Author: Gianluca Molla
(Università degli Studi dell'Insubria)


Author: Luciano Piubelli
(Università degli Studi dell'Insubria)
Degrading D-Aspartate

In mammals, D-aspartate (D-Asp) shows a peculiar spatial distribution and temporal pattern of emergence [Errico et al., 2015a]. It is mainly present in central nervous system, in the neuroendocrine system, testis and spermatozoa. Accordingly, this D-amino acid has been correlated to several crucial processes such as development and neurogenesis of several area of the brain (it is present at high concentrations in the central nervous system during the development of the foetus) and also in fertility [LINK to role of D-AAs].

This peculiar distribution of D-Asp requires an efficient and tightly controlled biochemical metabolism for its synthesis and degradation. The biochemical pathway for production of D-Asp in mammals is still debated, but there are few doubts that is the flavoprotein D-aspartate oxidase (DASPO, which had been identified for the first time in 1949 in rabbit kidney) is the principal enzyme responsible for the catabolic metabolism of D-Asp. [Katane et al., 2010a].

DASPO ( is a peroxisomal flavoenzyme which catalyses the oxidative deamination of D-aspartate to imino aspartate which, in water, spontaneously deaminates producing oxaloacetate and ammonia. The reduced cofactor is reoxidized by molecular oxygen with the production of a molecule of hydrogen peroxide (Fig. 1).

Distribution and physiological role

DASPO is present not only in mammals but almost in all animals, from marine invertebrates to primate; accordingly, DASPO activity was detected in several organisms: e.g., in cephalopods, gastropods, fishes, amphibians, birds, mammals, yeast [Setoyama et al., 1997; Negri et al., 1999]. Interestingly, DASPO is not present in bacteria or plants, an aspect that lumps this enzyme to D-amino acid oxidase [LINK to DAAO] which shows a similar distribution in Nature. Accordingly, phylogenetic analysis shows that genes coding for these two proteins are paralogues (i.e., they originated by a gene duplication event early in evolution).

In mammals, DASPO shows a distribution inversely correlated to the one of D-Asp; this enzyme is mainly present in heart muscle, kidney (epithelial cells of proximal renal tubules), liver (hepatocytes) and brain (Fig. 2) []. While in kidney the main physiological role of DASPO is probably the catabolic oxidation of D-Asp deriving from spontaneous racemization or diet intake, in brain the enzyme is probably involved in the fine tuning of D-Asp (and possibly NMDA) local concentration [Katane et al., 2010; Molla, unpublished].

Fig. 2. Tissues which express highest levels of DASPO (source: Genevisible).

Biochemical properties

Most DASPOs are monomeric proteins; only few DASPOs have been reported as tetrameric proteins. The protein binds in a non-covalent way a molecule of FAD with an affinity with a Kd value ranging from ~ 10-6 M (mouse) to 3x10-8 M (human) and even ~ 10-10 (yeast). The human enzyme is quite stable having a Tm of 49.4 °C and it is active in the 8-12 pH range. Its stability is increased by the presence of ligands at the active site, a feature common to other amino acid oxidases.

The substrate specificity among mammalian DASPOs is similar: the preferred substrates are D-aspartate (Km = 1.1 mM, kcat 43.3 s-1, human enzyme) and NMDA. The enzyme is also active, even to a very lower extent, on D-Glu and Asn, but with an efficiency that is less the 10% in comparison with D-asp [Katane et al., 2015a; Molla, unpublished]. Importantly, DASPO is not active on the other two most important free amino acids in brain: glycine and D-serine [LINK to role of D-AA]. Non-mammalian DASPOs shows a very different pattern of substrate specificity, probably related to different specific roles.


Mammalian DASPOs are very similar also from a structural point of view (Fig. 3). They share a degree of sequence identity which ranges from 75% to 91%. The experimental structure of mammalian DASPOs is lacking; anyway, homology models show that the general setup of the active site of DASPO is very similar to the one of DAAO [Umhau et al., 2000; Pollegioni et al., 2007]. The main difference is the presence of 2 additional arginines (Arg216 and Arg237) at the active site in substitution of a tyrosine (Tyr224) and a leucine (Leu215). This renders the active site of DASPO more positively charged allowing an efficient binding and oxidation of the negatively charged D-aspartate.

