Amino Acids

, Volume 47, Issue 7, pp 1465–1477 | Cite as

Biocatalytic synthesis, antimicrobial properties and toxicity studies of arginine derivative surfactants

  • M. Elisa Fait
  • Graciela L. Garrote
  • Pere Clapés
  • Sebastian Tanco
  • Julia Lorenzo
  • Susana R. MorcelleEmail author
Original Article


Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N α-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N α-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N α-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % the initial bacterial population after only 1 h of contact. The cytotoxic effect towards different cell types of both arginine derivatives was also measured. Bz-Arg-NHCn demonstrated lower haemolytic activity and were less eye-irritating than the commercial cationic surfactant cetrimide. A similar trend could also be observed when cytotoxicity was tested on hepatocytes and fibroblast cell lines: both arginine derivatives were less toxic than cetrimide. All these properties would make the two novel arginine compounds a promising alternative to commercial cationic surfactants, especially for their use as additives in topical formulations.


Arginine-based surfactants Papain Biocatalysis Antimicrobial activity Cytotoxic effect 



This research was supported by projects PIP 0150 (CONICET), X-613 (UNLP), CTQ2012-31605 and BIO2013-44973-R (MINECO, Spain). Mass spectra were performed in the Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (UMYMFOR), CONICET-FCEN-UBA, Buenos Aires, Argentina. The valuable contribution of Dr. Alicia S. Cánepa (LADECOR, Depto. de Química, Fac. Cs. Exactas, UNLP) in the analysis of NMR spectra is also acknowledged. MEF was awarded CONICET fellowship. GLG and SRM are members of CONICET Researcher Career.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2015_1979_MOESM1_ESM.docx (1.7 mb)
Supplementary material 1 (DOCX 1759 kb)


