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Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

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Abstract

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K m and V max values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 µmol/min/mg, respectively. PG was found to have temperature optimum at 65 °C and was totally stable at 45 °C for 90 min. Half-life at 55 °C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na+1, K+1, and Co+2 (1 mM) and Cu+2 (1 and 10 mM).

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Acknowledgments

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho de Desenvolvimento Científico e Tecnológico (CNPq). J.A. Jorge, H.F. Terenzi, and M.L.T.M. Polizeli are Research Fellows of CNPq. This work is part of the Master's degree dissertation of André Ricardo de Lima Damásio (A.R.L. Damásio is a FAPESP Fellowship recipient). We thank Ricardo Alarcon and Mauricio de Oliveira for technical assistance and also Desiree Qualls from Microbiology and Molecular Genetics Department—OSU, Stillwater, OK, USA and Abilio Borghi for the English manuscript review.

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  1. Departamento de Bioquímica e Imunologia—Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

    Andre Ricardo de Lima Damásio & Alexandre Maller

  2. Departamento de Biologia—Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14.040-901, Ribeirão Preto, SP, Brazil

    Tony Márcio da Silva, João Atílio Jorge, Hector Francisco Terenzi & Maria de Lourdes Teixeira de Moraes Polizeli

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  1. Andre Ricardo de Lima Damásio
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Correspondence to Maria de Lourdes Teixeira de Moraes Polizeli.

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de Lima Damásio, A.R., da Silva, T.M., Maller, A. et al. Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures. Appl Biochem Biotechnol 160, 1496–1507 (2010). https://doi.org/10.1007/s12010-009-8682-0

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