Expression, purification, and characterization of a membrane-bound d

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membrane-bound enzyme that uses FAD as a co- enzyme and catalyzes the dehydrogenation of D-amino acids producing NH3, 2-oxo acids and H2 (Satomura.
Biotechnol Lett DOI 10.1007/s10529-017-2388-0

ORIGINAL RESEARCH PAPER

Expression, purification, and characterization of a membrane-bound D-amino acid dehydrogenase from Proteus mirabilis JN458 Jinjin Xu

. Yajun Bai . Taiping Fan . Xiaohui Zheng . Yujie Cai

Received: 8 April 2017 / Accepted: 19 June 2017 Ó Springer Science+Business Media B.V. 2017

Abstract Objectives To characterize a novel membranebound D-amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD). Results The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with D-alanine the most preferred substrate. The Km and Vmax values for -1 D-alanine were 9 mM and 20 lmol min mg-1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H2O2 and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs. Electronic supplementary material The online version of this article (doi:10.1007/s10529-017-2388-0) contains supplementary material, which is available to authorized users. J. Xu  Y. Cai (&) The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China e-mail: [email protected] Y. Bai  T. Fan  X. Zheng College of Life Sciences, Northwest University, Xi’an 710069, Shanxi, China T. Fan Department of Pharmacology, University of Cambridge, Cambridge CB2 1T, UK

Conclusions D-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase. Keywords Characterization  D-amino acid  D-amino acid dehydrogenase  Proteus mirabilis

Introduction D-Amino acid dehydrogenase (EC 1.4.99.1; DAD) is a membrane-bound enzyme that uses FAD as a coenzyme and catalyzes the dehydrogenation of D-amino acids producing NH3, 2-oxo acids and H2 (Satomura et al. 2015; Tanigawa et al. 2009). The electrons resulting from these reactions are transferred to cytochromes in Escherichia coli (Franklin and Venables 1976). DAD was originally discovered in the Gram-negative E.coli B membrane. It contains two subunits and is an iron–sulfur protein (Olsiewski et al. 1980). DADs can perform conversions between Lamino acids and D-amino acids, and the enzyme can be used to produce pharmaceuticals and food additives, as well as biosensors of D-amino acid concentrations in the medical and food industries (Baek et al. 2011). Proteus mirabilis is a Gram-negative, facultatively anaerobic, roD-shaped bacterium and a member of the Enterobacteriaceae family. It displays swarming motility and potent urease activity and causes 90% of the infections associated with all Proteus sp. in

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humans as a leading agent of pyelonephritis, urolithiasis, prostatitis, and catheter-associated urinary tract infections. It is widely distributed in soil, polluted water and the intestinal tract (Armbruster and Mobley 2012; Drzewiecka 2016). DAD is a peripheral membrane protein associated with the bacterial inner cell membrane and plays two main roles (Olsiewski et al. 1980). First, it allows bacteria to grow on D-amino acids (especially D-Ala) as sole nitrogen, carbon, and energy source. DAD can be induced by D-Ala and even L-Ala due to a catabolic alanine racemase activity that converts the L- into the D-stereoisomer (Lobocka et al. 1994). Second, it prevents local over-accumulation of D-amino acids, which cause specific inhibitory effects on bacterial growth (Pollegioni et al. 2007). Two types of L-amino acid dehydrogenase have been identified in P. mirabilis (Baek et al. 2008, 2011). In this study, we found that P. mirabilis was could use various D-amino acids, but, until now, the D-biochemical characteristics of DADs from Proteus spp. have not been reported. We have analyzed the nucleotide sequence of P. mirabilis HI 4320 (GenBank: AM942759.1) and discovered the existence of a gene encoding a DAD. We then determined the function of this DAD by cloning the gene from P. mirabilis JN458 and purifying the encoded protein to high homogeneity from recombinant cells.

