Skip to main content
SpringerLink
Log in

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

  • Experimental Articles
  • Published:
Microbiology Aims and scope Submit manuscript

Abstract

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ∼55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T 1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K m 3.3 mM, K cat 0.04 s−1) followed by aromatic amino acids L-tyrosine (K m 5.3 mM, K cat 0.036 s−1) and L-phenylalanine (K m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC50 832.2 μg/mL) and MCF-7 (IC50, 370.6 μg/mL) tumor cells in vitro.

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

Similar content being viewed by others

References

  1. Ande, S.R., Fussi, H., Knauer, H., Murkovic, M., Ghisla, S., Frohlich, K.U., and Macheroux, P., Induction of apoptosis in yeast by L-amino 670 acid oxidase from the Malayan pit viper Calloselasma rhodostoma, Yeast, 2008, vol. 25, pp. 349–357.

    Article  CAS  PubMed  Google Scholar 

  2. Samel, M., Tonismagi, K., Ronnholm, G., Vija, H., Siigur, J., Kalkkinen, N., and Siigur, E., L-Amino acid oxidase from Naja oxiana venom, Comp. Biochem. Physiol., 2008, vol. 149, pp. 572–580.

    Article  Google Scholar 

  3. Ciscotto, P., Machado, R.A., de Avila, E.A., Coelho, J., Oliveira, C.G., Diniz-Farais, L.M., de Carvalho, M.A., Maria, W.S., Sanchez, E.F., Borges, A., and Chavez-Olortegui, C., Antigenic, microbial and parasitic properties of an L-amino acid oxidase isolated from Bothrops jararaca snake venom, Toxicon, 2009, vol. 53, pp. 330–341.

    Article  CAS  PubMed  Google Scholar 

  4. Kusakabe, H., Kodama, K., Kuninaka, A., Yoshino, H., Misono, H., and Soda, K., A new antitumor enzyme, L-lysine α-oxidase from Trichoderma viride: purification and enzymological properties, J. Biol. Chem., 1980, vol. 255, pp. 976–981.

    CAS  PubMed  Google Scholar 

  5. Treshalina, H.M., Lukasheva, E.V., Sedakova, L.A., Firsova, G.A., Guerassimova, G.K., Gogichaeva, N.V., and Berezov, T.T., Anticancer enzyme L-lysine α-oxidase. Properties and application prospectives, Appl. Biochem. Biotechnol., 2000, vol. 88, pp. 267–273.

    Article  CAS  Google Scholar 

  6. Toyama, M.H., Toyama, D.O., Passero, L.F.D., Laurenti, M.D., Corbett, C.E., Tomokane, T.Y., Fanseca, F.V., Antunes, M., Joazeiro, P.P., Beriam, L.O.S., Martins, M.A.C., Monteiro, H.S.A., and Fonteles, M.C., Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom, Toxicon, 2006, vol. 47, pp. 47–57.

    Article  CAS  PubMed  Google Scholar 

  7. Torri, S., Yamane, K., Mashima, T., Haga, N., Yamamoto, K., Fox, J.W., Naito, M., and Tsuruo, T., Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity, Biochemistry, 2000, vol. 39, pp. 3197–3205.

    Article  Google Scholar 

  8. Sakurai, Y., Shima, M., Matsumoto, T., Takatsuka, H., Nishiya, K., Kasuda, S., Fujimura, Y., and Yoshioka, A., Anticoagulant activity of M-LAO, L-amino acid oxidase purified from Agkistrodon halys blomhoffii, through detective inhibition of factor IX, Biochem. Biophys. Acta, 2003, vol. 1649, pp. 51–57.

    CAS  PubMed  Google Scholar 

  9. Jin, Y., Lee, W.-H., Zeng, L., and Zhang, Y., Molecular characterization of L-amino acid oxidase from king cobra venom, Toxicon, 2007, vol. 50, pp. 479–489.

