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ANCUT2, a Thermo-alkaline Cutinase from Aspergillus nidulans and Its Potential Applications

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Abstract

Biochemical characterization of purified ANCUT2 cutinase from Aspergillus nidulans is described. The identified amino acid sequence differs from that predicted in Aspergillus genomic databases in amino acids not relevant for catalysis. The enzyme is thermo-alkaline, showing its maximum activity at pH 9 and 60 °C, and it retains more than 60% of its initial activity after incubation for 1 h at 60 °C for pH values between 6 and 10. ANCUT2 is more active towards long-chain esters and it hydrolyzes cutin; however, it also hydrolyzes short-chain esters. Cutinase is inhibited by metal ions, PMSF, SDS, and EDTA (10 mM). It retains 50% of its activity in most of the solvents tested, although it is more stable in hydrophobic solvents. According to its found biochemical properties, preliminary assays demonstrate its ability to synthesize methyl esters from sesame oil and the most likely application of this enzyme remains in detergent formulations.

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Abbreviations

pI:

Isoelectric point

PMSF:

Phenylmethylsulfonyl fluoride

EDTA:

Ethylenediamine tetraacetic acid

SDS:

Sodium dodecyl sulfate

p-NPA:

p-Nitrophenyl acetate

min:

Minutes

h:

Hour

RT:

Room temperature

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

CEDE:

Continuous elution denaturing electrophoresis

CEH:

Carboxyl ester hydrolase

LC–MS/MS:

Liquid chromatography–mass spectrometry

NMWCO:

Nominal molecular weight cutoff

FTIR:

Fourier Transform Infrared Spectroscopy

TLC:

Thin layer chromatography

GC:

Gas chromatography

Ea:

Activation energy

p-NPE:

p-Nitrophenyl esters

MS:

Mass spectrometry

CAPS:

N-cyclohexyl-3-aminopropanesulfonic acid

References

  1. Acharya, P., Rajakumara, E., Sankaranarayanan, R., & Rao, N. M. (2004). Structural basis of selection and thermostability of laboratory-evolved Bacillus subtilis lipase. Journal of Molecular Biology, 341(5), 1271–1281.

    Article  CAS  Google Scholar 

  2. Adrio, J. L., & Demain, A. L. (2014). Microbial enzymes: tools for biotechnological processes. Biomolecules, 4(1), 117–139.

    Article  Google Scholar 

  3. Badenes, S. M., Lemos, F., & Cabral, J. M. (2010). Transesterification of oil mixtures catalyzed by microencapsulated cutinase in reversed micelles. Biotechnology Letters, 32(3), 399–403.

    Article  CAS  Google Scholar 

  4. Baptista, R. P., Cabral, J. M. S., & Melo, E. P. (2000). Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase. Biotechnology and Bioengineering, 70, 699–703.

    Article  CAS  Google Scholar 

  5. Bauer, S., Vasu, P., Person, S., Mort, A. J., & Somerville, C. R. (2006). Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. PNAS, 103(30), 11417–11422.

    Article  CAS  Google Scholar 

  6. BCC Research Report BIO030H (2014). Global markets for enzymes in industrial applications. Wellesley, MA: BCC Research.

    Google Scholar 

  7. Bornscheuer, U. T. (2002). Microbial carboxylesterases: classification, properties, and applications in biocatalysis. FEMS Microbiology Reviews, 26(1), 73–81.

    Article  CAS  Google Scholar 

  8. Bouzas, M. T., Barros-Velázquez, J., & González-Villa, T. (2006). Industrial applications of hyperthermophilic enzymes: a review. Protein Peptide Lett., 13, 645–651.

    Article  Google Scholar 

  9. Bustos-Jaimes, I., Mora-Lugo, R., Calcagno, M. L., & Farrés, A. (2010). Kinetic studies of Gly28: Ser mutant form of Bacillus pumilus lipase: changes in kcat and thermal dependence. Biochimica et Biophysica Acta, 1804, 2222–2227.

    Article  CAS  Google Scholar 

  10. Castro-Ochoa, D., Peña-Montes, C., González, A., Alva, A., Esquivel, R., Navarro, A., & Farrés, A. (2012). ANCUT2, an extracellular cutinase from Aspergillus nidulans induced by olive oil. Applied Biochemistry and Biotechnology, 166, 1275–1290.

