Purification and biochemical characterization of thermostable alkaline phosphatases produced by <Emphasis Type="Italic">Rhizopus microsporus</Emphasis> var. <Emphasis Type="Italic">rhizopodiformis</Emphasis> |
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Authors: | Jr" target="_blank">A BarbosaJr L H S GuimarÃes H F Terenzi J A Jorge F A Leone M L T M Polizeli |
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Institution: | 1.Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeir?o Preto,Universidade de S?o Paulo,S?o Paulo,Brazil;2.Departamento de Biologia, Faculdade Filosofia Ciências e Letras de Ribeir?o Preto,Universidade de S?o Paulo,S?o Paulo,Brazil;3.Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeir?o Preto,Universidade de S?o Paulo,S?o Paulo,Brazil |
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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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Keywords: | |
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