Skip to main content
SpringerLink
Log in

Extracellular alkaline phosphatase from the filamentous fungusAspergillus caespitosus: Purification and biochemical characterization

  • Published:
Folia Microbiologica Aims and scope Submit manuscript

Abstract

Among 30 species of filamentous fungi isolated from Brazilian soil,Aspergillus caespitosus produced and secreted the highest levels of alkaline phosphatase in culture medium supplemented with xylan. The extracellular alkaline phosphatase was purified by DEAE-cellulose and concanavalin A-sepharose chromatography. The enzyme was a glycoprotein containing up to 56 % sugar with molar mass of 134.8 kDa, according to gel filtration in Sepharose CL-6B, and 57 kDa according to SDS-PAGE. Non-denaturing electrophoresis (6 % PAGE) of the purified enzyme produced a single band, suggesting that the native enzyme was a homodimer. Optima of temperature and pH were 75 °C and 8.5, respectively. The enzyme was stable at 50 °C and its activity was enhanced by 95 % in the presence of Mg2+ (1 mmol/L). 4-Nitrophenyl phosphate was the preferentially hydrolyzed substrate withK m andv lim values of 74 µmol/L and 285 µmol/s, in the absence, and 90 µmol/L and 418 µmol/s, in the presence of Mg2+, respectively. The enzyme also hydrolyzed other phosphorylated amino acids (O-phosphothreonine,O-phosphotyrosine,O-phosphoserine).

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

  • Aquino A.C.M.M., Jorge J.A., Terenzi H.F., Polizeli M.L.T.M.: Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungusScytalidium thermophilum.Folia Microbiol. 46, 11–16 (2001).

    Article  CAS  Google Scholar 

  • Blum H., Beier H., Gross H.J.: Improved silver staining of plant protein, RNA and DNA in polyacrylamide gels.Electrophoresis 81, 93–99 (1987).

    Article  Google Scholar 

  • Buainain L.B., Kadowaki M.K., Polizeli M.L.T.M., Terenzi H.F., Jorge J.A.: Characterization of a conidial alkaline phosphatase from the thermophilic fungusHumicola grisea var.thermoidea.J.Basic Microbiol. 38, 85–94 (1998).

    Article  Google Scholar 

  • Davis B.J.: Disc electrophoresis: II. Method and application to human serum proteins.Ann.N.Y.Acad.Sci. 121, 407–427 (1964).

    Article  Google Scholar 

  • Denison S.H.: pH regulation of gene expression in fungi.Fungal Gen.Biol. 29, 61–71 (2000).

    Article  CAS  Google Scholar 

  • Dickman M.B., Yarden O.: Serine/threonine protein kinases and phosphatases in filamentous fungi.Fungal Gen.Biol. 26, 99–117 (1999).

    Article  CAS  Google Scholar 

  • Dong G., Zeikus G.: Purification and characterization of alkaline phosphatase fromThermotoga neapolitana.Enzyme.Microbiol.Technol. 21, 335–340 (1997).

    Article  CAS  Google Scholar 

  • Dubois M., Gilles K.A., Hamilton J.K., Rebers P.A., Smith F.: Colorimetric method for determination of sugars and related substances.Analyt.Chem. 28, 350–356 (1956).

    Article  CAS  Google Scholar 

  • Elzainy T.A., Ali T.H.: Participation of a proton-translocating plasma membrane ATPase degradation byAspergillus niger extracts.Biochim.Biophys.Acta 1239, 91–97 (1995).

    Article  PubMed  Google Scholar 

  • Guimarães L.H.S., Jorge J.A., Terenzi H.F., Polizeli M.L.T.M.: Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungusScytalidium thermophilum.J.Industr.Microbiol.Biotechnol. 27, 265–270 (2001).

    Article  Google Scholar 

  • Guimarães L.H.S., Jorge J.A., Terenzi H.F., Jamur M.C., Oliver C., Polizeli M.L.T.M.: Effect of carbon source on phosphatase production inAspergillus caespitosus.J.Basic Microbiol. 43, 200–207 (2003).

    Article  Google Scholar 

  • Heinonen J.K., Lahti R.J.: A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase.Analyt.Biochem. 113, 313–317 (1981).

