Characteristics of alanine racemase in Lactobacillus sakei ZH‐2 strain

Abstract Some d‐amino acid functions for food production are widely known: d‐alanine improves sensory evaluations of sake, beer, and fermented foods. Therefore, for the application of d‐amino acids, alanine racemase (ALRase) in Lactobacillus sakei ZH‐2, which has strong racemization, was analyzed using molecular biological methods. It had been hypothesized that ALRase coding DNA, alr, in ZH‐2 strain differs from those of other Lactobacillus sakei strains. However, complete genome sequencing by the National Center for Biotechnology (NCBI) revealed the amino acid sequence of alr in ZH‐2 strain to have homology of 99.4% similarity with the alr in Lactobacillus sakei 23K strain. However, it is considered that the sequence of alr was a unique amino acid sequence in the lactic acid bacteria group. DNA “alr” of ZH‐2 strain has a 1140 bp DNA base with 41 kDa molecular mass. Its molecular mass was inferred as approximately 38.0 kDa using SDS‐PAGE. Its optimum conditions are pH 9.0 at 30–40°C, showing stability at pH 9.0–10.0 and 4–40°C. Its cofactor is pyridoxal phosphate. Its activity is activated more by copper and zinc ions than by the lack of a metal ion. Additionally, its K m is 1.32 × 10−3 (mol), with V max of 4.27 × 10−5 (μmol−1 min−1). ALRase reacted against alanine most strongly in other substrates such as amino acids. The enzyme against serine was found to have 40% activity against alanine. The enzyme converted up to 54.5% of d‐alanine from l‐alanine ZH‐2 strain.

in regulating N-methyl-D-aspartate receptors in the human brain (Tsai et al., 1998). Moreover, d-amino acids are broadly applicable to functional foods intended for health improvement because they enhance the intestinal environment. Large amounts of d-amino acids have been found in fermented foods such as cheese (Bruckner & Hausch, 1989), beer (Bruckner & Hausch, 1989), sake (Gogami et al., 2011), wine (Erbe & Bruckner, 1998), and vinegar (Mutaguchi et al., 2013), which have more amino acids than fresh foods have (Marcone et al., 2020). Actually, d-amino acids have stronger tastes than l-amino acids. Sake containing d-amino acids such as d-alanine, d-aspartic acid, and d-glutamic acid is evaluated particularly highly in terms of sensory evaluation (Okada et al., 2013). Also, d-amino acids have been produced by microorganisms during fermentation processes taking place in cheese, bread, and vinegar. Such fermented foods, therefore, have strong umami or sweet tastes. During vinegar production, main fermenting microorganisms such as Acetobacter spp. and lactic acid bacteria are used to produce d-amino acids to enhance taste (Mutaguchi et al., 2013).
Reportedly, d-amino acids in sake, which are produced by lactic acid bacteria using Kimoto sake seed mash brewing, depend on the type or species of lactic acid bacteria used for sake moto brewing (Kobayashi, 2019). For example, Lactobacillus sakei NBRC 15893 converts d-alanine, d-glutamic acid, and d-aspartic acid using racemic enzymes (Kobayashi, 2019). The varieties and concentrations of racemized amino acids differ in the fermented solution because each microorganism's lactic acid bacteria have a varietal amino acid racemase. Therefore, for application to foods, many researchers should investigate varietal amino acid racemases from many lactic acid bacteria. Matsumoto and Kanauchi (2019) reported that d-amino acid assay developed rapidly and comprehensively using d-amino acid oxidase and lactic acid bacteria Lactobacillus sakei. The lactic acid bacteria were isolated for tasty sake brewing. Reportedly, the isolated Lactobacillus sakei ZH-2 strain had high racemization activity, with 14% conversion. The curd enzyme racemized l-alanine and l-serine. The findings indicate racemase as one kind of alanine racemase (ALRase). The produced d-alanine improves sake sensory evaluation (Okada et al., 2013). Moreover, d-amino acids improve the sensory evaluations of beer, wine, and several seasonings. During acid production, enzyme preparation for damino acid racemase addition might be undertaken to improve their taste and other characteristics. Moreover, d-serine, which facilitates human brain regulation, is a major gliotransmitter in mammalian central nervous systems (Hashimoto & Oka, 1997;Wolosker et al., 2008).
Applications of d-amino acids have broad potential for producing sake, beer, wine, medicines, and healthy functional foods.
Nevertheless, the racemase in ZH-2, which converts a high level of d-amino acid in a medium, has not been investigated for its characteristics, optimum conditions, or coding DNA base sequencing.
As described herein, the enzyme was expressed by recombinant Escherichia coli. Recombinant racemase was investigated for its enzymic characteristics because it was intended for the application of racemase in Lactobacillus sakei ZH-2 during food production, sake brewing, and beer brewing.

