Phytanic acid alpha-oxidation: accumulation of 2-hydroxyphytanic acid and absence of 2-oxophytanic acid in plasma from patients with peroxisomal disorders.

A stable isotope dilution method was developed for the measurement of 2-hydroxyphytanic acid and 2-oxophytanic acid in plasma. In plasma from healthy individuals and from patients with Refsum's disease, 2-hydroxyphytanic acid was found at levels less than 0.2 mumol/l, whereas the acid accumulated in plasma from patients with rhizomelic chondrodysplasia punctata, generalized peroxisomal dysfunction, and a single peroxisomal beta-oxidation enzyme deficiency. In plasma from both healthy controls and patients with peroxisomal disorders, 2-oxophytanic acid was undetectable. Four different groups of diseases were characterized with a defective phytanic acid alpha-oxidation and/or pristanic acid beta-oxidation: 1) Refsum's disease, with a defect at phytanic acid alpha-hydroxylation; 2) rhizomelic chondrodysplasia punctata, with a defect at 2-hydroxyphytanic acid decarboxylation; 3) generalized peroxisomal disorders, with defects at 2-hydroxyphytanic acid decarboxylation and at pristanic acid beta-oxidation; 4) single peroxisomal beta-oxidation enzyme deficiencies, with a defect at pristanic acid beta-oxidation, resulting in an impaired phytanic acid alpha-oxidation by inhibition. The results indicate that 2-hydroxyphytanic acid decarboxylation and pristanic acid beta-oxidation take place in peroxisomes.     

间苯二酚、水杨酸对绿豆下胚轴不定根形成的作用

２０—１００ｍｇＬ（－１）间苯二酚能明显地促进绿豆下胚轴不定根的形成,与２０ｍｇＬ（－１）ＩＢＡ混合处理具加成效应,其作用在于降低生根初期ＩＡＡ氧化酶和多酚氧化酶活性．１０—１００ｍｇＬ（－１）水杨酸抑制下胚轴不定根的形成,随处理浓度的加大,对生根数目、生根范围和根重的抑制作用增加．水杨酸处理后１－３ｄ,能提高ＩＡＡ氧化酶和多酚氧化酶的活性．     

间苯二酚、水杨酸对绿豆下胚轴不定根形成的作用

２０—１００ｍｇＬ（－１）间苯二酚能明显地促进绿豆下胚轴不定根的形成,与２０ｍｇＬ（－１）ＩＢＡ混合处理具加成效应,其作用在于降低生根初期ＩＡＡ氧化酶和多酚氧化酶活性．１０—１００ｍｇＬ（－１）水杨酸抑制下胚轴不定根的形成,随处理浓度的加大,对生根数目、生根范围和根重的抑制作用增加．水杨酸处理后１－３ｄ,能提高ＩＡＡ氧化酶和多酚氧化酶的活性．     

The interaction of ascorbate oxidase with L-dopa,L-tyrosine and 3,4-dihydroxycinnamic acid. Evidence for irreversible damage of the enzyme during catechol oxidase activity

The aerobic interaction between ascorbate oxidase and L-tyrosine, L-3,4-dihydroxyphenylalanine or 3,4-dihydroxycinnamic acid in 1:10 molar ratio was followed by optical absorption, CD and EPR spectroscopy in 0.1 M phosphate buffer at pH 5.0. While the spectra of the system ascorbate oxidase—L-tyrosine remain practically unaffected after several hours, indicating that no oxidation of the amino acid occurs in the conditions employed, rather drastic changes can be observed in the spectra of the ascorbate oxidase-catechol systems. In particular, while the optical absorption below 500 nm increases markedly due to the formation of the substrate oxidation products, an irreversible decrease in intensity of the absorption, CD and EPR spectral features associated with the blue copper(II) chromophores indicates that a partial loss of Type 1 copper by ascorbate oxidase has occurred during this secondary catechol oxidase activity. A copper species characterized by weak positive CD activity at 370 nm and EPR signal at intermediate field between those of the Type 2 and Type 1 coppers can be detected in the early stages of the reaction. The irreversible damage undergone by the protein during catechol oxidase activity may have biological significance and accounts for the low yield of purified enzyme obtained when the crude enzyme extract is left in prolonged contact with low molecular weight cell components, rich in σ-diphenolic compounds.     

