Molecular Characterization of Alanine Racemase from Bifidobacterium

Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213–222

Molecular characterization of alanine racemase from Biﬁdobacterium biﬁdum Tatsuyuki Yamashita a, Makoto Ashiuchi a, Kouhei Ohnishi b, Shin-ichiro Kato b, Shinji Nagata a, Haruo Misono a,b,∗ a Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan b Research Institute of Molecular Genetics, Kochi University, Nankoku, Kochi 783-8502, Japan Received 6 February 2003; received in revised form 15 April 2003; accepted 18 April 2003 Dedicated to Professor Dr. Kenji Soda in honor of his 70th birthday

Abstract Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties, and its peptidoglycans have the potential for applications in the fields of food science and medicine. We investigated the bifidobacterial alanine racemase, which is essential in the synthesis of d-alanine as an essential component of the peptidoglycans. Alanine racemase was purified to homogeneity from a crude extract of B. bifidum NBRC 14252. It consisted of two identical subunits with a molecular mass of 50 kDa. The enzyme required pyridoxal 5-phosphate (PLP) as a coenzyme. The activity was lost in the presence of a thiol-modifying agent. The enzyme almost exclusively catalyzed the alanine racemization; other amino acids tested, except for serine, were inactive as substrates. The kinetic parameters of the enzyme suggested that the B. bifidum alanine racemase possesses comparatively low affinities for both the coenzyme (9.1 ␮M for PLP) and substrates (44.3 mM for l-alanine; 74.3 mM for d-alanine). The alr gene encoding the alanine racemase was cloned and sequenced. The alr gene complemented the d-alanine auxotrophy of Escherichia coli MB2795, and an abundant amount of the enzyme was produced in cells of the E. coli MB2795 clone. The enzymologic and kinetic properties of the purified recombinant enzyme were almost the same as those of the alanine racemase from B. bifidum NBRC 14252. © 2003 Elsevier B.V. All rights reserved.

Keywords: Alanine racemase; d-Alanine; Peptidoglycan; Probiotic agent; Biﬁdobacterium biﬁdum

1. Introduction isms in the intestine is essential for good health; the occurrence of bifidobacteria in the large bowel is es- For many years, bifidobacteria have attracted par- pecially beneficial because bifidobacteria prevent the ticular attention because of their promising health- proliferation of pathogens that, for example, result promoting properties, including a decrease of total in diarrhea [7–9]. The nullification of Vero cytotoxin cholesterol and lipids in human serum [1–6].In from Escherichia coli O157:H7 in the coexistence newborns, the growth of nonpathogenic microorgan- of bifidobacteria has been also demonstrated [10]. Bifidobacteria probably repair and improve the mi- ∗ Corresponding author. Tel.: +81-888-64-5187; crobial communications in microflora of the human fax: +81-888-64-5200. gastrointestinal tracts (but the detailed mechanism E-mail address: [email protected] (H. Misono). has not been clarified) and are available practically as