    Article  CAS  PubMed  Google Scholar 

  10. Brearly, G.M., Price, C.P., Atkinson, T., and Hammond, P.M., Purification and partial characterisation of a broad-range L-amino acid oxidase from Bacillus carotarum 2Pfa isolated from soil, Appl. Microbiol. Biotechnol., 1994, vol. 41, pp. 670–676.

    Article  Google Scholar 

  11. Geueke, B. and Hummel, W., Heterologous expression of Rhodococcus opacus L-amino acid oxidase in Streptomyces lividans, Protein Expr. Purif., 2003, vol. 28, pp. 303–309.

    Article  CAS  PubMed  Google Scholar 

  12. Jin, Y., Lee, W.-H., Zeng, L., and Zhang, Y., Molecular characterization of L-amino acid oxidase from king cobra venom, Toxicon, 2007, vol. 50, pp. 479–489.

    Article  CAS  PubMed  Google Scholar 

  13. Gomez, D., Lucas-Elio, P., Sanchez-Amat, A., and Solano, F., A novel type of lysine oxidase: L-lysine-ɛ-oxidase, Biochem. Biophys. Acta, 2008, vol. 1764, pp. 15577–15585.

    Google Scholar 

  14. Samel, M., Tonismagi, K., Ronnholm, G., Vija, H., Siigur, J., Kalkkinen, N., and Siigur, E., L-Amino acid oxidase from Naja oxiana venom, Comp. Biochem. Physiol., 2008, vol. 149, pp. 572–580.

    Article  Google Scholar 

  15. Rodrigues, R.S., da Silva, J.F., Boldrini, F.J., Fonseca, F.P.P., Otaviano, A.R., Silva, F.H., Hamaguchi, A., Magro, A.J., Braz, A.S.K., dos Santos, J.I., Homsi-Brandeburgo, M.I., Fontes, M.R., Fuly, A.L., Soares, A.M., and Rodrigues, V.M., Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom, Biochemie, 2009, vol. 91, pp. 490–501.

    Article  CAS  Google Scholar 

  16. Geyer, A., Fitzpatrick T.B., Pawelek, P.D., Kitzing, K., Vrielink, A., Ghisla, S., and Macheroux, P., Structure and characterization of the glycan moiety of L-aminoacid oxidase from the Malayan pit viper Calloselasma rhodostoma, Eur. J. Biochem., 2001, vol. 268, pp. 4044–4053.

    Article  CAS  PubMed  Google Scholar 

  17. Yang, H., Ohnson, P.M., Ko, K.C., Kamio, M., German, M.W., Derby, C.D., and Tai, P.C., Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea have Aplysia californica, J. Exp. Biol., 2005, vol. 208, pp. 3609–3622.

    Article  CAS  PubMed  Google Scholar 

  18. El-Sayed, A.S.A., Microbial L-methioninase: production, molecular characterization and therapeutic applications. A review, Appl. Microbiol. Biotechnol., 2010, vol. 86, pp. 445–467.

    Article  CAS  PubMed  Google Scholar 

  19. Kusakabe, H., Kodama, K., Machida, H., Midorikawa, Y., Kuninaka, A., Misono, H., and Soda, K., Occurrence of a novel enzyme, L-lysine oxidase with antitumor activity in culture extract of Trichoderma viride, Agric. Biol. Chem., 1979, vol. 43, pp. 337–343.

    Article  CAS  Google Scholar 

  20. Davis, M.A., Askin, M.C., and Hynes, M.J., Amino acid catabolism by an areA-regulated gene encoding an L-amino acid oxidase with broad substrate specificity in Aspergillus nidulans, Appl. Environ. Microbiol., 2005, vol. 71, pp. 3551–3555.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. El-Sayed, A.S.A., Shindia, A.A., and Zaher, Y., L-Amino acid oxidase from filamentous fungi: screening and optimization, Ann. Microbiol., 2012, vol. 62, pp. 773–784.

