Amino Acid Racemase Research Articles

An Efficient Route to Both Enantiomers of allo‐Threonine by Simultaneous Amino Acid Racemase‐Catalyzed Isomerization of Threonine and Crystallization

AbstractWe present an efficient method for the production of D‐ and L‐allo‐threonine (allo‐Thr) with very high purity by enzymatic isomerization of L‐ or D‐threonine (Thr) and simultaneous crystallization. Isomerization of Thr to allo‐Thr is catalyzed by a purified amino acid racemase (AArac12996) from Pseudomonas putida NBRC12996, which can easily be obtained from a recombinant E. coli strain by secretion into the medium and subsequent anion exchange chromatography. Crystallization of D‐ and L‐allo‐Thr was performed in a repetitive batch mode over a period of up to 55 days at 30 °C. Total amounts of 30.8 g D‐allo‐Thr and 32.4 g L‐allo‐Thr were obtained with a very good diastereomeric excess of deD‐allo>99.2% and deL‐allo>98.4%, respectively, and in good yields (D‐allo‐Thr: 83%, L‐allo‐Thr: 79%). The enzyme’s remarkable high stability under process conditions resulted in enzyme specific yields of 2.56 g D‐allo‐Thr per mg AArac12996 and 1.62 g L‐allo‐Thr per mg AArac12996. In contrast to chemical multi‐step syntheses of allo‐Thr, our process consists of only one enzyme‐catalyzed reaction step with simultaneous product crystallization. The process is performed under low energy consumption (30 °C, atmospheric pressure) in water and avoids the use and production of any toxic or harmful compounds. Recovery of the enantiomerically pure products is performed by simple filtration which reduces downstream processing significantly compared to chromatographic methods which are usually applied.

A Novel Pyridoxal 5′-Phosphate-dependent Amino Acid Racemase in the Aplysia californica Central Nervous System

D-aspartate (D-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because D-Asp formation is not well understood, determining its function has proved challenging. Significant levels of D-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and D-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5'-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, D-serine and D-Asp signaling pathways.

Regulation and Characterization of the dadRAX Locus for d -Amino Acid Catabolism in Pseudomonas aeruginosa PAO1

D-amino acids are essential components for bacterial peptidoglycan, and these natural compounds are also involved in cell wall remodeling and biofilm disassembling. In Pseudomonas aeruginosa, the dadAX operon, encoding the D-amino acid dehydrogenase DadA and the amino acid racemase DadX, is essential for D- and L-Ala catabolism, and its expression requires a transcriptional regulator, DadR. In this study, purified recombinant DadA alone was sufficient to demonstrate the proposed enzymatic activity with very broad substrate specificity; it utilizes all D-amino acids tested as substrates except D-Glu and D-Gln. DadA also showed comparable k(cat) and K(m) values on D-Ala and several D-amino acids. dadRAX knockout mutants were constructed and subjected to analysis of their growth phenotypes on amino acids. The results revealed that utilization of L-Ala, L-Trp, D-Ala, and a specific set of D-amino acids as sole nitrogen sources was abolished in the dadA mutant and/or severely hampered in the dadR mutant while growth yield on D-amino acids was surprisingly improved in the dadX mutant. The dadA promoter was induced by several L-amino acids, most strongly by Ala, and only by D-Ala among all tested D-amino acids. Enhanced growth of the dadX mutant on D-amino acids is consistent with the finding that the dadA promoter was constitutively induced in the dadX mutant, where exogenous D-Ala but not L-Ala reduced the expression. Binding of DadR to the dadA regulatory region was demonstrated by electromobility shift assays, and the presence of L-Ala but not D-Ala increased affinity by 3-fold. The presence of multiple DadR-DNA complexes in the dadA regulatory region was demonstrated in vitro, and the formation of these nucleoprotein complexes exerted a complicated impact on promoter activation in vivo. In summary, the results from this study clearly demonstrate DadA to be the enzyme solely responsible for the proposed D-amino acid dehydrogenase activity of broad substrate specificity and the physiological functions of DadRAX in catabolism of several D-amino acids and support L-Ala as the signal molecule for induction of the dadAX genes through DadR binding to several putative operator sites.