  1. Adlercreutz P (1991) On the importance of the support material for enzymatic synthesis in organic media. Support effects at controlled water activity. Eur J Biochem 199:609–614. doi: 10.1111/j.1432-1033.1991.tb16161.x PubMedCrossRefGoogle Scholar
  2. Arnon R (1970) Papain. In: Perlmann GE, Lorand L (eds) Methods in Enzymology, vol 19., Proteolytic EnzimesAcademic Press, New York, pp 226–244Google Scholar
  3. Benavides T, Mitjans M, Martínez V et al (2004) Assessment of primary eye and skin irritants by in vitro cytotoxicity and phototoxicity models: an in vitro approach of new arginine-based surfactant-induced irritation. Toxicology 197:229–237. doi: 10.1016/j.tox.2004.01.011 PubMedCrossRefGoogle Scholar
  4. 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. doi: 10.1016/0003-2697(76)90527-3 PubMedCrossRefGoogle Scholar
  5. Carmona-Ribeiro AM, de Melo Carrasco LD (2013) Cationic antimicrobial polymers and their assemblies. Int J Mol Sci 14:9906–9946. doi: 10.3390/ijms14059906 PubMedCentralPubMedCrossRefGoogle Scholar
  6. Castillo JA, Pinazo A, Carilla J et al (2004) Interaction of antimicrobial arginine-based cationic surfactants with liposomes and lipid monolayers. Langmuir 20:3379–3387. doi: 10.1021/la036452h PubMedCrossRefGoogle Scholar
  7. Castillo JA, Infante MR, Manresa A et al (2006) Chemoenzymatic synthesis and antimicrobial and haemolytic activities of amphiphilic bis (phenylacetylarginine) derivatives. ChemMedChem 1:1091–1098. doi: 10.1002/cmdc.200600148 PubMedCrossRefGoogle Scholar
  8. Castillo-Expósito JA (2006) Studies on antimicrobial activity of arginine-based surfactants and chemo- enzymatic synthesis of novel amphiphiles based on l-arginine and d-fagomine. Ph D Thesis, Universidad Autónoma de BarcelonaGoogle Scholar
  9. Castro M, Griffiths D, Patel A et al (2004) Effect of chain length on transfection properties of spermine-based gemini surfactants. Org Biomol Chem 2:2814–2820. doi: 10.1039/B410240A PubMedCrossRefGoogle Scholar
  10. Chamani J, Heshmati M (2008) Mechanism for stabilization of the molten globule state of papain by sodium n-alkyl sulfates: spectroscopic and calorimetric approaches. J Colloid Interface Sci 322:119–127. doi: 10.1016/j.jcis.2008.03.001 PubMedCrossRefGoogle Scholar
  11. Chimutengwende-Gordon M, Pendegrass C, Bayston R, Blunn G (2014) Preventing infection of osseointegrated transcutaneous implants: incorporation of silver into preconditioned fibronectin-functionalized hydroxyapatite coatings suppresses Staphylococcus aureus colonization while promoting viable fibroblast growth in vitro. Biointerphases 9:031010. doi: 10.1116/1.4889977 PubMedCrossRefGoogle Scholar
  12. Clapés P, Morán C, Infante MR (1999) Enzymatic synthesis of arginine-based cationic surfactants. Biotechnol Bioeng 63:333–343. doi: 10.1002/(SICI)1097-0290(19990505)63:3<333:AID-BIT10>3.0.CO;2-G PubMedCrossRefGoogle Scholar
  13. Cornellas A, Perez L, Comelles F et al (2011) Self-aggregation and antimicrobial activity of imidazolium and pyridinium based ionic liquids in aqueous solution. J Colloid Interface Sci 355:164–171. doi: 10.1016/j.jcis.2010.11.063 PubMedCrossRefGoogle Scholar
  14. Fité M, Clapés P, López-Santín J et al (2002) Integrated process for the enzymatic synthesis of the octapeptide PhAcCCK-8. Biotechnol Prog 18:1214–1220. doi: 10.1021/bp0256465 PubMedCrossRefGoogle Scholar
  15. Florence AT, Attwood D (2006) Physicochemical principles of pharmacy, 4th edn. Pharmaceutical Press, LondonGoogle Scholar
  16. Hanefeld U, Gardossi L, Magner E (2009) Understanding enzyme immobilisation. Chem Soc Rev 38:453–468. doi: 10.1039/b711564b PubMedCrossRefGoogle Scholar
  17. Kelly JH, Darlington GJ (1989) Modulation of the liver specific phenotype in the human hepatoblastoma line Hep G2. Vitr Cell Dev Biol 25:217–222. doi: 10.1007/BF02626182 CrossRefGoogle Scholar