Fig. 3. 3D structure of mammalian DASPO.  Left: superimposition of the models of the 3D structures of DASPOs from rat (pink), mouse (blue) and human (green). Right: detail of the active site entrance of the human enzyme. The regions of the protein that slightly differ between the different mammalian DASPOs are highlighted in purple [Molla, unpublished].

DASPO as a novel drug target

Since D-Asp is involved in several crucial physiological and pathological processes of the central nervous system, it is not surprising that DASPO is considered a promising target for novel drug-based therapies for mental diseases [Errico et al., 2015b].
Inhibition of DASPO, in vivo, results into an increase of the concentration of free D-Asp, an effect that can be beneficial in the case of pathological states characterized by abnormal low concentrations of this amino acid [Errico et al., 2015b]. Activity of DASPO can be (competitively) inhibited by several classical amino acid oxidase inhibitors such as tartrate, anthranilate, 6-chloro-1,2-benzisoxazol-3(2H)-one (CBIO) although with a very low potency (the IC50 is in the mmolar range). Recently, novel inhibitors with a Ki even in the nmolar range have been identified: for example, thiolactomycin (a natural compound) or small aromatic compounds such as 5-aminonicotinic acid (5-ANA) or 3-hydroxyquinolin-2(1H)-one that have been discovered by a combined approach of in silico virtual screening and in vitro high-throughput screening [Katane et al., 2010b, Katane et al., 2015b, Duplantier et al., 2009) (Tab. 1).

Table 1. Selected inhibitors of DASPO.

Main properties (figures refers to human DASPO)

EC number:           
Abbreviation:                 DASPO, DDO
Length (residues):          341
Mass (Da):                       37535
Oligomerization state:   Monomeric

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D-Amino acid oxidase (DAAO, EC is a flavin adenine dinucleotide (FAD)-containing enzyme belonging to the dehydrogenase/oxidase class of flavoproteins. It catalyzes the dehydrogenation of the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD to FADH2. The imino acid then hydrolyzes spontaneously to give α-keto acid and ammonia; the reduced flavin cofactor reoxidizes on molecular oxygen, producing hydrogen peroxide (Fig. 1) [Pollegioni et al., 2008].

Figure 1: The reaction catalyzed by DAAO

DAAO shows an absolute stereospecificity (it does not oxidize L-amino acids) and a broad substrate specificity. It oxidizes aliphatic, aromatic and polar D-amino acids, whereas D-aspartic acid and D-glutamic acid are not substrates for DAAO [Pollegioni et al., 2008]. Instead, these two latter compounds are substrates of D-aspartate oxidase (DASPO or DDO).

DAAO was discovered in 1935 by Sir Hans Krebs and since then it has been the subject of a great mass of structural, functional and kinetic investigations (reviewed in [Curti et al., 1992; Pilone, 2000]) that make DAAO a model enzyme for the dehydrogenase/oxidase class of flavoproteins. A fundamental advance in the comprehension of the structure-function relationships in DAAO was made with the resolution of the 3D-structure of the enzyme from pig kidney (pkDAAO) [Mattevi et al., 1996; Mizutani et al., 1996], from the yeast Rhodotorula gracilis (RgDAAO) [Umhau et al., 2000], and from human (hDAAO) [Kawazoe et al., 2006]. A second fundamental factor that allowed conspicuous advances in DAAO studies was the availability of DAAO genes/cDNAs from different organisms. Both these aspects permitted the application of protein engineering techniques, such as site-directed mutagenesis and directed evolution [Pollegioni et al., 2007b; Pollegioni and Molla, 2011].

