Parasitica Protease Research Articles

The role of microbial coagulants on the physicochemical, proteolysis, microstructure and sensory properties of low-fat Edam cheese manufactured from ultrafiltered buffalo milk

This work investigates the influence of using microbial coagulants, including Rhizomucor miehei (MCR) protease and Cryphonectria parasitica (MCC) protease, on the quality characteristics of low-fat Edam cheese made from ultrafiltered buffalo milk (LFUE). Concurrently, a benchmark with calf rennet (CR) has been also performed. Throughout a 90-day ripening period, the cheeses were assessed for their physicochemical features, proteolysis, texture, free amino acid and free fatty acid content, microstructure, and sensory attributes. The study revealed that both microbial coagulants had no significant impact on the physicochemical composition and firmness of the cheeses while slightly affected the free fatty acids. Cheeses made with microbial coagulants displayed higher proteolysis, with MCR and MCC cheeses exhibiting greater levels of water-soluble nitrogen and 12% trichloroacetic acid-soluble nitrogen than CR cheese. MCR and MCC cheeses exhibited more extensive breakdown of αs- and β-caseins, as indicated by the SDS-PAGE electrophoretogram, compared to CR cheese during ripening. As for the proteolytic activity, the microbial coagulant contributed to shaping the free amino acid content, microstructure, and sensory qualities of the cheeses. Notably, MCC cheese outperformed MCR or CR cheeses in terms of free amino acid levels. MCR and MCC cheeses resulted in smooth microstructures with uniform protein networks as observed by microscopy, while CR cheese displayed rough, granular surfaces. With the highest scores for appearance, body, texture, and flavor, MCC cheese demonstrated superior sensory properties compared with MCR and CR cheeses.

Production of protease isozymes by Aphanomyces cochlioides and Aphanomyces euteiches

The production of protease activity by the sugarbeet pathogen Aphanomyces cochlioides, the legume pathogen A. euteiches, and the fish pathogen Saprolegnia parasitica was examined. Protease activity was readily detected in supernatants of water cultures of each organism using autoclaved host tissue as a nutrient source. Most of the protease isozymes extracted from sugarbeet and pea seedlings infected with A. cochlioides and A. euteiches, respectively, co-migrated with enzymes produced by the pathogens in culture. In inoculated sugarbeet seedlings, the protease activities were detected prior to or concommitent with the onset of disease symptoms and the activities were capable of digesting protein extracted from sugarbeet hypocotyls. Use of class-specific inhibitors indicated that a portion of the protease activity was of the trypsin-class. Trypsin-like isozymes that possessed a relatively fast electrophoretic migration were detected in the A. cochlioides, A. euteiches, and S. parasitica protease complements, whereas the remaining isozymes were not affected by any of the inhibitors tested. Proteinaceous trypsin inhibitors from the legumes lima bean ( Phaseolus lunatus) and soybean ( Glycine max) inhibited the trypsin-like isozymes from A. cochlioides, but not A. euteiches, whereas low molecular weight, synthetic trypsin inhibitors inhibited these isozymes from both pathogen sources. The potential role of protease inhibition in determining host range in phytopathogenic Aphanomyces species is discussed.

Combined use of chymosin and protease from Cryphonectria parasitica for control of meltability and firmness of cheddar cheese.

The combined use of chymosin and Cryphonectria parasitica protease was evaluated for manufacturing Cheddar cheese at different chymosin-to-C. parasitica protease ratios of 1:0, 0:1, 67:33, and 33:67. The degree of proteolysis over time was affected by the coagulant type. Proteolysis was thought to be the main cause of changes in functional properties of Cheddar cheeses. The meltability and hardness of cheese made with 100% C. parasitica enzyme was the highest, but it was high in bitterness. The chymosin-to-C. parasitica ratio between 0:1 to 67:33 was found suitable to independently control Cheddar cheese meltability and hardness without a significant level of bitterness.