sequencing was performed by Beijing Genomics Institute (Beijing, China), chemicals for biochemical analyses were purchased from Sigma-Aldrich (St. Louis, MO, USA) and H2O2 assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). E. coli expression of DAD Based on the nucleotide sequence of P. mirabilis HI 4320 dad (Pmdad), two primers were designed to construct plasmids expressing DAD. The ORF of the dad in P. mirabilis JN458 was PCR amplified using two primers. The forward primer contained the Nterminal region of dad and the SacI restrictiondigestion sequence (50 -GCCGGAGCTCATGAAAGT GATCATCTTAGGTGG-30 ), and the reverse primer contained the C-terminal region of dad and the XbaI restriction-digestion sequence (50 -GCCGTCTAGATTAATAAACAGCGTCAAGTTTTTG-30 ). PCR products were ligated into the plasmid vector, pcold II, and the recombinant plasmids were then transformed into E. coli DH5a and confirmed by sequencing. The sequenced plasmids were transformed into E. coli BL21 (DE3). Recombinant E. coli harboring the dad gene was induced by adding IPTG to 0.4 mM, followed by incubation at 15 °C for 24 h. Purification of PmDAD

D-Materials

and methods

Bacterial strains and plasmids Proteus mirabilis JN458 was stocked in our laboratory. E. coli strains DH5a and BL21 (DE3) were used as host strains for cloning and expression, respectively, of the DAD gene. E. coli and P. mirabilis were grown in Luria–Bertani medium. Plasmid pcold II was purchased from TaKaRa (Dalian, China), and ampicillin (50 lg ml-1) was used for plasmid selection. DNA manipulation and reagents PrimeSTAR HS DNA polymerase, restriction endonuclease, T4 DNA ligase, and other DNA-modifying enzymes were obtained from TaKaRa. The genomic DNA of P. mirabilis JN458 was isolated using a bacterial genome extraction kit (TaKaRa). DNA

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Purification procedures were performed at 4 °C. Recombinant E. coli cells harboring the dad gene were washed twice with 20 mM sodium phosphate buffer (pH 7.4), and induced cells were disrupted by sonication for 20 min. Whole cells and cells debris were centrifuged at 80009g for 10 min, and the cellfree extract (crude enzyme solution) in the soluble fraction was re-centrifuged at 160,0009g for 60 min. The insoluble fraction was used as the membrane fraction. The precipitate was solubilized in 20 mM Tris/HCl buffer (pH 8) consisting of 10% (v/v) glycerol, 0.03% Triton X-100, and 10 lM FAD. The mixture was then sonicated for 10 min, and the cell lysate was divided into soluble and insoluble fractions by centrifugation at 160,0009g for 60 min. The collected deposit was dissolved followed by sonication and repeat the steps again. [Triton X-100 can keep the protein stable, especially the membrane protein.] The resulting supernatant was used for protein

Biotechnol Lett

purification after centrifugation at 160,0009g for 60 min. The collected fractions were analyzed by 12% SDS-PAGE. Enzyme assays and measurement of PmDAD activity

buffer (pH 9–10). The optimal temperature for PmDAD activity and thermostability were determined for the range of temperatures from 20 to 80 °C at the optimal pH. Absorptions of reaction products were measured using a spectrophotometer. Determination of enzyme kinetics

The reaction system (500 ll) consisted of 20 mM sodium phosphate buffer (pH 7.4), 0.06% Triton X-100, 1.8 lM phenazine methosulfate (PMS), 0.5 mM 2,6-dichlorophenolindophenol (DCIP) and purified enzyme. The reaction was initiated by the addition of D-phenylalanine and the reduction of DCIP was determined by measuring absorbance at 600 nm. One unit of enzyme was defined as the amount catalyzing the reduction of 1 lmol DCIP per min at 37 °C. A millimolar absorption coefficient (emM) of 21.5 mM-1 cm-1 at 600 nm was used for DCIP.

Analysis of kinetic parameters was performed by measuring the corresponding keto acid titers using different concentrations of the substrates D-alanine, Dphenylalanine, and D-asparagine (1.25–80 mM) incubated at 45 °C for 5 min. Rates were determined as described previously and analyzed using the curve fitting to the Michaelis–Menten equation (OriginPro).