| Materials and chemicals
Chemicals for which no vendor is listed herein were purchased from Fujifilm Wako Pure Chemical Corp.

| Extraction of genomic DNA from Lactobacillus sakei ZH-2
Genomic DNA was extracted according to the published procedures (Ausubel et al., 2003).

| Inserting the alr to pMD20 T-Vector and its transformation to competent cells
Ligation of insert DNA and vector transformation of host cells were conducted using the reported methods (Ausubel et al., 2003).

| Cultivation of transformation to competent cells and extraction ALRase from Escherichia coli DH5α-pMD20-alr
Escherichia coli DH5α-pMD20-alr were cultivated in 10 mL of LB medium with 0.1 M IPTG (isopropylβ-D-thiogalactoside). After cell cultivation, the cell mass was washed using 0.9% sodium chloride. After the cell mass was gathered by centrifugation at 20,400 g for 5 min at 4°C, the cell mass was resuspended in 0.5 mL of cell digestion buffer (50 mM Tris (trishydroxymethylaminomethane)hydrogen chloride (HCl); pH 8.0, 150 mM sodium chloride, 1 mM dithiothreitol). Then, 0.5 mL of lysozyme solution (containing 0.4 mg/mL) was poured as the final concentration. The mixture was incubated at 4°C for 30 min. After centrifugation, the supernatant was a crude enzyme solution.

| Assaying ALRase activity
Using the modified method described by Matsumoto and Kanauchi (2019), the ALRase activity was assayed. Enzyme (50 μL) was added to 850 μL of 10 mM l-alanine solution in 50 mM Ncyclohexyl-2-aminoethanesulfonic acid (CHES, pH 9.0)-NaOH (sodium hydroxide) buffer containing 10 mM pyridoxal 5′-phosphate (PLP). Then, it was reacted at 37°C for 10 min. After the reaction, 50 μL of 100 mM hydrochloric acid solution was added to the reaction mixture to stop the reaction. It was then left to stand for 15 min at 37°C. After 50 μL of 100 mM sodium hydroxide solution was added to the reaction mixture to neutralize it, the mixture was centrifuged at 20,400 g for 5 min at 4°C. The supernatant (50 μL) and the amino acid oxidase solution (150 μL, 50 mM CHES-NaOH buffer, pH 9.0, containing 0.6 μmol min −1 mL −1 d-amino acid oxidase (DAO); 25 μmol min −1 mL −1 lactate dehydrogenase (LDH), and 0.2 mM nicotinamide adenine dinucleotide, reduced (NADH)) were poured to a 96-well microplate (#1860-096; AGC Techno Glass Co., Ltd.). The microplate absorbance was measured at 340 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific Inc.) after incubation at 37°C for 10 min. As Blank 1, assaying, phosphate buffer (50 mM, pH 6.0) instead of 50 μL of crude enzyme solution was added to 850 μL of 10 mM l-alanine solution in 50 mM CHES buffer containing 10 mM PLP. Then, after it was reacted, 50 μL of crude enzyme added to 850 μL of CHES buffer containing PLP without l-alanine was reacted as 'Blank 2' assaying to exclude effects of contaminating enzyme as LDH. After the reaction and centrifugation, the reaction solution was stopped. The solution was neutralized and centrifuged.
All blank solutions (50 μL) and DAO-LDH solution were poured into a 96-well microplate. The absorbance was measured at 340 nm using a microplate reader after incubation at 37°C for 10 min. The ΔA 340 was calculated according to the following formula.
A standard curve used 0-1.0 mM d-alanine dissolved in 50 mM CHES-NaOH buffer. Both ΔA 340 and the concentrations of d-alanine are shown on the graph.

| DNA sequencing
The nucleotide base sequence of the alr was found using a DNA auto-sequencer (ABI 3130; Applied Biosystems) with a kit (big Dye Terminator v 3.1 Cycle Sequencing; Applied Biosystems) according to the manufacturer's recommended protocols.

