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Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products

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Abstract

EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.

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Acknowledgements

The authors would like to thank Dr. Susan Liddell (Division of Agricultural and Environmental Sciences, University of Nottingham) for performing the amino acid sequence determinations. M. J. Benito was the recipient of a postdoctoral grant from the Spanish Ministerio de Educación y Ciencia. This work was supported by a grant from the Consejería de Educación, Ciencia y Tecnología de la Junta de Extremadura (2PR04C022) and Spanish Comisión Interministerial de Ciencia y Tecnología (AGL01-0804).

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  1. María J. Benito

    Present address: Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Carretera de Cáceres s/n, 06071-, Badajoz, Spain

Authors and Affiliations

  1. Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonnington Campus, Loughborough, LE12 5RD, UK

    María J. Benito & Ian F. Connerton

  2. Higiene de los Alimentos, Facultad de Veterinaria, Universidad de Extremadura, 10071, Cáceres, Spain

    Juan J. Córdoba

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  1. María J. Benito
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  3. Juan J. Córdoba
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Correspondence to María J. Benito.

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Benito, M.J., Connerton, I.F. & Córdoba, J.J. Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products. Appl Microbiol Biotechnol 73, 356–365 (2006). https://doi.org/10.1007/s00253-006-0498-z

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  • DOI: https://doi.org/10.1007/s00253-006-0498-z

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