Distribution and physiological role(s)

DAAO is a peroxisomal enzyme present in all eukaryotic taxa with the exception of plants; it is involved in a variety of different physiological functions (for an exhaustive review, see [Pollegioni et al., 2007a]). In yeasts, for example, oxidation of D-amino acids makes possible their use as carbon, nitrogen or energy source. In animals (both invertebrates and vertebrates), the role of DAAO is related to species-specific function. In mammals, DAAO is present in kidney where its role is related to the elimination of D-amino acids deriving from spontaneous racemization, diet or bacterial sources. However, a main role of mammalian (and especially human) DAAO is related to its presence in selected brain areas where it is devoted to the catabolism of D-serine, a neuromodulator that acts as a co-agonist of the N-methyl-D-aspartate receptors (NMDAr), which dysfunctions have been correlated to different neurodegenerative or psychiatric diseases, such as familial amyotrophic lateral sclerosis, Alzheimer’s disease and schizophrenia [Pollegioni and Sacchi, 2010; Sacchi et al., 2012]. Thus, by modulating D-serine levels, human DAAO plays a key role in regulating NMDAr activation state. The role of DAAO in human brain paves the way to the discovery of new drugs acting as DAAO inhibitors/modulators to be used for the treatment of the above mentioned diseases.

Biochemical and structural properties

Yeast and mammalian DAAOs show about 30% sequence identity, whereas pkDAAO shares with hDAAO 85% sequence identity. Despite the fact that DAAO from different sources share the same catalytic mechanism, they show important differences in many biochemical properties such as catalytic efficiency, substrate specificity, oligomeric state, stability, kinetic mechanism and FAD binding. All DAAOs bind the FAD cofactor in a non-covalent mode. The binding is tighter in yeast enzymes (Kd = 2x10-8 M for RgDAAO) than in mammalian ones (Kd = 2.2x10-7 M for pkDAAO and 8x10-6 M for hDAAO) [Pollegioni et al., 2007a; Molla et al., 2006].

DAAOs also differ in oligomeric state. RgDAAO and hDAAO are stable homodimers, whereas porcine DAAO exists in solution as a mixture of monomers, dimers or higher aggregates depending on the concentration and the presence of ligands [Curti et al., 1992; Pollegioni et al., 2007a]. Interestingly, only hDAAO is a homodimer even when the flavin cofactor is removed [Molla et al., 2006]: apoenzymes obtained from RgDAAO and pkDAAO are monomers. The 3D-structures of pkDAAO and hDAAO are very similar, whereas the mode of dimerization differs between mammalian and yeast enzymes. Both pkDAAO and hDAAO show a “head-to-head” dimerization mode, whereas RgDAAO shows a “head-to-tail” mode (Fig. 2).

Figure 2. Models of the two different monomer-monomer dimerization modes of DAAO from different sources:

hDAAO (panel A, pdb code: 2DU8) and RgDAAO (panel B, pdb code: 1C0I). (See text for details).

In all DAAOs, each monomer is divided into two domains [Mattevi et al., 1996; Mizutani et al., 1996; Umhau et al., 2000; Kawazoe et al., 2006]: the FAD-binding domain containing the typical βαβ dinucleotide-binding motif known as Rossmann fold, and the interface domain forming the contact surface with the second monomer. The FAD cofactor is buried inside the protein in an elongated conformation. The peculiar dimerization mode observed in RgDAAO is due to a positively charged long loop (not conserved in other DAAOs) that interacts with negatively charged residues belonging to two α-helices of the other monomer [Umhau et al., 2000; Pollegioni et al., 2002].

The different biochemical properties of DAAOs from diverse sources reflect their physiological role [Pollegioni et al., 2007a]: an efficient catabolic enzyme in yeast and an enzyme able to finely tune the levels of the neuromodulator D-Ser in mammalian brain.

Biotechnological applications of DAAO

Microbial DAAOs possess properties that render them suitable for biotechnological applications. They are stable enzymes, show broad substrate specificity, high turnover number and a tight binding with the FAD cofactor. Moreover, they can be produced at high levels, both as native and recombinant proteins. This made possible the production of enzyme variants with new and evolved properties by protein engineering techniques on the basis of the knowledge of the structure-function relationships. The main biotechnological applications of DAAO have been reviewed in details in [Pollegioni et al., 2007b, 2008, 2011] and comprise: a) production of 7-amino cephalosporanic acid (7-ACA) from cephalosporin C (CephC); b) resolution of racemic solutions of amino acids; c) detection and quantification of D-amino acids in biological samples; d) selective marker in plants.