Proteolytic activities of some milk clotting enzymes on ovine casein

Proteolytic activity of some milk clotting enzymes (calf and lamb rennets, bovine chymosin and pepsin, and proteases from Rhizomucor miehei and Cryphonectria parasitica) on ovine whole casein was determined by urea-PAGE and RP-HPLC. Microbial enzymes were more proteolytic than animal enzymes when acting on ovine whole casein. Lamb rennet and C. parasitica protease showed the lowest and the highest degree of proteolysis, respectively. Urea-PAGE results showed that all enzymes hydrolyzed ovine casein resulting in the formation of αs1-I and β-I as initial breakdown products of αs1-CN and β-CN. In addition to these products, C. parasitica protease produced a series of degradation products with lower mobilities than β-CN. Minor quantitative differences between coagulants from animal origin, but great quantitative and qualitative differences between microbial and animal coagulants (assessed throughout the study of the RP-HPLC peptide profiles), were observed.

Proteolytic activities of suparen and rennilase on buffalo, cow, and goat whole casein and beta-casein.

The proteolytic specificity and activity of Mucor miehei protease (Rennilase) and Endothia parasitica protease (Suparen) on buffalo, cow, and goat whole casein and beta-casein (CN) were studied by analyzing the degradation products. The results suggest that Rennilase hydrolyzes casein of the three species in a manner similar to that of chymosin, resulting in the formation of alpha(s1)-I and beta-I, -II, -III as initial degradation fragments of alpha(s1)- and beta-CN. alpha(s1)-I was also the initial breakdown product of alpha(s1)-CN by Suparen. Contrary to Rennilase, Suparen showed a higher affinity toward beta-CN and hydrolyzes beta-CN, giving rise to degradation products characterized by mobility lower than that of beta-CN. Increasing NaCl concentration (>3%) reduced the proteolysis of beta-CN of the three species by Rennilase but not by Suparen. The hydrolysis of alpha(s1)-CN and alpha(s1)-I by the two enzymes was enhanced in the presence of NaCl.

Relative Proteolytic Action of Milk‐Clotting Enzyme Preparations on Bovine α‐, β‐ and β‐casein

ABSTRACTConcentrations of seven milk‐clotting enzyme preparations were standardized to equal clot times. Portions of bovine αs‐, β‐ and κ‐casein were treated with enzymes. Proteolytic activity of the coagulants on each casein fraction was determined using the TNBS (2,4,6‐trinitrobenzene‐sulfonic acid) procedure. Recombinant chymosin showed the lowest degree of proteolysis on αs‐ and β‐caseins. Excessive proteolysis of calf rennet appeared to be due to the pepsin fraction. M. miehei and M. pusillus var Lindt proteases showed similar degradation of caseins, but M. pusillus var Lindt was more proteolytic when β‐casein was the substrate. C. parasitica protease showed the highest degree of proteolysis on αs‐ and β‐caseins but was the least proteolytic on κ‐casein.

Mozzarella Cheese: Impact of Coagulant Type on Chemical Composition and Proteolysis

The impact of different milk coagulants on initial chemical composition and proteolytic changes in Mozzarella cheese during refrigerated storage was determined. Three vats of cheese were made in 1 d, each using a different coagulant (i.e., Endothia parasitica protease, fermentation-derived chymosin, or Mucor miehei protease). Cheese making was replicated on 3 different d using a 3×3 Latin square design. Coagulant type did not affect pH, moisture, protein, or fat contents of cheese. Differences in salt content and fat on a dry weight basis of the cheese were slight. Nitrogen soluble in 12% TCA and in pH 4.6 acetate buffer increased significantly for cheeses made with all coagulants, but the increase was the greatest for cheese made with E. parasitica protease. The αs,-casein in cheeses made with each of the three coagulants was broken down during storage. Proteolysis of β-casein was only in the cheese made with E. parasitica protease. The milk coagulant used for Mozzarella cheese manufacture plays an important role in proteolysis during 50 d of storage at 4°C.

