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Dick B. Janssen - One of the best experts on this subject based on the ideXlab platform.
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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, 2004Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. JanssenAbstract: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, 2004Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. JanssenAbstract: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.
Sakayu Shimizu - One of the best experts on this subject based on the ideXlab platform.
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A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides
Applied and Environmental Microbiology, 2000Co-Authors: Chee-leong Soong, Jun Ogawa, Sakayu ShimizuAbstract: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, 1997Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Yoshie Fujiwara, Masahiko Goda, Sakayu ShimizuAbstract: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, 1993Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Hideaki Yamada, Toru Nagasawa, Makoto Nishiyama, Sueharu Horinouchi, Teruhiko Beppu, Sakayu ShimizuAbstract: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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Occurrence of Amidases in the Industrial Microbe Rhodococcus rhodochrous J1
Bioscience Biotechnology and Biochemistry, 1993Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Tofu Nagasawa, Hideaki Yamada, Sakayu ShimizuAbstract:Rhodococcus rhodochrous J1, of which the high-Mr nitrile hydratase has been used for the industrial manufacture of acrylamide from acrylonitrile, produced at least two Amidases differing in substrate specificity, judging from the effects of various amides on Amidase activity in this strain. These Amidases seemed to be inducible enzymes depending on amide compounds.
Esther M Gabor - One of the best experts on this subject based on the ideXlab platform.
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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, 2004Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. JanssenAbstract: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, 2004Co-Authors: Esther M Gabor, Erik F. J. De Vries, Dick B. JanssenAbstract: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.
Ken-ichi Kodaira - One of the best experts on this subject based on the ideXlab platform.
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molecular properties of the putative autolysin atlwm encoded by staphylococcus warneri m mutational and biochemical analyses of the Amidase and glucosaminidase domains
Gene, 2008Co-Authors: Kenji Yokoi, Ayanori Yamakawa, Akira Taketo, Kazuki Sugahara, Akinori Iguchi, Go Nishitani, Masahide Ikeda, Takako Shimada, Nobuya Inagaki, Ken-ichi KodairaAbstract:Abstract The putative autolysin AtlWM of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side Amidase (amiatlwm-R1-R2) and a C-terminal side glucosaminidase (R3-gluatlwm). Zymographic analysis of the protein overproduced in Escherichia coli showed that both enzymes were active toward 17 Gram-positive bacteria, including staphylococci, lactobacilli, lactococci, enterococci, and micrococci. The purified enzyme core amiatlwm (or gluatlwm) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) °C, respectively. amiatlwm was inactivated by EDTA, and was stimulated by such salts as CoCl2, MnCl2, CaCl2, or ZnCl2. Six mutations within amiatlwm, (H362A, E421A, H467A, H479, D481A, and Y491D) drastically reduced cell-lytic activity. Comparative analysis with other related Amidases suggested that the three residues H362, H467, and D481 likely act as ligands (and/or active sites). The lytic activity of gluatlwm markedly declined in four mutants (E1238A, E1238Q, T1239A, and Y1332A). For determination of the putative cell-recognition regions, four domains (R1-R2, R1, R2, and R3) were purified; all the proteins substantially bound to S. warneri M cells from exponential to stationary growth phases, and R1-R2 aggregated the cells. Protein sequencing and immunoblot analysis suggested that the extacellular AtlWM might be primarily processed at two specific sites (one between pro and amiatlwm, and the other between R2 and R3) to yield the mature Amidase and glucosaminidase.
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Molecular properties of the putative autolysin Atl(WM) encoded by Staphylococcus warneri M: mutational and biochemical analyses of the Amidase and glucosaminidase domains.
Gene, 2008Co-Authors: Kenji Yokoi, Ayanori Yamakawa, Akira Taketo, Kazuki Sugahara, Akinori Iguchi, Go Nishitani, Masahide Ikeda, Takako Shimada, Nobuya Inagaki, Ken-ichi KodairaAbstract:The putative autolysin Atl(WM) of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side Amidase (ami(atlwm)-R1-R2) and a C-terminal side glucosaminidase (R3-glu(atlwm)). Zymographic analysis of the protein overproduced in Escherichia coli showed that both enzymes were active toward 17 Gram-positive bacteria, including staphylococci, lactobacilli, lactococci, enterococci, and micrococci. The purified enzyme core ami(atlwm) (or glu(atlwm)) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) degrees C, respectively. ami(atlwm) was inactivated by EDTA, and was stimulated by such salts as CoCl(2), MnCl(2), CaCl(2), or ZnCl(2). Six mutations within ami(atlwm), (H362A, E421A, H467A, H479, D481A, and Y491D) drastically reduced cell-lytic activity. Comparative analysis with other related Amidases suggested that the three residues H362, H467, and D481 likely act as ligands (and/or active sites). The lytic activity of glu(atlwm) markedly declined in four mutants (E1238A, E1238Q, T1239A, and Y1332A). For determination of the putative cell-recognition regions, four domains (R1-R2, R1, R2, and R3) were purified; all the proteins substantially bound to S. warneri M cells from exponential to stationary growth phases, and R1-R2 aggregated the cells. Protein sequencing and immunoblot analysis suggested that the extacellular Atl(WM) might be primarily processed at two specific sites (one between pro and ami(atlwm), and the other between R2 and R3) to yield the mature Amidase and glucosaminidase.
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the two component cell lysis genes holwmy and lyswmy of the staphylococcus warneri m phage ϕwmy cloning sequencing expression and mutational analysis in escherichia coli
Gene, 2005Co-Authors: Kenji Yokoi, Akira Taketo, Kazuki Sugahara, Nobutaka Kawahigashi, Maiko Uchida, Masayuki Shinohara, Kenichi Kawasaki, Shogo Nakamura, Ken-ichi KodairaAbstract:Abstract From the genome library of Staphylococcus warneri M, the two successive cell-lysis genes ( holWMY and lytWMY ) were cloned and characterized. The lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and p I were 54 kDa and 8.95, respectively. When overproduced in Escherichia coli , lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative N -acetylmuramoyl- l -alanine Amidases encoded by the staphylococcal phages ϕ11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for d -alanyl-glycyl endopeptidase), Amidase ( l -muramoyl- l -alanine Amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and Amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys–His–Asn triad is crucial for the enzymatic function. On the other hand, the holWMY gene encoded a protein (HolWMY) with molecular mass and p I of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the S. aureus phage, ϕ11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (ϕWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the holWMY–lysWMY genes in the phage genome.
Michihiko Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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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, 1997Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Yoshie Fujiwara, Masahiko Goda, Sakayu ShimizuAbstract: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, 1993Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Hideaki Yamada, Toru Nagasawa, Makoto Nishiyama, Sueharu Horinouchi, Teruhiko Beppu, Sakayu ShimizuAbstract: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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Occurrence of Amidases in the Industrial Microbe Rhodococcus rhodochrous J1
Bioscience Biotechnology and Biochemistry, 1993Co-Authors: Michihiko Kobayashi, Hidenobu Komeda, Tofu Nagasawa, Hideaki Yamada, Sakayu ShimizuAbstract:Rhodococcus rhodochrous J1, of which the high-Mr nitrile hydratase has been used for the industrial manufacture of acrylamide from acrylonitrile, produced at least two Amidases differing in substrate specificity, judging from the effects of various amides on Amidase activity in this strain. These Amidases seemed to be inducible enzymes depending on amide compounds.