细胞外ATP通过刺激NADPH氧化酶缓解水杨酸诱导的细胞死亡

水杨酸（SA）是植物重要的信号分子,低浓度的SA能够诱导植物的抗病反应,而高浓度的SA导致植物细胞死亡。本文采用500μmol·L-1的SA处理烟草悬浮细胞BY-2,研究了细胞外ATP在SA诱导的细胞死亡中的作用及可能的机制。结果显示,外源ATP可缓解SA诱导的细胞死亡水平的上升。另外,SA导致NADPH氧化酶活性下降,而外源ATP则刺激其活性上升。外源ATP能缓解SA对NADPH氧化酶活性的抑制,且这种缓解作用可被NADPH氧化酶的抑制剂——二亚苯基碘（DPI）所消除。DPI还可部分消除外源ATP对SA所诱导的细胞死亡的缓解作用。上述结果表明,胞外ATP通过刺激NADPH氧化酶活性缓解SA诱导的细胞死亡。     

An in Vitro System of Indole-3-Acetic Acid Formation from Tryptophan in Maize (Zea mays) Coleoptile Extracts

The formation of a product from tryptophan that had the same retention time as that of authentic indole-3-acetic acid (IAA) on high performance liquid chromatography was detected in crude extracts of maize (Zea mays) coleoptiles. The product was identified as IAA by mass spectrometry. The IAA-forming activity was co-purified with an indole-3-acetaldehyde (IAAld) oxidase activity by chromatography on hydrophobic and gel filtration (GPC-100) columns. During purification, the IAA-forming activity, rather than that of IAAld oxidase, decreased; but when hemoprotein obtained from the same tissue was added, activity recovered to the same level as that of IAAld oxidase. The promotive activity of the hemoprotein was confirmed by the result that the activity coincided with amounts of the hemoprotein after GPC-100 column chromatography. The hemoprotein was characterized and identified as a cytosolic ascorbate peroxidase (T. Koshiba [1993] Plant Cell Physiol [in press]). The reaction of the IAA-forming activity was apparently one step from tryptophan. The activity was inhibited by 2-mercaptoethanol. The optimum temperature for the IAA-forming system as well as for the IAAld oxidase was 50 to 60[deg]C, and the acitivity at 30[deg]C was one-third to one-half of that at 60[deg]C. The system did not discriminate the L- and D-enantiomers of tryptophan.     

Omega-hydroxylation of phytanic acid in rat liver microsomes: implications for Refsum disease

The 3-methyl-branched fatty acid phytanic acid is degraded by the peroxisomal alpha-oxidation route because the 3-methyl group blocks beta-oxidation. In adult Refsum disease (ARD), peroxisomal alpha-oxidation is defective, which is caused by mutations in the gene coding for phytanoyl-CoA hydroxylase in the majority of ARD patients. As a consequence, phytanic acid accumulates in tissues and body fluids. This study focuses on an alternative route of phytanic acid degradation, omega-oxidation. The first step in omega-oxidation is hydroxylation at the omega-end of the fatty acid, catalyzed by a member of the cytochrome P450 multienzyme family. To study this first step, the formation of hydroxylated intermediates was studied in rat liver microsomes incubated with phytanic acid and NADPH. Two hydroxylated metabolites of phytanic acid were formed, omega- and (omega-1)-hydroxyphytanic acid (ratio of formation, 5:1). The formation of omega-hydroxyphytanic acid was NADPH dependent and inhibited by imidazole derivatives. These results indicate that phytanic acid undergoes omega-hydroxylation in rat liver microsomes and that an isoform of cytochrome P450 catalyzes the first step of phytanic acid omega-oxidation.     

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.     

Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.     

The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

模拟酸雨胁迫下钙对龙眼光合功能的调节作用

以福建省主栽品种乌龙岭龙眼为材料研究了模拟酸雨胁迫下钙对龙眼光合功能的调节作用 .结果表明 ,叶绿体的反应介质中Ca2 + 浓度在 0～ 5mmol·L-1范围内可增加其光还原活性 ,以 3 .5mmol·L-1的活性最高 ,比对照增加 41.90 % ;而 2mmol·L-1EGTA会使其活性下降 2 6 .0 6 % ;Mn2 + 和Mg2 + 对光还原活性均有抑制作用 .0～ 6mmol·L-1的Ca2 + 可提高叶绿体的光合磷酸化活性 ,最适的浓度为 4.5mmol·L-1;0～ 8mmol·L-1的Ca2 + 可提高叶绿体超氧物歧化酶 (SOD)活性 ,最适浓度为 6mmol·L-1.pH 3 .0的酸雨胁迫下 ,10mmol·L-1和 15mmol·L-1Ca(NO3) 2 均可减缓叶绿素的降解 ,稳定叶圆片的膜结构 ,降低膜透性 ,以 15mmol·L-1的效果较佳 ;Ca(NO3) 2 浓度超过 2 0mmol·L-1加剧了酸雨对叶片的伤害作用 .喷15mmol·L-1Ca(NO3) 2 可增加叶片光合速率 ,表现出模拟酸雨胁迫下钙对龙眼叶片的良好保护作用 .     