Production of antibiotics

7-ACA is the starting point for the production of a number of semi-synthetic cephalosporins. 7-ACA is industrially produced in a well-established two-step enzymatic process starting from CephC: the first step is the conversion of CephC into glutaryl-7-amino cephalosporanic acid (Gl-7-ACA) catalyzed by DAAO; then, the enzyme glutaryl acylase converts Gl-7-ACA into 7-ACA [Pilone, 2000; Pollegioni et al., 2008]. This process is currently the most important industrial application of DAAO. During the years, some improvements of the process have been obtained by protein engineering studies. These studies include: a) site-directed mutagenesis of Trigonopsis variabilis DAAO (TvDAAO) to obtain a 6-fold increased maximal activity [Wong et al. 2010]; b) production of RgDAAO and TvDAAO variants fused with hemoglobin [Khang et al., 2003; Ma et al., 2009] or more active at low O2 concentration [Rosini et al., 2009; Rosini et al., 2010]; c) production of a chimeric enzyme made of Pseudomonas glutaryl acylase and TvDAAO [Luo et al., 2004]; d) coexpression of both DAAO and glutaryl acylase in E. coli cells [Zheng et al., 2007 ]. In c) and d) a so called “one-pot” (i.e., a single reactor) conversion of CephC into 7-ACA was obtained.

Resolution of racemic mixtures of amino acids

Availability of enantiomerically pure α-amino acids or unnatural amino acids are of increasing interest for the fine chemical and pharmaceutical industries, especially for drug discovery. This aim has been obtained by rational design of RgDAAO together with the use of multi-enzymatic systems. The use of the RgDAAO/M213G variant allowed the complete resolution of racemic unnatural amino acids texted in a short time (up to 10-times faster) and using low amounts of enzyme (down to 60-fold less) [Caligiuri et al., 2006a]. The complete recovery of L-enantiomers was obtained combining RgDAAO/M213G variant with L-aspartate amino transferase and catalase [Caligiuri et al., 2006b]. Similarly, in a four-enzymes system including DAAO from Arthrobacter protophormiae, D-methionine was fully converted to the L-isomer [
Findrik and Vasiæ-Racki, 2007].

DAAO-based biosensors

Detection of D-amino acids in biological samples is an important issue: the presence of D-amino acids in food (especially in dairy products) represents an indication of bacterial contamination (D-amino acids are components of the bacterial cell wall) and D-serine is a neuromodulator acting on NMDA receptors in the mammalian brain (see above).

Biosensors for different D-amino acids based on commercial pkDAAO appeared between 2001 and 2003. DAAO was employed in combination with horseradish peroxidase for the determination of several common D-amino acids [Domínguez et al., 2001] and D-pipecolic acid (a marker for peroxisomal disorders) [Stefan et al., 2003a], or in combination with pyruvate oxidase for the determination of D-alanine [Inaba et al., 2003]. DAAO-based biosensors have been also employed for determination of clinically relevant molecules such as D-methotrexate [Stefan et al., 2003b] and D-thyroxine [Stefan van Staden et al., 2010].

Biosensors with significant improvements in terms of sensitivity and substrate specificity have been obtained substituting commercial pkDAAO with RgDAAO. This allowed the determination of D-serine levels in vivo [Pernot et al., 2008; Polcari et al., 2014] and, using an evolved RgDAAO obtained combining rational design and directed-evolution approaches, the determination of the total concentration of all D-amino acids present in a biological sample [Rosini et al., 2008]. Very recently, a bienzymatic biosensors based on commercial pkDAAO and horse radish peroxidase coimmobilized on carbon nanotubes and gold nanoparticles has been set up. This innovative device allowed the determination of total D-amino acids content in complex biological matrices, such as bacterial samples [Moreno-Guzmán et al., 2017].