    Article  CAS  Google Scholar 

  11. Chen, Z., Franco, C. F., Baptista, R. P., Cabral, J. M. S., Coelho, A. V., Rodrigues Jr., C. J., & Melo, E. P. (2007). Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides. Applied Microbiology and Biotechnology, 73, 1306–1313.

    Article  CAS  Google Scholar 

  12. Chen, S., Su, L., Billig, S., Zimmermann, W., Chen, J., & Wu, J. (2010). Biochemical characterization of the cutinases from Thermobifida fusca. J Mol Catal B-Enzym., 63(1), 121–127.

    Article  CAS  Google Scholar 

  13. Chen, S., Su, L., Chen, J., & Wu, J. (2013). Cutinase: characteristics, preparation, and application. Biotechnology Advances, 31(8), 1754–1767.

    Article  CAS  Google Scholar 

  14. Cherif, S., Mnif, S., Hadrich, F., Abdelkafi, S., & Sayadi, S. (2011). A newly high alkaline lipase: an ideal choice for application in detergent formulations. Lipids in Health and Disease., 10(1), 1–8.

    Article  Google Scholar 

  15. Chevallet, M., Luche, S., & Rabilloud, T. (2006). Silver staining of proteins in polyacrylamide gels. Nature Protocols, 1(4), 1852–1858.

    Article  CAS  Google Scholar 

  16. Chin, J. T., Wheeler, S. L., & Klibanov, A. M. (1994). On protein solubility in organic solvent. Biotechnology and Bioengineering, 44(1), 140–145.

    Article  CAS  Google Scholar 

  17. Corpet, F. (1988). Multiple sequence alignment with hierarchical clustering. Nucl Acids Res., 16(22), 10881–10890.

    Article  CAS  Google Scholar 

  18. Creighton, T. E. (1997). Protein structure. A practical approach (2nd ed.pp. 349–364). Oxford: IRL Press.

    Google Scholar 

  19. Creveld, J. M., Meijberg, W., Berendsen, H. J. C., & Pepermans, H. A. M. (2001). DSC studies of Fusarium solani pisi cutinase: consequences for stability in the presence of surfactants. Biophysical Chemistry, 92(1), 65–75.

    Article  CAS  Google Scholar 

  20. Das, A. K., Bellizzi, J. J., Tandel, S., Biehl, E., Clardy, J., & Hofmann, S. L. (2000). Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride. The Journal of Biological Chemistry, 275(31), 23847–23851.

    Article  CAS  Google Scholar 

  21. Dutta, K., Sen, S., & Veeranki, V. D. (2009). Production, characterization, and applications of microbial cutinases. Proc Biochem., 44, 127–134.

    Article  CAS  Google Scholar 

  22. Dutta, K., Hegde, K., & Veeranki, V. D. (2014). Synthesis of methyl esters by transesterification catalyzed by cutinase from Pseudomonas cepacia NRRL B 2320 and kinetic analysis. Curr Trends Biotechnol Pharm., 8(1), 1–10.

    Google Scholar 

  23. Elleuche, S., Schröder, C., & Antranikian, G. (2016). Lipolytic extremozymes from psychro-and (hyper-)thermophilic prokaryotes and their potential for industrial applications. In P. H. Rampelotto (Ed.), Biotechnology of extremophiles (pp. 351–374). Switzerland: Springer.

    Chapter  Google Scholar 

  24. Fett, W., Wijey, C., Moreau, R., & Osman, S. (1999). Production of cutinolytic esterase by filamentous bacteria. Letters in Applied Microbiology, 31(1), 25–29.

    Article  Google Scholar 

  25. Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L. J., Wortman, J. R., et al. (2005). Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature, 438, 1105–1115.

    Article  CAS  Google Scholar 

  26. García-Lepe, R., Nuero, O., & Reyes, F. (1997). Esterases in autolysed cultures of filamentous fungi. Letters in Applied Microbiology, 25, 127–130.

