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Production of polygalacturonase from Coriolus versicolor grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates

  • Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by “in situ” detection of enzyme activity.

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Acknowledgments

MRF is grateful to Évora University for financial support and the authors thank Fundação para a Ciência e Tecnologia for partial financial support (Research Unit 702, POCTI/EQU/48812/2002) as well as Instituto Politécnico de Lisboa (Research Grant No. 25/2003).

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Authors and Affiliations

  1. Department of Chemistry, Universidade de Évora, Rua Ramalho Ortigão N°59, Évora, Portugal

    Maria do Rosário Freixo, Amin Karmali & José Maria Arteiro

  2. Chemical Engineering and Biotechnology Research Center of Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1950-062, Lisbon, Portugal

    Maria do Rosário Freixo & Amin Karmali

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  1. Maria do Rosário Freixo
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  2. Amin Karmali
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Correspondence to Amin Karmali.

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do Rosário Freixo, M., Karmali, A. & Arteiro, J.M. Production of polygalacturonase from Coriolus versicolor grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates. J Ind Microbiol Biotechnol 35, 475–484 (2008). https://doi.org/10.1007/s10295-008-0305-1

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  • DOI: https://doi.org/10.1007/s10295-008-0305-1

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