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.     

Differential effect of sunflower seed oil on hepatic and renal D-amino acid oxidase in the rat.

1. The specific activity of hepatic and renal peroxisomal D-amino acid oxidase (D-AAOX) was measured in rats fed diets containing various quantities of vegetable oil. 2. Increasing the amount of dietary sunflower seed oil (SSO) from 10 to 25% (w/w) reduced the specific activity of hepatic D-AAOX by up to 30% after 10 days. 3. In both tissues, the enzyme activity was moderately decreased during the first two-day period after administration of the 25% SSO diet was begun. Unlike hepatic D-AAOX, renal D-AAOX returned to its baseline level in the kidney after the third day. 4. In contrast to SSO, hydrogenated coconut oil (HCO) did not evoke alterations of D-AAOX activity. 5. The activity levels of another peroxisomal enzyme, L-2-hydroxy acid oxidase (L-HAOX), in the liver of rats fed the high-SSO diet vs those fed the control diet were similar. 6. The subcellular distribution of D-AAOX and L-HAOX was not altered in the liver of rats fed the 25% SSO diet during the 10-day period.     

NaCl胁迫对甘薯苗期生长、IAA代谢的影响及其与耐盐性的关系

随NaCl胁迫浓度的提高 ,甘薯苗期株高和叶面积生长受抑制 ;叶片IAA水平下降 ,而IAA氧化酶和POD活性提高 .不耐盐品种生长受抑制程度及IAA水平下降幅度均大于中耐盐品种和耐盐品种 ,叶片IAA氧化酶和POD活性上升的幅度大于中耐盐及耐盐品种 .同时浓度NaCl胁迫还引起了叶片绿原酸含量的增加 ,中耐盐品种栗子香和不耐盐品种胜利百号在 170mmol·L-1的NaCl浓度胁迫下 ,绿原酸含量上升达到最高值 ,耐盐品种徐薯 18绿原酸含量达到最高值的浓度是 2 5 5mmol·L-1,而后随NaCl胁迫浓度的提高 ,绿原酸含量均有下降 ,但仍高于对照 .     

Evidence for the occurrence of polyamine oxidase in the dicotyledonous plant Medicago sativa L. (alfalfa)

Polyamine oxidase (EC 1.5.3.3) activity has not been detected previously in cells of dicotyledonous plants, although it has been characterized extensively in monocotyledonous plants. Evidence is presented in this report for the occurrence of polyamine oxidase in dialyzed crude extracts of the dicotyledonous plant, Medicago sativa L. (alfalfa). Three enzyme assays were used to quantitate the formation of the three products of the reaction catalyzed by polyamine oxidase. 1-Pyrroline formation was measured colorimetrically as a yellow quinazolinium complex with o-aminobenzaldehyde. Hydrogen peroxide formation was measured spectrophotometrically with a coupled peroxidase assay system by peroxidative oxidation of guaiacol. [³H]1,3-Diaminopropane formation was measured by using [1,8-³H]spermidine as the substrate and separating the radiolabelled reaction product from the substrate by paper electrophoresis. This latter assay provided evidence that a polyamine oxidase of type [EC 1.5.3.3] catalyzed the cleavage reaction between a secondary nitrogen atom and an adjacent carbon of the butyl moiety of spermidine. Significant polyamine oxidase activity was detected in floral tissues, cortex tissues of the root, young leaves, and young germinated seedlings of alfalfa. The occurrence of polyamine oxidase in alfalfa accounts for the formation of the essential substrate, 1,3-diaminopropane, required for the biosynthesis of the uncommon polyamines, norspermidine and norspermine, which we have recently detected in alfalfa.Abbreviations PAO polyamine oxidase - MOPS [3-(N-morpholino)propanesulfonic acid] - MES [2-(N-morpholino)ethanesulfonic acid] - TES [N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid] - BICINE [N,N-bis (2-hydroxyethyl)glycine] - DTC diethyldithiocarbamic acid - R_m the distance of migration of a polyamine relative to putrescine after electrophoresis on paper     

Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa.

A microorganism capable of degrading DL-mandelic acid was isolated from sewage sediment of enrichment culture and was identified as Pseudomonas convexa. It was found to metabolize mandelic acid by a new pathway involving 4-hydroxymandelic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid as aromatic intermediates. All the enzymes of the pathway were demonstrated in cell-free extracts. L-Mandelate-4-hydroxylase, a soluble enzyme, requires tetrahydropteridine, nicotinamide adenine dinucleotide phosphate, reduced form, and Fe2+ for its activity. The next enzyme, L-4-hydroxymandelate oxidase (decarboxylating), a particulate enzyme, requires flavine adenine dinucleotide and Mn2+ for its activity. A nicotinamide adenine dinucleotide-dependent, as well as a nicotinamide adenine dinucleotide phosphate-dependent, benzaldehyde dehydrogenase has been resolved and partially purified.     

Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora

Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe(2+) and Mn(2+), and inhibited by metal chelating agents.     

L-pipecolic acid oxidation in the rabbit and cynomolgus monkey. Evidence for differing organellar locations and cofactor requirements in each species

L-Pipecolic acid oxidation was studied in the rabbit and cynomolgus monkey. Tissue homogenates from both species incubated with L-[2,3,4,5,6-3H]pipecolic acid produced a single radioactive product identified as alpha-aminoadipic acid. In the rabbit, L-pipecolic acid oxidation was greatest in kidney cortex with progressively lesser specific activities in liver, heart, and brain. When rabbit kidney cortex was fractionated by differential centrifugation or on Percoll gradients, activity paralleled that of the mitochondrial marker, glutamate dehydrogenase. In sonicated mitochondria, 92% of the activity was in the soluble fraction. Activity was inhibited by both rotenone and antimycin A and was maximal when FAD, phenazine ethosulfate, and glycerol were included in the assay; Km,app was 0.74 +/- 0.16 mM. Nipecotic acid, piperidine, and cis-2,4-piperidine dicarboxylic acid did not inhibit L-pipecolic acid oxidation, while L-proline had a Ki greater than or equal to 10 mM. D-Alanine and kojic acid, substrate and inhibitor of D-amino acid oxidase, respectively, were also not inhibitory. When monkey kidney cortex was fractionated on Percoll gradients, L-pipecolic acid oxidation activity paralleled that of the peroxisomal marker, catalase. After organellar subfractionation, the activity was membrane-associated and maximal at pH 8.5; Km,app was 4.22 +/- 0.30 mM. L-Pipecolic acid oxidation produced hydrogen peroxide, suggesting involvement of an oxidase in alpha-aminoadipic acid formation. Antimycin A did not inhibit the reaction. No specific cofactor requirements were identified and phenazine ethosulfate inhibited the reaction. D-Pipecolic acid, L-proline, and the other compounds cited above did not significantly inhibit the activity.     

Enzymatic synthesis of L-6-hydroxynorleucine

L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.     

Detection and substrate selectivity of new microbial D-amino acid oxidases

In order to screen for new microbial D-amino acid oxidase activities a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide was developed. Catalase, which coexists with oxidases in the peroxisomes or the microsomes and, which competes with peroxidase for hydrogen peroxide, was completely inhibited by o-dianisidine up to a catalase activity of 500 nkat ml(-)(1). Thus, using the peroxidase/o-dianisidine assay and employing crude extracts of microorganisms in a microplate reader, a detection sensitivity for oxidase activity of 0.6 nkat ml(-)(1) was obtained.Wild type colonies which were grown on a selective medium containing D-alanine as carbon, energy and nitrogen source were examined for D-amino acid oxidase activity by the peroxidase/o-dianisidine assay. The oxidase positive colonies possessing an apparent oxidase activity > 2 nkat g dry biomass(-)(1) were isolated. Among them three new D-amino acid oxidase-producers were found and identified as Fusarium oxysporum, Verticilium lutealbum and Candida parapsilosis. The best new D-amino oxidase producer was the fungus F. oxysporum with a D-amino acid oxidase activity of about 900 nkat g dry biomass(-)(1) or 21 nkat mg protein(-)(1). With regard to the use as a biocatalytic tool in biotechnology the substrate specificities of the three new D-amino acid oxidases were compared with those of the known D-amino acid oxidases from Trigonopsis variabilis, Rhodotorula gracilis and pig kidney under the same conditions. All six D-amino acid oxidases accepted the D-enantiomers of alanine, valine, leucine, proline, phenylalanine, serine and glutamine as substrates and, except for the D-amino acid oxidase from V. luteoalbum, D-tryptophane, D-tyrosine, D-arginine and D-histidine were accepted as well. The relative highest activities (>95%) were measured versus D-alanine (C. parapsilosis, F. oxysporum, T. variabilis), D-methionine (V. luteoalbum, R. gracilis), D-valine (T. variabilis, R. gracilis) and D-proline (pig kidney). The D-amino oxidases from F. oxysporum and V. luteoalbum were able to react with the industrially important substrate cephalosporin C although the D-amino acid oxidase from T. variabilis was at least about 20-fold more active with this substrate.As the results of our studies, a reliable oxidase assay was developed, allowing high throughput screening in a microplate reader. Furthermore, three new microbial D-amino acid oxidase-producers with interesting broad substrate specificities were introduced in the field of biotechnology.