Abstract
The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5′-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of l-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at −20°C and 4°C. It was observed that the K m for l-allo-threonine was 38-fold higher than that for l-threonine, suggesting this enzyme can be classified as a specific l-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6–7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible β-hydroxy-α-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of β-hydroxy-α-amino acids.
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Acknowledgements
The authors wish to acknowledge financial support from CICYT (project PPQ 2002-04625-C02-01). The Department of Chemical Engineering is the Unit of Biochemical Engineering of the Centre de Referència en Biotecnologia de la Generalitat de Catalunya (CeRBa). We thank Dr. Tzanetakis (Aristotle University of Thessaloniki, Greece) for providing us with the S. thermophilus YKA-184 strain.
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Vidal, L., Calveras, J., Clapés, P. et al. Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst. Appl Microbiol Biotechnol 68, 489–497 (2005). https://doi.org/10.1007/s00253-005-1934-1
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DOI: https://doi.org/10.1007/s00253-005-1934-1