A novel N-carbamoyl-l-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-l-cysteine amidohydrolase expressed in Escherichia coli

Ohmachi, Tetsuo; Narita, Megumi; Kawata, Maki; Bizen, Akiko; Tamura, Yoshiharu; Asada, Yoshihiro

doi:10.1007/s00253-004-1687-2

Cited by 13 publications

(19 citation statements)

References 26 publications

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“…3747 [Wilms et al, 1999] and N-carbamoyl-L -cysteine amidohydrolase from Pseudomonas sp. ON-4a [Ohmachi et al, 2004]. The optimum temperature rises to 70 ° C for the thermostable microorganisms of B. stearothermophilus NS 1122A [Ishikawa et al, 1994] and B. kaustophilus CCRC 11223 [Hu et al, 2003].…”

Section: Discussionmentioning

confidence: 99%

“…Optimal pH values for the other L -N-carbamoylases range from pH 7.4 for B. kaustophilus CCRC 11223 to 8.5 for A. aurescens DSM 3747, while for N-carbamoyl-L -cysteine amidohydrolase from Pseudomonas sp. ON-4a it is more alkaline (pH 9.0) [Ohmachi et al, 2004].…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, N-carbamoyl-L -cysteine amidohydrolase from Pseudomonas sp. ON-4a has recently been characterized as a homotetramer [Ohmachi et al, 2004].…”

Section: Discussionmentioning

confidence: 99%

“…Likewise, metal ions restore the activity of L -N-carbamoylases from B. kaustophilus CCRC 11223, A. xylosoxidans and P. putida IFO 12996 as well as N-carbamoyl-Lcysteine amidohydrolase from Pseudomonas sp. ON-4a previously treated with the chelating agent EDTA [Hu et al, 2003;Ogawa and Shimizu, 1994;Ogawa et al, 1995;Ohmachi et al, 2004]. There is no information available about divalent metal ion requirement for L -N-carbamoylase from A. aurescens DSM 3747.…”

Section: +mentioning

confidence: 99%

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Molecular Cloning and Biochemical Characterization of L-N-Carbamoylase from Sinorhizobium meliloti CECT4114

et al. 2005

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An N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) from Sinorhizobium meliloti CECT 4114 was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl α-amino acid to the corresponding free amino acid, and its purification has shown it to be strictly L-specific. The enzyme showed broad substrate specificity, and it is the first L-N-carbamoylase that hydrolyses N-carbamoyl-L-tryptophan as well as N-carbamoyl L-amino acids with aliphatic substituents. The apparent K_m values for N-carbamoyl-L-methionine and tryptophan were very similar (0.65 ± 0.09 and 0.69 ± 0.08 mM, respectively), although the rate constant was clearly higher for the L-methionine precursor (14.46 ± 0.30 s^–1) than the L-tryptophan one (0.15 ± 0.01 s^–1). The enzyme also hydrolyzed N-formyl-L-methionine (k_cat/K_m = 7.10 ± 2.52 s^–1·mM^–1) and N-acetyl-L-methionine (k_cat/K_m = 12.16 ± 1.93 s^–1·mM^–1), but the rate of hydrolysis was lower than for N-carbamoyl-L-methionine (k_cat/K_m = 21.09 ± 2.85). This is the first L-N-carbamoylase involved in the ‘hydantoinase process’ that has hydrolyzed N-carbamoyl-L-cysteine, though less efficiently than N-carbamoyl-L-methionine. The enzyme did not hydrolyze ureidosuccinic acid or 3-ureidopropionic acid. The native form of the enzyme was a homodimer with a molecular mass of 90 kDa. The optimum conditions for the enzyme were 60°C and pH 8.0. Enzyme activity required the presence of divalent metal ions such as Ni²⁺, Mn²⁺, Co²⁺ and Fe²⁺, and five amino acids putatively involved in the metal binding were found in the amino acid sequence.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…In contrast, N-carbamoyl-L -cysteine amidohydrolase from Pseudomonas sp. ON-4a has recently been characterized as a homotetramer [Ohmachi et al, 2004].…”

