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ABSTRACT: An L-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with L-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with L-lysine as ligand. The enzyme oxidized L-lysine, L-ornithine and L-arginine, but not other L-amino acids and d-amino acids. The oxidase activity for L-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for L-ornithine and L-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5 kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of L-amino acid oxidases.
Journal of Bioscience and Bioengineering 06/2012; 114(3):257-61. · 1.79 Impact Factor
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ABSTRACT: Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.
Protein Expression and Purification 05/2011; 77(1):92-7. · 1.59 Impact Factor
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ABSTRACT: PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.
Bioscience Biotechnology and Biochemistry 02/2011; 75(2):214-20. · 1.28 Impact Factor
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ABSTRACT: The genes encoding omega-laurolactam hydrolases from Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, and Sphingomonas sp. U238 were cloned and sequenced. Nucleotide and amino acid sequence analysis of the four genes indicated that the primary structures of these omega-laurolactam hydrolases are significantly similar to the 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12). These genes were expressed in Escherichia coli, and the omega-laurolactam hydrolysing activity of the recombinant enzymes was compared with that of 6-aminohexanoate-cyclic-dimer hydrolase from Arthrobacter sp. KI72. The enzyme from Acidovorax sp. T31 was most successfully expressed in E. coli. Cell-free extract of the recombinant strain was used for the synthesis of 12-aminolauric acid from omega-laurolactam by "enzymatic transcrystallization," because crystalline omega-laurolactam added into the enzyme solution was converted to crystalline 12-aminolauric acid (> or =97.3% yield). Under the optimum conditions, 208 g/l of 12-aminolauric acid was produced in 17 h. The resulting pure product was identical to authentic 12-aminolauric acid.
Bioscience Biotechnology and Biochemistry 05/2009; 73(5):980-6. · 1.28 Impact Factor
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ABSTRACT: Several omega-laurolactam degrading microorganisms were isolated from soil samples. These strains were capable of growing in a medium containing omega-laurolactam as sole source of carbon and nitrogen. Among them, five strains (T7, T31, U124, U224, and U238) were identified as Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, Rhodococcus sp. U224, and Sphingomonas sp. U238, respectively. The omega-laurolactam hydrolyzing enzyme from Rhodococcus sp. U224 was purified to homogeneity, and its enzymatic properties were characterized. The enzyme acts on omega-octalactam and omega-laurolactam, but other lactam compounds, amides and amino acid amides, cannot be substrates. The enzyme gene was cloned, and the deduced amino acid sequence showed high homology with 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12) from Arthrobacter sp. KI72 and Pseudomonas sp. NK87. Enzymatic synthesis of 12-aminolauric acid was performed using partially purified omega-laurolactam hydrolase from Rhodococcus sp. U224.
Bioscience Biotechnology and Biochemistry 09/2008; 72(8):2141-50. · 1.28 Impact Factor
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ABSTRACT: Rhodococcus sp. strain Oct1 utilizing ω-octalactam as a sole source of carbon and nitrogen was isolated from soil. ω-Octalactam hydrolyzing enzyme was purified to homogeneity. The purified enzyme has a molecular weight of approximately 48,100 by SDS polyacrylamide gel electrophoresis and 99,100 by gel filtration, indicating that the enzyme consists of 2 subunits. The purified enzyme catalyzed the hydrolysis of ω-octalactam to form 8-aminooctanoic acid at a rate of 3.95 U/mg. The purified enzyme also acted on ω-heptalactam, ω-laurolactam, nitroacetoanilide substitutions, and various aliphatic amides. The most suitable substrate was o-nitroacetanilide for the enzyme (11.6 U/mg). The enzyme belongs to aryl acylamidase. The gene for the enzyme was cloned and the deduced amino acid sequence showed similarity to ω-laurolactam hydrolase from Rhodococcus sp. U224 (51%) and putative aryl acylamidase from Nocardia farcinica IFM 10152 (98%), and N-terminal amino acid sequence (28 residues) of aryl acylamidase from Nocardia globerula IFO 13510 (92%). Aryl acylamidases and 6-aminohexanoate-cyclic-dimer hydrolases are in the same phylogenic lineage. These enzymes were mostly active toward non-natural amides. From phylogenic analysis, these enzymes were classified into amidase signature family. The enzyme was produced in a soluble form as a fusion protein (extension of 13 amino acids at C-terminal) in Escherichia coli.
Enzyme and Microbial Technology.