Results

Detecting the peroxide production of PmDAD

Cloning and sequencing of P. mirabilis dad

The production of H2O2 was detected by FeIIIXO agar assay as described previously (Yu et al. 2013). The ferrous-XO (FeIIXO) agar plates were prepared by mixing 1.5% agar, 6 mM H2SO4, 0.25 mM FeSO4, 0.15 Mm Xylenol Orange (XO), and 0.1 mM Dsorbitol. The reaction (100 ll) was added to circular wells that were made by a hole puncher, and incubated at 37 °C for 60 min to allow purplish red halos to form. To determine the concentration of H2O2, a correlation was extracted between H2O2 concentrations ranging from 0 to 160 lM and the diameters of the purplish red halos. Phosphate buffer, substrate without PmDAD, purified PmDAD without substrate, and 40 lM H2O2 were used as controls. In an alternative measurement, the peroxide production was detected by spectrophotometer using an H2O2 assay kit.

P. mirabilis JN458 was sequenced by Sangon (Shanghai, China) and annotated through the Rapid Annotations using Subsystems Technology (RAST) server (http://rast.nmpdr.org/). To identify the dad nucleotide sequence, we searched the complete genomic sequence of P. mirabilis HI 4320 (Accession No. AM942759) for nucleotide sequences similar to those of genes encoding DAD. A gene of 1305 bp was successfully cloned by PCR and ligated into the pcold II plasmid. An ORF for dad was analyzed using BLAST and deposited in GenBank (Accession No. KY364844). The predicted amino acid sequence is 434 amino acids in length, with a molecular mass of 47.7 kDa.

Characterization of PmDAD Initial reaction velocity was determined by measuring the production of phenylpyruvic acid under optimal reaction conditions. The reaction mixture (pH 7.4) consisted of 1 g D-phenylalanine l-1 and 0.05 g purified enzyme l-1. The optimal pH and pH stability for PmDAD activity were determined by experiments at a range of pH values from 3 to 10 at 37 °C using three different buffers: Na2HPO4/citric acid buffer (pH 3–8), Tris/HCl buffer (pH 8–9), and carbonate

Expression and purification of PmDAD The dad gene from P. mirabilis JN458 was expressed in E. coli BL21 (DE3) cells. The membrane fraction of induced E. coli cells exhibited DAD activity, and the enzyme was then purified to a single band according to SDS-PAGE analysis after ultracentrifugation for several times. The resulting molecular mass of the purified DAD from P. mirabilis JN458 (PmDAD) was 48 kDa according to SDS-PAGE analysis, which was consistent with the molecular mass predicted based on the primary structure of dad (Supplementary Fig. 1). The specific activity of PmDAD was 3.7 lmol min-1 mg-1, which was

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determined using D-phenylalanine as a substrate (Supplementary Table 1). Characterization of PmDAD Optimal activity of PmDAD was at pH 8 (Fig. 1a) and at 45 °C (Fig. 1c). The enzyme retained [80% activity over 1 h at pH 6–8 (Fig. 1b). It retained [50% of its maximal activity between 20 and 40 °C over 1 h (Fig. 1d). PmDAD had high activity against a wide range of D-amino acids (Table 1). The preferred substrate was D-alanine, which gave nearly two-fold higher conversion rates as compared with those for D-phenylalanine and D-asparagine. However, the enzyme exhibited activity against several other D-amino acids (Table 1). Production of peroxide by PmDAD The correlation between the concentration of H2O2 and the diameters of purplish red halos can be fitted by

Fig. 1 Effect of pH and temperature on enzyme activity and stability. a Effect of pH on the activity of the purified D-amino acid dehydrogenase from P. mirabilis JN458 (PmDAD) using Dalanine as substrate at 37 °C. The maximal activity (16.3 lmol min-1 mg-1) was taken as 100%. b The test on pH stability involved the incubation of the purified PmDAD for 1 h at 37 °C and pH 3–10 before measurement of residual activity. The activity of the enzyme without pre-incubation

