Production of Recombinant Caffeine Synthase from Guarana (Paullinia cupana var. sorbilis) in Escherichia coli

  • Flávia Camila Schimpl
  • Eduardo Kiyota
  • Paulo MazzaferaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1405)


Caffeine synthase (CS) is a methyltransferase responsible for the last two steps of the caffeine biosynthesis pathway in plants. CS is able to convert 7-methylxanthine to theobromine (3,7-dimethylxanthine) and theobromine to caffeine (1,3,7-trimethylxanthine) using S-adenosyl-l-methionine as the methyl donor in both reactions. The production of a recombinant protein is an important tool for the characterization of enzymes, particularly when the enzyme has affinity for different substrates. Guarana has the highest caffeine content among more than a hundred plant species that contain this alkaloid. Different from other plants, in which CS has a higher affinity for paraxanthine (1,7-dimethylxanthine), caffeine synthase from guarana (PcCS) has a higher affinity for theobromine. Here, we describe a method to produce a recombinant caffeine synthase from guarana in Escherichia coli and its purification by affinity chromatography. The recombinant protein retains activity and can be used in enzymatic assays and other biochemical characterization studies.

Key words

Caffeine synthase Guarana Paullinia cupana Recombinant expression Alkaloid 


  1. 1.
    Mazzafera P (2012) Which is the by‐product: caffeine or decaf coffee? Food Energy Secur 1:70–75CrossRefGoogle Scholar
  2. 2.
    Schimpl FC, da Silva JF, Gonçalves JFC, Mazzafera P (2013) Guarana: revisiting a highly caffeinated plant from the Amazon. J Ethnopharmacol 150:14–31CrossRefPubMedGoogle Scholar
  3. 3.
    Ashihara H, Ogita S, Crozier A (2011) Purine alkaloid metabolism. In: Ashihara H, Crozier A, Komamine A (eds) Plant metabolism and biotechnology. Wiley, Chichester, pp 163–189CrossRefGoogle Scholar
  4. 4.
    Schimpl FC, Kiyota E, Mayer JLS, Gonçalves JFC, da Silva JF, Mazzafera P (2014) Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit. Phytochemistry 105:25–36CrossRefPubMedGoogle Scholar
  5. 5.
    Kato M, Mizuno K, Crozier A, Fujimura T, Ashihara H (2000) Caffeine synthase gene from tea leaves. Nature 406:956–957CrossRefPubMedGoogle Scholar
  6. 6.
    Mizuno K, Okuda A, Kato M, Yoneyama N, Tanaka H, Ashihara H, Fujimura T (2003) Isolation of a new dual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-methylxanthine to caffeine from coffee (Coffea arabica L.). FEBS Lett 534:75–81CrossRefPubMedGoogle Scholar
  7. 7.
    Uefuji H, Ogita S, Yamaguchi Y, Koizumi N, Sano H (2003) Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol 132:372–380PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Ângelo PCS, Nunes-Silva CG, Brígido MM, Azevedo JSN, Assunção EN, Sousa ARB, Patrício FJB, Rego MM, Peixoto JCC, Oliveira WP Jr, Freitas DV, Almeida ERP, Viana AMHA, Souza AFPN, Andrade EV, Acosta POA, Batista JS, Walter MEMT, Leomil L, Anjos DAS, Coimbra RCM, Barbosa MHN, Honda E, Pereira SS, Silva A, Pereira JO, Silva ML, Marins M, Holanda FJ, Abreu RMM, Pando SC, Gonçalves JFC, Carvalho ML, Leal-Mesquita ERRBP, Da Silveira MA, Batista WC, Atroch AL, França SC, Porto JIR, Schneider MPC, Astolfi-Filho S (2008) Guarana (Paullinia cupana var. sorbilis), an anciently consumed stimulant from the Amazon rain forest: the seeded-fruit transcriptome. Plant Cell Rep 27:117–124CrossRefPubMedGoogle Scholar
  9. 9.
    Rezaian MA, Krake LR (1987) Nucleic acid extraction and virus detection in grapevine. J Virol Methods 17:277–285CrossRefPubMedGoogle Scholar
  10. 10.
    Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LC, Panda AK (2000) Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expr Purif 18:182–192CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Flávia Camila Schimpl
    • 1
  • Eduardo Kiyota
    • 1
  • Paulo Mazzafera
    • 1
    Email author
  1. 1.Departamento de Biologia Vegetal-IBUniversidade Estadual de CampinasCampinasBrazil

Personalised recommendations