Mozzarella Cheese: Impact of Coagulant Type on Functional Properties

The objective of this study was to determine the impact of coagulant type and refrigerated storage on functional properties of unmelted and melted Mozzarella cheese. A “no-brine” Mozzarella cheese-making method was used to produce cheese with homogeneous chemical composition. Cultured Mozzarella cheeses were made with three different coagulants (Endothia parasitica protease, chymosin derived by fermentation, and Mucor miehei protease) in 1 d, and cheese making was replicated on 3 different d. During 50 d of storage at 4°C, texture profile analysis parameters (hardness, cohesiveness, and springiness) of unmelted cheese decreased, meltability increased, apparent viscosity of melted cheese decreased, and free oil formation from melted cheese increased. Overall, the Mozzarella cheese made using E. parasitica protease was softer in unmelted cheese texture, was more meltable, and had lower apparent viscosity and more free oil release on melting than other cheese. In general, cheeses made with chymosin and Mucor miehei protease were similar in functional characteristics.

Affinity Partition of Acid Proteases in Aqueous Two-phase Systems: Modeling and Protein Purification.

Aqueous two-phase systems consisting of dextran and hydroxypropyidextran (HPD) were used in protein partition studies. The phase diagram of such systems was determined by fluorescein-labelled dextran or HPD. The phase envelope was close to symmetrical, consistent with the Flory-Huggins theory for polymer solutions owing to the similarities between the two phase-forming polymers. A new mathematical model for affinity partition was developed. Simulation results from this model indicated that the ratio of dissociation constant of protein-ligand complex to total ligand concentration should be lower than 10–3, and the ratio of total protein concentration to total ligand concentration should be kept below 1 to get the best partition results. Partitions of pepsin, chymosin, and Endothia parasitica protease were studied in affinity dextran/HPD aqueous two-phase systems with pepstatin attached to dextran as the ligand. The proteins strongly preferred the bottom phase into which the pepstatyl-dextran segregated. Data from the partition experiments can be satisfactorily correlated with the model developed. Purification of chymosin was carried out in this affinity system with a five-step liquid-liquid extraction. The purification factor was 6.2 with a yield of 83%.

Effect of Calcium Addition on Yield of Cheese Manufactured with Endothia parasitica Protease

Yield of stirred curd cheese from milk containing 0, .02, .03, and .05% added calcium chloride and coagulated with prolease from Endothia parasitica was compared with the yield of curd coagulated with calf rennet. Five treatments replicated five times in a randomized block design were manufactured to produce 25 vats of cheese. Milk was analyzed for fat, total solids, total N, and non-casein N. Cheese was analyzed for fat, total solids, and total N. Whey was analyzed for fat and total N. Dry matter cheese yield data adjusted for covariants of milk fat and casein were lower for cheese manufactured from E. parasitica protease than from calf rennet. Addition of calcium chloride to cheese milk clotted with E. parasitica protease increased cheese yield. Calcium needed to be higher than .02% to produce maximum yields when cheese was manufactured with Endothia parasitica protease.

Effect of Milk-Clotting Enzymes on Cheese Yield

Stirred curd cheese was manufactured to determine the effects of six milk-clotting enzymes on yield. Concentrations of calf rennet, recombinant chymosin, 50:50 blend of calf rennet and bovine pepsin, and proteases from Mucor miehei, Mucor pusillus var. Lindt, and Endothia parasitica were adjusted to give equal clot times. Forty-eight vats of cheese were manufactured with eight replications of each treatment in a randomized block design. Milk was analyzed for fat, total solids, total N, and noncasein N. Cheese was analyzed for fat, total solids, and total N. Whey was analyzed for fat and total N. Dry matter cheese yield adjusted for milk lot changes of milk fat and casein was lower for cheese made with Endothia parasitica protease than for calf rennet. Cheese made with recombinant chymosin or a 50:50 blend of calf rennet and bovine pepsin resulted in yields that were not significantly different from those of calf rennet Cheeses manufactured with protease from Mucor miehei had slightly lower yields than did rennets containing chymosin.

