Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

Purification and properties of a novel enzyme,N-acylamino acid racemase,from Streptomyces atratus Y-53

A novel enzyme, N-acylamino acid racemase, was purified to homogeneity from Streptomyces atratus Y-53 and characterized. This enzyme catalyzes the interconversion of optically active N-acylamino acids. The relative molecular mass (M_r) of the enzyme was estimated to be about 41 000 and 244 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of six subunits with an equal M_r. The enzyme showed a broad substrate specificity toward N-acylamino acids, such as N-acetylmethionine, N-chloroacetylphenylalanine and N-chloroacetylvaline. The apparent Michaelis constant (K_m) values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 15.2 and 5.6 mm, respectively. Enzyme activity was markedly enhanced by divalent metal ions, such as Co²⁺, Mg²⁺ and Mn²⁺, and was inhibited by metal-chelating reagent, indicating that the enzyme is a metalloenzyme. We propose to name the enzyme N-acylamino acid racemase (acylamino acid racemase). Correspondence to: S. Tokuyama     

Enantioselective synthesis ofN-acetyl-l-methionine with aminoacylase in organic solvent

We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl₂, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids.     

Regulation of aspartokinase activity in non-fermentative,marine eubacteria

Summary Cell-free extracts of gram-negative, non-fermentative, marine eubacteria were assayed for aspartokinase activity. The organisms tested included polarly flagellated species and groups which had GC contents in their DNAs of 46 to 64 moles % (Alteromonas, Pseudomonas) as well as species which had peritrichous flagellation and moles % GC contents of 53 to 68 (Alcaligenes). The results of these studies suggested that in all the strains tested, aspartokinase activity was catalyzed by a single enzyme. On the basis of the effect ofl-threonine,l-lysine,l-methionine, andl-isoleucine on activity, five different types of aspartokinases (designated I through V) were delineated. In aspartokinase types I through IV,l-threonine andl-lysine inhibited activity by means of a concerted feedback inhibition; in type V, activity was inhibited byl-threonine but unaffected byl-lysine. In types I, III, and IV,l-threonine andl-lysine alone were inhibitory, while in type II these effectors had virtually no effect on activity when tested singly. Three distinct responses were observed in the presence of two other end products of the aspartate pathway,l-methionine andl-isoleucine. In types I and II, these two amino acids usually stimulated activity and overcame the inhibition byl-threonine andl-lysine; in types IV and V,l-methionine andl-isoleucine had no effect; and in type III these amino acids inhibited activity. The results of this study indicate that the aspartokinases of a number of species and groups of marine bacteria have similarities and differences which should be of use in making future taxonomic groupings.     

Overexpression of the gene forN-acylamino acid racemase fromAmycolatopsis sp. TS-1-60 inEscherichia coli and continuous production of optically active methionine by a bioreactor

A DNA sequence encodingN-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c. The recombinant plasmid was introduced intoEscherichia coli MM194 lysogenized with a bacteriophage having a T7 RNA polymerase gene. The amount of AAR produced by theE. coli transformant was 1100-fold more than that produced byAmycolatopsis sp. TS-1-60, the DNA donor strain. The AAR was purified to homogeneity from the crude extract of theE. coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography. Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR andd- orl-aminoacylase.d- orl-methionine was continuously produced with a high yield fromN-acetyl-dc-methionine by the bioreactor.     

Regulation of biosynthesis of thiolutin and aureothricin inStreptomyces kasugaensis

l-Methionine anddl-ethionine decreased production of thiolutin and aureothricin inStreptomyces kasugaensis. In the presence ofl-methionine the culture also produced 3-methylthioacrylic acid, 3-methylthiopropionic acid and 3,6-bis-(2-methylthioethyl)-2,5-dioxopiperazine. Production of the metabolites depended on the concentration ofl-methionine in the medium.     

Altered <Emphasis Type="SmallCaps">d</Emphasis>-methionine kinetics in rats with renal impairment

d-Amino acids are now recognized to be widely present in mammals. In rats, exogenously administered d-methionine is almost converted into the l-enantiomer via 2-oxo-4-methylthiobutylic acid as an intermediate. d-Amino acid oxidase is associated with conversion of d-methionine into the 2-oxo acid. Since d-amino acid oxidase is present at the highest activity in the kidney compared to other organ, kidney injury is suggested to cause accumulation of d-methionine. The purpose of the present study is to assess the role of kidney in the elimination of d-methionine and metabolic conversion into l-methionine in rats using a stable isotope methodology. After a bolus i.v. administration of d-[²H₃]methionine to 5/6-nephrectomized rats, plasma concentrations of d-[²H₃]methionine, l-[²H₃]methionine, and endogenous l-methionine were determined by a stereoselective GC–MS method. Renal mass reduction slowed down the elimination of d-[²H₃]methionine. The clearance values of conversion of d-[²H₃]methionine into the l-enantiomer in 5/6-nephrectomized rats were one-sixth of those in sham-operated rats. The elimination behavior of d-[²H₃]methionine observed in rats demonstrated that kidney was the principal organ responsible for chiral inversion of d-methionine.     

d-Methionine preparation from racemic methionines by Proteus vulgaris IAM 12003 with asymmetric degrading activity

