title: Characterization of a heat-resistant extracellular protease from Pseudomonas fluorescens 07A shows that low temperature treatments are more effective in deactivating its proteolytic activity.
abstract: This work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu(2+), Ni(2+), Zn(2+), Hg(2+), Fe(2+), and Mg(2+), but induced by Mn(2+). After incubation at 100°C for 5min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca(2+)- and Zn(2+)-binding domains, indicating that Ca(2+), the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry.
authors: Inovaleite Laboratory, Department of Food Technology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil, 36570000.
authors: Maura P
authors: MP
authors: Alves
authors: Department of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil, 36570000.
authors: Rafael L
authors: RL
authors: Salgado
authors: Department of Food Technology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil, 36570000.
authors: Monique R
authors: MR
authors: Eller
authors: Núcleo de Análise de Biomoléculas (NuBioMol), Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil, 36570000.
authors: Pedro Marcus P
authors: PMP
authors: Vidigal
authors: Inovaleite Laboratory, Department of Food Technology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil, 36570000. Electronic address: antoniofernandes@ufv.br.
authors: Antonio
authors: A
authors: Fernandes de Carvalho
mesh_terms: D000818:Animals; D003080:Cold Temperature; D010450:Endopeptidases / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008892:Milk; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013696:Temperature
chemical_list: D010450:Endopeptidases
keywords: enzyme characterization; heat treatment; milk proteolysis; protease