2. Experimental

2.1. Materials

All restriction enzymes used, T4 DNA ligase, and isopropyl-␤-d-thiogalactopyranoside (IPTG) were purchased from Takara Shuzo, Kyoto, Japan; N-tert-but- Fig. 1. Structure of B. bifidum peptidoglycans. GlcNAc, N-acetyl- yloxycarbonyl-l-cysteine (Boc-l-Cys) from Nova- glucosamine; MurNAc, N-acetylmuramic acid; l-Ala, l-alanine; biochem, Läufelfingen, Switzerland; o-phthalaldehyde d d l l d d -Glu, -glutamic acid; -Orn, -ornithine; -Ala, -alanine; (OPA) from Nacalai Tesque, Kyoto, Japan; 5,5-dithi- d-Asp, d-aspartic acid; and d-Ser, d-serine. obis-(2-nitrobenzoic acid) (DTNB) from Sigma, St. ␮ probiotic agents in fermented diary products, such as Louis, MO, USA; a 4 m Nova-Pack C18 column yogurt. from Waters, MA, USA; DEAE-Toyopearl resin slurry Exceedingly interesting functions of bifidobacterial and a TSK gel G3000SW column from Tosoh, Tokyo, peptidoglycans, i.e. a decrease of harmful bacteria and Japan; Gigapite resin slurry from Toh’a, Tokyo, Japan; toxic compounds in the intestine, antitumorigenic ac- Superose 12 HR10/30, phenyl-Superose HR5/5, and tivities, and effects as immunological enhancers, were MonoQ HR5/5 columns for FPLC from Amersham recently reported [11–14]. How such bioavailable Pharmacia Biotech, Buckingham, UK; a protein assay peptidoglycans can be mass produced remains to kit from Bio-Rad, CA, USA; and a PRISM kit from l be determined. Fig. 1 illustrates the structure of the Perkin-Elmer, CA, USA. -Alanine dehydrogenase peptidoglycans from Bifidobacterium bifidum. Gener- was prepared as described previously [33]. All other ally, bacterial peptidoglycans (alternatively, mureins) chemicals were of analytical grade. contain several kinds of d-amino acids [15] and are thought to protect cells from protease actions. As 2.2. Bacteria and vectors shown in Fig. 1, d-alanine is the essential component d of peptidoglycans. It is assumed that d-alanine is syn- E. coli MB2795, the auxotroph of -alanine, was a thesized by alanine racemase, a pyridoxal 5-phos- kind gift of Dr. Michael J. Benedik, professor at the phate (PLP)-dependent enzyme catalyzing the racem- University of Houston, TX, USA. A plasmid, pUC18, ization of l- and d-alanine (Fig. 2). Alanine racemase was purchased from Takara Shuzo. has been purified and characterized from bacteria [16–26], yeasts [27], fungi [28], and invertebrates 2.3. Culture conditions [29–31]. It was used for the synthesis of d-amino ◦ acids from the corresponding 2-keto acids [32]. The B. bifidum NBRC 14252 was cultured at 37 C for gene (alr) encoding alanine racemase is ubiquitously 48 h in GAM broth (pH 7.1) comprising 1% peptone, inherited in almost all bacteria, but the enzyme has 0.3% soy peptone, 1% protease peptone, 1.35% di- not been identified from bifidobacteria. Here, we gested serum, 0.5% yeast extract, 0.22% meat extract, 0.12% liver extract, 0.3% glucose, 0.25% KH2PO4, report the purification and characterization of the ala- l nine racemase from B. bifidum NBRC 14252 and the 0.3% NaCl, 0.5% soluble starch, 0.03% -cysteine– cloning and overexpression of the enzyme gene. HCl, and 0.03% sodium thioglycolate (Nissui, Tokyo, Japan). E. coli MB2795 was cultured at 37 ◦C for 24 h in Luria–Bertani (LB) broth containing d-alanine (fi- nal concentration, 200 ␮g/ml).

2.4. Enzyme and protein assays

Alanine racemase was assayed as follows. The assay mixture comprising 100 mM N-cyclohexyl-3-amino- Fig. 2. Enzymatic alanine racemization. propanesulfonic acid (CAPS) buffer (pH 10.5), 10 ␮M T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222 215