Heat‐Stability of Milk‐Clotting Enzymes in Conditions Encountered in Swiss Cheese Making

ABSTRACTChymosin is inactivated in gelled milk at pH 6.50 at 53°C according to a biphasic kinetic. Both phases appear to follow first‐order kinetics. The D‐value for the first phase is 30 min. In the same medium, the Mucor miehei and Mucor pusillus proteases are much more stable (D53°C > 100 min) while bovine pepsin, a heat‐labile Mucor miehei protease and the Endothia parasitica protease are rapidly inactivated (D53°C < 10 min). pH appears to be the most important parameter for heat stability. Protein and calcium concentrations affect the resistance to heat treatment. A residual activity of chymosin in Swiss‐type curd will be very weak at the most unless the curd is cooked at pH < 6.50.

Residual Milk Coagulants in Whey — Their Inactivation and Effects on Ultrafiltration Flux and Nitrogen Yields

Ten commercial milk coagulants were used to produce Cheddar cheese wheys. Wheys were then concentrated fourfold by ultrafiltration following pasteurization, separation, and prewarming. Fluxes were monitored. Wheys, final concentrates, and pooled permeates were tested for total nitrogen, nonprotein nitrogen, and fat. Wheys and concentrates were also tested for residual milk clotting activity. Residual activity was observed in the wheys and concentrates originating from the unmodified proteases of Mucor miehei and Mucor pusillus but not from the two modified proteases of Mucor miehei, an Endothia parasitica protease, two bovine pepsins, a bovine pepsin-swine pepsin blend, a Mucor pusillus-swine pepsin blend, or calf rennet, which served as the control. No significant differences in flux or nitrogen yields were observed.

ACYL TRANSPEPTIDATION AND HYDROLYTIC REACTIONS CATALYZED BY ENDOTHIA PARASITICA PROTEASE WITH SMALL PEPTIDE SUBSTRATES

Endothia parasitica protease shows hysteretic behavior (a lag phase) when acting upon L-leucyl-L-leucine amide at pH 4.5. Initially, the products are L-leucyl-L-leucine, L-leucyl-L-leucyl-L-leucine and L-leucine amide with little formation of L-leucine and no ammonia. The relative concentration of L-leucyl-L-leucine is about one-fourth that of L-leucine amide. As the reaction continues the relative concentration of L-leucine increases slowly until it is equal to the concentration of L-leucyl-L-leucine near the end of the reaction with each being one-fourth that of L-leucine amide. Formation of these products can only be explained by a combination of transpeptidation and hydrolytic reactions. By itself, L-leucyl-L-leucine is cleaved very slowly but when mixed with L-leucyl-L-leucine amide the rate is several fold greater than the combined rates of the two alone. L-Leucyl-L-leucyl-L-leucine and L-leucyl-L-leucyl-L-leucine amide as substrates of E. parasitica protease give normal kinetic behavior with no evidence of formation of higher molecular weight compounds. Addition of as little as 1 μM L-leucyl-L-leucyl-L-leucine (Km= 0.348 mM) to a reaction containing 1 mM L-leucyl-L-leucine amide increases the rate of reaction with a shortening of the lag phase. Addition of 100 μM L-leucyl-L-leucyl-L-leucine to such a reaction eliminates the hysteretic behavior. It is proposed that E. parasitica protease is initially in an inactive form which can be activated by substrates as small as L-leucyl-L-leucyl-L-leucine but not by L-leucyl-L-leucine amide. During the lag phase observed with substrates such as L-leucyl-L-leucine amide, the enzyme produces, via acyl transpeptidation, larger compounds which can rapidly activate the enzyme. While in the activated form, E. parasitica protease cleaves L-leucyl-L-leucine amide via normal kinetic behavior; however, both acyl transpeptidation and hydrolysis still occur as shown by the relative concentrations of L-leucine, L-leucyl-L-leucine and L-leucine amide of 1:1:4 near end of the reaction. E. parasitica protease cannot cleave carbobenzoxy-L-leucyl-L-leucine indicating that, at least with this substrate, the enzyme cannot perform amino transpeptidation reactions.

