title: Comparing two commercial enzymes to estimate in vitro proteolysis of purified or semi-purified proteins.
abstract: Two experiments compared the suitability of two commercial enzymes to estimate in vitro proteolysis of different proteins. Experiment 1 compared the proteolytic activity over various incubation times of a microbial enzyme (protease from Streptomyces griseus) with a plant enzyme (papain from Papaya latex) by using either 1.33 (high, H) or 0.4 (low, L) units (U, amount) of each enzyme per mg crude protein (CP) of purified proteins including bovine (BA) or egg albumin (EA). Experiment 2 compared the activity of 0.66 U of each of these enzymes per mg CP of semi-purified proteins including casein, wheat gluten (WG) and maize gluten meal (MG). Each incubation was terminated by adding trichloroacetic acid (TCA) and the TCA soluble supernatant collected to estimate concentration of total amino acids (AA) as the measure of proteolysis of each protein over each time. The data on proteolysis over time were fitted into a non-linear model to derive constants for solubility (a) and rate (c) and extents (a + b) of proteolysis of each food by each amount of each enzyme. All data on proteolysis over time and the derived constants were statistically analysed to study the effect of food, enzyme, amount and their interactions. Significant differences were observed between foods, enzymes, enzyme amount (H vs. L for Experiment 1 only) for the proteolysis at most incubation times in both experiments (p<0.001). The mean proteolysis over all times for BA was 1.6 (SD, 0.52) times greater than EA (p<0.001). While the protease gave about four times (SD, 2.1) more proteolysis than papain (p<0.001), the high amount of enzyme gave only about two times (SD, 0.28) greater proteolysis than that of the low amount (p<0.001). On average, the protease was over three times faster (c) than papain (p<0.001) and high amount was two times faster than the low amount of enzyme (p<0.001). While both purified foods were similar in solubility (p>0.05), they differed in the rate and extent of proteolysis (p<0.001). The BA was degraded about four times faster than EA (p < 0.001). Amongst semi-pure proteins, casein gave the highest but MG the lowest proteolysis at each incubation (p<0.001). However, the magnitude of proteolysis depended upon enzyme, food and hours of incubation. On average, casein was degraded at a much faster rate than WG or MG by both enzymes. It appeared that the protease and not papain can be used to estimate in vitro proteolysis of pure and semi-pure food proteins. However, further studies are needed to standardise the relevant procedures when using protease to estimate proteolysis of ruminant foods.
authors.affiliation: School of Agriculture, Food and Rural Development, University of Newcastle-upon-Tyne, UK. a.s.chaudhry@ncl.ac.uk
authors.forename: A S
authors.initials: AS
authors.lastname: Chaudhry
mesh_terms: D000418:Albumins / Q000378:metabolism; D000821:Animal Feed; D000818:Animals; D002364:Caseins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005983:Glutens / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D010047:Ovalbumin / Q000378:metabolism; D010206:Papain / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D012418:Ruminants; D012995:Solubility
chemical_list: D000418:Albumins; D002364:Caseins; D010446:Peptide Fragments; D005983:Glutens; D010047:Ovalbumin; D010447:Peptide Hydrolases; D010206:Papain