Eur. J. Biochem. 271, 4798–4803 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04445.x Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum Tatsuyuki Yamashita1, Makoto Ashiuchi1, Kouhei Ohnishi2, Shin’ichiro Kato2, Shinji Nagata1 and Haruo Misono1,2 1Department of Bioresources Science, Kochi University, Nankoku, Kochi, Japan; 2Research Institute of Molecular Genetics, Kochi University, Nankoku, Kochi, Japan Bifidobacterium bifidum is a useful probiotic agent exhibiting enzyme, however, showed a novel characteristic, namely, health-promoting properties and contains D-aspartate as an that its thermal stability significantly increased in the pres- essential component of the cross-linker moiety in the pepti- ence of aspartate, especially the D-enantiomer. The gene doglycan. To help understand D-aspartate biosynthesis in encoding the monomeric aspartate racemase was cloned and B. bifidum NBRC 14252, aspartate racemase, which cata- overexpressed in Escherichia coli cells. The nucleotide lyzes the racemization of D-andL-aspartate, was purified to sequence of the aspartate racemase gene encoded a peptide homogeneity and characterized. The enzyme was a mono- containing 241 amino acids with a calculated molecular mass mer with a molecular mass of 27 kDa. This is the first report of 26 784 Da. The recombinant enzyme was purified to showing the presence of a monomeric aspartate racemase. homogeneity and its properties were almost the same as Its enzymologic properties, such as its lack of cofactor those of the B. bifidum enzyme. requirement and susceptibility to thiol-modifying reagents Keywords: aspartate racemase; Bifidobacterium bifidum; in catalysis, were similar to those of the dimeric aspartate D-aspartate; peptidoglycan; probiotic agent. racemase from Streptococcus thermophilus. The monomeric Bifidobacteria, including Bifidobacterium bifidum, have been Most bacterial alanine racemases assemble in a dimer applied widely as probiotic agents exhibiting health-promo- structure [5], whereas glutamate racemases are mainly ting properties. Recent research suggested very interesting characterized as monomeric enzymes [6]. On the other functions of bifidobacterial peptidoglycans e.g. reduction hand, aspartate racemase is found in limited organisms of harmful bacteria and toxic compounds in the intestine, [7–11], which encompass even peptidoglycan-less species, antitumorigenic activities, and immunological enhancement such as archaea and mollusks. Recent studies showed two properties [1–3]. Bacterial peptidoglycans contain several distinct characteristics of aspartate racemases in the co- kinds of D-amino acids [4] and are thought to protect cells enzyme requirement in catalysis [11,12]. Among them, the from protease actions. D-Alanine and D-glutamate occur in PLP-independent aspartate racemase is considered to share the main chains of bifidobacterial peptidoglycans [5]. The structural features and catalytic properties with the gluta- cross-linker moieties of B. bifidum contain D-aspartate as mate racemase [12]. Nevertheless, aspartate racemases are the essential component [5]. Two kinds of amino acid typically dimeric, and neither monomeric nor multimeric racemases, alanine racemase [5] and glutamate racemase [6], aspartate racemase has been identified yet. have been identified ubiquitously from bacteria, and it has This paper presents the first identification of been assumed that the former, a pyridoxal 5¢-phosphate PLP-independent monomeric aspartate racemase from (PLP)-dependent amino acid racemase [5], is involved in B. bifidum NBRC 14252 and its enzymologic characteris- D-alanine biosynthesis and the latter, a PLP-independent tics, as well as cloning and overexpression of its gene in racemase [6], participates in the supply of D-glutamate. Escherichia coli. Materials and methods Correspondence to M. Ashiuchi, Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Materials Japan. Fax: +81 88 8645200, Tel.: +81 88 8645215, E-mail: [email protected] N-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) was pur- Abbreviations:Boc-L-cys, N-tert-butyloxycarbonyl-L-cysteine; chased from Novabiochem, La¨ufelfingen, Switzerland; BTP, bis-trispropane; IPTG, isopropyl thio-b-D-galactoside; o-phthalaldehyde (OPA), from Nacalai Tesque, Kyoto, OPA, o-phthalaldehyde; PLP, pyridoxal 5¢-phosphate; Tes, Japan; a 4-lm Nova-Pack C18 column, from Waters, N-tris(hydroxymethyl)-methyl-2-aminoethansulfonic acid. Milford, MA, USA; a HiPrep Sephacryl S-200 column Enzymes: alanine racemase (EC 5.1.1.1); glutamate racemase (1.6 · 60 cm) and a HiTrap Butyl FF column (1.0 mL), (EC 5.1.1.3); aspartate racemase (EC 5.1.1.13). from Amersham Bioscience, Uppsala, Sweden; a Bio-Scale (Received 25 August 2004, revised 30 September 2004, Q20 column (20 mL) and a protein assay kit, from Bio-Rad, accepted 19 October 2004) Richmond, CA, USA; a TSK gel G3000SW column, from Ó FEBS 2004 Bifidobacterial aspartate racemase (Eur. J. Biochem. 