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23 Congress of the International Union for Biochemistry and Molecular Biology
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44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
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Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
PARTIAL CHARACTERIZATION OF AN EXTRACELLULAR PROTEASE OF
Pseudomonas fluorescens
Alves, M.P.1; Salgado, R.L.2; Eller, M.R.1; Carvalho, A.F.1
1
Departamento de Tecnologia de Alimentos da Universidade Federal de Viçosa,
Minas Gerais, Brasil; 2Departamento de Bioquímica Agrícola da Universidade
Federal de Viçosa, Minas Gerais, Brasil.
Psychotropic bacteria play an important role in the deterioration of milk and dairy
products since they produce heat-resistant proteolytic enzymes, which may
remains active after heat treatments applied throughout their production. This
study aimed the partial characterization of an extracellular protease produced by a
proteolytic strain of P. fluorescens. The enzyme was partially purified from the
supernatant of the culture of the bacteria P. fluorescens 07A by fractioned
precipitation with ammonium sulfate, followed by size exclusion chromatography
and concentrated using Amicon® Ultra-15. The effect of temperature and pH on
protease activity was assessed using azocasein as substrate. The enzyme was
incubated at temperatures ranging from 4 to 60 °C, and different buffers at pH
values ranged from 4.0 to 10.0. By SDS-PAGE it was observed that the protease
has a molecular mass of 50 kDa. It shows activity in a wide temperature range, 460 °C, with maximum activity at 37 °C. Moreover, more than 95% of the protease
activity is maintained at 50 °C and above this temperature it is considerably
reduced. The protease is also active over a wide pH range, from 5.0 to 10.0, with
maximum activity at pH 7.5. Between pH 8.0 and 10.0 the protease activity
remains constant, approximately 69% of the maximum activity. The wide pH and
temperature ranges tested in this work in which the protease retains much of its
activity include many of the storage conditions of milk and dairy products. The
knowledge about the characteristics of proteolytic enzymes in order to control their
production and activity in raw milk is essential to reduce the various technological
problems caused by psychrotrophic bacteria in dairy products.
Keywords: enzyme characterization, milk proteolysis, protease
Acknowledges: CNPq, FAPEMIG, CAPES.
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)