Skip to main content
SpringerLink
Log in

Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032)

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD+ release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD+ to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Bezbradica D, Mijin D, Mihailovic M, Knezevic-Jugovic Z (2009) Microwave-assisted immobilization of lipase from Candida rugosa on Eupergit® supports. J Chem Technol Biotechnol 84:1642–1648

    Google Scholar 

  • Borchardt RT, Huber JA, Wu YS (1976) A convenient preparation of S-adenosylhomocysteine and related compounds. J Org Chem 6:565–567

    Article  Google Scholar 

  • Boller T, Meier C, Menzler S (2002) EUPERGIT Oxirane Acrylic Beads: How to Make Enzymes Fit for Biocatalysis. Org Process Res Dev 6:509-519

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Chabannes B, Gharib A, Cronenberger L, Pacheco H (1982) S-Adenosyl-l-homocysteine: simplified enzymatical preparation with high yield. Prep Biochem 12:395–415

    Article  CAS  Google Scholar 

  • Clark DS (1994) Can immobilization be exploited to modify enzyme activity? Trends Biotechnol 12:439–443

    Article  CAS  Google Scholar 

  • Desphande PB, Senthilkumar UP, Padmanabhan R (2002) Chemical synthesis of S-adenosyl-l-methionine with enrichment of (S,S)-isomer. US Patent 2002/0188116 A1

  • Duerre JA, Schlenck F (1962) Formation and metabolism of S-adenosyl-l-homocysteine in yeast. Arch Biochem Biophys 69:575

    Article  Google Scholar 

  • Edwards AL, Reyes FE, Héroux A, Batey RT (2010) Structural basis for recognition of S-adenosylhomocysteine by riboswitches. RNA 16:2144–2155

    Article  CAS  Google Scholar 

  • Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95

    Article  CAS  Google Scholar 

  • Fonlupt P, Roche M, Cronenberger L, Pacheco H (1979) S-Adenosyl-l-homocysteine and salts thereof for use in therapy. UK Patent GB 2 019720 A

  • Fonlupt P, Roche M, Cronenberger L, Pacheco H (1980a) S-adenosyl-l-homocysteine: 1. Influence on sleep. Can J Physiol Pharmacol 58:160–166

    Article  CAS  Google Scholar 

  • Fonlupt P, Roche M, Andre AC, Cronenberger L, Pacheco H (1980b) S-adenosyl-l-homocysteine: 2. An anticonvulsant. Can J Physiol Pharmacol 58:493–498

    Article  CAS  Google Scholar 

  • Gaetani GF, Ferraris AM, Sanna P, Kirkman HN (2005) A novel NADPH:(bound) NADP+ reductase and NADH:(bound) NADP+transhydrogenase function in bovine liver catalase. Biochem J 385:763–768

    Google Scholar 

  • Hayden DM, Rolletschek H, Borisjuk L, Corwin J, Kliebenstein DJ, Grimberg A, Stymne S, Dehesh K (2011) Cofactome analyses reveal enhanced flux of carbon into oil for potential biofuel production. Plant J 67:1018–1028. doi:10.1111/j.1365-313X.2011.04654.x

    Article  CAS  Google Scholar 

  • Heiland PC, Hill FF (1993) Accumulation of S-Adenosylhomocysteine and S-Adenosylmethionine by an Ethionine-Resistant Mutant of Bakers’ Yeast. Process Biochem 28:171–177

    Google Scholar 

  • Hildesheim J, Hildesheim R, Lederer E (1971) Synthesis of methyl-transferase inhibitors: analogs of S-adenosyl homocysteine. Biochimie 53:1067–1071

    Article  CAS  Google Scholar 

  • Hu Y, Yang X, Yin DH, Mahadevan J, Kuczera K, Schowen RL, Borchardt RT (2001) Computational characterization of substrate binding and catalysis in S-adenosylhomocysteine hydrolase. Biochemistry 40:15143–15152

    Article  CAS  Google Scholar 

  • Ikeda M, Nakagawa S (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109

    Article  CAS  Google Scholar 

  • Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Kramer R, Linke B, McHardy AC, Meyer F, Mockel B, Pfefferle W, Puhler A, Rey DA, Ruckert C, Rupp O, Sahm H, Wendisch VF, Wiegrabe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104:5–25

    Article  CAS  Google Scholar 

  • Katchalski-Katzir E, Kraemer DM (2000) Eupergit® C, a carrier for immobilization of enzymes of industrial potential. J Mol Catal B—Enzym 10:157–176

    Article  CAS  Google Scholar 

  • Knezevic Z, Milosavic N, Bezbradica D, Jakovljevic Z, Prodanovic R (2006) Immobilization of lipase from Candida rugosa on Eupergit® C supports by covalent attachment. Biochem Eng J 30:269–278

    Google Scholar 

  • Lauchtenberger W (1996) Amino acids—technical production and use. In: Rehm HJ, Reed G, Pühler A, Stadler P (eds) Biotechnology, vol 6. Weinheim, Germany, pp 465–502

    Google Scholar 

  • Lozada-Ramírez JD, Martínez-Martínez I, Sánchez-Ferrer A, García-Carmona F (2006) A colorimetric assay for S-adenosylhomocysteine hydrolase. J Biochem Biophys Meth 67:131–140

