Applied Microbiology and Biotechnology

, Volume 68, Issue 4, pp 489–497

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

  • L. Vidal
  • J. Calveras
  • P. Clapés
  • P. Ferrer
  • G. Caminal
Biotechnologically Relevant Enzymes and Proteins


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 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 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.


  1. Bolotin A, Quinquis B, Renault P, Sorokin A, Ehrlich D, Kulakauskas S, Lapidus A, Glotsman E, Mazur M, Pusch G, Fonstein M, Overbeek R, Kyprides N, Purnelle B, Prozzi D, Ngui K, Masuy D, Hancy F, Burteau S, Boutry M, Delcour J, Goffeau A, Hols P (2004) Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus. Nat Biotechnol 22:1554–1558CrossRefPubMedGoogle Scholar
  2. Chavez ACSD, Fernandez M, Lerayer ALS, Mierau I, Kleerebezem M, Hugenholtz J (2002) Metabolic engineering of acetaldehyde production by Steptococcus thermophilus. Appl Environ Microbiol 68:5656–5662PubMedCrossRefGoogle Scholar
  3. Espelt L, Clapés P, Esquena J, Manich A, Solans C (2003) Enzymatic carbon–carbon bond formation in water-in-oil highly concentrated (gel emulsions). Langmuir 19:1337–1346CrossRefGoogle Scholar
  4. Gijsen H, Qiao L, Fitz W, Wong CH (1996) Recent advances in chemoenzymatic synthesis of carbohydrates and carbohydrates mimetics. Chem Rev 96:443–473CrossRefPubMedGoogle Scholar
  5. Kataoka M, Ikemi M, Morikawa T, Miyoshi T, Nishi K, Wada M, Yamada H, Shimizy S (1997a) Isolation and characterization of d-threonine aldolase, a pyridoxal-5′-phosphate-dependent enzyme from Arthrobacter sp. DK-38. Eur J Biochem 248:385–393CrossRefPubMedGoogle Scholar
  6. Kataoka M, Wada M, Nishi K, Yamada H, Shimizu S (1997b) Purification and characterization of l-allo-threonine aldolase from Aeromonas jandaei DK-39. FEMS Microbiol Lett 151:245–248CrossRefPubMedGoogle Scholar
  7. Kimura T, Vassilev VP, Shen GJ, Wong CH (1997) Enzymatic synthesis of beta-hydroxy-alpha-aminoacids based on recombinant d- and l-threonine aldolases. J Am Chem Soc 119:11734–11742CrossRefGoogle Scholar
  8. Liu JQ, Nagata S, Dairi T, Misono H, Shimizu S, Yamada H (1997) The GLY1 gene of Saccharomyces cerevisiae encodes a low-specific l-threonine aldolase that catalyses cleavage of l-allo-threonine and l-threonine to glycine. Expression of the gene in Escherichia coli and purification and characterization of the enzyme. Eur J Biochem 245:289–293CrossRefPubMedGoogle Scholar
  9. Liu JQ, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H (1998a) Gene cloning of a thermostable low-specificity l-threonine aldolase from Escherichia coli. Eur J Biochem 255:220–226CrossRefPubMedGoogle Scholar
  10. Liu JQ, Ito S, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H (1998b) Gene cloning, nucleotide sequencing and purification and characterization of the low-specificity l-threonine aldolase from Pseudomonas sp. strain NCIMB 10558. Appl Environ Microbiol 64:549–554PubMedGoogle Scholar
  11. Liu JQ, Ito S, Dairi T, Itoh N, Shimizu S, Yamada H (1998c) Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-aminoacids synthesis. Appl Microbiol Biotechnol 49:702–708CrossRefGoogle Scholar
  12. Liu JQ, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H (2000a). Diversity of microbial threonine aldolases and their application. J Mol Catal B Enzym 10:107–115CrossRefGoogle Scholar