Thermal Inactivation of Residual Milk Clotting Enzymes in Whey

Cheddar cheese whey from milk clotted with six milk clotting enzymes (Mucor miehei, Mucor pusillus var Lindt, and Endothia parasitica proteases; calf rennet, porcine pepsin, and chymosin-free bovine pepsin) were adjusted from pH 5.2 to 7.0 prior to heating at 68.3 and 73.9C for .25, .5, 1, 5, and 10min. Enzyme activities were measured before and after heating. Mucor miehei protease was the most heat stable at all pH values followed by Mucor pusillus protease, calf rennet, bovine pepsin, Endothia parasitica protease, and porcine pepsin. The heat stability of all enzymes increased with decreasing pH except Endothia parasitica protease was most heat stable at pH 7.0.Whey from cheese milk clotted with Mucor miehei protease was adjusted from pH 4.2 to 6.4 at .2 intervals of pH and subjected to high-temperature short-time pasteurization at 73.9, 76.6, and 79.5C for 25 s. Milk clotting activity was determined before and after pasteurization. All measurable activity was destroyed above pH 5.4 by pasteurization at 79.5C, above pH 5.8 at 76.6C, and above pH 6.0 at 73.9C.

Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl- l-leucine amide and l-leucyl- l-phenylalanine amide at the peptide bond. l-Phenylalanyl- l-leticine amide, N-carbobenzoxy- l-leucyl- l-phenylalanine amide, N-carbobenzoxy- l-leucyl- l-pheml-alanine, N-carbobenzoxy- l-phenylalanyl- l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl- l-leucine amide and l-leucyl- l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, viz l-leucyl- l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl- l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl- l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.

Rates and Extents of Hydrolysis of Several Caseins by Pepsin, Rennin, Endothia parasitica Protease and Mucor pusillus Protease

The initial rates of hydrolysis, rates of hydrolysis and extents of hydrolysis after 1,440min reaction of whole, α-, β-, and κ-caseins by crystallized rennin, crystallized pepsin and purified M. pusillus protease and E. parasitica protease at pH 3.0, 3.5, 5.5 and 6.0 have been determined. The number of peptide bonds hydrolyzed was determined by the trinitrobenzene sulfonic acid method. E. parasitica protease generally had more activity than the other three enzymes on all substrates at all pHs. With whole, α- and κ-caseins the initial rates and extents of hydrolysis decreased from pH 3.0 to 6.0 with a few exceptions. With all four enzymes the extent of hydrolysis of β-casein was lower at pH 3.0 than at pH 3.5. At pH 6.0, a11 four enzymes had the fastest initial rates of hydrolysis on κ-casein followed in turn by α-casein and β-casein. The initial rates of hydrolysis of β-casein at pH 6.0 were very low by all four enzymes. The extents of hydrolysis at pH 6.0 were in the order of α-, κ- and β-casein except for E. parasitica protease where the order was α-, β- and κ-casein. With a11 four enzymes the extent of hydrolysis of β-casein at pH 5.5 was much greater than at pH 6.0.

