• KSBB Journal
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• v.14 no.2
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• pp.235-240
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• 1999

Simple and rapid screening methods were developed to screen mutant strains of Trigonopsis variabilis ATCC10679 (TW). D-amino acid oxidase (D-AAO) from a mutant strain, T26, showed about 30% higher specific activity against cephalosporin C than from its wild type, TW. D-AAO genes from both TW and T26 strains were cloned and sequenced. There was one nucleotide changed from T to C at 811 position, resulting in an amino acid codon changed from Phe-258 to Ser-258.

• Journal of Microbiology
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• v.39 no.3
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• pp.229-231
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• 2001

Different DL-amino acids were studied as inducers of D-amino acid oxidase (DAAO) and for their influence on the growth of Trigonopsis variabilis. DL-amino acids with non-polar side groups were found to be the befit inducers of DAAO. Maximum increase in the growth of Trigonopsis variabilis (gram dry weight per liter culfure) was observed with DL-methionine (2.39 g/l) followed by DL-serine (2.22 g/l) and DL-alanine (2.21 g/l).

• Journal of Microbiology
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• v.42 no.4
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• pp.336-339
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• 2004

The antibacterial actions of two amino acid oxidases, a D-amino acid oxidase from hog kidney and a L-amino acid oxidase from the venom of Agkistrodon halys, were investigated, demonstrating that both enzymes were able to inhibit the growth of both Gram-positive and Gram-negative bacteria, and that hydrogen peroxide, a product of their enzymatic reactions, was the antibacterial factor. However, hydrogen peroxide generated in the enzymatic reactions was not sufficient to explain the degree to which bacterial growth was inhibited. A fluorescence labeling assay showed that both of these two enzymes could bind to the surfaces of bacteria. To the best of our knowledge, this is the first report regarding the antibacterial activity of the D-amino acid oxidases.

• KSBB Journal
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• v.10 no.3
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• pp.264-270
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• 1995

Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

• Journal of Plant Biotechnology
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• v.34 no.1
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• pp.31-36
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• 2007

Though higher plants car not metabolize D-amino acid, many prokaryotes and eukaryotes have the D-amino acid metabolism. Therefore, we transformed tobacco plants with D-amino acid oxidase (DAO), which can metabolize D-amino acid, and confirmed that transgenic tobacco plants might metabolize D-amino acid. Transgenic tobacco plants were survived a high concentration of D-serine, however non-transgenic plants were not grown on D-serine medium. From Southern and Northern blot analysis, transgenic tobacco plants selected on D-serine medium were confirmed by insert and expression of transgene. $T_{1}$ tobacco seeds derived $T_{0}$ tobacco plants selfing were grown on D-serine medium and showed normal phenotype compared to wild tobacco plants. Transgenic tobacco plants displayed the metabolic capability of D-serine. Therefore, we suggested that DAO is useful selectable marker gene for plant transformation.

• Microbiology and Biotechnology Letters
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• v.22 no.3
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• pp.296-303
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• 1994

An immobilized Trigonopsis variabilis cells having an high activity of D-amino acid oxidase(DAO) was used to convert CPC into GL-7-ACA. The optimal pH of the reaction system was 8.0-8.5, and the optimal temperature was 40$\circ$C. When immobilized cell was used repeatedly in semi-batchwise reaction, the system retained 80% of the initial activity after used of 12 times for over 12 hours. The storage stability of the immobilized cell was maintained for 30 days at 4$\circ$C. The CPC concentration for the maximal reaction rate was about 30 mM and 40 mM for free and immobilized cells, respectively. Substrate inhibition of CPC concentration more than 50 mM was overcomed by 20~25% by immobilization. Pure oxygen supply into reaction system was most efficient in D-amino acid oxidase reaction. Continuous conversion to GL-7-ACA from CPC has been developed with an bioreactor system containing immobilized T variabilis cells. By opera- tion of the reactor for 5 hours, the average conversion yield of >80% and GL-7-ACA production of 40~45 mM per hour could be obtained.

• Journal of Pharmacopuncture
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• v.16 no.2
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• pp.28-32
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• 2013

Objective: This study was performed to analyze the single-dose toxicity of D-amino acid oxidase (DAAO) extracts. Methods: All experiments were conducted at the Korea Testing & Research Institute (KTR), an institution authorized to perform non-clinical studies, under the regulations of Good Laboratory Practice (GLP). Sprague-Dawley rats were chosen for the pilot study. Doses of DAAO extracts, 0.1 to 0.3 cc, were administered to the experimental group, and the same doses of normal saline solution were administered to the control group. This study was conducted under the approval of the Institutional Animal Ethics Committee. Results: In all 4 groups, no deaths occurred, and the $LD_{50}$ of DAAO extracts administered by IV was over 0.3 ml/kg. No significant changes in the weight between the control group and the experimental group were observed. To check for abnormalities in organs and tissues, we used microscopy to examine representative histological sections of each specified organ, the results showed no significant differences in any organs or tissues. Conclusion: The above findings suggest that treatment with D-amino acid oxidase extracts is relatively safe. Further studies on this subject should be conducted to yield more concrete evidence.

• Proceedings of the Korean Society of Life Science Conference
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• 2006.09a
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• pp.28.1-28.1
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• 2006

• Toxicological Research
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• v.11 no.2
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• pp.187-192
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• 1995

The cytotoxic effects of hydrogen peroxide and neuroprotective effects of a variety of agents were investigated in newborn mouse forebrain tissue culture. In our experiments, oxygen radical was generated enzymatically by glucose oxidase and the values were expressed as a percentage of number of living cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cytotoxicity of oxygen radicals was prevented by catalase and (N, N, N', N', -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), but N-tetra-ot-butyl-phenylnitrone (PBN), and deferoxamine (DFX), failed to show protective effects against oxygen radicals. Antagonists of the N-methyl-D-aspartate (NMDA) receptor, D-2-amino-5-phosphonovaleric acid (APV), 7-chlorokynurenic acid (CKA), and MK801 (a non-competitive NMDA antagonist) were also not effective in blocking neurotoxicity induced by glucose oxidase generated oxygen radicals.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.4
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• pp.996-1001
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• 2003

It is well known that oxidative stress of reactive oxygen species(ROS) may be a causative factor in the pathogenesis of bone disorder. The purpose of this study was to evaluate the cytotoxicity of oxidative stress. Cell viability by MTS assay or INT assay, activity of glutathione peroxidase(GPx), lipid peroxidation(LPO) activity and cell viablity. And also protctive effect of glutamate receptors against ROS-induced osteotoxicity was examined by protein synthesis, alkaline phosphatase (ALP) activity and lactate dehydrogenase (LDH) activity in cultured rat osteoblasts and osteoclasts. XO/HX decreased cell viability and GPx activity, protein synthesis and ALP activity, but increased LPO activity and LDH activity. In the protective effect, N-methyl-D-aspartate (NMDA) receptor antagonists or AMPA/kainate receptor antagonists such as D-2-amino-5-phosphonovaleric acid (APV), 7-chlorokynurenic acid (CKA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), NMDA receptor antagonists but AMPA/kainate receptor antagonists showed protective effect on xanthine oxidase (XO) and hypoxanthine (HX) in these cultures by the increse of protein synthesis, ALP activity.