  13. Liu JQ, Odani M, Yasuoka T, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H (2000b) Gene cloning and overproduction of low-specificity d-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for Parkinson drug. Appl Microbiol Biotechnol 54:44–51CrossRefPubMedGoogle Scholar
  14. Ogawa H, Fujioka M (1981) Purification and characterization of cytosolic and mitochondrial serine hydroxymethyltransferases from rat liver. J Biochem 90:381–390PubMedGoogle Scholar
  15. Ogawa H, Gomi T, Fujioka M (2000) Serine hydroxymethyltransferase and threonine aldolase: are they identical? Int J Biochem Cell Biol 32:289–301CrossRefPubMedGoogle Scholar
  16. Plamann MD, Stauffer GV (1983) Characterization of the Escherichia coli gene for serine hydroxymethyltransferase. Gene 22:9–18CrossRefPubMedGoogle Scholar
  17. Plamann MD, Stauffer LT, Urbanowsky ML, Stauffer GV (1983) Complete nucleotide sequence of the Escherichia coli glyA gene. Nucleic Acids Res 11:2065–2075PubMedCrossRefGoogle Scholar
  18. Saeed A, Young DW (1992) Synthesis of l-beta-hydroxy amino acids using serine hydroxymethyltransferase. Tetrahedron 48:2507–2514CrossRefGoogle Scholar
  19. Sambrook J, Maniatis T, Fritsch EF (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  20. Schirch LG, Mason M (1962) Serine transhydroxymethylase. Spectral properties of the enzyme-bound pyridoxal-5-phosphate. J Biol Chem 237:2578–2581Google Scholar
  21. Schirch V, Hopkins S, Villar E, Angelaccio S (1985) Serine hydroxymethyltransferase from Escherichia coli: purification and properties. J Bacteriol 163:1–7PubMedGoogle Scholar
  22. Stadtman ER, Levine RL (2003) Free radical-mediated oxidation of free amino acids and amino residues in proteins. Amino Acids 25:207–218CrossRefPubMedGoogle Scholar
  23. Usha R, Savithri HS, Rao NA (1994) The primary structure of sheep liver cytosolic serine hydroxymethyl transferase and analysis of the evolutionary relationships among serine hydroxymethyl transferases. Biochim Biophys Acta 1204:75–83PubMedGoogle Scholar
  24. Viaplana E, Villaverde A (1996) Polylinker-encoded peptides can confer toxicity to recombinant proteins produced in Escherichia coli. Biotechnol Prog 12:723–727CrossRefPubMedGoogle Scholar
  25. Vidal L, Durany O, Suau T, Ferrer P, Benaiges MD, Caminal G (2003) High-level production of recombinant His-tagged rhamnulose 1-phosphate aldolase in Escherichia coli. J Chem Technol Biotechnol 78:1171–1179CrossRefGoogle Scholar
  26. Wilkins DW, Schmidt RH, Kennedy L (1986) Threonine aldolase activity in yogurt bacteria as determined by headspace gas chromatography. J Agric Food Chem 34:150–152CrossRefGoogle Scholar
  27. Xanthopoulos V, Petridis D, Tzanetakis N (2001) Characterization and classification of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains isolated from traditional Greek yogurts. J Food Sci 66:747–752CrossRefGoogle Scholar
  28. Yadav JS, Chandrasekhar S, Ravindra Reddy Y, Rama Rao AV (1995) Synthesis of (2S,3R)-3-hydroxy leucine: a constituent of lysobactin. Tetrahedron 51:2749–2754CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • L. Vidal
    • 1
  • J. Calveras
    • 2
  • P. Clapés
    • 2
  • P. Ferrer
    • 1
  • G. Caminal
    • 1
  1. 1.Unitat de Biocatalisi Aplicada Associada al IIQAB (CSIC-UAB), Departament d’Enginyeria Química, Escola Tècnica Superior d’EnginyeriaUniversitat Autònoma de BarcelonaBarcelonaSpain
  2. 2.Institute for Chemistry and Environmental Research—CSICBarcelonaSpain

Personalised recommendations