  18. Klein JU, Cerovský V (1996) Protease-catalyzed synthesis of Leu-enkephalin in a solvent-free system. Int J Pept Protein Res 47:348–352PubMedCrossRefGoogle Scholar
  19. Lang A, Hatscher C, Kuhl P (2007) Papain-catalysed synthesis of Z-l-aminoacyl-antipyrine amides from Z-protected amino acid esters and 4-aminoantipyrine. Tetrahedron Lett 48:3371–3374. doi: 10.1016/j.tetlet.2007.03.067 CrossRefGoogle Scholar
  20. Lartigue DJ (1975) Characteristics of free vs. immobilized enzymes. In: Messing RA (ed) Immobilized enzymes for industrial reactors. Academic Press, New York, pp 125–127CrossRefGoogle Scholar
  21. Martinez V, Corsini E, Mitjans M et al (2006) Evaluation of eye and skin irritation of arginine-derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays. Toxicol Lett 164:259–267. doi: 10.1016/j.toxlet.2006.01.005 PubMedCrossRefGoogle Scholar
  22. Messing RA (1975) Introduction and general history of immobilized enzymes. In: Messing RA (ed) Immobilized enzymes for industrial reactors. Academic Press, New York, pp 2–3Google Scholar
  23. Miletić N, Nastasović A, Loos K (2012) Immobilization of biocatalysts for enzymatic polymerizations: possibilities, advantages, applications. Bioresour Technol 115:126–135. doi: 10.1016/j.biortech.2011.11.054 PubMedCrossRefGoogle Scholar
  24. Mitin Y, Braun K, Kuhl P (1997) Papain catalyzed synthesis of glyceryl esters of N-protected amino acids and peptides for the use in trypsin catalyzed peptide synthesis. Biotechnol Bioeng 54:287–290. doi: 10.1002/(SICI)1097-0290(19970505)54:3<287:AID-BIT9>3.0.CO;2-B PubMedCrossRefGoogle Scholar
  25. Morán C, Infante MR, Clapés P (2001a) Synthesis of glycero amino acid-based surfactants. Part 1. Enzymatic preparation of rac-1-O-(Nα-acetyl-l-aminoacyl) glycerol derivatives. J Chem Soc Perkin Trans l 1:2063–2070. doi: 10.1039/b103132p CrossRefGoogle Scholar
  26. Morán C, Clapés P, Comelles F et al (2001b) Chemical structure/property relationship in single-chain arginine surfactants. Langmuir 17:5071–5075. doi: 10.1021/la010375d CrossRefGoogle Scholar
  27. Morcelle SR, Liggieri CS, Bruno MA et al (2009) Screening of plant peptidases for the synthesis of arginine-based surfactants. J Mol Catal B Enzym 57:177–182. doi: 10.1016/j.molcatb.2008.08.013 CrossRefGoogle Scholar
  28. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. doi: 10.1016/0022-1759(83)90303-4 PubMedCrossRefGoogle Scholar
  29. Nakaoka H, Miyajima Y, Morihara K (1998) papain-catalyzed synthesis of aspartame precursor: a comparison with thermolysin. J Ferment Bioeng 85:43–47. doi: 10.1016/S0922-338X(97)80351-0 CrossRefGoogle Scholar
  30. Narai-Kanayama A, Koshino H, Aso K (2008) Mass spectrometric and kinetic studies on slow progression of papain-catalyzed polymerization of l-glutamic acid diethyl ester. Biochim Biophys Acta 1780:881–891. doi: 10.1016/j.bbagen.2008.03.009 PubMedCrossRefGoogle Scholar
  31. Nogueira DL, Mitjans M, Infante MR, Vinardell MP (2011) Comparative sensitivity of tumor and non-tumor cell lines as a reliable approach for in vitro citotoxicity screening of lysine-based surfactants with potential pharmaceutical applications. Int J Pharm 420:51–58. doi: 10.1016/j.ijpharm.2011.08.020 PubMedCrossRefGoogle Scholar
  32. Ota S, Moore S, Stein WH (1964) Preparation and Chemical Properties of Purified Stem and Fruit Bromelains. Biochemistry 3:180–185. doi: 10.1021/bi00890a007 PubMedCrossRefGoogle Scholar
  33. Pape WJW, Pfannenbecker U, Hoppe U (1987) Validation of the red blood cell test system as in vitro assay for the rapid screening of irritation potential of surfactants. Mol Toxicol 1:525–536PubMedGoogle Scholar
  34. Patiny L, Borel A (2013) ChemCalc: a building block for tomorrow’s chemical infrastructure. J Chem Inf Model 53:1223–1228. doi: 10.1021/ci300563h PubMedCrossRefGoogle Scholar