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an exponential equation y = 1.284ex/0.315 - 12.983 (R2 = 0.996), where x is the diameter and y is the H2O2 concentration. Further plotting demonstrated that the change in diameter was a function of the logarithm of the concentration of H2O2 within the range of 5–160 lM with a linear fit to the equation y = 1.747x - 0.472 (R2 = 0.998), where x is the diameter and y is the logarithm of H2O2 concentration. The results show that purified PmDAD produced H2O2, yielding purplish red halos with diameters of 1.07 cm. The corresponding concentration of H2O2 was 25 lM (Fig. 2), which is similar to that (21 lM) detected spectrophotometrically. PmDAD kinetic parameters Km and Vmax were determined in the presence of Dalanine, D-phenyalanine, and D-asparagine under optimal conditions. The corresponding turnover rate (kcat) and catalytic efficiency (kcat/Km) were also calculated (Table 2).

(14.7 lmol min-1 mg-1) was defined as 100%. c Effect of temperature on the activity of PmDAD was determined at pH 8. The maximal activity (16.9 lmol min-1 mg-1) was taken as 100%. d To determine the thermal stability of PmDAD, the enzyme was incubated at 20–60 °C for 60 min. The activity of the enzyme without pre-incubation (15.8 lmol min-1 mg-1) was defined as 100%

Biotechnol Lett

We cloned the gene encoding a DAD from the Gramnegative bacterium P. mirabilis JN458. Subsequent expression in E. coli and purification by ultracentrifugation allowed determination of DAD activity, which was observed in the membrane fraction. The

primary structure inferred from the DAD from P. mirabilis JN458 (PmDAD) (Fig. 3) displayed 99% similarity with the DAD from P. mirabilis HI 4320, and membrane-bound enzyme showed broad substrate specificity for various D-amino acids, with the highest affinity for D-alanine. DADs exhibiting broad substrate specificities have been found in other Gram-negative bacteria, including P. fluorescens (Tsukada 1966), Pseudomonas aeruginosa (He et al. 2011), E. coli B (Olsiewski et al. 1980) and Salmonella typhimurium (Wild et al. 1974). All exhibit the highest affinities for D-alanine, similar to DAD from P. mirabilis. DADs from pig kidney (PkDAO) (Dixon and Kleppe 1965), Pyrobaculum islandicum (Satomura et al. 2002), and Helicobacter pylori (Tanigawa et al. 2009) exhibit three- to fourfold higher activity in the presence of D-proline compared to D-alanine, indicating that the substrate specificity of P. mirabilis DAD is similar to that of other bacterial DADs. Comparison of PmDAD with EcDAD and PaDAD revealed sequence identities of 68 and 60%, respectively, whereas the identities between PmDAD and PdDAD, HpDAD, PiProD, and RmDAD were only 30, 25, 21 and 32%, respectively (Fig. 3). PmDAD and PaDAD share high degrees of similarity because both organisms are common pathogens that often form biofilms together in CAUTIs (Li et al. 2016). These findings suggest P. mirabilis DAD is a novel D-alanine dehydrogenase. Similarly, other D-alanine dehydrogenases share a common optimal temperature and pH profiles for PmDAD activity, with values of alkaline pH (8) and a temperature of 45 °C (Satomura et al. 2015).

Fig. 2 The purified PmDAD produces H2O2 in vitro. a Detection of H2O2 by FeIIIXO agar assay. 100 ll of each reactions were added to the wells of FeIIXO agar plate, and purplish red halos were generated by reaction with PmDAD. b Correlation

between the detected H2O2 concentration and the corresponding diameter of purplish red FeIIIXO halo. c The changes in H2O2 concentration over time. The enzyme was incubated at 37 °C with D-Ala and without D-Ala

Table 1 Substrate specificity of DAD from P. mirabilis (PmDAD) Relative activity (%)a