| Homology analysis
The homology characteristics of each sequence were assessed using a Basic Local Alignment Search Tool (BLAST; NCBI, 2022).

| Phylogenetic tree analysis of alanine racemase
Data of the nucleotide base sequences of 30 strains of genes of lactic acid bacteria were selected from the homology analysis by BLAST (NCBI, 2022), they were analyzed using neighbor-joining method with software 11 (Molecular Evolutionary Genetics Analysis; Pennsylvania State University, 2022).

| High-level expression of recombinant alanine racemase (rALRase) by Escherichia coli LB21-pCold-alr
After the transformation of Escherichia coli LB21, SOC medium (100 μL) was added to the transformation cell mass. Then, they were incubated at 37°C for 60 min. After the cells were mass inoculated to LB plate medium (containing 100 μg/mL ampicillin), the medium was cultivated at 37°C for 16 h. Cells from each colony were inoculated to 10 mL of LB medium containing 100 μg/mL ampicillin using the medium cultivated until O.D. 600 (= 0.5). They were incubated and recultivated in LB plate medium (containing 0.1 mM IPTG). The colony having the highest ALRase activity was selected as Escherichia coli LB21-pCold-alr.
The protein was run at 20 mA for 75 min. After it was gel dyed using Ez Stain Aqua (#AE-1340; Atto Corp.) standard marker, SDS-PAGE Molecular Weight Standards (Broad Range, #161-0317) was used for electrophoresis. Escherichia coli BL21 competent cells were used to prepare negative control extraction.
Blank 1 and Blank 2 were assayed to calculate ΔA 340 under each pH or each temperature condition according to the described method.
Furthermore, each produced d-alanine was assayed.

| Metal ion and inhibitor effects against rALRase activity
Metal ions (sodium chloride, potassium chloride, magnesium sulfate, zinc sulfate, copper sulfate, cobalt sulfate, iron dichloride, calcium chloride, mercury chloride, and manganese sulfate) were dissolved to a final concentration of 1 mM in 50 mM CHES-NaOH

| Kinetics analysis of rALRase
Kinetics of rALRase as V max and K m values were calculated using Lineweaver-Burk plotting after assaying ALRase within an l-alanine solution (0.1-50 mM).

| Substrate specificity of ALRase
The d-amino acids produced by ALRase were assayed using peroxide from d-amino acid by d-alanine oxidase using peroxidase (Matsumoto & Kanauchi, 2019). First, for the main sample assay, were incubated at 37°C for 30 min. The microplate absorbance was measured at 590 nm using a microplate reader. All standard curves for 0-1.0 mM amino acids were produced to show their activity.

| Cloning of alanine racemase (ALRase)
For this study, the primers for PCR amplification of alr were prepared from genomic information in Lactobacillus sakei with complete genome sequencing from the National Center for Biotechnology Information (NCBI, 2022). After amplification by PCR, the alr was analyzed using pMD20 T-Vector for TA cloning. Consequently, the amplification of DNA containing coding alr was found on the gel for approximately 1300 bp (Data not shown). Then, T-vector pMD20 and alr were ligated. After running electrophoresis, the bands of the DNA ligated T-vector pMD20 and alr (Escherichia coli DH5α-pMD20-alr) were apparent at approx. 4000 bp on agarose gel (data not shown), indicating transgenesis ligating alr and pMD20.
Escherichia coli DH5α without transformation as negative control had no ALRase activity. On the other hand, Escherichia coli DH5α-pMD20-alr has 12.1 (μmol min −1 mL −1 ) ALRase activity (data not shown). Xue et al. (2013) reported that the competent Escherichia coli did not find ALRase. Their data agreed with those of earlier studies. Furthermore, Hols et al. (1997) reported that no ALRase-deleting gram-positive bacteria strain can be grown without d-alanine medium because it does not produce cell walls, which are necessary components for d-amino acids. Palumbo et al. (2004)  At the amino acid sequence, the 117th amino acid in alr, threonine in Lactobacillus sakei 23K strain, was replaced by alanine in the ZH-2 strain. It is considered that 'TTATTTT' is a Promoter residue found 16 bp upstream from the initiator codon (data not shown).
For several reasons, the ZH-2 strain produces large amounts of d-alanine in the medium. For example, ZH-2 strain expressed highlevel ALRase or some other. There might be racemase in ZH-2 cells F I G U R E 1 Amino acid sequence of ALRase of ZH2 strain.
that is not this ALRase. Lactobacillus sakei had bifunctional aminoacid racemase with multiple substrate specificities: MalY (Kato & Oikawa, 2018). Future studies must be conducted to analyze other racemization enzymes and ALRase expression levels using rtPCR and its secretion levels.
Alanine racemase findings related to phylogenetic tree analysis are presented in Figure 2. To analyze alanine racemase in L. sakei Lacticaseibacillus which belongs to the Lactobacillus genus as L. casei or L. paracasei was also found to have a weak relation. Results suggest that alanine racemase in L. sakei ZH-2 was unique not only in the same L sakei group but also in other lactic acid bacteria groups.