    Article  CAS  Google Scholar 

  22. Kebeish, R.M. and El-Sayed, A.S.A., Morphological and molecular characterization of L-methioninase producing Aspergillus species, Afr. J. Biotechnol., 2012, vol. 11, pp. 15280–15290.

    CAS  Google Scholar 

  23. El-Sayed, A.S.A., Khalaf, S.A., and Ahmed, H.A., Characterization of homocysteine Γ-lyase from submerged and solid cultures of Aspergillus fumigatus ASH, J. Microbiol. Biotechnol., 2013, vol. 23, pp. 499–510.

    Article  CAS  PubMed  Google Scholar 

  24. El-Sayed, A.S.A., L-Methioninase production by Aspergillus flavipes under solid state fermentation, J. Basic. Microbiol., 2009, vol. 49, pp. 331–341.

    Article  CAS  PubMed  Google Scholar 

  25. El-Sayed, A.S.A., Purification and characterization of a new L-methioninase from the solid cultures of Aspergillus flavipes, J. Microbiol., 2011, vol. 49, pp. 130–140.

    Article  CAS  PubMed  Google Scholar 

  26. El-Sayed, A.S.A. and Shindia, A.A., Characterization and immobilization of purified Aspergillus flavipes L-methioninase: continuous production of methanethiol, J. Appl. Microbiol., 2011, vol. 111, pp. 54–69.

    Article  CAS  PubMed  Google Scholar 

  27. Bergmeyer, H.U., Gawehn, K., and Grassl, M., in Methods of Enzymatic Analysis, vol. 1, Bergmeyer, H.U., Ed., New York: Academic Press, 1974, pp. 495–496.

  28. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 1951, vol. 193, pp. 265–275.

    CAS  PubMed  Google Scholar 

  29. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacterio-phage T4, Nature, 1970, vol. 227, pp. 680–685.

    Article  CAS  PubMed  Google Scholar 

  30. Trinder, P., Determination of blood glucose using an oxidase-peroxidase system with non-carcinogenic chromogen, J. Clin. Pathol., 1969, vol. 22, pp. 158–161.

    Article  CAS  PubMed  Google Scholar 

  31. Bergmeyer, H.U., Horder, M., and Rej, R., Approved recommendation of IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase, Eur. J. Clin. Chem. Clin. Biochem., 1985, vol. 24, pp. 418–489.

    Google Scholar 

  32. Wei, X.-L., Wei, J.-F., Li, T., Qiao, L.-Y., Liu, Y.-L., Huang, T., and He, S.-H., Purification, characterization and potent lung lesion activity of an L-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom, Toxicon, 2007, vol. 50, pp. 1126–1139.

    Article  CAS  PubMed  Google Scholar 

  33. Tay, S.T., Devi, S., Puthcheary, S.D., and Kautner, I.M., Detection of haemolytic activity of Campylobacter by agarose haemolysis and microplate assay, J. Med. Microbiol., 1995, vol. 42, pp. 175–180.

    Article  CAS  PubMed  Google Scholar 

  34. Hansen, M.B., Nielsen, S.E., and Berg, K., Re-Examination and further development of a precise and rapid dye method for measuring cell growth/cell kill, J. Immunol. Methods, 1989, vol. 119, pp. 203–210.

    Article  CAS  PubMed  Google Scholar 

  35. Khaduev, S.K.H., Dzhukova, O.S., Dobrinin, J.V., Soda, K., and Berezov, T.T., Comparative study of the effect of L-lysine-L-oxidase from Trichoderma harzianum Rifai and Trichoderma viride on nucleic acid synthesis in human tumor cells in vitro, Bull Exper. Biol. Med., 1986, vol. 101, pp. 603–604.

    Article  CAS  Google Scholar 

  36. Koyama, H., Purification and characterization of a novel L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501, J. Biochem., 1982, vol. 92, pp. 1235–1240.

    CAS  PubMed  Google Scholar 

  37. Tan, N.H. and Swaminathan, S., Purification and properties of the L-amino acid oxidase from monocellate cobra (Naja naja kaouthia) venom, Int. J. Biochem., 1992, vol. 24, pp. 967–973.

