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Purification and properties of Saccharomyces cerevisiae S-adenosylmethionine synthetase expressed in recombinant Pichia pastoris

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Abstract

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K m of 120 and 330 μM and V max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.

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References

  • Berger BJ, Knodel MH (2003) Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis. BMC Microbio 3:12

    Article  Google Scholar 

  • 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 

  • Cabrero C, Puerta J, Alemany S (1987) Purification and comparison of two forms of S-adenosylmethionine synthetase from rat liver. FEBS 170:299–304

    CAS  Google Scholar 

  • Cereghion JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  Google Scholar 

  • Chen XL, Wang YS, Zhen YG (2006) Study on the extraction and purification of S-adenosylmethionine synthetase (Chinese). Biotechnol 15(1):61–63

    Google Scholar 

  • Chiang PK, Cantoni GL (1977) Activition of methionine for transmethylation. purification of the S-adenosylmethionine synthetase of Bakers’ yeast and its separation into two forms. J Biol Chem 252(13):4506–4513

    CAS  Google Scholar 

  • Chiang PK, Gordon RK, Zeng GC, Doctor BP, Pardhasaradhi K, (1996) S-Adenosylmethionine and methylation. FASEB J 10:471–480

    CAS  Google Scholar 

  • Chou TC, Talalay P (1972) The mechanism of S-adenosylmethionine synthesis by purified preparations of Baker’s yeast. Biochem 11:1065–1073

    Article  CAS  Google Scholar 

  • Cui W, Huang L, Li W (2006) Cloning and expression of rat liver S-adenosylmethionine synthetase. Chem Res Chinese 22:236–238

    Article  CAS  Google Scholar 

  • Li DY, Yu J, Tian L, Ji XS, Yuan ZY, (2002) Production of SAM by recombinant Pichia pastoris. Chin J Biotechnol 3:295–299

    Google Scholar 

  • López-Vara MC, Gasset M, Pajares MA (2000) Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies. Protein Expr Purif 19:219–226

    Article  CAS  Google Scholar 

  • Markham CD, Hafner EW, Tabor CW, Tabor H, (1980) Adenosylmethionine synthetase: supplemental material to S-adenosylmethionine synthetase from Escherichia coli. J Biol Chem 255:982–9092

    Google Scholar 

  • Markham GD (1981) Spatial proximity of two divalent metal ions at the active site of S-adenosylmethionine synthetase. J Biol Chem 256:1903–1909

    CAS  Google Scholar 

  • Mathur M, Sachar RC (1991) Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls. FEBS 287:113–117

    Article  CAS  Google Scholar 

  • Mcqueney MS, Markham GD (1995) Investigation of monovalent cation activation of S-adenosylmethionine synthetase using mutagenesis and uranyl inhibition. J Biol Chem 270:18277–18284

    Article  CAS  Google Scholar 

  • Mincheva K, Kamburova V, Balutzov V (2002) Production of S-adenosyl-lmethionine by a mutant strain of Kluyveromyces lactis. Biotechnol Lett 24:985–988

    Article  CAS  Google Scholar 

  • Mitsui K, Teraoka I, Tsukada K (1988) Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain. J Biol Chem 263:11211–11216

    CAS  Google Scholar 

  • Ochi K (1987) Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to A factor. J Bacteriol 169:3608–3616

    CAS  Google Scholar 

  • Okada G, Teraoka H, Tsukada K (1981) Multiple species of mammalian S-adenosylmethionine synthetase: partial purification and characterization. Biochem 20:934–940

    Article  CAS  Google Scholar 

  • Omura S, Ikeda H, Ishikawa J (2001) Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci USA 98:12215–2220

    Article  CAS  Google Scholar 

  • Patrick SM, Fazenda ML, Mcneil B, Harvey LM (2005) Heterologous protein production using Pichia pastoris expression system. Yeast 22:249–270

    Article  CAS  Google Scholar 

  • Schroder G, Eichel J, Breinig S, Schroder J (1997) Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization. Plant Mol Biol 33:211–22

    Article  CAS  Google Scholar 

  • Shiomi N, Fukuda H, Fukuda Y, Schroder J (1990) Production of S-adenosyl-l-methionine by Saccharomyces cerevisiae cells carrying a gene for ethionine resistance. Biotechnol Bioeng 35:1120–1124

    Article  CAS  Google Scholar 

  • Yoon GS, Ko KH, Kang HW, Suh J, Kim GS, Ryu YW (2006) Characterization of S-adenosylmethionine synthetase from Streptomyces avermitilis NRRL 8165 and its effect on antibiotic production. Enzyme Microb Technol 39:466–473

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to National High Technology Research and Development Program of China (863 Program) (No. 2002aa217021; 2002aa2z3451) for its financial support. We are also indebted to Dr. Xiao-Qing Hu for some advice during the course of this work.

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, 130 Meilong Road, Shanghai, 200237, P. R. China

    Jin Zhou, Ju Chu, Yong-Hong Wang, Si-Liang Zhang, Ying-Ping Zhuang & Zhong-Yi Yuan

  2. National Research Center for Biotechnology (Shanghai), East China University of Science & Technology, Shanghai, 200237, P. R. China

    Jin Zhou, Ju Chu, Yong-Hong Wang, Si-Liang Zhang, Ying-Ping Zhuang & Zhong-Yi Yuan

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  1. Jin Zhou
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  2. Ju Chu
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Correspondence to Ju Chu.

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Zhou, J., Chu, J., Wang, YH. et al. Purification and properties of Saccharomyces cerevisiae S-adenosylmethionine synthetase expressed in recombinant Pichia pastoris . World J Microbiol Biotechnol 24, 789–796 (2008). https://doi.org/10.1007/s11274-007-9538-9

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