Many bacteria have racemases such as ALRase and glutamic
acid. PLP, a co-factor for ALRase, forms a Schiff base in an active center site on PLP-dependent enzyme as ALRase, as reported by Hayashi (1995). Furthermore, Watanabe et al. (1999) reported that an active site in ALRase of Bacillus stearothermophilus had lysine in a 39-residue amino acid. Some ALRases in Bacillus sp. had the amino acid present in the activity center (Shaw et al., 1997;Tanizawa et al., 1988).
In fact, four amino acids have been reported as being in the ac- Lactobacillus, Bacillus, and Geobacillus sp. However, ALRase in the ZH-2 strain, the lysine or alanine next to histidine-leucine at approximately the 130th amino acid residue, had neither. Serine S134 is located at the next H132 and L133 in the enzyme. The 134th amino acid in the ZH-2 strain replaced serine (S134) from lysine or alanine. However, lysine residue and PLP bind to form a Schiff base.
Therefore, some amino acids such as K158, R141, M318, and D319 are thought to have some relation with racemase activity. Future F I G U R E 2 Phylogenetic tree analysis of alanine racemase.
studies will be conducted to investigate amino acid residues at the active center site.

| Characteristics of rALRase
Next, pCold-alr ligated alr coding ALRase was transferred to Escherichia coli BL21 for high expression of ALRase. Its molecular weight according to SDS-PAGE was found to be 35.7-38.7 kDa (Figure 3), which is less than that of the amino acid sequence ( Figure 1). Seow et al. (2000) reported the ALRase of Thermus thermophilus as having 38 kDa molecular mass. Kobayashi et al. (2015) reported ALRase of L. salivarius as approx. 41 kDa. Their data agreed with molecular mass data of ALR from this amino acid sequence.
Next, optimum pH and temperature stability of purified rALRase are presented in Figure 4a,b. Actually, rALRase had the highest activity, at pH 9.0. The highest condition was calculated as having 100% as the relative activity. The rALRase was 90% of the relative activity at pH 8.0, 74% of the relative activity at pH 9.5, and 57%-69% of the relative activity at pH 10. Furthermore, in acid pH ranges, it was 44%-47% of the relative activity at pH 6.0 and 32% of the relative activity at pH 5.0. Results demonstrate that the enzyme reacted more in an alkaline condition than in an acid condition.
Lactic acid bacteria are grown under acidic conditions. But intercellular pH of some bacteria generally keeps at 7.5-8.0 by cell homeostasis (Booth, 1985). And some intercellular enzymes had optimum pH at alkaline condition (Hutkins & Nannen, 1993). In particular, X-prolyl-dipeptidyl peptidase in Lactobacillus sakei reacted under alkaline condition, and it was approx. 80% of the relative activity at pH 8.0 (Sanz & Toldrá, 2001). Therefore, it is considered that some intercellular enzymes such as rALRase also had optimum pH at alkaline to react in the cytoplasm of the bacteria.
The enzyme reacted at 15-50°C. The highest temperature of 40°C was the optimum temperature of rALRase. In fact, the activity at 40°C was calculated as 100% of the relative activity. Activity at 50°C was calculated as 72% of the relative activity. The activity at 60°C was only 4% of the relative activity. As one might expect, the enzyme activity was nonexistent at temperatures higher than 70°C.
Generally, ALRase having lactic acid bacteria was stable in alkaline conditions. For instance, for ALRase in L. sakei NBRC15893 isolated from Kimoto as sake seed mash, Kato and Oikawa (2018)  The activity persisted after holding at 4°C: it was calculated to have 100% as the relative activity. The enzyme retained 96% of its activity after holding at 15°C: 92%-95% of the relative activity was retained after holding at 30-40°C. However, only 3% of the relative activity remained after holding at 60°C. Results indicate that the rALRase is stable at 4-40°C. In addition, L. salivarius with ALRase had heat tolerance for 30 min at 50°C: 70% of its activity was retained, as reported by Kobayashi et al. (2015). Furthermore, ALRase having Bacillus anthracis had heat tolerance, even at 60°C, at which 60% of its activity remained (Kanodia et al., 2009). These results indicate that rALRase has no heat tolerance.

