Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

Abstract

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.Dry-cured ham is an uncooked meat product obtained after 8 to 24 months of ripening. During this time, an uncontrolled microbial population grows on the surface (18). Molds, yeasts, and gram-positive catalase-positive cocci have been reported as the dominant organisms on the surface of dry-cured ham (17,18,22). Most molds and some cocci growing spontaneously on dry-cured ham are toxigenic (18, 23). However, they show an interesting hydrolytic activity on myofibrillar proteins, particularly molds (13,14).A decisive role has been attributed to proteolysis in the taste (15) and in the generation of flavor (28) in dry-cured meat products. For this reason, the use of proteolytic molds or their purified enzymes would increase protein hydrolysis in drycured meat products, which is of special interest in shortening their ripening time.Several proteolytic enzymes from fungi and bacteria have been purified and characterized, but only a limited number of proteases from microorganisms isolated from dry-cured meat products have been reported (24). On the other hand, the intracellular cell extract of Penicillium aurantiogriseum isolated from dry-cured meat products has been demonstrated elsewhere to show an increase of proteolysis during the ripening of these products (2). A selected nontoxigenic strain of Penicillium chrysogenum (Pg222) has shown a high proteolytic activity on controlled ripening of pork loins (14), but its proteases have not been characterized. Since these microorganisms are able to grow on dry-cured meat products, with 2 to 4% NaCl and quite restrictive water activity values (4, 22), their proteases could be very efficient at hydrolyzing meat proteins ...

Effect of the fungal protease EPg222 on proteolysis and texture in the dry fermented sausage ‘salchichón’

“…This finding is in accordance with the low activity observed for EPg222 against sarcoplasmic proteins in in-vitro assays. 21 Proteolytic activity of EPg222 against myofibrillar proteins is also demonstrated by a higher accumulation of NPN in treated than control samples in the first 20 days of ripening, which is in accordance with a higher reduction of myofibrillar proteins at this ripening period in treated sausages. At day 35 production of NPN in treated samples stopped and no differences from control were found.…”

“…24 The optimum working conditions of this protease are 45-55 • C, pH 6 and 0.25 M NaCl. 21 For this assay purified extract of EPg222 was obtained after DEAE-sepharose, ultrafiltration and HPLC analysis according to the protocol described by Benito et al 21 Preparation of dry fermented sausages The mixture for dry fermented sausages 'salchichón' was prepared using the following formula: 75% Iberian pork, 25% Iberian pork fat, 1 g kg −1 white pepper, 1.5 g kg −1 black pepper, 30 g kg −1 NaCl and a mixture of dextrin, dextrose and lactose named SALAVI from ANVISA (Madrid, Spain) at concentrations recommended by the manufacturers. Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus TEXEL SA-201 from Rhodia (Boulogne-Billancourt, France) were used as starter cultures following the instructions of the manufacturers.…”

“…Protease EPg222 purified from Penicillium chrysogenum Pg222 isolated from a dry-cured meat product has shown high proteolytic activity against myofibrillar proteins at those conditions of temperature, pH and NaCl concentration found in dry-cured meat products 21 and in a meat model system. 22 EPg222 also showed collagenolytic activity which could favour tenderness because meat toughness is attributed to both myofibrillar proteins and connective tissue.…”

Purification and Characterization of Two Novel Halotolerant Extracellular Proteases fromBacillus subtilisStrain FP-133

“…Some of them are produced by Filobacillus sp. RF2-5 isolated from fish sauce, 3) Penicillium chrysogenum Pg222 from cured ham, 4) and Pseudoalteromonas sp. strain CP7 from soil, 5) but there have been few reports on halotolerant proteases from the main industrial protease producer Bacillus subtilis.…”

Molecular Cloning and Sequence Analysis of Two Distinct Halotolerant Extracellular Proteases fromBacillus subtilisFP-133

“…On the other hand, moderately halophilic bacteria possess halotolerant proteases that retain their activity not only under normal but also relatively high salt conditions. Some halotolerant proteases have been purified and characterized, in order to apply them to the fermentation of certain foods, [4][5][6] but the genetic structure of the proteases have not been sufficiently investigated, and the only example to date is the molecular cloning of a metalloprotease of Bacillus vietnamensis 11-4, which hydrolyzes gelatin, collagen, and elastin in 0% and 15% NaCl. 7) Previously, two distinct halotolerant proteases, Expro-I and Expro-II, from Bacillus subtilis FP-133 were characterized and classified as a serine protease and a metalloprotease respectively.…”