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Leone F.A., Degreve L., Baranauskas J.A.: SIGRAF: a versatile computer program for fitting assay kinetics data.Biochem.Educ. 20, 94–96 (1992).

    Article  CAS  Google Scholar 

  • Leone F.A., Baranauskas J.A., Ciancaglini P.: Enzyplot: a microcomputer-assisted program for teaching enzyme kinetics.Biochem.Educ. 23, 35–37 (1995).

    Article  CAS  Google Scholar 

  • MacKintosh C., MacKintosh R.W.: Inhibitors of protein kinases and phosphatases.Trends Biochem.Sci. 19, 444–448 (1994).

    Article  PubMed  CAS  Google Scholar 

  • McComb R.B., Bowers G.N. Jr.,Posen S.:Alkaline Phosphatase, pp. 229–287. Plenum Press, New York 1979.

    Google Scholar 

  • Morales A.C., Nozawa S.R., Thedei-Jr. G., Maccheroni W., Rossi A.: Properties of a constitutive alkaline phosphatase from strain 74A of moldNeurospora crassa.Braz.J.Med.Biol.Res. 33, 905–912 (2000).

    Article  PubMed  CAS  Google Scholar 

  • Negrete-Urtasun S., Reiter W., Diez E., Denison S.H., Tilburn J., Espeso E.A., Peñalva M.A., Arst H.N. Jr.: Ambient pH signal transduction inAspergillus: completion of gene characterization.Mol.Microbiol. 33, 994–1003 (1999).

    Article  PubMed  CAS  Google Scholar 

  • Oshima Y.: The phosphatase system inSaccharomyces cerevisiae.Gen.Genet.Syst. 72, 323–334 (1997).

    Article  CAS  Google Scholar 

  • Pereira M., Pereira-Jr. H., Thedei-Jr. G., Rossi A., Martinez-Rossi N.M.: Purification ofNeurospora crassa alkaline phosphatase without DNAse activity for use in molecular biology.World J.Microbiol.Biotechnol. 11, 505–507 (1995).

    Article  CAS  Google Scholar 

  • Raper K.B., Fennell D.I.:The Genus Aspergillus. Williams & Wilkins, Baltimore 1965.

    Google Scholar 

  • Rizzatti A.C.S., Jorge J.A., Terenzi H.F., Rechia C.G.V., Polizeli M.L.T.M.: Purification and properties of a thermostable extracellular β-d-xylosidase produced by thermotolerantAspergillus phoenicis.J.Industr.Microbiol.Biotechnol. 26, 156–160 (2001).

    Article  CAS  Google Scholar 

  • Say J.C., Furriel R.P.M., Ciancaglini P., Jorge J.A., Polizeli M.L.T.M., Pizauro J.M., Terenzi H.F., Leone F.A.: Conidial alkaline phosphatase fromNeurospora crassa.Phytochemistry 41, 71–75 (1996).

    Article  PubMed  CAS  Google Scholar 

  • Vincent J.B., Crowder M.W., Averill B.A.: Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions.Trends Biol.Sci. 17, 105–110 (1992).

    Article  CAS  Google Scholar 

  • Watanabe T., Takeuchi T., Otsuka M., Tanaka S., Umezawa K.: Synthesis and protein tyrosine phosphatase inhibitory activity of dephostatin analogs.J.Antibiot. 48, 1460–1466 (1995).

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departmento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040 901, Ribeirâo Preto, São Paulo, Brazil

    L. H. S. Guimarães, H. F. Terenzi, J. A. Jorge, F. A. Leone & M. L. T. M. Polizeli

Authors
  1. L. H. S. Guimarães
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. F. Terenzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. A. Jorge
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. A. Leone
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. L. T. M. Polizeli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. L. T. M. Polizeli.

Additional information

This work was supported by grants fromFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) andConselho de Desenvolvimento Cientifico e Tecnológico (CNPq). This work is part of the DSc Thesis of the first author who is a recipient of a fellowship from CNPq. The other authors are research fellows of CNPq.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guimarães, L.H.S., Terenzi, H.F., Jorge, J.A. et al. Extracellular alkaline phosphatase from the filamentous fungusAspergillus caespitosus: Purification and biochemical characterization. Folia Microbiol 48, 627–632 (2003). https://doi.org/10.1007/BF02993469

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02993469

Keywords

Search

Navigation