PLP, 20 mM d-alanine, 4 mM NAD+, l-alanine de- containing 180–220 mM NaCl. The active fractions hydrogenase (20 U/ml), and enzyme was used. The were combined, dialyzed against the standard buffer enzyme activity was determined by measurement of overnight, and concentrated by ultrafiltration with an an increase in absorbance at 340 nm during incuba- Amicon PM-10 membrane. tion of the mixture at 37 ◦C. Alternatively, the activity was estimated by determination of the antipode 2.5.2. Step 2: Gygapite column chromatography formed from either enantiomer of alanine by HPLC. The enzyme solution was put on a Gygapite col- The reaction mixture composed of 100 mM CAPS umn (4.0cm× 14 cm) equilibrated with the standard buffer (pH 10.5), 10 ␮M PLP, 20 mM l-alanine, buffer, and the column was washed with the buffer. and enzyme was incubated at 37 ◦C. After termina- The enzyme was eluted after impure proteins. The ac- tion of the reaction, the products were incubated at tive fractions were collected and concentrated by ul- 25 ◦C for 2 min with a 300 mM borate solution (pH trafiltration with an Amicon PM-10 membrane. 9.0) containing 0.2% Boc-l-Cys and 0.2% OPA. A 10 ␮l-aliquot of the resulting mixture was subjected 2.5.3. Step 3: Superose 12 column chromatography to a Shimadzu LC-10 HPLC system (Kyoto, Japan), The enzyme solution was subjected to an Amersham composed of an LL-10AD dual pump, a CBM-10A Pharmacia FPLC system equipped with a Superose 12 control box, an RF-10A spectrofluorometer, and a column (1.0cm× 30 cm) that had been equilibrated DGU-14A degasser, with the 4 ␮m Nova-Pack C18 with the standard buffer containing 100 mM NaCl. The column (3.9mm × 300 mm). Other conditions were column was developed at the flow rate of 0.3 ml/min, the same as those described by Hashimoto et al. [34]. and the enzyme activity was found in the fractions at One unit of the enzyme was defined as the amount the elution volume of 12–12.5 ml. The active fractions of enzyme that catalyzes the formation of 1 ␮mol of were combined, concentrated by ultrafiltration with l-alanine from d-alanine per min. an Amicon PM-10 membrane, and dialyzed overnight Protein concentrations were determined by means against the standard buffer containing 15% saturated of the protein assay kit with bovine serum albumin as ammonium sulfate. a standard. 2.5.4. Step 4: phenyl-Superose column 2.5. Enzyme purification chromatography The enzyme solution was subjected to the FPLC Harvested cells of B. bifidum NBRC 14252 (wet system equipped with a phenyl-Superose column weight, 45 g) were suspended in 90 ml of a standard (0.5cm × 5 cm) that had been equilibrated with the buffer [10 mM potassium phosphate buffer (pH 7.2), standard buffer containing ammonium sulfate (15% 1 mM dithiothreitol, 10 ␮M PLP, and 10% glycerol] saturation). After the column had been washed with supplemented with 0.1 mM phenylmethanesulfonyl the same buffer, the enzyme was eluted with a linear fluoride, and then disrupted by sonication on ice for gradient of ammonium sulfate (15–0% saturation) in 10 min. The suspension was centrifuged at 12,000 × g the buffer. The column was developed at the flow rate for 30 min, and the resulting supernatant was used as of 0.3 ml/min, and the enzyme activity was found in the cell extract. All the purification procedures were the fractions containing ammonium sulfate (7–0% performed at 4 ◦C. saturation). The active fractions were combined, dialyzed against the standard buffer overnight, and 2.5.1. Step 1: DEAE-Toyopearl column concentrated by ultrafiltration with an Amicon PM-10 chromatography membrane. The cell extract was put on a DEAE-Toyopearl column (4.0cm× 28 cm) equilibrated with the standard 2.5.5. Step 5: MonoQ column chromatography buffer containing 100 mM NaCl. After the column was The enzyme solution was subjected to an FPLC sys- washed with the same buffer, the enzyme was eluted tem equipped with a MonoQ column (0.5cm× 5 cm) with a linear gradient of 100–250 mM NaCl in the that had been equilibrated with the standard buffer buffer. The enzyme activity was found in the fractions containing 100 mM NaCl. After the column had been 216 T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222 washed with the same buffer, the enzyme was