Use of DAAO in agriculture

Due to the absence of DAAO activity in plants, it can be used as innovative system both for positive and negative selection of transgenic plants. This strategy has been applied for the first time to an economically relevant species (apple) in 2009 [Hättasch et al., 2009]. A second application of DAAO in agriculture is the production of hybrid F1 seeds. For this purpose, a selective destruction of the male reproductive organs is mandatory to avoid undesired self-pollination. To this aim, an evolved variant of Rhodosporidium toruloides DAAO (RtDAAO) has been employed in tobacco plants. This system is based on the conversion of the inactive D-enantiomer of the herbicide glufosinate into its toxic L-enantiomer by the evolved RtDAAO (selectively expressed in anthers) in conjunction with the endogenous enzyme L-glutamate aminotransferase [Hawkes et al., 2011]. Evolved RtDAAO is much more active on D-glufosinate with respect to the wild-type enzyme and the combined action of the two enzymes produces transgenic lines exhibiting complete male sterility persisting for more than two weeks after foliar treatment with D-glufosinate [Hawkes et al., 2011].


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References for D-Aspartase Oxidase:

  • Duplantier AJ, Becker SL, Bohanon MJ, Borzilleri KA, Chrunyk BA, Downs JT, Hu  LY, El-Kattan A, James LC, Liu S, Lu J, Maklad N, Mansour MN, Mente S, Piotrowski MA, Sakya SM, Sheehan S, Steyn SJ, Strick CA, Williams VA, Zhang L. Discovery, SAR, and pharmacokinetics of a novel 3-hydroxyquinolin-2(1H)-one series of potent D-amino acid oxidase (DAAO) inhibitors. J Med Chem. 2009 Jun 11;52(11):3576-85.
  • Errico F, Mothet JP, Usiello A. D-Aspartate: An endogenous NMDA receptor agonist enriched in the developing brain with potential involvement in schizophrenia. J Pharm Biomed Anal. 2015 Dec 10;116:7-17.
  • Errico F, D'Argenio V, Sforazzini F, Iasevoli F, Squillace M, Guerri G, Napolitano F, Angrisano T, Di Maio A, Keller S, Vitucci D, Galbusera A, Chiariotti L, Bertolino A, de Bartolomeis A, Salvatore F, Gozzi A, Usiello A. A role for D-aspartate oxidase in schizophrenia and in schizophrenia-related symptoms induced by phencyclidine in mice. Transl Psychiatry. 2015 Feb 17;5.
  • Katane M, Homma H. D-aspartate oxidase: the sole catabolic enzyme acting on free D-aspartate in mammals. Chem Biodivers. 2010 Jun;7(6):1435-49.
  • Katane M, Saitoh Y, Hanai T, Sekine M, Furuchi T, Koyama N, Nakagome I, Tomoda H, Hirono S, Homma H. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment. Biochimie. 2010 Oct;92(10):1371-8.
  • Katane M, Kawata T, Nakayama K, Saitoh Y, Kaneko Y, Matsuda S, Saitoh Y, Miyamoto T, Sekine M, Homma H. Characterization of the enzymatic and structural properties of human D-aspartate oxidase and comparison with those of the rat and mouse enzymes. Biol Pharm Bull. 2015;38(2):298-305.
  • Katane M, Yamada S, Kawaguchi G, Chinen M, Matsumura M, Ando T, Doi I, Nakayama K, Kaneko Y, Matsuda S, Saitoh Y, Miyamoto T, Sekine M, Yamaotsu N, Hirono S, Homma H. Identification of Novel D-Aspartate Oxidase Inhibitors by in Silico Screening and Their Functional and Structural Characterization in Vitro. J Med Chem. 2015 Sep 24;58(18):7328-40.
  • Negri A, Tedeschi G, Ceciliani F, Ronchi S. 1999. Purification of beef kidney D-aspartate oxidase overexpressed in Escherichia coli and characterization of its redox potentials and oxidative activity towards agonists and antagonists of excitatory amino acid receptors. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1431(1):212-222.
  • Pollegioni L, Piubelli L, Sacchi S, Pilone MS, Molla G. Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci. 2007 Jun;64(11):1373-94.
  • Umhau S, Pollegioni L, Molla G, Diederichs K, Welte W, Pilone MS, Ghisla S. The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation. Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12463-8.
References for D-Amino Acid Oxidase:

