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Purification and biochemical characterization of thermostable alkaline phosphatases produced by Rhizopus microsporus var. rhizopodiformis

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Abstract

The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1–2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3×, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn2+, Na+ and Mg2+ stimulated the activity, while Al3+ and Zn2+ activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 °C and pH 8.0, respectively. The enzymes were stable at 50 °C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K m 0.28 and 0.22 mmol/L, with υ lim 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.

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Abbreviations

AcP(s):

acid phosphatase(s)

AlP(s):

alkaline phosphatase(s)

ConA:

concanavalin A

4-NPP:

4-nitrophenyl phosphate

P i :

inorganic phosphate

SbmF:

submerged fermentation

SSF:

solid state fermentation

Tris-HCl:

Tris(hydroxymethyl)aminomethane

UDP-Glc:

uridine 5’-diphosphoglucose

UDP-Glc:

uridine 5’-diphosphoglucose

UDP-GlcNAc:

uridine 5’-diphospho-N-acetylglucosamine

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Authors and Affiliations

  1. Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil

    A. Barbosa Jr

  2. Departamento de Biologia, Faculdade Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil

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

  3. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil

    F. A. Leone

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  1. A. Barbosa Jr
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  2. L. H. S. GuimarÃes
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  3. H. F. Terenzi
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  4. J. A. Jorge
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  5. F. A. Leone
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  6. M. L. T. M. Polizeli
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Correspondence to M. L. T. M. Polizeli.

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Barbosa, A., GuimarÃes, L.H.S., Terenzi, H.F. et al. Purification and biochemical characterization of thermostable alkaline phosphatases produced by Rhizopus microsporus var. rhizopodiformis . Folia Microbiol 53, 509–516 (2008). https://doi.org/10.1007/s12223-008-0080-4

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  • DOI: https://doi.org/10.1007/s12223-008-0080-4

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