| Effects of rALRase against metal ions and inhibitors
Effects of rALRase against metal ions and inhibitors are presented in Table 1. For this enzyme assay method, ALRase, amino acid oxidase, and lactic acid dehydrogenase were used. Therefore, to assay rAL-Rase within ions or inhibitors precisely, a standard curve of d-alanine was produced using the d-alanine solution, oxidase, and lactic acid dehydrogenase within each metal ion or inhibitor. The rALRase was assayed and compared with and without PLP in the reaction mixture system. The rALRase with PLP in the reaction mixture had 1.5-2 times the activity of the reaction mixture without PLP (data not shown). It is readily apparent that its cofactor was PLP.
Generally, enzymes of many kinds have an active center site combining metal ions, which allow for effective enzymic catalysis.
Standard curves of d-alanine using solutions containing each metal ion (final concentration 0.1 mM) or inhibitor (final concentration 0.1 mM) and the enzyme system using two enzymes as DAO and LDH might be inhibited by some metal ions or inhibitors. Consequently, the standard curve of d-alanine was produced under existing metal ion or inhibitor conditions. Their metal and inhibitor concentrations were regarded as being of low levels, such as 0.1 mol/L (data not shown), after rALRase reaction solutions. Therefore, all standard curves were made without metal ion or inhibitor inhibition of DAO or LDH. Their relative activity (100%) was recorded as the activity without the metal ion or inhibitor (Table 1). The enzyme activity was activated by copper and zinc ions. It was 133% of the relative activity by copper ion and 327% of the relative activity by zinc ion. It was significantly highest activity.
One kind of PLP-dependent enzyme was activated by the metal ion. For instance, Yoshimura and Goto (2008) reported that serine dehydrogenase was activated by zinc ion. Moreover, they described that its ion combined cysteine amino acid residues at its active center sites. Furthermore, some enzymes were deactivated by mercury ion binding with cysteine residue at the active site (Ynalvez et al., 2016).
Although rALRase had five cysteine residues in components, only one cysteine, C317, is located by M318 and D319 at the main active site. Probably, mercury ions are deactivated from binding with C317.
Moreover, the effects of inhibitors, chelating reagents of EDTA and EGTA, inhibited rALRase, suggesting the respective activities of 59.6% and 55.6%. These findings demonstrate that the enzyme requires metal ions for enzymes. Yamashita et al. (2003) reported ALRase of Bifidobacterium spp.
as inhibited by DTNB, which reacts with sulfhydryl group in amino acid residues (Ajsuvakova et al., 2020;Bamforth et al., 2009). The ALRase was inhibited by divalent ions as a mercury ion sulfhydryl group in amino acid residues. Both enzymes are regarded as having a sulfhydryl group in amino acid residues in the active center.
The rALR had lysine, arginine, methionine, and aspartic acid in