    Article  CAS  PubMed  Google Scholar 

  38. Piedras, P., Pineda, M., Munoz, J., and Cardenas, J., Purification and characterization of an L-amino-acid oxidase from Chlamydomonas reinhardtii, Planta, 1992, vol. 188, pp. 13–18.

    Article  CAS  PubMed  Google Scholar 

  39. MacHeroux, P., Seth, O., Bollschweile, R.C., Schwarz, M., Kurfurst, M., Au, L.C., and Ghisla, S., L-Amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme, Eur. J. Biochem., 2001, vol. 268, pp. 1679–1686.

    Article  CAS  PubMed  Google Scholar 

  40. Wei, J.-F., Yang, H.-W., Wei, X.-L., Qiao, L.-Y., Wang, W.-Y., and He, S.-H., Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom, Toxicon, 2009, vol. 54, pp. 262–272.

    Article  CAS  PubMed  Google Scholar 

  41. Bohmer, A., Muller, A., Passarge, M., Liebs, P., Honeck, H., and Muller, H.G., A novel L-glutamate oxidase from Streptomyces endus: purification and properties, Eur. J. Biochem., 1989, vol. 182, pp. 327–332.

    Article  CAS  PubMed  Google Scholar 

  42. Braun, M., Kim, J.M., and Schmid, R.D., Purification and some properties of an extracellular L-amino acid oxidase from Cellulomonas cellulans AM8 isolated from soil, Appl. Microbiol. Biotechnol., 1992, vol. 37, pp. 594–598.

    Article  CAS  Google Scholar 

  43. Isobe, K., Sugawara, A., Domon, H., Fukuta, Y., and Asano, Y., Purification and characterization of an L-amino acid oxidase from Pseudomonas sp. AIU 813, J. Biosci. Bioeng., 2012, vol. 114, pp. 257–261.

    Article  CAS  PubMed  Google Scholar 

  44. Fujisawa, S., Hori, K., Miyazawa, K., and Ito, K., Occurence of L-amino acid oxidase in the marine red alga Gymnogongrus flabelliformis, Bull. Jap. Soc. Scient. Fish., 1982, vol. 48, pp. 97–103.

    Article  CAS  Google Scholar 

  45. Ito, K., Hori, K., and Miyazawa, K., Purification and some properties of L-amino acid oxidase from Amphiroa crassissima Yen-do, Hydrobiologica, 1987, vol. 151, pp. 563–569.

    Article  Google Scholar 

  46. Pistorius, E.K. and Voss, H., Some properties of basic L-amino acid oxidase from Anacystis nidulans, Biochem. Biophys. Acta, 1980, vol. 611, pp. 227–240.

    CAS  PubMed  Google Scholar 

  47. Pistorius, E.K. and Gau, A.E., Presence of a flavoprotein in O2-evolving photosystem II preparations from the cyanobacterium Anacystis nidulans, Biochem. Bophys. Acta, 1986, vol. 849, pp. 203–210.

    CAS  Google Scholar 

  48. Pratt, D.S. and Kaplan, M.M., Evaluation of abnormal liver enzyme results in asymptomatic patients, N. Engl. J. Med., 2000, vol. 342, pp. 1266–1271.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Microbiology and Botany Department, Faculty of Science, Zagazig University, Zagazig, Egypt

    Ashraf S. El-Sayed, Ahmed A. Shindia & Yomna A. Zaher

Authors
  1. Ashraf S. El-Sayed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ahmed A. Shindia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yomna A. Zaher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ashraf S. El-Sayed.

Additional information

The article is published in the original.

Rights and permissions

Reprints and permissions

About this article

Cite this article

El-Sayed, A.S., Shindia, A.A. & Zaher, Y.A. Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH. Microbiology 82, 762–771 (2013). https://doi.org/10.1134/S0026261713060143

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0026261713060143

Keywords

Search

Navigation