Amino acids D-Phe

52

D-Leu

14

D-Ile

3

D-Met

43

D-Val

8

D-Ser

28

D-Pro

3

D-Thr

6

D-Ala

100

D-Tyr

40

D-His

6

D-Gln

1

D-Asn

50

D-Lys

1

D-Glu D-Cys

1 28

D-Trp

4

D-Arg

2

D-Gly

2

D-Asp a

2 -1

100% activity = 18.4 lmol min

-1

mg

Discussion

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Biotechnol Lett Table 2 Kinetic parameters for the PmDAD using D-Ala, D-Phe and D-Asn as substrate Substrate D-alanine

Vmax (U mg-1)

Km (mM)

kcat (s-1)

kcat/Km (s-1 mM-1)

20 ± 0.5

9.1 ± 0.3

16

1.8

D-phenyalanine

7.3 ± 0.2

19 ± 0.4

5.8

0.31

D-asparagine

4.3 ± 0.1

15 ± 0.3

3.4

0.22

Fig. 3 Linear alignment of the amino acid sequence of P. mirabilis DAD (PmDAD) with those of other bacterial D-amino acid dehydrogenases. The DADs are: EcDAD of E. coli (NCBI accession No. AAC74273); PaDAD of Pseudomonas aeruginosa (AAG08689); PdDAD of Paracoccus denitrificans (SDI58620); HpDAD of Helicobacter pylori (AAD07988); Pk-DAO of pig kidney D-amino acid oxidase (AAA31025);

PiProD of Pyrobaculum islandicum D-proline dehydrogenase (BAB88883); RmDAD of Rhodothermus marinus (BAR71661). The yellow frames indicate identical residues among eight sequences; The green frames indicate block similar residues; The blue frames indicate conservative residues. The nucleotidebinding motif (GxGxxG) is shown with a rectangular frame

However, the enzyme measured in our study exhibited better stability, with PmDAD retaining 50% of its maximal activity at 40 °C over 1 h and[90% activity from pH 6 to pH 8 over 1 h. The D-alanine

dehydrogenases from E. coli and P. aeruginosa are labile losing more than 90% activity after incubation at 37 and 42 °C for 10 min, respectively (Satomura et al. 2015).

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Electrochemical approaches using DADs have been developed to determine the preferred D-amino acid substrates (Satomura et al. 2015). PmDAD showed a broader substrate specificity than the reported DADs in that it used 17 amino acids, while DAD from H. pylori, P. islandicum, S. typhimurium and E. coli B could function with 10 (Tanigawa et al. 2009), 13 (Satomura et al. 2002), 11 (Wild et al. 1974) and 9 (Olsiewski et al. 1980), respectively. PmDAD based on biosensors shows potential for the sensitive and easy determination of D-amino acids in biological tissues and foodstuffs and for monitoring procedures for D-amino acid production. PmDAD exhibited catalytic activities toward DAla, D-Phe, and D-Asn. Compared with DAD from P. aeruginosa (He et al. 2011), the Km value of PmDAD for D-Ala was higher, but the kcat/Km value was higher, and the kcat value was consequently 22-fold higher than that of the former. The Km value of PmDAD for DPhe was higher than the DADs from P. aeruginosa and E. coli B (He et al. 2011; Satomura et al. 2015), indicating its affinity toward D-Phe is lower. In some bacteria, L-alanine is sufficient for conversion by alanine racemase to D-alanine, followed by Dalanine conversion to pyruvate through the action of DAD in the metabolic cycle (He et al. 2011). Alanine racemase (complementary genomic region in P. mirabilis HI4320: 1601233–1602345) and DAD (complementary genomic region in P. mirabilis HI4320: 1602359–1603663) share a functional relationship based on their proximity within the same operon in the P. mirabilis genome. DADs might also function as enzymes involved in the H. pylori respiratory chain, playing a similar role to that of NADH dehydrogenase in mitochondria (Saito et al. 2007; Tanigawa et al. 2009). However, the roles of DAD in P. mirabilis respiration remain unknown. Additionally, P. mirabilis contains L-amino acid deaminase, which converts L-amino acids to ammonia and keto acids. The coexistence of these two paths, as well as which plays a major role in metabolic processes, will be the focus of our future work. Acknowledgments We are grateful for the financial support of this work by the National Key Scientific Instrument and Equipment Development Project of China (2013YQ17052504), the Program for Changjiang Scholars and Innovative Research Team in the University of the Ministry of Education of China (IRT_15R55), and the seventh group of the Hundred-Talent Program of Shanxi Province (2015).

