• BMB Reports
• /
• v.33 no.1
• /
• pp.82-88
• /
• 2000

A homologous gene to alanine racemase was cloned from a hyperthermophilic bacterium, Aquifex pyrophilus. The cloned gene encodes a protein of 341 amino acids, which has a significant homology to alanine racemase of Bacillus stearothermophilus, Lactobacillus brevis, and E. coli. When the gene was expressed in Escherichia coli, it produced a 40 kDa protein. The purified protein contains one mole pyridoxal 5-phosphate per one mole of protein, which is essential for catalytic activity of alanine racemase. The purified protein catalyzed racemization of L-alanine to D-alanine, or vice versa, indicating that the cloned gene encoded alanine racemase. It also showed significant racemization activity against L-serine and ${\alpha}-aminobutylic$ acid. The A. pyrophilus alanine racemase showed strong thermostability, and it maintained catalytic activity in the presence of organic solvents.

• Journal of Applied Biological Chemistry
• /
• v.53 no.3
• /
• pp.132-138
• /
• 2010

A gene encoding a putative alanine racemase in B. pseudomycoides was cloned and expressed in Escherichia coli BL21(DE3) using a pET-21 vector harbouring 6xHistidine tag. Affinity purification of the recombinant alanine racemase with a nickel resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 46 kDa. The enzyme was the most active toward L-alanine and secondly D-alanine, implying that the enzyme is an alanine racemase. D-cysteine significantly inhibited the enzyme activity and also L-cysteine to a lesser extent. The enzyme was considerably activated by addition of pyridoxal-5'-phosphate (PLP), showing that 73% increase in activity was observed at 0.3 mM, compared to control. The enzyme was the most active at pH 9.0 and more stable at alkaline pHs than acidic pHs.

• Journal of Applied Biological Chemistry
• /
• v.53 no.4
• /
• pp.195-201
• /
• 2010

A gene encoding an alanine racemase in B. pseudomycoides was cloned and one (Cys316) or both of two cysteines (Cys316 and Cys365) was (were) substituted with alanine. The cysteine (-) alanine racemases were expressed in E. coli BL21 (DE3) using a pET-21 vector. The expressed enzymes were purified through affinity chromatography using 6xHis ligand. The purified enzymes all showed major one bands by SDS-PAGE analysis, corresponding to 46 kDa. The cysteine (-) alanine racemases as well as the wild type enzyme showed alanine racemase activities, indicating that the enzyme is an alanine racemase and the cysteines in the enzyme may not be involved in the catalysis and/or substrate binding. Thermal stabilities of Cys (-) alanine racemases decreased considerably and half-lives were 26 (wild type), 21 (C316A) and 18 min (C316-365A), respectively at $60^{\circ}C$ pH 8.0, suggesting that cysteine is considerably contributive to the thermal stability of the alanine racemase.

• Journal of Applied Biological Chemistry
• /
• v.54 no.4
• /
• pp.231-237
• /
• 2011

A gene encoding a putative alanine racemase in Xanthomonas. oryzae pv. oryzae was cloned, expressed and characterized. Expression of the cloned gene was performed in Escherichia coli BL21(DE3)pLys using a pET-21(a) vector harbouring $6{\times}histidine$ tag. Purification of the recombinant alanine racemase by affinity chromatography resulted in major one band by sodium dodecyl sulfate polyacryl amide gel electrophoresis analysis, showing about 45 kDa of molecular weight. The alanine racemase gene, cloned in this experiment, appears to be constitutively expressed in X. oryzae, as analyzed by reverse transcriptase polymerase chain reaction. The enzyme was the most active toward L-alanine and secondly D-alanine, showing a racemic reaction, thus the enzyme is considered as an alanine racemase. The enzyme was considerably activated by addition of pyridoxal-5-phosphate (PLP), showing that 75% increase in activity was observed at 0.3 mM, compared with control. D-Cysteine as well as L-cysteine significantly inhibited the enzyme activity. The inhibitions by cysteines were more prominent in the absence of PLP, showing 9 and 5% of control activity at 2 mM of addition, respectively. The enzyme was the most active at pH 8.0 and more stable at alkaline pHs than acidic pH condition.

• BMB Reports
• /
• v.42 no.1
• /
• pp.47-52
• /
• 2009

Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent $K_m$ of 3 mM for L-alanine, and a $V_{max}$ of $295\;{\mu}moles/min/mg$, with the optimum activity occurring at $37^{\circ}C$ and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a $K_i$of $160\;{\mu}M$ and 30 mM, respectively.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.1
• /
• pp.55-58
• /
• 2008

Alanine racemase, a bacterial enzyme belonging to the fold-type III group of pyridoxal 5'-phosphate (PLP)-dependent enzymes, has been shown to catalyze the interconversion between L- and D-alanine. The alanine racemase from the pathogenic bacterium Enterococcus faecalis v583 has been overexpressed in E. coli and was shown to crystallize an enzyme at 295 K, using polyethylene glycol (PEG) 8000 as a precipitant. X-ray diffraction data to $2.5{\AA}$ has been collected using synchrotron radiation. The crystal is a member of the orthorhombic space group, $C222_1$ with unit cell parameter of a=94.634, b=156.516, $c=147.878{\AA},\;and\;{\alpha}={\beta}={\gamma}=90{\AA}$. Two or three monomers are likely to be present in the asymmetric unit, with a corresponding $V_m\; of\;3.38{\AA}^3\;Da^{-1}\;and\;2.26{\AA}^3\;Da^{-1}$ and a solvent content of 63.7% and 45.5%, respectively.

• Bulletin of the Korean Chemical Society
• /
• v.35 no.7
• /
• pp.2186-2188
• /
• 2014

• Bulletin of the Korean Chemical Society
• /
• v.35 no.6
• /
• pp.1720-1726
• /
• 2014

(S)-Alanine Racemase Chiral Analogue ((S)-ARCA) was used as an efficient adsorbent for the selective separation of D-amino acids (D-AAs), which are industrially important as chiral building blocks for the synthesis of pharmaceutical intermediates. The organic phase, containing (S)-ARCA adsorbent and phase transfer reagents, such as ionic liquid type molecules (Tetraphenylphosphonium chloride (TPPC), Octyltriphenylphosponium bromide (OTPPBr)), were coated on the surfaces of mesoporous carbon supports. For the immobilization of chiral adsorbents, meso/macroporous monolithic carbon (MMC), having bimodal pore structures with high surface areas and pore volumes, were fabricated. The separation of chiral AAs by adsorption onto the heterogeneous (S)-ARCA was performed using a continuous flow type packed bed reactor system. The effects of loading amount of ARCA on the support, the molar ratio of AA to ARCA, flow rates, and the type of phase transfer reagent (PTR) on the isolation yields and the optical purity of product D-AAs were investigated. D-AAs were selectively combined to (S)-ARCA through imine formation reaction in an aqueous basic solution of racemic D/L-AA. The (S)-ARCA coated MMC support showed a high selectivity, up to 95 ee%, for the separation of D-type phenylalanine, serine and tryptophan from racemic mixtures. The ionic liquids TPPC and OTPPBr exhibited superior properties to those of the ionic surfactant Cetyltrimethyl ammonium bromide (CTAB), as a PTR, showing constant optical purities of 95 ee%, with high isolation yields for five repeated reuses. The unique separation properties in this heterogeneous adsorption system should provide for an expansion of the applications of porous materials for commercial processes.