eluted mixed primers were designed from the determined N- with a linear gradient of 100–300 mM NaCl in the terminal and internal amino acid sequences of the en- buffer. The column was developed at the flow rate of zyme: the sense primer [5-AA(C,T)GC(A,T,G,C)GC- 0.6 ml/min, and the enzyme activity was found in the (A,T,G,C)(A,T)(G,C)(A,T,G,C)GA(A,G)(C,T)T(A,T, fractions containing 180–220 mM NaCl. The active G,C)CA(A,G)TT-3] and the antisense primer [5-CT- fractions were combined, dialyzed against the stan- (A,G)CA(A,T,G,C)CT(C,T)CG(A,T,G,C)CC(A,T,G,- dard buffer overnight, and then concentrated by ultra- C)GT(A,G)CC(A,T,G,C)CA-3]. PCR was performed filtration with an Amicon PM-10 membrane. as described previously [39]. The nucleotide sequence of the amplified DNA fragment (850 bp) was deter- 2.6. Electrophoresis mined by means of the PRISM kit with an Applied Biosystems 373A DNA sequencer (CA, USA). Next, Sodium dodecyl sulfate-polyacrylamide gel elec- the inverse PCR of the B. bifidum chromosomal DNA trophoresis (SDS-PAGE) was carried out with a 12.5% was carried out as follows. The chromosomal DNA polyacrylamide gel by the method of Laemmli [35]. was digested with the restriction enzyme NcoI, and the digested fragments were incubated with T4 DNA 2.7. Molecular mass determination ligase so that they could undergo self-circulation (or self-ligation). Two single primers were further de- The molecular mass was determined at 25 ◦Cby signed from the determined nucleotide sequence of the HPLC on a TSK gel G3000SW column (0.75 cm × amplified DNA fragment: RACE1 (5-GTCGACGA- 60 cm) at a flow rate of 0.7 ml/min with 0.1 M potas- TCGCCTGCGCGGGGTACCGGCG-3) and RACE2 sium phosphate buffer (pH 7.0) containing 0.3 M (5-ATTGCAGGCGCAGCTCGGTACGGTCAAGG- NaCl [36]. A calibration curve was made with the fol- A-3). PCR was conducted by the use of the self- lowing proteins: glutamate dehydrogenase (290 kDa), circulated fragments (as a template DNA) and the two lactate dehydrogenase (142 kDa), enolase (67.0 kDa), primers RACE1 and RACE2 under the conditions adenylate kinase (32.0 kDa), and cytochrome c reported previously [40]. Eventually, the nucleotide (12.4 kDa). The molecular mass of the subunit was sequence of the 2.3 kb region containing a proba- estimated by SDS-PAGE. The following marker pro- ble structural gene of the enzyme was determined. teins (Amersham Pharmacia Biotech, Buckingham, Two single primers were newly designed from the UK) were used: myosin (200 kDa), ␤-galactosidase nucleotide sequences at both the immediate up- and (116 kDa), bovine serum albumin (66.2 kDa), oval- downstreams of the structural gene of the enzyme: the bumin (45.0 kDa), carbonic anhydrase (31.0 kDa), sense primer ARN (5-GGGGATCCCAGGAAACA- trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), GACCATGACTTTGAACGCAGCATCCGAA-3), in and aprotinin (6.5 kDa). which a BamHI site (underlined) and the ribosome- binding site (bold types) are designed, and the anti- 2.8. Determination of the partial amino acid sequences of the enzyme sence primer ARC (5 -GGAAGCTTCTACAGCAGG- CTCGCCGGATCGAGCTTGGC-3), in which a The N-terminal amino acid sequence of the en- HindIII site (underlined) is designed. The alanine zyme was analyzed with an Applied Biosystems model racemase gene of B. bifidum was amplified by PCR 492 protein sequencer linked to a phenylthiohydan- using the two primers ARN and ARC and was desig- toin derivative analyzer. Internal peptide fragments of nated alr. The DNA fragment (1.4 kb) containing the the enzyme were isolated and sequenced as described alr gene was digested with both restriction enzymes previously [37]. BamHI and HindIII and ligated into the BamHI– HindIII site of pUC18. The constructed plasmid was 2.9. PCR amplification, DNA sequencing, and gene named pBALR1. The sequence of the alr gene for cloning the enzyme in the plasmid was analyzed in both directions with an Applied Biosystems 373 DNA se- First, the chromosomal DNA of B. bifidum was quencer and a DNA sequencing kit. The nucleotide prepared by the method of Saito and Miura [38].Two sequence data appear in the DDBJ/EMBL/GenBank T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222 217 nucleotide sequence databases under the accession number AB101584.