    Article  Google Scholar 

  27. Gupta R, Jung E, Brunak S (2004) Prediction of N-glycosylation sites in human proteins. In preparation. Available: http://www.cbs.dtu.dk/services/NetNGlyc/

  28. Hama, S., & Kondo, A. (2013). Enzymatic biodiesel production: an overview of potential feedstocks and process development. Bioresource Technology, 135, 386–395.

    Article  CAS  Google Scholar 

  29. Hiol, A., Jonzo, M., & Rugani, N. (2000). Purification and characterization of an extracellular esterase from a thermophilic Rhizopus oryzae strain isolated from a palm fruit. Enzyme and Microbial Technology, 26, 421–430.

    Article  CAS  Google Scholar 

  30. Hynes, M. J., Murray, S. L., Duncan, A., Khew, G. S., & Davis, M. A. (2006). Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans. Eukaryotic Cell, 5(5), 794–805.

    Article  CAS  Google Scholar 

  31. Jeong, G. T., & Park, D. T. (2008). Lipase-catalyzed transesterification of rapeseed oil for biodiesel production with tert-butanol. Applied Biochemistry and Biotechnology, 148, 131–139.

    Article  CAS  Google Scholar 

  32. Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., & Baratti, J. (2000). Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans. Applied Microbiology and Biotechnology, 54(2), 173–179.

    Article  CAS  Google Scholar 

  33. Karpushova, A., Brümmer, F., Barth, S., Lange, S., & Schmid, R. D. (2005). Cloning, recombinant expression and biochemical characterization of novel esterases from Bacillus sp. associated with the marine sponge Aplysina aerophoba. Applied Microbiology and Biotechnology, 67, 59–69.

    Article  CAS  Google Scholar 

  34. Kim, Y. H., Ahn, J. Y., Moon, S. H., & Lee, J. (2005). Biodegradation and detoxification of organophosphate insecticide, malathion by Fusarium oxysporum f. sp. pisi cutinase. Chemosphere, 60(10), 1349–1355.

    Article  CAS  Google Scholar 

  35. Käfer, E. (1977). Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Advances in Genetics, 19, 33–131.

    Google Scholar 

  36. Kolattukudy, P. E. (1970). Biosynthesis of a lipid-polymer, cutin: the structural component of plant cuticle. Biochem Biophys Res., 41, 299–305.

    Article  CAS  Google Scholar 

  37. Kolattukudy, P. E. (1985). Enzymatic penetration of the plant cuticle by fungal pathogens. Ann Rev Phytopathol., 23(1), 223–250.

    Article  CAS  Google Scholar 

  38. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  39. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., & McGettigan, P. A. (2007). ClustalW and ClustalX version 2. Bioinformatics, 23(21), 2947–2948.

    Article  CAS  Google Scholar 

  40. Leach, M. D., Tyc, K. M., Brown, J., & Klipp, E. (2012). Modelling the regulation of thermal adaptation in Candida albicans, a major fungal pathogen of humans. PloS One, 7(3), e32467.

    Article  CAS  Google Scholar 

  41. Liu, L., Gosser, Y., Baker, P. J., Ravee, Y., Lu, Z., Alemu, G., Li, H., Butterfoss, G. L., Kong, X., Gross, R., & Montclare, J. K. (2009). Structural and functional studies of Aspergillus oryzae cutinase: enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation. Journal of the American Chemical Society, 131(1), 15711–15716.

    Article  CAS  Google Scholar 

  42. Lyon, C. K. (1972). Sesame: current knowledge of composition and use. JAOCS., 49(4), 245–249.

    CAS  Google Scholar 

  43. Machado, M. F., & de Castro-Prado, M. A. (2001). Differential esterase expression in developmental mutants of Aspergillus nidulans. Biochemical Genetics, 39, 357–368.

    Article  CAS  Google Scholar 

  44. Maeda, H., Youhei, Y., Keietsu, A., Fumihiko, H., Masayuki, M., Ryoji, I., Katsuya, G., & Tasuku, N. (2002). Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae. Applied Microbiology and Biotechnology, 67, 778–788.

    Article  Google Scholar 

  45. Mayordomo, I., Randez-Gil, F., & Prieto, J. (2000). Isolation, purification, and characterization of a cold-active esterase from Aspergillus nidulans. Journal of Agricultural and Food Chemistry, 48, 105–109.