  35. Pérez L, Torres JL, Manresa A et al (1996) Synthesis, aggregation, and biological properties of a new class of gemini cationic amphiphilic compounds from arginine, bis (Args). Langmuir 12:5296–5301. doi: 10.1021/la960301f CrossRefGoogle Scholar
  36. Pérez L, García MT, Ribosa I et al (2002) Biological properties of arginine-based gemini cationic surfactants. Environ Toxicol Chem 21:1279–1285PubMedCrossRefGoogle Scholar
  37. Pérez L, Pinazo A, Pons R, Infante M (2014) Gemini surfactants from natural amino acids. Adv Colloid Interface Sci 205:134–155. doi: 10.1016/j.cis.2013.10.020 PubMedCrossRefGoogle Scholar
  38. Piera E, Comelles F, Erra P, Infante MR (1998) New alquil amide type cationic surfactants from arginine. J Chem Soc Perkin Trans 2:335–342. doi: 10.1039/a705565j CrossRefGoogle Scholar
  39. Piera E, Infante MR, Clapés P (2000) Chemo-enzymatic synthesis of arginine-based gemini surfactants. Biotechnol Bioeng 70:323–331. doi: 10.1002/1097-0290(20001105)70:3<323:AID-BIT9>3.0.CO;2-N PubMedCrossRefGoogle Scholar
  40. Sanchez L, Mitjans M, Infante MR, Vinardell MP (2004) Assessment of the potential skin irritation of lysine-derivative anionic surfactants using mouse fibroblasts and human keratinocytes as an alternative to animal testing. Pharm Res 21:1637–1641PubMedCrossRefGoogle Scholar
  41. Sanchez L, Mitjans M, Infante MR, Vinardell MP (2006) Potential irritation of lysine derivative surfactants by hemolysis and HaCaT cell viability. Toxicol Lett 161:53–60. doi: 10.1016/j.toxlet.2005.07.015 PubMedCrossRefGoogle Scholar
  42. Schechter I, Berger A (1967) On the size of the active site in proteases. I Papain Biochem Biophys Res Commun 27:157–162. doi: 10.1016/S0006-291X(67)80055-X CrossRefGoogle Scholar
  43. Scudiero DA, Shoemaker RH, Paul KD et al (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48:4827–4833PubMedGoogle Scholar
  44. Singh A, Van Hamme JD, Ward OP (2007) Surfactants in microbiology and biotechnology: part 2. Application aspects. Biotechnol Adv 25:99–121. doi: 10.1016/j.biotechadv.2006.10.004 PubMedCrossRefGoogle Scholar
  45. Tischer W, Wedekind F (1999) Biocatalysis—from discovery to application 200:95–126. doi: 10.1007/3-540-68116-7 CrossRefGoogle Scholar
  46. Torres JL, Piera E, Infante MR, Clapés P (2001) Purification of non-toxic, biodegradable arginine-based gemini surfactants, bis (Args), by ion exchange chromatography. Prep Biochem Biotechnol 31:259–274. doi: 10.1081/PB-100104908 CrossRefGoogle Scholar
  47. Valivety R, Jauregi P, Gill I, Vulfson E (1997) Chemo-enzymatic synthesis of amino acid-based surfactants. J Am Oil Chem Soc 74:879–886. doi: 10.1007/s11746-997-0232-8 CrossRefGoogle Scholar
  48. Vieira OV, Hartmann DO, Cardoso CMP et al (2008) Surfactants as microbicides and contraceptive agents: a systematic in vitro study. PLoS One 3:e2913. doi: 10.1371/journal.pone.0002913 PubMedCentralPubMedCrossRefGoogle Scholar
  49. Walsh KA, Wilcox PE (1970) Proteolytic enzymes. Methods Enzymol 19:31–41. doi: 10.1016/0076-6879(70)19005-7 Google Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • M. Elisa Fait
    • 1
  • Graciela L. Garrote
    • 2
  • Pere Clapés
    • 3
  • Sebastian Tanco
    • 4
    • 5
    • 6
  • Julia Lorenzo
    • 4
  • Susana R. Morcelle
    • 1
    Email author
  1. 1.Centro de Investigación de Proteínas Vegetales (CIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias ExactasUniversidad Nacional de La Plata (UNLP)La PlataArgentina
  2. 2.Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA-CONICET-UNLP)La PlataArgentina
  3. 3.Biotransformation and Biomolecules GroupCatalonia Institute of Advanced Chemistry (IQAC-CSIC)BarcelonaSpain
  4. 4.Institut de Biotecnologia i Biomedicina, Departament de Bioquímica i de Biologia MolecularUniversitat Autònoma de BarcelonaBarcelonaSpain
  5. 5.Department of Medical Protein ResearchVIBGhentBelgium
  6. 6.Department of BiochemistryGhent UniversityGhentBelgium

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