    Article  Google Scholar 

  • Lozada-Ramírez JD, Sánchez-Ferrer A, García-Carmona F (2008) S-Adenosylhomocysteine hydrolase from Corynebacterium glutamicum: cloning, over-expression, purification, and biochemical characterization. J Mol Microbiol Biotechnol 15:277–286

    Article  Google Scholar 

  • Martín MT, Plou FJ, Alcalde M, Ballesteros A (2003) Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of immobilized biocatalyst. J Mol Catal B-Enzym 21:299–308

    Article  Google Scholar 

  • Miles RW, Nielsen LP, Ewing GJ, Yin D, Borchardt RT, Robins MJ (2002) S-homoadenosyl-l-cysteine and S-homoadenosyl-l-homocysteine. Synthesis and binding studies of non-hydrolyzed substrate analogues with S-adenosyl-l-homocysteine hydrolase. J Org Chem 67:8258–8260

    Article  CAS  Google Scholar 

  • Pignot M, Pljevaljcic G, Weinhold E (2000) Efficient synthesis of S-adenosyl-l-homocysteine natural product analogues and their use to elucidate the structural determinant cofactor binding of the DNA methyltransferase M HhaI. Eur J Org Chem 3:549–555

    Article  Google Scholar 

  • Porter DJT (1993) S-Adenosylhomocysteine hydrolase. Stereochemistry and kinetics of hydrogen transfer. J Biol Chem 268:66–73

    CAS  Google Scholar 

  • Porter DJT, Boyd FL (1992) Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3′-ketoadenosine, adenosine, and adenine. J Biol Chem 267:3205–3213

    CAS  Google Scholar 

  • Richards HH, Chiang PK, Cantoni GL (1978) Adenosylhomocysteine hydrolase. Crystallization of the purified enzyme and its properties. J Biol Chem 253:4476–4480

    CAS  Google Scholar 

  • Ruckert C, Puhler A, Kalinowski J (2003) Genome-wide analysis of the l-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation. J Biotechnol 104:213–228

    Article  CAS  Google Scholar 

  • Serafinowski P (1987) Synthesis of some S-3′-deoxyadenosyl-l-homocysteine analogues. Nucleic Acids Res 15:1121–1137

    Article  CAS  Google Scholar 

  • Shapiro SK, Yphantis DA, Almenas A (1964) Biosynthesis of methionine in Saccharomyces cerevisiae. Partial purification and properties of S-adenosylmethionine: homocysteine methyltransferase. J Biol Chem 239:1551–1556

    CAS  Google Scholar 

  • Sheldon RA (2007) Enzyme Immobilization: The Quest for Optimum Performance. Adv Synth Catal 349:1289–1307

    Google Scholar 

  • Shimizu S, Yamada H (1984) Microbial and enzymatic processes for the production of pharmacologically important nucleosides. Trend Biotech 2:137–141

    Article  CAS  Google Scholar 

  • Takata Y, Yamada T, Huang Y, Komoto J, Gomi T, Ogawa H, Fujioka M, Takusagawa F (2002) Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J Biol Chem 277:22670–22676

    Article  CAS  Google Scholar 

  • Tu M, Zhang X, Kurabi A, Gilkes N, Mabee W, Saddler J (2006) Immobilization of b-glucosidase on Eupergit C for lignocellulose hydrolysis. Biotechnol Lett 28:151–156.

    Google Scholar 

  • Ueland PM (1982) Pharmacological and biochemical aspects of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase. Pharmacol Rev 34:223–253

    CAS  Google Scholar 

  • Usdin E, Borchardt RT, Creveling CR (1979) Transmethylation. Elsevier, Amsterdam

    Google Scholar 

  • Usdin E, Borchardt RT, Creveling CR (1982) Biochemistry of adenosylmethionine and related compounds. Macmillan, London

    Google Scholar 

  • Wellheim EH, Percha GW (1972) Process for preparing S-adenosylhomocysteine. US Patent 3,642,772

  • Yamada H, Tani Y, Shimizu S (1991) Process for producing S-adenosyl-l-homocysteine. US Patent 5,008,188

Download references

Acknowledgments

This work was partially supported by Programa de Ayuda a Grupos de Excelencia de la Región de Murcia, SÉNECA Foundation (04541/GERM/06, Plan de Ciencia y Tecnología 2007–2010) and by Consejo Nacional de Ciencia y Tecnología Proyecto Ciencia Básica 133949/2011-2013.

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Sciences, School of Sciences, Universidad de las Américas Puebla, Santa Catarina Mártir Cholula, 72810, Puebla, Mexico

    J. D. Lozada-Ramírez

  2. Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, 30071, Murcia, Spain

    A. Sánchez-Ferrer & F. García-Carmona

Authors
  1. J. D. Lozada-Ramírez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Sánchez-Ferrer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F. García-Carmona
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. D. Lozada-Ramírez.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lozada-Ramírez, J.D., Sánchez-Ferrer, A. & García-Carmona, F. Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032). Appl Microbiol Biotechnol 93, 2317–2325 (2012). https://doi.org/10.1007/s00253-011-3769-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-011-3769-2

Keywords

Search

Navigation