    Article  CAS  Google Scholar 

  46. Melo, E. P., Baptista, R. P., & Cabral, J. M. S. (2003). Improving cutinase stability in aqueous solution and reverse micelles by media engineering. J Mol Catal B-Enzym., 22(3), 299–306.

    Article  CAS  Google Scholar 

  47. Niehaus, F., Bertoldo, C., Kähler, M., & Antranikian, G. (1999). Extremophiles as a source of novel enzymes for industrial application. Applied Microbiology and Biotechnology, 51(6), 711–729.

    Article  CAS  Google Scholar 

  48. Nyyssölä, A. (2015). Which properties of cutinases are important for applications? Applied Microbiology and Biotechnology, 99(12), 4931–4942.

    Article  Google Scholar 

  49. Peña-Montes, C., González, A., Castro-Ochoa, D., & Farrés, A. (2008). Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans. Applied Microbiology and Biotechnology, 78, 603–612.

    Article  Google Scholar 

  50. Petersen, S. B., Fojan, P., Petersen, E. I., & Neves-Petersen, M. T. (2001). The thermal stability of the Fusarium solani pisi cutinase as a function of pH. Journal of Biomedicine & Biotechnology, 1(2), 62–69.

    Article  CAS  Google Scholar 

  51. Petersen, T. N., Brunak, S., von Heijne, G., & Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods., 8, 785–786.

    Article  CAS  Google Scholar 

  52. Plou, F. J., Ferrer, M., Nuero, O. N., Calvo, M. V., Alcalde, M., Reyes, F., & Ballesteros, A. (1998). Analysis of Tween 80 as an esterase/lipase substrate for lipolytic activity assay. Biotechnology Techniques, 12(3), 183–186.

    Article  CAS  Google Scholar 

  53. Pocalyko, D. J., & Tallman, M. (1998). Effects of amphipaths on the activity and stability of Fusarium solani pisi cutinase. Enz Microb Technol., 22(7), 647–651.

    Article  CAS  Google Scholar 

  54. Prim, N., Sánchez, M., Ruiz, C., Pastor, F. J., & Diaz, P. (2003). Use of methylumbelliferyl-derivative substrates for lipase activity characterization. J Mol Catal B-Enzym., 22(5–6), 339–346.

    Article  CAS  Google Scholar 

  55. Purdy, R. E., & Kolattukudy, P. E. (1973). Depolymerization of a hydroxy fatty acid biopolymer, cutin, by an extracellular enzyme from Fusarium solani f. pisi isolation and some properties of the enzyme. Archives of Biochemistry and Biophysics, 159, 61–69.

    Article  CAS  Google Scholar 

  56. Ranganathan, S. V., Narasimhan, S. L., & Muthukumar, K. (2008). An overview of enzymatic production of biodiesel. Bioreosur Technol., 99(10), 3975–3981.

    Article  CAS  Google Scholar 

  57. Schmidt-Dannert, C., Rua, M. L., Wahl, S., & Schmid, R. D. (1997). Bacillus thermocatenulatus esterase: a thermo alkalophilic esterase with interesting properties. Biochemical Society Transactions, 25, 178–182.

    Article  CAS  Google Scholar 

  58. Sinchaikul, S., Sookkheo, B., Phutrakul, S., Pan, F., & Chen, S. (2001). Optimization of a thermostable esterase from Bacillus stearothermophilus P1: overexpression, purification, and characterization. Protein Expression and Purification, 22, 388–398.

    Article  CAS  Google Scholar 

  59. Speranza, P., Carvalho, P. D. O., & MacEdo, G. A. (2011). Effects of different solid state fermentation substrate on biochemical properties of cutinase from Fusarium sp. J Mol Catal B-Enzym., 72(3–4), 181–186.

    Article  CAS  Google Scholar 

  60. Stöcklein, W., Sztajer, H., Menge, U., & Schmid, R. D. (1993). Purification and properties of a lipase from Penicillium expansum. Biochem Biophys Acta., 1168, 181–189.

