title: Characterization of a new oxidant-stable serine protease isolated by functional metagenomics.
abstract: A novel serine protease gene, SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent (Tween 20, Tween 80, or Triton X-100). Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H2O2.
authors.affiliation: Unité de Microbiologie et Génomique, Gembloux Agro-Bio Tech, Université de Liège, Avenue Maréchal Juin 6, B-5030 Gembloux, Belgium.
authors.forename: Sophie
authors.initials: S
authors.lastname: Biver
authors.forename: Daniel
authors.initials: D
authors.lastname: Portetelle
authors.forename: Micheline
authors.initials: M
authors.lastname: Vandenbol
keywords: Calcium; Functional metagenomics; Oxidant; Serine protease
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