Insights into the structural properties of d-serine dehydratase from Saccharomyces cerevisiae: An FT-IR spectroscopic and in silico approach

d-serine dehydratase (Dsd) from baker’s yeast is a recently discovered enzyme catalyzing the oxidation of d-serine to pyruvate and ammonia. The reaction depends on the cofactors pyridoxal-5′-phosphate (PLP) and Zn2+, featuring a very high selectivity towards the d-enantiomer of the amino acid serine. In humans, altered levels of d-serine in the cerebrospinal fluid (CSF) and blood correlate with the onset and evolution of a number of neurodegenerative diseases. Up to date very little is known on the structure of Dsd. Hence, we have investigated the structure of this enzyme by means of Fourier Transform infrared (FT-IR) spectroscopy and used the structural data derived thereof to validate a homology model of Dsd. In this model, Dsd adopts a fold that is characteristic of type III pyridoxal-dependent enzymes. This consists of an α/β (TIM) barrel and a β-sandwich domain at the N- and C-termini, respectively. Analysis of the Amide I and Amide III infrared bands revealed that the amounts of α (24%), β (29%) and unordered structures (47%) correlate well with those derived from the model (25%, 29% and 46% respectively), suggesting reliability of the latter. In addition, the model of Dsd was further refined by recreating the PLP- and zinc-restored active site based on a PLP- and zinc-dependent bacterial amino acid racemase recently crystallized, allowing us to identify the potential cofactor and metal binding residues of Dsd.

Detection and Function of the Intramolecular Disulfide Bond in Arginine Racemase: An Enzyme with Broad Substrate Specificity

We found that a single intramolecular disulfide bond between the cysteines C47 and C73 exists in the primary structure of arginine racemase (ArgR) from Pseudomonas taetrolens NBRC 3460, and this is the first example of a pyridoxal phosphate (PLP)-dependent amino acid racemase that contains a disulfide bond. The amino acid racemase activity was still detected, when the disulfide bond of ArgR was disrupted by site-directed mutagenesis or reduced with dithiothreitol (DTT). The thermal and pH profiles and the quaternary structure of ArgR did not change when the disulfide bond of ArgR was disrupted by site-directed mutagenesis. The substrate specificity and the overall structure did not change when the disulfide bond of ArgR was reduced with DTT after the protein was matured. However, these properties changed when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before protein maturation. The total activity of ArgR decreased when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before the protein was matured or when ArgR was expressed in the cytoplasm. Based on these results, we can conclude that the disulfide bond of ArgR is essential for ArgR to fold and mature as an amino acid racemase with broad substrate specificity.

Medium/High Throughput D-Amino Acid Oxidase Colorimetric Method for Determination of D-Amino Acids. Application for Amino Acid Racemases

Free amino acids detection and quantification in samples still calls for elaborated, powerful and usually high cost and sometimes time-consuming technologies such as gas chromatography, HPLC or highly selective and sensitive column-switching techniques. Here, we undertook to develop a simple, one-step, microtiter plate method to detect and/or quantify most D-amino acids in solutions based on the enzyme D-amino acid oxidase and the generation of stoechiometric amounts of hydrogen peroxide. Inclusion of horseradish peroxidase and OPD allows the determination of D- amino acid concentration through the absorbance of the reaction products as compared to serial dilutions of equimolar amounts of L/D amino acid standards. The methodology is easily adapted for a medium/high throughput analysis of amino acid racemase activities.

Enzyme‐assisted physicochemical enantioseparation processes—Part III: Overcoming yield limitations by dynamic kinetic resolution of asparagine via preferential crystallization and enzymatic racemization

The application of enantioseparation methods alone can only yield up to 50% of the desired chiral product. Thus enantioseparation becomes more attractive when accompanied by the racemization of the counter-enantiomer. Here we present first results of dynamic kinetic resolution of L-asparagine (L-Asn) via preferential crystallization and enzymatic racemization from a racemic, supersaturated solution on a 20 mL scale. An enzyme lyophilisate (WT amino acid racemase from P. putida KT2440 (E.C. 5.1.1.10), overexpressed in E. coli BL21(DE3)) was used for in situ racemization (enzyme concentrations varying from 0 to 1 mg/mL). When preferential crystallization was applied without any enzyme, a total of 31 mg of L-Asn monohydrate could be crystallized, before crystal formation of d-Asn started. Crystallization experiments accompanied by enzymatic racemization led to a significant increase of crystallized L-Asn (198 mg L-Asn monohydrate; >92%ee) giving the first experimental proof for this new process concept of dynamic kinetic resolution via preferential crystallization and enzymatic racemization. Measurements of the racemase activity before and after the crystallization process showed no significant differences, which would allow for enzyme recovery and recycling.

A periplasmic, pyridoxal-5′-phosphate-dependent amino acid racemase in Pseudomonas taetrolens

The pyridoxal-5'-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of D: -arginine and D: -lysine by P. taetrolens.