Activity Assay for Endothia parasitica Protease Using Trypsinogen as Substrate

A sensitive activity assay method for Endothia parasitica protease based on activation of trypsinogen has been developed. Activation of trypsinogen was performed at pH 4.0 and 30.0C using 3.0 mg/ml of bovine trypsinogen and 1.25 μ/ml of purified Endothia parasitica protease in .04M sodium formate −.3M KCl buffer. At intervals of 1, 2, 3, 5, and 7 minutes the amounts of trypsin formed were determined on .05ml aliquots with 1.95ml of 1% casein in .2M Tris −.01M CaCl2 buffer, pH 8.0 and 30C for 10min. The trichloroacetic acid-soluble products were determined at 280nm. Enzyme activity was determined from the initial rate of trypsinogen activation. The pH optimum for activation of trypsinogen by Endothia parasitica protease was 3.5 to 3.6, Vmax, 2,950±177 micromoles trypsinogen activated per minute per micromole Endothia parasitica protease, and Km was 8.32±.30×10−5 M. Sensitivity of the trypsinogen activation assay was 11,400 times that of direct determination of Endothia parasitica protease activity on casein at pH 3.0 and 30C and 600 times more sensitive than a milk clotting assay at pH 5.1 and 30C.

Hydrolysis of peptide bonds of the oxidized B-chain of insulin by Endothia parasitica protease

The relative rates of hydrolysis of the peptide bonds of the oxidized B-chain of insulin by Endothia parasitica protease have been determined at pH 3.60 and at a substrate concentration of 1.07 × 10 −3 m and an enzyme concentration of 1.95 × 10 −6 m. The Phe 24Phe 25, bond is hydrolyzed at a maximum rate followed by hydrolysis of the Tyr 16Leu 17 and Gln 4His 5 bonds. The Leu 11Val 12 and Asn 3Gln 4 bonds are hydrolyzed at slower rates. The LeunVal 12 bond appears to be considerably more resistant to hydrolysis in the peptide 5–16 than in the intact oxidized B-chain. The Leu 15-Tyr 16 bond is very slowly hydrolyzed in peptide 5–16 but there is no evidence for hydrolysis of this bond in the intact oxidized B-chain. Phe 25 is slowly hydrolyzed from the peptide 25–30 and the bond involving Gly 20Glu 21 is slowly hydrolyzed in peptide 12–24 and/or peptide 17–24. Thus the enzyme has specificity for the hydrophobic regions of the oxidized B-chain. The specificity of Endothia parasitica protease on the oxidized B-chain of insulin is similar to that of pepsin in that bonds involving Gln 4His 5, Leu 11Val 12, Leu 15Tyr 16, Tyr 16Leu 17, Phe 24Phe 25 and Phe 25Tyr 26 are hydrolyzed by both enzymes. Endothia parasitica protease hydrolyzes bonds involving Asn 3Gln 4 and Gly 20Glu 20, not attacked by pepsin, but fails to hydrolyze bonds involving Phe 1Val 2, Glu 13Ala 14, Ala 14Leu 15 and Gly 23Phe 24, hydrolyzed by pepsin. Thus, Endothia parasitica protease is more specific in its action on the oxidized B-chain of insulin than is pepsin.

Endothia parasitica Protease. Parameters Affecting Activity of the Rennin-like Enzyme

Parameters expected to influence the activity of Endothia parasitica protease were investigated. Under the assay conditions using 20% nonfat dry milk dispersed in 0.2M acetate buffer, pH 5.10 and 35.0, the primary phase (initial enzymatic splitting of peptide bonds) is the rate-determining step in the clotting of milk by E. parasitica protease. Thus, the clotting time is directly proportional to the reciprocal of enzyme concentration. The higher the substrate concentration, the longer the clotting time. Between 10 and 20% substrate the relationship between the two is linear but at lower substrate concentrations the clotting time is longer than expected. The pH optimum of E. parasitica protease on acid-denatured hemoglobin and casein is 2.0 and 2.5, respectively. Over the pH range of 5.1 to 6.5 the clotting activity of E. parasitica protease is much less sensitive to the pH of the reaction medium than is that of rennin. Endothia parasitica protease has a different, and a broader, specificity than rennin and pepsin as determined on the oxidized B chain of insulin and its inability to hydrolyze the synthetic substrate, α-n-benzyloxycarbonyl-l-glutamyl-l-tyrosine.