3. Results

3.1. Puriﬁcation of alanine racemase from B. biﬁdum

The alanine racemase was purified to homogeneity from cell extracts of B. bifidum NBRC 14252. A summary of the purification is presented in Table 1. The enzyme was purified about 9000-fold from the crude extract with a 5.5% yield. The purified enzyme showed a single band on SDS-PAGE (Fig. 3).

3.2. Molecular mass, subunit structure, and Fig. 3. SDS-PAGE of alanine racemase from B. bifidum. Lane M, the molecular marker proteins; and lane E, the purified alanine N-terminal amino acid sequence racemase from B. bifidum (1 ␮g of protein).

The molecular mass was estimated to be 100 kDa phate buffer (pH 6.0–8.0), N-tris(hydroxymethyl)me- by gel ﬁltration on a TSK gel G3000SW column. The thyl-3-aminopropanesulfonate buffer (pH 8.0–9.0), molecular mass of the subunit was calculated to be and CAPS buffer (pH 9.0–12.0). The enzyme showed 50 kDa by SDS-PAGE. These results indicate that the the maximum activity at pH 10–10.5. enzyme is a homodimer. The N-terminal and the internal amino acid sequences of the enzyme were TLN- 3.4. Coenzyme AASELQFSSPAGEANYR and DVEAGHGVSYGR- TYLTPDNT, respectively. The initiator methion- The absorption spectrum of the enzyme showed ine, which is not observed in the N-terminal amino a maximum at 420 nm in addition to 280 nm, as acid sequence of the enzyme, may be removed by a shown in Fig. 4. The absorption peak at 420 nm is post-translational modiﬁcation in vivo. probably derived from an internal Schiff-base formed between PLP and ε-amino group of the lysine residue 3.3. Stability and optimum pH in the active site. The PLP content of the enzyme was 2.0 mol/mol of the enzyme by the phenylhy- The enzyme was fully stable up to 40 ◦C. It was drazine method [41]. To obtain the apoenzyme, the most stable in the pH range of 7.0–10.5 when kept at enzyme was incubated with 10 mM hydroxylamine 40 ◦C for 10 min in the following buffers (100 mM): at 37 ◦C for 10 min, followed by dialysis against sodium acetate buffer (pH 4.0–6.0), potassium phos- 10 mM potassium phosphate buffer containing 0.01%

Table 1 Purification of alanine racemase from B. bifidum NBRC 14252 Steps Total protein (mg) Total activity (units) Specific activitya (units/mg) Yield (%)

Cell extract 1210 5.79 0.005 100 DEAE-Toyopearl 83.9 10.1 0.12 174 Gygapite 6.49 4.99 0.77 86.2 Superose 12 1.26 3.25 2.58 56.1 Phenyl-Superose 0.08 1.74 21.0 30.1 MonoQ 0.01 0.32 44.9 5.53 a The activity was measured by the l-alanine dehydrogease-coupled assay. 218 T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222

strates. As shown in Fig. 1, d-serine is the peculiar compound of the B. biﬁdum peptidoglycans. It, however, remains to be investigated whether the enzyme participates in d-serine synthesis. The Km and Vmax values of the enzyme for l-alanine were 44.3 mM and 159 ␮mol/min/mg, whereas those for d-alanine were 74.3 mM and 233 ␮mol/min/mg. Although their values for d- and l-alanine are different, the Vmax/Km values for both enantiomers of alanine are similar: the Keq value of the enzyme reaction is nearly one.

