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.
Vidal
L
L
Unitat de Biocatalisi Aplicada Associada al IIQAB (CSIC-UAB), Departament d'Enginyeria Química, Escola Tècnica Superior d'Enginyeria, Universitat Autònoma de Barcelona, Barcelona, 08193, Spain.
Calveras
J
J
Clapés
P
P
Ferrer
P
P
Caminal
G
G
D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D048249:Streptococcus thermophilus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase