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Properties of Glutamate Racemase from Bacillus Subtilis IFO 3336 Producing Poly-Ƒá-Glutamate1

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Properties of Glutamate Racemase from Bacillus Subtilis IFO 3336 Producing Poly-Ƒá-Glutamate1 J. Biochem. 123, 1156-1163 (1998) Properties of Glutamate Racemase from Bacillus subtilis IFO 3336 Producing Poly-ƒÁ-Glutamate1 Makoto Ashiuchi,* Kazuhiko Tani,t Kenji Soda,' and Haruo Misono*,•õ,2 * Research Institute of Molecular Genetics and •õDepartment of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502; and Faculty of Engineering, Kansai University, Suita, Osaka 564-0073 Received for publication, January 13, 1998 We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-ƒÁ-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or in hibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D- enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransfer ase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-ƒÁ-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-ƒÁ-glutamate production in B. subtilis IFO 3336. Key words: glutamate racemase, Bacillus subtilis, poly-ƒÁ-glutamate producer, poly-ƒÁ -glutamate. Several strains of Bacillus extracellularly produce poly-ƒÁ- traditional Japanese fermented food made from soybeans, glutamate in large quantities (1-5) in the stationary phase and found high glutamate racemase activity and little n- (2, 6, 7). The polymer usually consists of 70 to 80% D- and amino acid aminotransferase activity in the cell extracts. 20 to 30% L-glutamate (6, 7). Cells of Bacillus poly-ƒÁ- We describe the purification and characterization of glutamate producers must produce one or more enzymes glutamate racemase from B. subtilis IFO 3336 (B. natto), catalyzing the synthesis of a large amount of D-glutamate. cloning of the enzyme gene into Escherichia coli cells, and Thorne et al. (8) suggested that D-glutamate was formed overproduction of the gene product. We further discuss the from L-glutamate through coupling of the D-amino acid physiological function of the enzyme. aminotransferase reaction with the alanine racemase reac tion in Bacillus subtilis ATCC 9945. However, Liu et al. (9) MATERIALS AND METHODS reported that n-amino acid aminotransferase activity was not found in cell extracts of Bacillus pumilus, whose Materials-The DNAs of both the probe and primers for characteristics are very close to those of B. subtilis (10). To PCR were obtained from Hokkaido System Science, elucidate the synthetic pathway of D-glutamate in B. Hokkaido. pUC19, ƒÀ-agarase, 5-bromo-4-chloro-3-indolyl- subtilis cells, we examined the activities of both glutamate ƒÀ-D-galactoside (X-Gal) , isopropyl-ƒÀ-D-thiogalactopyrano racemase and D-amino acid aminotransferase in cell ex- side (IPTG), and SeaPlaque GTG low-melt agarose were tracts of B. subtilis IFO 3336 (formerly Bacillus natto, B. purchased from Takara Shuzo, Kyoto. The restriction subtilis var. natto), a potent producer of poly-ƒÁ-glutamate, enzyme, TspEI, was obtained from Toyobo, Osaka. L- which is industrially used for the production of "natto," a Glutamate dehydrogenase (GDH) was purchased from Boehringer Mannheim, Germany. Homocysteinesulfinate was obtained from Tocris Cookson, UK. All other chemicals 1 This work was partly supported by a Takano Agriculture Research were of analytical grade. Grant from The Takano Life Science Research Foundation. 2 To whom correspondence should be addressed. E-mail: hmisono@ Bacteria-B. subtilis CU741 [leuC7, trpC2] (11) was a cc.kochi-u.ac.jp kind gift from Professor Teruo Tanaka of Tokai University, Abbreviations: X-Gal, 5-bromo-4-chloro-3-indolyl-ƒÀ-D-galactoside; Shizuoka. E. coli JM109 was purchased from Takara IPTG, isopropyl-ƒÀ-n-thiogalactopyranoside; GDH, L-glutamate de Shuzo. Other bacteria were obtained from the Institute for hydrogenase; DIG, digoxigenin; NTCB, 2-nitro-5-thiocyanatobenzo Fermentation, Osaka. ate. Culture Conditions for B. subtilis NO 3336-After a c 1998 by The Japanese Biochemical Society. spore suspension of B. subtilis IFO 3336 had been prepared 1156 J. Biochem. Properties of Bacillus subtilis Glutamate Racemase 1157 (2), a 0.1-ml portion of the suspension was inoculated into (pH 8.0) at 4•Ž overnight. The dialyzed enzyme solution 5 liters of a growth