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Molecular Characterization of Alanine Racemase from Bifidobacterium

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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 1381-1177/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1381-1177(03)00083-3 214 T. Yamashita et al. / Journal of Molecular Catalysis B: Enzymatic 23 (2003) 213–222 2. Experimental 2.1. Materials All restriction enzymes used, T4 DNA ligase, and isopropyl-␤-d-thiogalactopyranoside (IPTG) were pu- rchased 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) 213–222 215 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 activ- ity 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).
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