  • Caligiuri A., D’Arrigo P., Rosini E., Tessaro D., Molla G., Servi S., Pollegioni L. (2006a) Enzymatic conversion of unnatural amino acids by yeast D-amino acid oxidase. Adv. Synth. Catal. 348, 2183-2190.
  • Caligiuri A., D’Arrigo P., Gefflaut T., Molla G., Pollegioni L., Rosini E., Rossi C., Servi S. (2006b) Multistep enzyme catalysed deracemisation of 2-naphthylalanine. Biocatal. Biotransform. 24, 409-413.
  • Curti B., Ronchi S., Pilone M.S. (1992) D- and L-amino acid oxidase. In: Chemistry and Biochemistry of Flavoproteins, vol. 3, pp. 69-94, Müller F. (Ed.), CRC Press, Boca Raton (FL).
  • Domínguez R., Serra A., Reviejo A.J., Pingarrón J.M. (2001) Chiral analysis of amino acids using electrochemical composite bienzyme biosensors. Anal. Biochem. 298, 275-282.
  • Findrik Z., Vasiæ-Racki D. (2007) Biotransformation of D-methionine into L-methionine in the cascade of four enzymes. Biotechnol. Bioeng. 98(5), 956-967.
  • Hawkes T., Pline-Srnic W., Dale R., Friend E., Hollinshead T., Howe P., Thompson P., Viner R., Greenland A. (2011) D-glufosinate as a male sterility agent for hybrid seed production. Plant Biotechol. J. 9, 301-314.
  • Hättasch C., Hanke M.-V., Flachowsky H. (2009) Preliminary results to establish the DAAO system as an alternative selection strategy on apple. Acta Horticulturae 814, 267-272.
  • Inaba Y., Mizukami K., Hamada-Sato N., Kobayashi T., Imada C., Watanabe E. (2003) Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase. Biosens. Bioelectron. 19, 423-431.
  • Kawazoe T., Tsuge H., Pilone M.S., Fukui K. (2006) Crystal structure of human D-amino acid oxidase: context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring. Protein Sci. 15(12), 2708-2717.
  • Khang Y.-H., Kim I.-W., Hah Y.-R., Hwangbo J.-H., Kang K.-K. (2003) Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C. Biotechnol. Bioeng. 82, 480-488.
  • Luo H., Li Q., Yu H., Shen Z. (2004) Construction and application of fusion proteins of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid. Biotechnol. Lett. 26, 939-945.
  • Ma X.-F., Yu H.-M., Wen C., Luo H., Li Q., Shen Z.-Y. (2009) Triple fusion of D-amino acid oxidase Trigonopsis variabilis with polyhistidine and Vitreoscilla hemoglobin. World. J. Microb. Biotechnol. 25,1353-1361.
  • Mattevi A., Vanoni M.A., Todone F., Rizzi M., Teplyakov A., Coda A., Bolognesi M., Curti B. (1996) Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2. Proc. Natl. Acad. Sci. USA 93(15), 7496-7501.
  • Mizutani H., Miyahara I, Hirotsu K., Nishina Y., Shiga K., Setoyama C., Miura R. (1996) Three-dimensional structure of porcine kidney D-amino acid oxidase at 3.0 A resolution. J. Biochem. (Tokyo) 120(1), 14-17.
  • Molla G., Sacchi S., Bernasconi M., Pilone M.S., Fukui K., Pollegioni L. (2006) Characterization of human D-amino acid oxidase. FEBS Lett. 580(9), 2358-2364.
  • Moreno-Guzmán M., García-Carmona L., Molinero-Fernández Á., Cava F., López Gil M.Á., Escarpa A. (2017) Bi-enzymatic biosensor for on-site, fast and reliable electrochemical detection of relevant D-amino acids in bacterial samples. Sens. Actuators B. Chem. 242, 95-101.
