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Publisher Summary This chapter discusses the determination of alanine racemase from Streptococcus faecalis and glutamic acid racemase from Laclobacillus arabinosus . For deriving the alanine racemase from Streptococcus faecalis , all assay procedures are based on the measurement of one stereoisomer of alanine, since the substrate and end product differ only in configuration. A manometric measurement of the rate of racemization may be made with L-alanine as the substrate and an excess of D-amino acid oxidase. Under these conditions the rate of racemization limits the rate of oxygen uptake. In addition, the removal of the reaction product prevents the formation of a racemic mixture the approach toward which is accompanied by a decrease in reaction velocity. The stoichiometry of the reaction is dependent upon the presence of catalase. In its presence 1 micromole of D-alanine is equivalent to 11.2 μl. of oxygen, whereas in its absence 1 micromole of D-alanine is equivalent to 22.4 μl. of oxygen. In case of deriving the glutamic acid racemase from Laclobacillus arabinosus , the rate of racemization is measured by the rate of L-glutamate formation from D-glutamate. Aliquots of the reaction mixture are assayed manometrically at desired intervals for L-glutamate with L-glutamic acid decarboxylase.

Publisher Summary This chapter describes the assay methods and mechanism of action of amino acid racemases and epimerases. Amino acid racemases catalyze the formation of a racemic mixture from either the D or L form of a free amino acid by equilibrating configuration at the α-carbon. Amino acid epimerases also catalyze the equilibration of configuration at the α-carbon and because epimerases act on compounds possessing an additional asymmetric carbon, a diastereomer or epimer of the substrate is formed rather than its antipode. Both groups of enzymes are frequently referred to as “racemases” because they share a common reaction at the carbon directly involved in the catalytic change. The most general assay principle applied is the enzymic detection of a specific antipode formed as a product. Most commonly, this has involved incubating the L antipode as a substrate and measuring the D antipode with D -amino acid oxidase. The latter step has been carried out manometrically in the studies of alanine racemase of phenylalanine racemization or of the racemization of other amino acids. The absence of pyridoxal phosphate, pyridine or flavin nucleotides, or of any phosphate-containing prosthetic group, has been demonstrated for homogeneous hydroxyproline-2-epimerase by a variety of methods, including spectral and fluorometric investigation, the content of 32 P after purification from cultures containing this isotope, and the ineffectiveness of a variety of inhibitors, including excess borohydride.

Publisher Summary This chapter discusses the methods of preparation of Amino Acid Racemase ( Pseudomonas striata ). Amino acid racemase catalyzes the conversion of either D - or L -amino acids to the racemates. All assay methods are based on the measurement of one stereoisomer of the substrate. When the L -amino acids whose D -isomer are susceptible to D -amino acid oxidase are used as the substrates, the amino acid racemase activity is followed by manometric determination of the D -amino acids formed with D -amino acid oxidase. L -Glutamate decarboxylase or L -lysine decarboxylase is used to determine the activity in the reaction mixtures containing D -glutamate or D -lysine, respectively, as the substrate. Determination of the activity catalyzing the racemization of arginine is performed by measuring the formation of L -arginine from the D -isomer with arginase. The substrate specificity of the enzyme is extremely low in comparison with usual amino acid racemases thus far reported—for example, proline racemase. The enzyme activity is inhibited by D -cycloserine and hydroxylamine.

Analysis of the growing number of available fully-sequenced genomes has shown that Horizontal Gene Transfer (HGT) in eukaryotes is more common than previously thought. It has been proposed that genes with certain functions may be more prone to HGT than others, but we still have a very poor understanding of the selective forces driving eukaryotic HGT. Recent work uncovered that d-amino acid racemases have been commonly transferred from bacteria to fungi, but their role in the receiving organisms is currently unknown. Here, we set out to assess whether d-amino acid racemases are commonly transferred to and between eukaryotic groups. For this we performed a global survey that used a novel automated phylogeny-based HGT-detection algorithm (Abaccus). Our results revealed that at least 7.0% of the total eukaryotic racemase repertoire is the result of inter- or intra-domain HGT. These transfers are significantly enriched in plant-associated fungi. For these, we hypothesize a possible role for the acquired racemases allowing to exploit minoritary nitrogen sources in plant biomass, a nitrogen-poor environment. Finally, we performed experiments on a transferred aspartate-glutamate racemase in the fungal human pathogen Candida glabrata, which however revealed no obvious biological role.

9 pages, 5 figures, 4 tables, 30 references., Isopenicillin N (IPN) epimerase, an enzyme involved in cephalosporin and cephamycin biosynthesis that converts IPN into penicillin N, was extracted from Nocardia lactamdurans and purified 88-fold. The enzyme was unstable but could be partially stabilized by addition of pyridoxal phosphate. The purified enzyme did not require ATP for activity in contrast to other amino acid racemases. The enzyme had an Mr of 59000 as determined by gel filtration; IPN epimerase from Streptomyces clavuligerus had an Mr of 63000. A protein band of Mr 59000 was found to be enriched in SDS-PAGE of active fractions from N. lactamdurans. The optimal temperature of the epimerase was 25°C and the optimal pH 7.0. The apparent Km for IPN was 270 μM. Fe2+, Cu2+, Hg2+ and Zn2+ strongly inhibited enzyme activity. α-Aminoadipic acid, valine, glutamine, glycine, aspartic acid and glutathione do not affect enzyme activity, whereas ammonium sulphate was inhibitory. The epimerase activity was partially inhibited by several thiol-specific reagents., This research was funded by grants from the CICYT (83-2039) (Madrid, Spain) and Gist-Brocades (Delft, The Netherlands). L. Láiz was supported by a fellowship of the Diputación de León (Spain) and J. M. Castro by a PFPI fellowship of the Ministry of Education and Science (Madrid, Spain).

Integration of racemization and a resolution process is an attractive way to overcome yield limitations in the production of pure chiral molecules. Preferential crystallization and other crystallization-based techniques usually produce low enantiomeric excess in solution, which is a constraint for coupling with racemization. We developed an enzymatic fixed bed reactor that can potentially overcome these unfavorable conditions and improve the overall yield of preferential crystallization. Enzyme immobilization strategies were investigated on covalent-binding supports. The amino acid racemase immobilized in Purolite ECR 8309F with a load of 35 mg-enzyme/g-support showed highest specific activity (approx. 500 U/g-support) and no loss in activity in reusability tests. Effects of substrate inhibition observed for the free enzyme were overcome after immobilization. A packed bed reactor with the immobilized racemase showed good performance in steady state operation processing low enantiomeric excess inlet. Kinetic parameters from batch reactor experiments can be successfully used for prediction of packed bed reactor performance. Full conversions could be achieved for residence times above 1.1 min. The results suggest the potential of the prepared racemase reactor to be combined with preferential crystallization to improve resolution of asparagine enantiomers.

Modest stereoselectivity (15–20% enantiomeric excess) is observed in the protonation of the aminoacrylate product that forms in kinetic competition with suicidal capture of the pyridoxal phosphate (PLP) cofactor by that aminoacrylate in the inactivation of a racemase (Pseudomonas striata) with O-acetylserines.