Section: Discussionmentioning

confidence: 99%

Section: +mentioning

confidence: 99%

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Molecular Cloning and Biochemical Characterization of L-N-Carbamoylase from Sinorhizobium meliloti CECT4114

et al. 2005

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“…ON4a, which is more alkaline (pH 9.0). 25 Thermal stability studies performed on BsLcar have shown that maximun activity gradually decreases at temperatures over 65 C for 60 min, with 60% activity remaining after incubation at 70 C for 60 min. b-Ureidopropionase with L-N-carbamoylase activity from P. putida IFO 12996, which showed high thermostability, retaining 80% of initial activity after incubation at 65 C for 30 min.…”

Section: Temperature and Ph Dependence In The Three Kinetic Resolutiomentioning

confidence: 99%

Evaluation of substrate promiscuity of anL‐carbamoyl amino acid amidohydrolase fromGeobacillus stearothermophilusCECT43

Pozo-Dengra

Martínez-Gómez

Martínez‐Rodríguez

et al. 2010

Biotechnology Progress

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N-carbamoyl-amino-acid amidohydrolase (also known as N-carbamoylase) is the stereospecific enzyme responsible for the chirality of the D- or L-amino acid obtained in the "Hydantoinase Process." This process is based on the dynamic kinetic resolution of D,L-5-monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L-N-carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N-acetyl and N-formyl-L-amino acids as well as the known N-carbamoyl-L-amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N-formyl-L-amino acids than for N-carbamoyl and N-acetyl-L-derivatives, with a catalytic efficiency (k(cat)/K(m)) of 8.58 x 10(5), 1.83 x 10(4), and 1.78 x 10(3) (s(-1) M(-1)), respectively, for the three precursors of L-methionine. Optimum reaction conditions for BsLcar, using the three N-substituted-L-methionine substrates, were 65 degrees C and pH 7.5. In all three cases, the metal ions Co(2+), Mn(2+), and Ni(2+) greatly enhanced BsLcar activity, whereas metal-chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co(2+)-dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations.

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Trimeric l‐N‐carbamoylase from newly isolated Brevibacillus reuszeriHSN1: A potential biocatalyst for production of l‐α‐amino acids

Nandanwar

Vohra²,

Hoondal

2013

Biotech and App Biochem

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l-N-carbamoylase was isolated from Brevibacillus reuszeri HSN1 and purified to homogeneity in three steps, which is a reasonably short protocol for native l-N-carbamoylase. The enzyme purification protocol resulted in ≈60-fold purification of l-N-carbamoylase with specific activity of 145 µmol/Min/mg. The subunit and native molecular mass were found to be 44.3 and 132 kDa, respectively. Temperature and pH optima were determined as 50°C and 8.5, respectively. The enzyme had retained ≈86% activity at 50°C when incubated for 60 Min and the half-life was determined as 180 Min at 50°C. N-carbamoyl-l-methionine (l-N-CMet) was found to be a preferred substrate with Km and Vmax values of ≈13.5 mM and ≈103 µmol/Min/mg, respectively. The broad substrate specificity with derivatives of N-carbamoyl amino acids is advantageous to be a better biocatalyst for production of corresponding l-α-amino acids. The enzyme activity was enhanced by 73% in the presence of 0.8 mM Mn(2+) ion during the biotransformation. In the batch experiment, ≈97% conversion of 5.0% l-N-CMet into enantiomerically pure l-methionine was achieved in 10 H when carried out at pH 8.0, 45°C, and 15% wet (w/v) cell loading, under controlled conditions. The overall merits of this enzyme show promise as a potential biocatalyst for l-α-amino acid production.

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A novel N-carbamoyl-l-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-l-cysteine amidohydrolase expressed in Escherichia coli

Cited by 13 publications

References 26 publications

Molecular Cloning and Biochemical Characterization of <i>L</i>-N-Carbamoylase from <i>Sinorhizobium meliloti</i> CECT4114

Molecular Cloning and Biochemical Characterization of <i>L</i>-N-Carbamoylase from <i>Sinorhizobium meliloti</i> CECT4114

Evaluation of substrate promiscuity of anL‐carbamoyl amino acid amidohydrolase fromGeobacillus stearothermophilusCECT43

Trimeric l‐N‐carbamoylase from newly isolated Brevibacillus reuszeriHSN1: A potential biocatalyst for production of l‐α‐amino acids

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