medium (pH 7 .0) comprising 1% (12 ml) was concentrated with Centriprep-10 and Cen glucose, 0.5% polypepton, 0.25% yeast extract, 0.5% NaCl, tricon-10 concentrators (Amicon, USA). The enzyme solu and 0.05% MgSO4 • 7H2O . Cultures were grown at 30•Ž for tion (1 ml) was applied to a Bio-Scale DEAE 2 anion- 30 h. exchange column (volume, 2 ml; Bio-Rad) equilibrated Preparation of Cell Extracts of B . subtilis-Harvested with the standard buffer (pH 8.0) containing 0.1 M NaCl. cells (40 g, wet weight) of B . subtilis IFO 3336 were The enzyme was eluted with a linear gradient (50 ml + 50 suspended in 20 ml of the standard buffer [0 .1 M Tris-HCl ml) of NaCl (0.1-0.5 M) in the standard buffer (pH 8.0) at buffer (pH 8.0) containing 0 .2% 2-mercaptoethanol and the flow rate of 0.5ml/min. The active fractions (4 ml) 10% glycerol] supplemented with 0 .1 mM phenylmeth- were dialyzed against the standard buffer (pH 8.0) at 4°C anesulfonyl fluoride, and then disrupted by sonication at overnight and then concentrated to 0.2 ml with the above 4•Ž. The cell debris was removed by centrifugation. The concentrators. The solution was passed through a Super supernatant solution was dialyzed against 4 liters of the ose12 HR 10/30 gel filtration column (1Ox300mm, standard buffer (pH 8.0) at 4°C overnight. The dialyzed Pharmacia) equilibrated with the standard buffer (pH 8.0) solution was used as the cell extract . containing 0.1 M NaCl. The column was developed with 30 Enzyme and Protein Assays-Glutamate racemase was ml of the same buffer at the flow rate of 0.2 ml/min. The assayed at 37•Ž with GDH (12). The mixture (1.0 ml) active fractions (1.6 ml) were dialyzed against 10mM comprised 100ƒÊmol of Tris-HCl buffer (pH 8 .0), 10ƒÊmol potassium phosphate buffer (pH 7.0) containing 0.2% 2- of D-glutamate, 5ƒÊmol of NAD+, 10 units of GDH , and mercaptoethanol and 10% glycerol at 4•Ž overnight. The enzyme. The reaction was started by the addition of enzyme solution was concentrated to 0.2 ml with the D-glutamate. The activity was estimated from the initial Centricon-10 concentrator and then put on a Bio-Scale rate of formation of L-glutamate determined by following CHT-1 hydroxyapatite column (volume, 2 ml; Bio-Rad) the increase in the absorbance at 340 nm with a Shimadzu equilibrated with the dialysis buffer (pH 7.0). The column UV-2200A spectrophotometer. One unit of the racemase was washed with 8 ml of the buffer and then eluted with a was defined as the amount of enzyme that catalyzed the linear gradient (12 ml + 12 ml) of potassium phosphate formation of 1ƒÊmol of L-glutamate per min. The activity buffer (0.01-0.5 M) at the flow rate of 0.2 ml/min. The was alternatively measured by determinationnnnnnnnnn of each active fractions were combined, and then dialyzed against antipode formed from D- or L-glutamate by HPLC with a 0.1 M Tris-HCl buffer (pH 7.0) containing 0.2% 2-mercap chiral carrier as follows. The reaction mixture (0.1 ml) toethanol and 10% glycerol at 4•Ž overnight. The enzyme comprising 10ƒÊmol of Tris-HCl (pH 8.0), 1 umol of D- or solution (0.4 ml) was applied to a Mono Q HR 5/5 anion- 20ƒÊmol of L-glutamate, and enzyme was incubated at 37°C exchange column (5 x 50 mm, Pharmacia) equilibrated for 1 min. The reaction was terminated by the addition of 8 with the dialysis buffer (pH 7.0). The enzyme was eluted ƒÊl of 12 M HCl. The mixture was neutralized by the with a linear gradient (10 ml+10 ml) of NaCl (0-0.5 M) in addition of 16ƒÊl of 6 M NaOH. After 5-fold dilution of the the buffer at the flow rate of 0.5 ml/min. The enzyme mixture with a 2mM CuSO4 solution, a 5-ƒÊl aliquot was solution (0.4 ml) was dialyzed against the standard buffer withdrawn and then applied to a CHIRALPAK MA(+) (pH 8.0), and then concentrated to 80ƒÊl. column (4.6 x 50 mm, DAICEL, Tokyo) in a Shimadzu Molecular Mass Determination-The molecular mass of LC-10A HPLC system. The column was developed with 2 the enzyme was determined with the Bio-Logic chroma mM CuSO4 solution at the flow rate of 1 ml/min, with tography system on a Superosel2 HR 10/30 gel filtration monitoring of the absorbance at 235 nm. Standard curves column (10 x 300 mm) equilibrated with the standard were prepared by estimation of the peak areas. The kinetic buffer (pH 8.0) containing 0.1 M NaCl.
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