abstr. Annu. Meet. ’99 Soc. Biosci. Bioeng., abstr. 182, 1999 [in Japanese]). Amidase also has a potential for the chiral resolution of dicarboxylates through the stereoselective hydrolysis of half-amides (Fig. ​(Fig.1C).1C).

    FIG. 1

    Proposed pathway for cyclic imide degradation in Blastobacter sp. strain A17p-4.

    We report here an Amidase catalyzing the second step of cyclic imide transformation. This Amidase, named half-Amidase, was distinct from known Amidases especially in substrate specificity. We also confirmed the physiological role of imidase and half-Amidase in cyclic imide transformation by investigating their induction/expression profiles.

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  • identification of active sites in Amidase evolutionary relationship between amide bond and peptide bond cleaving enzymes
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Yoshie Fujiwara, Masahiko Goda, Sakayu Shimizu

    Abstract:

    Mainly based on various inhibitor studies previously performed, Amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular Amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant Amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 → Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the Amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 → Asn substitution as well as Ser195 → Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all Amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various Amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus Amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

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  • Amidase coupled with low molecular mass nitrile hydratase from rhodococcus rhodochrous j1 sequencing and expression of the gene and purification and characterization of the gene product
    FEBS Journal, 1993
    Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Hideaki Yamada, Toru Nagasawa, Makoto Nishiyama, Sueharu Horinouchi, Teruhiko Beppu, Sakayu Shimizu

    Abstract:

    The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23–33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI–SphI region located 1.9-kb downstream of the L-NHase gene was found to be essential for the expression of Amidase activity in Escherichia coli; the gene arrangement of the Amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-deter-mined sequence indicated that the Amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the Amidase had high similarity to those of Amidases from Rhodococcus species including N-774 and P. chlororaphis B23 and to indole-3-acetamide hydrolase from Pseudomonas savastanoi.

    The Amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of Amidase activity in cell-free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This Amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This Amidase was highly specific for the S-enantiomer of 2-phenylpropionamide, but could not recognize the configuration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.

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Esther M Gabor – One of the best experts on this subject based on the ideXlab platform.

  • construction characterization and use of small insert gene banks of dna isolated from soil and enrichment cultures for the recovery of novel Amidases
    Environmental Microbiology, 2004
    Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. Janssen

    Abstract:

    Summary To obtain new Amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting Amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli , and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an Amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin Amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of b -lactam antibiotics.

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  • construction characterization and use of small insert gene banks of dna isolated from soil and enrichment cultures for the recovery of novel Amidases
    Environmental Microbiology, 2004
    Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. Janssen

    Abstract:

    Summary To obtain new Amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting Amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli , and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an Amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin Amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of b -lactam antibiotics.

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