Characterization of an Immobilized Amino Acid Racemase for Potential Application in Enantioselective Chromatographic Resolution Processes

Enantioselective resolution processes can be improved by integration of racemization. Applying environmentally friendly enzymatic racemization under mild conditions is in particular attractive. Owing to the variety of enzymes and the progress in enzyme engineering, suitable racemases can be found for many chiral systems. An amino acid racemase (AAR) from P. putida KT2440 is capable of processing a broad spectrum of amino acids at fast conversion rates. The focus of this study is the evaluation of the potential of integrating ARR immobilized on Purolite ECR 8309 to racemize L- or D-methionine (Met) within an enantioselective chromatographic resolution process. Racemization rates were studied for different temperatures, pH values, and fractions of organic co-solvents. The long-term stability of the immobilized enzyme at operating and storage conditions was found to be excellent and recyclability using water with up to 5 vol% ethanol at 20 °C could be demonstrated. Packed as an enzymatic fixed bed reactor, the immobilized AAR can be coupled with different resolution processes; for instance, with chromatography or with preferential crystallization. The performance of coupling it with enantioselective chromatography is estimated quantitatively, exploiting parametrized sub-models. To indicate the large potential of the AAR, racemization rates are finally given for lysine, arginine, serine, glutamine, and asparagine.

Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis

The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Members of Thermococcales harbor a number of genes encoding putative aminotransferase Class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis. The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell-surface glycoprotein gene TK0895 (Pcsg). The purified protein did not display aminotransferase activity, but exhibited racemase activity. An examination on most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal media supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo. When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase, but displayed cell yield similar to that observed in medium with l-Leu. By contrast, the ΔTK1211 strain displayed growth in medium with l-Leu, but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells, and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensis. IMPORTANCE Phylogenetic analysis of aminotransferase Class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups can now be presumed to function as a broad substrate specificity amino acid racemase (Subgroup 1), alanine/serine racemase (Subgroup 2), ornithine ω-aminotransferase (Subgroup 3), or Leu/Met racemase (Subgroup 4).