Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst |
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Authors: | L Vidal J Calveras P Clapés P Ferrer G Caminal |
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Institution: | (1) 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;(2) Institute for Chemistry and Environmental Research—CSIC, Jordi Girona 18-26, Barcelona, 08034, Spain |
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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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