Enzyme-assisted physicochemical enantioseparation processes—Part II: Solid–liquid equilibria, preferential crystallization, chromatography and racemization reaction

This contribution addresses the design and investigation of two hybrid enantioseparation processes including an enzymatic racemization step in order to enhance the overall performance. Complementary to part I where the manufacturing and the characterization of an amino acid racemase (EC 5.1.1.10) was emphasized [Würges, K., Petrusevska, K., Serci, S., Wilhelm, S., Wandrey, C., Seidel-Morgenstern, A., Elsner, M.P., Lütz, S., 2009. Enzyme-assisted physicochemical enantioseparation processes—part I: production and characterization of a recombinant amino acid racemase. J. Mol. Cat. B (in print), online available: doi:10.1016/j.molcatb.2008.10.006.], the work presented in this paper tends more towards developing a data base for potential process schemes for the manufacture of selected amino acids. The first proposed process concept (P-I) couples preferential crystallization (PC) and racemization for the production of l-asparagine ( l-Asn) using racemic mixture of dl-asparagine (conglomerate-forming system) as a starting material, while the second concept (P-II) integrates chromatography and racemization for the preparation of l-methionine ( l-Met) starting with racemic mixture of dl-methionine (compound-forming system). As mentioned in part I, a racemization unit, where the unwanted enantiomer will be converted into racemate, is incorporated into the hybrid processes for the sake of 100% yield, theoretically. Besides the basic investigation according to the solid–liquid equilibria, PC and chromatography, the focus of this paper is mainly on the kinetic studies of the racemization reaction. Initially, the solubility ternary phase diagrams of both examined systems were determined, leading into the idea of combination of the proposed process schemes. For P-I the concept of PC of l-Asn was experimentally proven and the kinetics of the racemization was examined for d- and l-Asn in water using purified lyophilizate (PL). Concerning P-II, for the chromatographic unit the impact on the separation of dl-Met on eremomycin based stationary phase using KPi buffer and MeOH as mobile phase was evaluated in terms of resolution and selectivity at three different temperatures by varying the content of methanol (MeOH) in the mobile phase and the pH. The experiments for determination of the racemization kinetics were done for a compromised parameter set using crude lyophilizate (CL). In both cases a Michaelis–Menten three-step model was used to describe the enzymatic reaction.

Arginine racemization by coupled catabolic and anabolic dehydrogenases

D-amino acids exist in living organisms as specialized components of many different machineries. Biosynthesis of D-amino acids from racemization of predominant L-enantiomers is catalyzed by a single enzyme. Here, we report the finding of a novel 2-component amino acid racemase for D-to-L inversion in D-arginine metabolism of Pseudomonas aeruginosa. From DNA microarray analysis, the putative dauBAR operon (for D-arginine utilization) of unknown functions was found to be highly induced by D-arginine. The importance of the dau operon in D-arginine metabolism was demonstrated by the findings that strains with a lesion at dauA or dauB failed to use D-arginine as sole carbon source. Two lines of evidence suggest that DauA and DauB are required for D-to-L racemization of arginine. First, growth complementation of an L-arginine auxotroph by D-arginine was abolished by a lesion at dauA or dauB. Second, D-arginine induced L-arginine-specific genes in the parental strain PAO1 but not in its dauA or dauB mutants. This hypothesis was further supported by activity measurements of the purified enzymes: DauA catalyzes oxidative deamination of D-arginine into 2-ketoarginine and ammonia, and DauB is able to use 2-ketoarginine and ammonia as substrates and convert them into L-arginine in the presence of NADPH or NADH. Thus, we propose that DauA and DauB are coupled catabolic and anabolic dehydrogenases to perform D-to-L racemization of arginine, which serves as prerequisite of D-arginine utilization through L-arginine catabolic pathways.

Crystal Structure of Diaminopimelate Epimerase from Arabidopsis thaliana, an Amino Acid Racemase Critical for l-Lysine Biosynthesis

Diaminopimelate (DAP) epimerase is a key enzyme for the biosynthesis of lysine in plants. Lysine is an essential dietary nutrient for mammals. In both plants and bacteria, DAP epimerase catalyzes the interconversion of ll-DAP and dl( meso)-DAP. The absence of a mammalian homolog makes DAP epimerase a promising target for the design of novel herbicides and antibacterials. This enzyme requires no cofactors and it functions through an unusual mechanism involving two cysteine residues acting in concert and alternating as a base (thiolate) and as an acid (thiol). The present study reports the crystal structures of two enzyme–inhibitor complexes of DAP epimerase from Arabidopsis thaliana with different isomers of the irreversible inhibitor and substrate mimic, 2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate, at 1.95 and 2.3 Å resolution. These structures provide the first atomic details of a plant amino acid racemase. Structural analysis reveals that ligand binding to a cleft between the two domains of the enzyme is accompanied by domain closure with two strictly conserved cysteine residues, Cys99 and Cys254, optimally positioned to perform acid/base catalysis via a carbanion stabilization mechanism on the stereogenic α-carbon atom of the amino acid. Stereochemical control in catalysis is achieved by means of a highly symmetric catalytic site that can accommodate both the l and d stereogenic centers of DAP at the proximal site, whereas specific interactions at the distal site require only the l configuration. Structural comparisons of the plant enzyme with its bacterial counterpart from Haemophilus influenzae reveal significant conservation of amino acid residues around the active site that extends to their three-dimensional structures and catalytic mechanism.

Enzyme-assisted physicochemical enantioseparation processes: Part I. Production and characterization of a recombinant amino acid racemase

The demand for enantiopure substances, e.g. for pharmaceutical applications or fine chemical production, continues to increase. This has led to the development of numerous stereoselective synthesis methods. Nevertheless a large number of chemical syntheses still result in racemic mixtures making a subsequent enantioseparation step necessary and thus are restricted to a maximum yield of 50%. Our work focuses on strategies to overcome this limitation by combining physicochemical separation processes with enzymatic racemization of the unwanted enantiomer in order to produce enantiopure amino acids. This paper deals with the production and characterization of a suitable amino acid racemase with broad substrate specificity (EC 5.1.1.10) from Pseudomonas putida which we cloned into Escherichia coli. Two enzyme lyophilizates of different purity were obtained from which the crude ( CL) was sufficient for the racemization of methionine (Met) and the pure ( PL) was used for asparagine (Asn). Racemization reactions of D-/ L-Asn in H 2O and D-/ L-Met in 95 vol.% 100 mM KP i-buffer, 5 vol.% methanol (MeOH) at different pH values and temperatures were characterized. The studied range of reaction parameters was chosen in dependency on planned enantioseparation processes. We found increasing V max values when temperature was risen stepwise from 20 to 40 °C for both systems and when pH was shifted from 6 to 8 for the Met system. The presented results provide the basis for engineering enzyme-assisted physicochemical enantioseparation processes.

Environmental Microbiology meets Microbial Biotechnology

<i>Environmental Microbiology</i> meets <i>Microbial Biotechnology</i>

Glutamate racemase as a target for drug discovery.

SummaryThe bacterial cell wall is a highly cross‐linked polymeric structure consisting of repeating peptidoglycan units, each of which contains a novel pentapeptide substitution which is cross‐linked through transpeptidation. The incorporation of d‐glutamate as the second residue is strictly conserved across the bacterial kingdom. Glutamate racemase, a member of the cofactor‐independent, two‐thiol‐based family of amino acid racemases, has been implicated in the production and maintenance of sufficient d‐glutamate pool levels required for growth. The subject of over four decades of research, it is now evident that the enzyme is conserved and essential for growth across the bacterial kingdom and has a conserved overall topology and active site architecture; however, several different mechanisms of regulation have been observed. These traits have recently been targeted in the discovery of both narrow and broad spectrum inhibitors. This review outlines the biological history of this enzyme, the recent biochemical and structural characterization of isozymes from a wide range of species and developments in the identification of inhibitors that target the enzyme as possible therapeutic agents.

D‐Amino acids in the brain: structure and function of pyridoxal phosphate‐dependent amino acid racemases

D-serine serves as a co-agonist of the N-methyl D-aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer's disease and amyotrophic lateral sclerosis. D-Serine is synthesized by a pyridoxal 5'-phosphate (PLP)-dependent serine racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP-dependent amino acid racemase on the basis of its 3D structure. We compared the eukaryotic serine racemase with bacterial alanine racemase, the best-studied enzyme among the PLP-dependent amino acid racemases, and thus suggested a putative reaction mechanism for mammalian D-serine synthesis.