| Kinetic analyses and kinetic analyses of rALRase
Results of kinetic analyses of rALRase are presented in Figure 5.
After rALRase was assayed against each concentration of alanine solution, the data and alanine concentrations were presented as a Lineweaver-Burk plot. There, K m , representing the affinity of the substrate and enzyme, was 1.32 × 10 −3 (M). In addition, V max , signifying the reaction speed, was 4.27 × 10 −5 (μmol −1 min −1 ). ALRase having L. salivarius was 5.33 × 10 −3 (M), demonstrating that the ALRase from ZH-2 has low K m . It had high-affinity characteristics with substrates such as l-alanine.
Substrate specificity of rALR was analyzed. ALRase assay specifically decreased NADH by lactic acid dehydrogenase and pyruvic acid produced from d-alanine by d-alanine oxidase. Using this method, ALRase was not useful for assay against other substrates.
Therefore, ALRase for other substrates was assayed with peroxide from d-amino acid by d-alanine oxidase using peroxidase according to the method reported by Matsumoto and Kanauchi (2019). That earlier report described that the crude ALRase from ZH-2 strain reacted most against l-alanine in other amino acids. Moreover, rAL-Rase reacted most against l-alanine in other amino acids.
The activity of rALRase (13.8 μmol min −1 mL −1 ) was calculated as 100% of the relative activity. The data are portrayed in Figure 6.
The enzyme reacted against serine was 40% of the relative activity. The enzyme reacted against arginine was 15%. The enzyme reacted against proline was 14%. The enzyme reacted against leucine was 10% because d-alanine conversion by ALRase included the peptidoglycan layer in the cell wall (Hols et al., 1997). d-Alanine and d-glutamic acid included all four classified peptidoglycan types.
Lactobacillus sakei has A4β type peptidoglycan composing d-alanine, d-glutamic acid, and d-aspartic acid (Lund et al., 1999). However, this racemase did not react against aspartic acid or glutamic acid. Kato and Oikawa (2018) reported that amino acid racemase in Lactobacillus sakei has activity against glutamic acid, but it has very low activity. Furthermore, they reported that alanine is reacted most by the enzyme in others. These data agree with those presented in their report. Yoshimura and Goto (2008) reported that serine racemase in the brain has 31% similar homology to ALRase of B. stearothermophilus. They also reported that important residues are conserved for the catalysis racemase reaction. Therefore, we infer that ALRase in ZH-2 strain also has activity against serine and other amino acids. However, this ALRase has no activity against histidine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, or tyrosine.

| Substrate-product equilibrium of rALRase
Finally, the substrate-product equilibrium of rALRase was analyzed to assay d-alanine (Figure 7). The enzyme converted d-alanine from l-alanine quickly for up to 10 min. Then, it converted it slowly after F I G U R E 5 Lineweaver-Burk plot of rALRase activity from Escherichia coli BL21-pCold-alr.
10 min. For more than 30 min, the conversion ratio of d-alanine from lalanine was 56.0%. ALRase in Aeromonas hydrophila was keq(L/D)1.0 (Liu et al., 2015). The enzyme will convert 50% of the substrate: until l-alanine:d-alanine (1:1). Results show that Lactobacillus sakei ZH-2 strain has some potential to produce d-amino acid as d-alanine for the production of good sake. For this study, this rALRase was not assayed to convert l-alanine from d-alanine. Assay of K m of racemase against conversion of d-alanine to l-alanine will be undertaken in future studies.

| CON CLUS ION
The Lactobacillus sakei ZH-2 strain with alanine racemase (ALRase) was analyzed for application to food production. The ALRase was expressed using transformed Escherichia coli DH5α-pMD20-alr strain. The transduction Escherichia coli DH5α-pMD20-alr had 13.9 (μmol −1 min −1 ). The alr DNA was 1140 bp; ALRase has 41 kDa molecular weight. However, its molecular weight was found to be 35.7-38.7 kDa by SDS-PAGE. Results showed 99.4% similarity of homology of amino acid sequence with complete genome-sequenced Lactobacillus sakei 23K. For high expression of ALRase, the transformed strain was designated as Escherichia coli BL21-pCold-alr strain. Consequently, expressing ALRase was obtained. Its optimum pH is 9.0, but results indicate that it is stable at pH 9.0 and pH 10.0.
Its optimum temperature is 30-40°C, but it is stable at 4-40°C.
Heating to temperatures higher than 60°C eliminates its activity. Its co-factor was found to be PLP. It is activated by copper ion and zinc ions such as bivalent ions. This PLP-dependent and metalrequiring enzyme has K m of 1.32 × 10 −3 (mol). Its V max is 4.27 × 10 −5 (μmol −1 min −1 ). Results obtained from this study demonstrate that this racemase has higher affinity with the substrate than others.
Substrate specificity of the ALRase was assayed using d-amino acid oxidase, which can assay many d-amino acids. Alanine was reacted most by rALRase in other substrates such as amino acids. This rAL-Rase also reacted weakly with serine, arginine, and proline. The enzyme converted 54.5% of d-alanine from l-alanine while demonstrating potential to produce d-amino acid as d-alanine.

ACK N OWLED G M ENTS
We appreciate financial support for part of this work from a grant provided by the Institute for Fermentation, Osaka, 2021.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.