The microbial degradation of l-methionine was investigated in order to develop a practical process for d-methionine production from racemic methionines. Among the 1000 culture strains tested, microorganisms belonging to the Achromobacter, Bacillus, Micrococcus, Morganella, Proteus, Providencia, Pseudomonas and Sarcina genera exhibited a high l-methionine-degrading activity. Proteus vulgaris IAM 12003 was determined to be the best strain and was used as a biocatalyst for eliminating the l-isomer. The degradation of l-isomer in this P. vulgaris IAM 12003 cell was assured by the action of l-amino acid oxidase. The maximum rate of l-isomer degradation was obtained at 30 °C and pH 8.0. Under these optimal conditions, the l-isomer in a 100 g/l mixture of racemic methionines was almost degraded within 20 h, with 46.5 g d-methionine/l remaining in the reaction mixture. Crystalline d-methionine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 30% from the reaction mixture by simple purification. Received: 17 June 1996 / Received last revision: 11 September 1996 / Accepted: 29 September 1996     

Distribution of the glycoconjugate oligosaccharides in the human placenta from pregnancies complicated by altered glycemia: lectin histochemistry

The aim of this study was to investigate the distribution of the oligosaccharides of the glycoconjugates in placentas from pregnancies complicated by different degree of altered glycaemia. Placentas from women with physiological pregnancies (group 1), with pregnancies complicated by minor degree of glucose intolerance (group 2) and with pregnancies complicated by gestational diabetes mellitus (GDM) treated with insulin (group 3) were collected. Ten lectins were used (ConA, WGA, PNA, SBA, DBA, LTA, UEA I, GSL II, MAL II and SNA) in combination with chemical and enzymatic treatments. The data showed a decrease of sialic acid linked α(2–6) to galactose/N-acetyl-d-galactosamine and an increase of N-acetyl-d-glucosamine in the placentas of the pathological groups, in particular the group 3, comparing to the group 1. A decrease of l-fucose (LTA) and d-galactose-(β1–3)-N-acetyl-d-galactosamine, and an increase and/or appearance of l-fucose (UEA I) and N-acetyl-d-galactosamine were observed in both the pathological groups, particularly in the group 2, with respect to the group 1. In GDM, and even in pregnancies with a simple alteration of maternal glycaemia, the changes in the distribution of oligosaccharides could be related to alteration of the structure and functionality of the placenta.     

D-Amino acids in protein de novo design. II. Protein-diastereomerism versus protein-enantiomerism

Summary With a few exceptions, proteins in our biosphere are based exclusively onl-amino acids. The inversion of configuration of all the stereogenic centers in a protein leads to anall-d compound with ‘mirror image’ properties and ‘mirror image’ structure. We propose to use the termprotein-enantiomerism to describe the relationship between two proteins that have the same sequence but whose amino acids have opposite configuration. We will use the termprotein-diastereomerism to define the relationship between two proteins that have the same sequence in which some amino acids have opposite configurations. A classification of type I, II, III, and IV protein-diastereomerism is proposed. By extension, a diastereoprotein is a protein where some amino acids have the same configuration (l ord) while others have the opposite one (d orl). A particular case of diastereoproteins aremesoproteins, also analyzed in this article. In addition to the goal of making proteins resistant to protease degradation, the use ofd-amino acids in protein de novo design may give rise to proteins with structures, and perhaps properties, very different to those of nativeall-l-proteins.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Genetic control of amino acid transport in sheep erythrocytes

An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr ^H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr ^h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr ^H gene behaves as if codominant to Tr ^h. Erythrocytes from sheep homozygous for the Tr ^H gene exhibit rapid saturable l-alanine influx (apparent K _m ,21.6mm; V _max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr ^h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V _max (apparent K _m, 19.1mm; V _max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr ^H, Tr^H sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant.     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts l- or d-glutamate to d- or l-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K _m and k_cat values of the overexpressed A. pyrophilus glutamate racemase for the racemization of l-glutamate to the d-form and the conversion of d-glutamate to the l-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06 s⁻¹ or 0.50 ± 0.07 mM and 0.25 ± 0.01 s⁻¹, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min. Received: September 11, 1998 / Accepted: January 12, 1999     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Overproduction and characterization of a recombinant <Emphasis Type="SmallCaps">D</Emphasis>-amino acid oxidase from <Emphasis Type="Italic">Arthrobacter protophormiae</Emphasis>

A screening of soil samples for d-amino acid oxidase (d-AAO) activity led to the isolation and identification of the gram-positive bacterium Arthrobacter protophormiae. After purification of the wild-type d-AAO, the gene sequence was determined and designated dao. An alignment of the deduced primary structure with eukaryotic d-AAOs and d-aspartate oxidases showed that the d-AAO from A. protophormiae contains five of six conserved regions; the C-terminal type 1 peroxisomal targeting signal that is typical for d-AAOs from eukaryotic origin is missing. The dao gene was cloned and expressed in Escherichia coli. The purified recombinant d-AAO had a specific activity of 180 U mg protein⁻¹ for d-methionine and was slightly inhibited in the presence of l-methionine. Mainly, basic and hydrophobic d-amino acids were oxidized by the strictly enantioselective enzyme. After a high cell density fermentation, 2.29 × 10⁶ U of d-AAO were obtained from 15 l of fermentation broth.