3.6. Inhibitor Fig. 4. Absorption spectra of alanine racemase from B. bifidum. Solid line, the holoenzyme (the native form of the enzyme); and dotted line, the apoenzyme. The enzyme was inhibited completely by 1 mM hydroxylamine. The enzyme was also inhibited by 1 mM DTNB, suggesting the existence of cysteine residues 2-mercaptoethanol. The enzyme thus treated had no essential for the activity. It, however, was not affected activity in the absence of added PLP and did not by 1 mM ethylenediamine tetraacetic acid. The en- show an absorption maximum at 420 nm (Fig. 4). zyme, thus, requires no metal ions in catalysis. Activity was almost fully restored by addition of 50 ␮M PLP. The active enzyme reconstituted with 3.7. Cloning of the alanine racemase gene PLP showed the same spectrum as the native holoenzyme. The Michaelis constant for PLP was estimated By the application of PCR techniques, the DNA as 9.1 ␮M. fragment that carries the alr gene encoding the probable alanine racemase of B. bifidum was cloned and 3.5. Substrate specificity sequenced (Fig. 5). The alr gene encodes a protein consisting of 452 amino acid residues. The first 20 l d The enzyme exclusively acted on - and -alanine predicted amino acid sequence except for methionine (Table 2). Other amino acids, such as both enan- was identical with that of the enzyme purified from B. tiomers of aspartate and glutamate, were inactive as bifidum. Its predicted molecular mass (48,084 Da) is substrates. Both enantiomers of serine were poor sub- in good agreement with that of the alanine racemase purified from B. bifidum. To identify the physiological Table 2 function of the alr gene product, the growth of E. coli Substrate specificity of alanine racemase from B. bifidum MB2795 harboring pBALR1 (MB2795/pBALR1) Amino acids Relative activity (%)a on a plate of an LB medium containing ampicillin ␮ l (50 g/ml) and IPTG (1 mM) was examined. The -Alanine 100 d d-Alanine 88.9 result showed that the -alanine auxotrophy of E. l-Serine 1.5 coli MB2795 was complemented by the expression d-Serine 1.3 of the alr gene and indicated that the alr gene en- l-Aspartate 0 codes an enzyme responsible for d-alanine synthesis. d -Aspartate 0 Fig. 6 shows a linear alignment of the amino acid l-Tyrosineb 0 l-Tryptophan 0 sequences of bacterial alanine racemases. The amino l-Methionine 0 acid sequence of the B. bifidum enzyme shows the l-Glutamate 0 highest similarity (42%) to that of the Streptomyces a The racemization activities for the indicated amino acids coelicolor alanine racemase. According to the se- (20 mM) were measured by HPLC as described in Section 2. quence similarity, alanine racemases are classified b 0.2 mM. into three groups A, B, and C [27]. The enzymes T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222 219

Fig. 5. Nucleotide sequence of the alr gene and the deduced amino acid sequence of the alanine racemase of B. biﬁdum. The N-terminal and internal amino acid sequences determined are indicated by single underlines. The putative ribosome binding sequence is shown by a double underline. The putative promoter region is described by bold types. 220 T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222

