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

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.

Influence of starter culture and a protease on the generation of ACE-inhibitory and antioxidant bioactive nitrogen compounds in Iberian dry-fermented sausage “salchichón”

“…In previous studies, it has been found that the addition of proteases during the meat dry-curing process releases peptides and free amino acids (Díaz et al, 1993 , 1997 ; Naes et al, 1995 ; Ordóñez et al, 1999 ; Zapelena et al, 1997 ; 1999 ), but the potential bioactivity of these compounds has not been studied. The use of EPg222 with high proteolytic activity (Benito et al, 2002 , 2003 , 2004 , 2006 ; Casquete et al, 2011c ) is important due to its activity on meat proteins. Previously, it has been observed that the batches in which this enzyme was added showed a higher concentration of nitrogen compounds ( Table 1 ).…”

“…Starter culture P200S34 composed by Pediococcus acidilactici (MS200) and Staphylococcus vitulus (RS34) was used in the dry-fermented sausages ( Casquete et al, 2011a ). Proteolytic enzyme EPg222 was obtained according to Benito et al (2006) .…”

“…Similarly, the use of proteases obtained from microorganisms isolated from dry-fermented meat products could be more appropriate than other proteases, since they may be more suitable and adapted to the traditional ripening process. EPg222 protease purified from Penicillium chrysogenum Pg222, which was isolated from dry-cured meat products, has high proteolytic activity against myofibrillar proteins under the conditions of temperature, pH and NaCl concentration used for dry-cured meat products (Benito et al, 2002 , 2006 ). This protease has been reported in pieces of pork loins and in fermented sausages (Benito et al, 2003 , 2004 ; Casquete et al, 2011c ).…”

Industrial Fungal Enzymes: An Occupational Allergen Perspective

“…Pg222 is a novel extracellular protease produced by P. chrysogenum (Pg222). The enzyme was isolated from dry-cured hams and was found to have a broad range of applications in industries that produce dry-cured meat products [89]. Although no occupational sensitization has been reported to this enzyme, it demonstrates that the introduction of any new enzyme could potentially represent an occupational hazard.…”

Potential antimicrobial and antiproliferative activities of autochthonous starter cultures and protease EPg222 in dry-fermented sausages

“…To date, antihypertensive and antioxidant properties in peptides from meat and fermented meat products have been widely investigated; however, other beneficial effects such as antimicrobial and antiproliferative potential have been scarcely addressed. 13,24,25 In this study, we applied starter cultures and protease EPg222, with a demonstrated proteolytic activity, [26][27][28] generate bioactive nitrogen compounds during ripening of dry cured sausages 29,30 The aim of this work was to investigate about the presence of nitrogen compounds with antimicrobial and antiproliferative properties in dry-cured Iberian pork sausages manufactured with different autochthonous starter cultures and protease EPg222.…”