Enhancement of the activity, stability and reusability of an extracellular protease from Pseudomonas fluorescens 07A via
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ORIGINAL PAPER
Enhancement of the activity, stability and reusability of an extracellular protease from Pseudomonas fluorescens 07A via three different strategies of immobilization Kimberly Freitas Cardoso1 · Juliana Severo Miranda1 · Jane Aparecida de Paula2 · Thiago de Paula Carmo2 · Monique Renon Eller2 Received: 11 October 2019 / Revised: 27 March 2020 / Accepted: 14 May 2020 © Associação Brasileira de Engenharia Química 2020
Abstract The bacterium Pseudomonas fluorescens 07A produces a protease with potential for industrial application. In order to remedy problems associated with the use of free enzymes and allow its reuse, the protease was immobilized on DEAE S ephacel® resin via three different strategies based on ionic interaction and covalent bonding. The matrix-bound enzymes were characterized in relation to their activity (pH, temperature and stability), reuse and storage. Immobilization raised the optimum temperature of activity from 37 °C to 50 °C, whereas the pH of highest activity changed from 7.5 to 7.0 or 8.0, depending on the immobilization strategy. Immobilization proved to be efficient for successive reuses, even leading to an increase in the enzymatic activity along the use. The immobilized enzyme also presented greater stability to high temperatures and storage conditions and has potential as a biocatalyst for industrial applications due to its high efficiency, stability and easy recovery. Keywords Enzyme immobilization · Biocatalysts · Pseudomonas fluorescens 07A · Proteolysis
Introduction Proteases hydrolyze peptide bonds, being widely used in the food and pharmaceutical industries (Kumar and Takagi 1999), such as for beverage clarification and production of Kimberly Freitas Cardoso and Juliana Severo Miranda have equally contributed to this paper. * Monique Renon Eller [email protected] Kimberly Freitas Cardoso [email protected] Juliana Severo Miranda [email protected] Jane Aparecida de Paula [email protected] Thiago de Paula Carmo [email protected] 1
Department of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Viçosa, Brazil
Department of Food Technology, Universidade Federal de Viçosa, Av. Peter Henry Rolfs, s/n, Campus Universitário, Viçosa, MG CEP 36570‑900, Brazil
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bioactive peptides, respectively. The limitations involving the use of proteases are based on their high cost, on their mild properties (that could be incompatible with the conditions of processing) and difficult or no recovery at the end of the process. During immobilization, the enzyme is physically or chemically associated with a support through interactions or entrapment (Khan et al. 2017; Kumari and Kayastha 2011; Shrinivas et al. 2012). This process promotes changes in the catalytic properties and allows the reuse of the enzyme, reducing costs (Mehdi et al. 2017; Shi et al. 2010). Moreover, immobilization leads to the retention of the enzyme in high concentrations in a specific and limited environment, allowing control of enzymatic catalysis
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