Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst.

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 degrees C and 4 degrees C. It was observed that the Km 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 beta-hydroxy-alpha-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of beta-hydroxy-alpha-amino acids.