    Article  Google Scholar 

  61. Sugihara, A., Ueshima, M., Shimada, Y., Tsunasawa, S., & Tominaga, Y. (1992). Purification and characterization of a novel thermostable esterase from Pseudomonas cepacia. Journal of Biochemistry, 112, 598–603.

    Article  CAS  Google Scholar 

  62. Sundaram, P. V., & Srimathi, S. (2004). Analysis of catalytic and structural stability of native and covalently modified enzymes. In A. Svendsen (Ed.), Enzyme functionality: design, engineering, and screening (pp. 632–661). New York: Marcel Dekker.

    Google Scholar 

  63. Taiz, L., & Zeiger, E. (2002). Plant physiology (3a ed.pp. 320–332). USA: Freeman.

    Google Scholar 

  64. Takao, M., Nakaniwa, T., Yoshikawa, K., Terashita, T., & Sakai, T. (2000). Molecular cloning, DNA sequence, and expression of the gene encoding for thermostable pectate lyase from thermophilic Bacillus sp. TS 47. Bioscience, Biotechnology, and Biochemistry, 65(2), 322–329.

    Article  Google Scholar 

  65. Thomsen, M. S., & Nidetzky, B. (2008). Microfluidic reactor for continuous flow biotransformations with immobilized enzymes: the example of lactose hydrolysis by a hyperthermophilic β-glycoside hydrolase. Engineering in Life Sciences, 8(1), 40–48.

    Article  CAS  Google Scholar 

  66. Turner, P., Mamo, G., & Nordberg-Karlsson, E. (2007). Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microbial Cell Factories, 6(1), 9.

    Article  Google Scholar 

  67. Vertommen, M. A., Nierstrasz, V. A., Veer, M., & Warmoeskerken, M. M. (2005). Enzymatic surface modification of poly(ethylene terephthalate). Journal of Biotechnology, 120, 376–386.

    Article  CAS  Google Scholar 

  68. Walton, T. J., & Kolattukudy, P. E. (1972). Determination of the structure of cutin monomers by a novel depolymerization procedure and combined gas chromatography and mass spectrometry. The Biochemist, 11(10), 1885–1897.

    Article  CAS  Google Scholar 

  69. Wang, P., Wang, Q., Fan, X. R., Cui, L., Yuan, J. G., Chen, S., & Wu, J. (2009). Effects of cutinase on the enzymatic shrink-resist finishing of wool fabrics. Enzyme and Microbial Technology, 44(5), 302–310.

    Article  Google Scholar 

  70. Yu, J., Bhatnagar, D., & Cleveland, T. E. (2004). Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus. FEBS letters., 564(1–2), 126–130.

    Article  CAS  Google Scholar 

  71. Zhang, S. B., Pei, X. Q., & Wu, Z. L. (2012). Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger. Bioresource Technology, 117, 140–147.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was partially supported by CONACyT project 153500 and DGAPA IN231311. Eva Bermudez received a scholarship from CONACyT, Mexico. We thank Dr. César Batista, from the Proteomics Unit, Biotechnology Institute, UNAM, for LC–MS/MS Analysis and American Journal Experts for English revisions; Dra. Hilda E. Calderón Villagómez for the technical assistance in the use of GC equipment; and Sacnité González, who evaluated enzyme stability in commercial detergents.

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  1. Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Avenida Universidad, 3000 Ciudad Universitaria, 04510, Mexico City, Mexico

    Eva Bermúdez-García, Carolina Peña-Montes, José Augusto Castro-Rodríguez, Arturo Navarro-Ocaña & Amelia Farrés

  2. Departamento de Medicina Experimental, Facultad de Medicina, Hospital General de México, Universidad Nacional Autónoma de México (UNAM), Dr. Balmis, 148, 06726, Mexico City, D.F, Mexico

    Augusto González-Canto

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  1. Eva Bermúdez-García
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  2. Carolina Peña-Montes
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Correspondence to Amelia Farrés.

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Bermúdez-García, E., Peña-Montes, C., Castro-Rodríguez, J.A. et al. ANCUT2, a Thermo-alkaline Cutinase from Aspergillus nidulans and Its Potential Applications. Appl Biochem Biotechnol 182, 1014–1036 (2017). https://doi.org/10.1007/s12010-016-2378-z

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