Fig. 6. Linear alignment of amino acid sequences of bacterial alanine racemases. Bb Alr, the alanine racemase of B. bifidum; Sc Alr, a probable alanine racemase from S. coelicolor (DDBJ/EMBL/GenBank accession number, AL939121); Ec DadX, the alanine racemase of E. coli (for catabolism; accession number, AF081283); Ec Alr, the alanine racemase of E. coli (for biosynthesis, accession number, AE000478); Bs Alr, the alanine racemase of Bacillus subtilis (accession number, AB001488). Asterisks indicate identical residues in the five sequences; double and single dots indicate conservative substitutions and functionally similar residues, respectively. Two highly conserved catalytic bases of alanine racemase, the lysine and tyrosine residues, are indicated by open circles. T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222 221 from Gram-positive bacteria and Gram-negative bac- 4. Discussion teria belong to the group A and the group C, respectively. The enzymes from the coryneform of Bifidobacteria generally display a very low growth bacteria and actinomycetes are included in the group rate and various nutrition requirements, such as pep- B. The B. bifidum enzyme belongs to the group B. tides and amino acids [43,44]. PLP, the bioavailable The octapeptide (KA(D/N)AYGHG) containing the form of vitamin B6, plays an important role as a PLP-binding lysine residue is conserved in alanine coenzyme of many enzymes involved in amino acid racemases studied so far. Recently, the alanine racem- metabolisms. As described earlier, d-alanine synthe- ization was proposed to proceed through a two-base sis is an essential step for peptidoglycan production mechanism involving the lysine and tyrosine residues (eventually, for cell division) in almost all eubacteria [42]. The B. bifidum enzyme also conserves the including bifidobacteria. Thus, some characteristics probable catalytic base, Tyr 303, in addition to Lys of alanine racemase operating in d-alanine synthesis 63. should influence bacterial cell proliferation. The physical, enzymologic, and kinetic properties 3.8. Overproduction of alanine racemase of the B. bifidum alanine racemase are summarized in E. coli clone cells in Table 3. The B. bifidum enzyme is a homodimer as well as the enzymes from Bacillus stearothermophilus The B. bifidum alanine racemase was abundantly [20], Pseudomonas fluorescens [21], and Bacillus produced in the E. coli MB2795/pBALR1 clone cells psychrosaccharolyticus [22], Acidophilium organovo- and was purified to homogeneity in the same way rum [25], whereas the enzymes from Streptococcus as described above (Fig. 7). The molecular mass of faecalis [18], Salmonella typhimurium [19], Thermus the recombinant enzyme, which was estimated to be thermophilus [23], Schizosaccharomyces pombe [27] 50 kDa by SDS-PAGE, was identical to that of the B. and crayfish muscle [29] are monomers. Molecular bifidum enzyme. The enzymologic and kinetic proper- size of the subunit of the B. bifidum alanine racemase ties of the recombinant enzyme were almost the same (50 kDa) is bigger than those of other bacterial alanine as those of the alanine racemase from B. bifidum (data racemases (33–43 kDa) [18–26], but is smaller than not shown). that of the crayfish enzyme (58 kDa) [29]. The catalytic activity, in the direction from l-tod-alanine, of the B. bifidum enzyme (159 U/mg) is lower than those

Table 3 Properties of alanine racemase from B. bifidum Molecular mass (kDa) 100 (homodimer) Thermostability (◦C) 40 pH stability 7–10.5 Optimal pH 10–10.5 Inhibitors Hydroxylamine, DTNB Coenzyme PLP (Km, 9.1 ␮M) Kinetic parameters [l-Alanine] Km (mM) 44.3 Vmax (␮mol/min/mg) 159 Vmax/Km 3.59 [d-Alanine] Fig. 7. Overproduction of alanine racemase in E. coli cells. Lane Km (mM) 74.3 M: the molecular marker proteins; lane 1: the cell extract of Vmax (␮mol/min/mg) 233 E. coli MB2795 (10 ␮g); lane 2: the cell extract of the E. coli Vmax/Km 3.14 MB2795/pBALR1 clone (10 ␮g); and lane 3: the enzyme purified [Keq (l/d)] 1.14 from E. coli MB2795/pBALR1 clone cells (10 ␮g). 222 T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213Ð222 of the dimeric enzymes from B. stearothermophilus [14] T. Sasaki, Y. Samegai, S. Namioka, J. Vet. Med. Sci. 58 (2550 U/mg) [20], P. fluorescens (2400 U/mg) [21], (1996) 85. and B. psychrosaccharolyticus (2000 U/mg) [22],but [15] S.H. 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