Supporting information Supplementary Table 1—Purification of P. mirabilis DAD from recombinantEscherichia coli cells. Supplementary Fig. 1—SDS-PAGE (12%) analysis of recombinant P. mirabilis DADfrom E. coli cells.

Compliance with Ethical Standards Conflicts of interest conflict of interest.

The authors declare that they have no

References Armbruster CE, Mobley HL (2012) Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis. Nat Rev Microbiol 10:743–754 Baek JO, Seo JW, Kwon O, Seong SI, Kim IH, Kim CH (2008) Heterologous expression and characterization of L-amino acid deaminases from Proteus mirabilis in Escherichia coli. J Biotechnol 136(Supplement):S300 Baek JO, Seo JW, Kwon O, Seong SI, Kim IH, Kim CH (2011) Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli. J Basic Microb 51:129–135 Dixon M, Kleppe K (1965) D-amino acid oxidase II. Specificity, competitive inhibition and reaction sequence. Biochim Biophys Acta 96:368–382 Drzewiecka D (2016) Significance and roles of Proteus spp. bacteria in natural environments. Microb Ecol 72:741–759 Franklin FCH, Venables WA (1976) Biochemical, genetic, and regulatory studies of alanine catabolism in Escherichia coil K12. Mol Gen Genet 149:229–237 He WQ, Li CR, Lu CD (2011) Regulation and characterization of the dadRAX locus for D-amino acid catabolism in Pseudomonas aeruginosa PAO1. J Bacteriol 193:2107–2115 Li XB, Lu NX, Brady HR, Packman AI (2016) Biomineralization strongly modulates the formation of Proteus mirabilis and Pseudomonas aeruginosa dual-species biofilms. FEMS Microbiol Ecol 92 Lobocka M, Hennig J, Wild J, Klopotowski T (1994) Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of D-amino-acid dehydrogenase and the catabolic alanine racemase. J Bacteriol 176:1500–1510 Olsiewski PJ, Kaczorowski GJ, Walsh C (1980) Purification and properties of D-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli B. J Biol Chem 255:4487–4494 Pollegioni L, Piubelli L, Sacchi S, Pilone MS, Molla G (2007) Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci 64:1373–1394 Saito M, Nishimura K, Hasegawa Y, Shinohara T, Wakabayashi S, Kurihara T, Ishizuka M, Nagata Y (2007) Alanine racemase from Helicobacter pylori NCTC 11637: Purification, characterization and gene cloning. Life Sci 80:788–794

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Biotechnol Lett Satomura T, Kawakami R, Sakuruba H, Ohshima T (2002) Dyelinked D-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum is a novel FAD-dependent amino acid dehydrogenase. J Biol Chem 277:12861–12867 Satomura T, Sakuraba H, Suye S, Ohshima T (2015) Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology. Appl Microbiol Biotechnol 99:9337–9347 Tanigawa M, Shinohara T, Saito M, Nishimura K, Hasegawa Y, Wakabayashi S, Ishizuka M, Nagata Y (2009) D-amino acid dehydrogenase from Helicobacter pylori NCTC 11637. Amino Acids 38:247–255

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Tsukada K (1966) D-amino acid dehydrogenases of Pseudomonas fluorescence. J Biol Chem 241:4522–4528 Wild J, Walczak W, Krajewska Grynkiewicz K, Klopotowski T (1974) D-amino acid dehydrogenase: the enzyme of the first step of D-histidine and D-methionine racemization in Salmonella typhimurium. Mol Gen Genet 128:131–146 Yu ZL, Wang J, Zhou N, Zhao CT, Qiu JP (2013) A highly sensitive method for quantitative determination of L-amino acid oxidase activity based on the visualization of ferricxylenol orange formation. PloS One 8(12):e82483