Dynamics of catalysis revealed from the crystal structures of mutants of diaminopimelate epimerase

Diaminopimelate (DAP) epimerase catalyzes the stereoinversion of ll-DAP to meso-DAP, a precursor of l-lysine and an essential component of the bacterial peptidoglycan. This function is vital to bacteria and the enzyme therefore represents an attractive target for the design of novel anti-bacterials. DAP epimerase belongs to the group of PLP-independent amino acid racemases that function through a rather unusual mechanism involving two cysteines acting in concert as a base (thiolate) and an acid (thiol). We have solved the crystal structures of the apo-forms of DAP epimerase mutants (C73S and C217S) from Haemophilus influenzae at 2.3 Å and 2.2 Å resolution, respectively. These structures provide a snapshot of the enzyme in the first step of the catalytic cycle. Comparisons with the structures of the inhibitor-bound form reveal that the enzyme adopts an ‘open conformation’ in the absence of substrates or inhibitors with the two active site cysteines existing as a thiol–thiolate pair. Substrate binding to the C-terminal domain triggers the closure of the N-terminal domain coupled with tight encapsulation of the ligand, stabilization of the conformation of an active site loop containing Cys73 and expulsion of water molecules with concomitant desolvation of the thiolate base. This structural rearrangement is critical for catalysis.

Prediction and assignment of function for a divergent N-succinyl amino acid racemase

The protein databases contain many proteins with unknown function. A computational approach for predicting ligand specificity that requires only the sequence of the unknown protein would be valuable for directing experiment-based assignment of function. We focused on a family of unknown proteins in the mechanistically diverse enolase superfamily and used two approaches to assign function: (i) enzymatic assays using libraries of potential substrates, and (ii) in silico docking of the same libraries using a homology model based on the most similar (35% sequence identity) characterized protein. The results matched closely; an experimentally determined structure confirmed the predicted structure of the substrate-liganded complex. We assigned the N-succinyl arginine/lysine racemase function to the family, correcting the annotation (L-Ala-D/L-Glu epimerase) based on the function of the most similar characterized homolog. These studies establish that ligand docking to a homology model can facilitate functional assignment of unknown proteins by restricting the identities of the possible substrates that must be experimentally tested.

Model‐based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium‐constrained continuous processes

The amino acid racemase with broad substrate specificity from Pseudomonas putida DSM 3263 was overproduced and characterized with respect to application in an integrated multi-step process (e.g., dynamic kinetic resolution) that--theoretically--would allow for 100% chemical yield and 100% enantiomeric excess. Overexpression of the racemase gene in Escherichia coli delivered cell free extract with easily sufficient activity (20-50 U mg(-1) total protein) for application in an enzyme membrane reactor (EMR) setting. Model-based experimental analysis of a set of enzyme assays clearly indicated that racemization of the model substrates D- or L-methionine could be accurately described by reversible Michaelis-Menten kinetics. The corresponding kinetic parameters were determined from progress curves for the entire suitable set of aqueous-organic mixtures (up to 60% methanol and 40% acetonitrile) that are eligible for an integrated process scheme. The resulting kinetic expression could be successfully applied to describe enzyme membrane reactor performance under a large variety of settings. Model-based calculations suggested that a methanol content of 10% and an acetonitrile content of 20% provide maximum productivity in EMR operations. However product concentrations were decreased in comparison to purely aqueous operation due to decreasing solubility of methionine with increasing organic solvent content. Finally, biocatalyst stability was investigated in different solvent compositions following a model-based approach. Buffer without organic content provided excellent stability at moderate temperatures (20-35 degrees C) while addition of 20% acetonitrile or methanol drastically reduced the half-life of the racemase.

New Insights into the Reaction Mechanism Catalyzed by the Glutamate Racemase Enzyme: pH Titration Curves and Classical Molecular Dynamics Simulations

The mechanism of the reactions catalyzed by the pyridoxal-phosphate-independent amino acid racemases and epimerases faces the difficult task of deprotonating a relatively low acidicity proton, the amino acid's alpha-hydrogen, with a relatively poor base, a cysteine. In this work, we propose a mechanism for one of these enzymes, glutamate racemase (MurI), about which many controversies exist, and the roles that its active site residues may play. The titration curves and the pK1/2 values of all of the ionizable residues for different structures leading from reactants to products have been analyzed. From these results a concerted mechanism has been proposed in which the Cys70 residue would deprotonate the alpha-hydrogen of the substrate while, at the same time, being deprotonated by the Asp7 residue. To study the consistency of this mechanism classical molecular dynamics (MD) simulations have been carried out along with pK1/2 calculations on the MD-generated structures.

Synthesis of dl-tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive L-amino acids to DL-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of D-amino acids, especially D-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. L-Trp (20 mM) was successfully racemized, and the proportion of D-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial L-Trp.