271) 4799 Tosoh, Tokyo, Japan; a PRISM kit, from PerkinElmer, (Amersham Bioscience, Uppsala, Sweden) equipped with a Fremont, CA, USA; restriction enzymes, T4 DNA ligase, Bio-Scale Q20 column (20 mL) that had been equilibrated and isopropyl thio-b-D-galactoside (IPTG), from Takara with the standard buffer (pH 6.5). After the column was Shuzo, Kyoto, Japan; and a plasmid pATE19, from washed with the same buffer and a buffer containing 0.15 M BioLeaders Corporation, Daejeon, Korea. All other chem- NaCl, the enzyme was eluted with the buffer containing icals were of analytical grade. 0.3 M NaCl. The active fractions were combined, dialyzed against the standard buffer (pH 6.5) overnight, and con- centrated by ultrafiltration with an Amicon PM-10 mem- Bacteria and culture conditions brane. The enzyme solution was dialyzed against the B. bifidum NBRC 14252 was cultured at 37 °Cfor48hin standard buffer (pH 6.5) containing ammonium sulfate GAM broth (pH 7.1) comprising 1% peptone, 0.3% soy (15% saturation) and subjected to the FPLC system peptone, 1% protease peptone, 1.35% digested serum, equipped with a HiTrap Butyl FF column (1.0 mL) that 0.5% yeast extract, 0.22% meat extract, 0.12% liver extract, had been equilibrated with the standard buffer (pH 6.5) 0.3% glucose, 0.25% KH2PO4, 0.3% NaCl, 0.5% soluble containing ammonium sulfate (15% saturation). After the starch, 0.03% L-cysteine/HCl, and 0.03% sodium thiogly- column had been washed with the same buffer, the enzyme colate (Nissui, Tokyo, Japan). was eluted with a linear gradient of ammonium sulfate (15% to 0% saturation) in the buffer. The active fractions were combined, dialyzed against the standard buffer Enzyme and protein assays (pH 6.5) overnight, and concentrated by ultrafiltration with The aspartate racemase activity was estimated by determin- an Amicon PM-10 membrane. NaCl was added to the ation of the antipode formed from either enantiomer of enzyme solution (final concentration, 0.15 M NaCl), and the aspartate by HPLC. The reaction mixture (200 lL) com- enzyme solution (2.2 mL) was subjected to the FPLC posed of 0.1 M bis-trispropane (BTP) buffer (pH 7.0), system equipped with a HiPrep Sephacryl S-200 column 50 mML-aspartate, 4 mM dithiothreitol, 1 mM EDTA, (1.6 · 60 cm) that had been equilibrated with the standard and enzyme was incubated at 45 °C for 10 h. The reaction buffer (pH 6.5) containing 0.15 M NaCl. The column was )1 was terminated by addition of 50 lLof2M HCl. After developed at the flow rate of 1.0 mLÆmin with the neutralization of the reaction mixture, it was incubated at standard buffer (pH 6.5) containing 0.15 M NaCl. The 25 °C for 2 min with a 0.3 M borate solution (pH 9.0) active fractions were combined, dialyzed against the stand- containing 0.2% Boc-L-Cys and 0.2% OPA. A 2-lL aliquot ard buffer overnight, and concentrated by ultrafiltration of the resulting mixture was subjected to a Shimadzu LC-10 with an Amicon PM-10 membrane. HPLC system (Kyoto, Japan) composed of an LL-10AD dual pump, a CBM-10 A control box, an RF-10 A Electrophoresis spectrofluorometer, and a DGU-14 A degasser, with a 4-lm Nova-Pack C18 column (3.9 · 300 mm). Other SDS/PAGE was carried out with 12.5% polyacrylamide by conditions were the same as those described by Hashimoto the method of Laemmli [14]. et al. [13]. One unit of the enzyme was defined as the amount of enzyme that catalyzes the formation of 1 lmol of Molecular mass determination D-aspartate from L-aspartate per hour. Protein concentrations were determined using a protein The molecular mass was determined by HPLC on a TSK gel assay kit with bovine serum albumin as a standard. G300SW column (0.75 · 60 cm) at a flow rate of 0.7 mLÆ )1 min with the standard buffer (pH 6.5) containing 50 mM D-aspartate and 0.15 M NaCl. A calibration curve was Enzyme purification made with the following proteins: glutamate dehydrogenase Harvested cells of B. bifidum NBRC 14252 (wet weight, (290 kDa), lactate dehydrogenase (142 kDa), enolase 104 g) were suspended in 200 mL of a standard buffer (67.0 kDa), adenylate kinase (32.0 kDa), and cytochrome c [10 mM N-tris(hydroxymethyl)-methyl-2-aminoethansulf- (12.4 kDa). The molecular mass of the subunit was estimated onic acid (Tes) buffer (pH 6.5), 4 mM dithiothreitol, and by SDS/PAGE. The following marker proteins (Amersham 1mM EDTA] supplemented with 0.1 MD-aspartate and Bioscience, Uppsala, Sweden) were used: rabbit muscle 0.1 mM phenylmethanesulfonyl fluoride and then disrupted phosphorylase b (97 kDa), bovine serum albumin (66 kDa), by sonication on ice for 20 min. The suspension was ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin centrifuged at 12 000 g for 30 min, and the resulting inhibitor (20.1 kDa), and a-lactalbumin (14.4 kDa). supernatant was dialyzed against the standard buffer (pH 6.5) and used as the cell extract.