  • Pernot P., Mothet J.-P., Schuvailo O., Soldatkin A., Pollegioni L., Pilone M., Adeline M.T., Cespuglio R., Marinesco S. (2008) Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system. Anal. Chem. 80, 1589-1597.
  • Pilone M.S. (2000) D-Amino acid oxidase: new findings. Cell. Mol. Life Sci. 57, 1732-1747.
  • Polcari D., Kwan A., Van Horn M.R., Danis L., Pollegioni L., Ruthazer E.S., Mauzeroll J. Disk-shaped amperometric enzymatic biosensor for in vivo detection of D-serine. Anal. Chem. 86, 3501-3507.
  • Pollegioni L, Piubelli L, Sacchi S, Pilone MS, Molla G. Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci. 2007 Jun;64(11):1373-94.
  • Pollegioni L., Sacchi S., Caldinelli L., Boselli A., Pilone M.S., Piubelli L., Molla G. (2007b) Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach. Curr. Protein Pept. Sci. 8(6), 600-618.
  • Pollegioni L., Molla G., Sacchi S., Rosini E., Verga R., Pilone M.S. (2008) Properties and applications of microbial D-amino acid oxidases: current state and perspectives. Appl. Microbiol. Biotechnol. 78(1), 1-16.
  • Pollegioni L., Sacchi S. (2010) Metabolism of the neuromodulator D-serine. Cell. Mol. Life Sci. 67, 2387-2404.
  • Pollegioni L., Molla G. (2011) New biotech applications from evolved D-amino acid oxidases. Trends Biotechnol. 29(6), 276-283.
  • Rosini E., Molla G., Rossetti C., Pilone M.S., Pollegioni L., Sacchi S. (2008) A biosensor for all D-amino acids using evolved D-amino acid oxidase. J. Biotechnol. 135, 377-384.
  • Rosini E., Pollegioni L., Ghisla S., Orrù R., Molla G. (2009) Optimization of D-amino acid oxidase for low substrate concentrations towards a cancer enzyme therapy. FEBS J. 276(17), 4921-4932.
  • Rosini E., Molla G., Ghisla S., Pollegioni L. (2010) On the reaction of D-amino acid oxidase with dioxygen: O2 diffusion pathways and enhancement of reactivity. FEBS J. 278(3), 482-492.
  • Sacchi S., Caldinelli L., Cappelletti P., Pollegioni L., Molla G. (2012) Structure–function relationships in human D-amino acid oxidase. Amino acids 43(5), 1833-1850.
  • Stefan R.-I., Nejema R’.M., van Staden J.F., Aboul-Enein H.Y. (2003a) Biosensors for the enantioselective analysis of pipecolic acid. Sens. Actuators B. Chem. 94, 271-275.
  • Stefan R.-I., Bokretsion R.G., van Staden J.F., Aboul-Enein H.Y. (2003b) Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system. Biosens. Bioelectron. 19, 261-267.
  • Stefan van Staden R.-I., van Staden J.F., Aboul-Enein H.Y., Balcu I. (2010) Simultaneous determination of L- and D-T4 using a sequential injection analysis/sensors system. Comb. Chem. High Throughput Screen. 13(6), 497-501.
  • Umhau S, Pollegioni L, Molla G, Diederichs K, Welte W, Pilone MS, Ghisla S. The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation. Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12463-8.
  • Wong K.-S., Fong W.-P., Tsang P.W.-K. (2010) A single Phe54Tyr substitution improves the catalytic activity and thermostability of Trigonopsis variabilis D-amino acid oxidase. New Biotechnol. 27, 78-84.
  • Zheng H., Zhu T., Chen J., Zhao Y., Jiang W., Zhao G., Yang S., Yang Y. (2007) Construction of recombinant Escherichia coli D11/pMSTO and its use in enzymatic preparation of 7-aminocephalosporanic acid in one-pot. J. Biotechnol. 129(3), 400-405.