Protease activity casein

Jones LJ, Upson RH, Haugland RP, Panchuk-Voloshina N, Zhou M, Haugland RP. Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement. Anal Biochem 1997 251(2) 144-152. 

A succinylated casein derivative that has nearly all its amines blocked can be used as a substrate in protease assays (Hatakeyama etal., 1992). As the casein is degraded by a protease, free amines are created from a-chain cleavage and release of a-amino groups. The creation of amines can be monitored by an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS Section 4.3). The procedure forms the basis for a highly sensitive assay for protease activity. 

Blocking of amine groups on proteins also has been used to create a sensitive reagent for measuring protease activity (Hatakeyama etal., 1992). With nearly all the primary amines of casein blocked, an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS) will only minimally react with the protein and form its typical orange derivative. As proteases cleave the protein, however, primary a-amines are created from cleavage of the a-chain peptide bonds, and TNBS can react with them. The more protease activity present, the more color is formed. 

Application and Principle This procedure is used to determine protease activity, expressed as PC units, of preparations derived from Bacillus subtilis var. and Bacillus licheniformis var. The assay is based on a 30-min proteolytic hydrolysis of casein at 37° and pH 7.0. Unhydrolyzed casein is removed by filtration, and the solubilized casein is determined spectro-photometrically. 

Among the first applications of this type of experimental strategy were screenings for protease activity [45,46], Here, agar plates containing a turbid suspension of casein... 

Serum albumin labeled with an iodine radionuclide was firstly used as a substrate for determining protease activity by Absolon This method was later on modified several times and applied for assaying various proteolytic activities in different materials. Mego et al. injected denaturated I-human %rum albumin into the tail vein of rats and measured the rate of intralysosomal proteolysis on isolated lysosomes containing endocytosed substrate. This method was also used for the determining the intralysosomal pH on the basis of differences found in the rate of I-albumin breakdown in intact and lysed lysosomes C-bovine serum albumin, I-casein or I-albumin have been alternatively used as substrate for measuring the activity of trypsin, chymotrypsin and papain - ). 

Protease activity was measured using 1% (w/v) casein as substrate dissolved in 0.1 M phosphate buffer (pH 3.0). Aliquots (2 ml) of diluted supernatant were mixed with 2 ml of 1% (w/v) casein and the reaction mixtures incubated at 40 °C for 10 min. An equal volume of trichloroacetic acid (0.4 1) was added to the reaction mixture, after filtration, the tyrosine released was quantified by the absorbance of the filtrate at 275 nm (A275). 

M. pusillus var. Lindt protease has given satisfactory results as a chymosin substitute in the manufacture of a number of cheese varieties, but not all varieties of M. pusillus var. Lindt are capable of producing acceptable cheese (Babel and Somkuti 1968). The clotting activity of M. pusillus var. Lindt protease is more sensitive to pH changes between 6.4 and 6.8 than chymosin, but is much less sensitive than that of porcine pepsin (Richardson et al 1967). The same authors reported that CaCL added to milk affected the clotting activity of M. pusillus var. Lindt rennet more than it did that of chymosin rennet. They also reported that this rennet was more stable than chymosin between pH 4.75 and 6.25. M. pusillus var. Lindt rennet is not destroyed during the manufacture of Cheddar cheese, although less than 2% of the enzyme added to the milk remains in the curd. Nearly all of it is found in the whey (Holmes et al. 1977). Mickelsen and Fish (1970) found M. pusillus var. Lindt rennet to be much less proteolytic than E. parasitica rennet but more proteolytic than chymosin rennet on whole casein, a8-casein and /3-casein at pH 6.65. 

The other major casein in cheese is /3-casein, but it is generally not hydrolyzed by rennet in low-pH cheeses. Alkaline milk protease (plas-min) plays the major role in the hydrolysis of /3-casein (Richardson and Pearce 1981). The plasmin level in cheese is related to the pH of the curd at whey drainage, since plasmin dissociates from casein micelles as the pH is decreased. Richardson and Pearce (1981) found two or three times more plasmin activity in Swiss cheese than in Cheddar cheese. Swiss cheese curds are drained at pH 6.4 or higher, while Cheddar cheese curds are drained at pH 6.3 or lower. Proteolysis of /3-casein is significantly inhibited by 5% sodium chloride. The inhibitory influence of sodium chloride is most likely due to alteration of /3-casein or a reduction in the attractive forces between enzyme and substrate (Fox and Walley 1971). 

The gross proteolysis of casein is probably due solely to rennet and plasmin activity (O Keeffe et al. 1978). Bacterial proteases and peptides are responsible for subsequent breakdown of the large peptides produced by rennet and plasmin into successively smaller peptides and finally amino acids (O Keeffe et al. 1978). If the relative rate of proteinase activity by rennet, plasmin, and bacterial proteases exceeds that of the bacterial peptidase system, bitterness in the cheese could result. Bitter peptides can be produced from a,-,- or /3-casein by the action of rennet or the activity of bacterial proteinase on /3-casein (Visser et al. 1983). The proteolytic breakdown of /3-casein and the subsequent development of bitterness are strongly retarded by the presence of salt (Fox and Walley 1971 Stadhouders et al. 1983). The principal source of bitter peptides in Gouda cheese is 3-casein, and more particularly the C-terminal region, i.e., 3(193-209) and 3(193-207) (Visser et al. 1983). In model systems, bitter peptides are completely debittered by a peptidases system of S. cremoris (Visser et al. 1983). 

Micrococci comprise approximately 78% of the nonlactic bacteria in raw milk Cheddar cheese (Alford and Frazier 1950). The proteolytic system of Micrococcus freudenreichii functions optimally at 30 °C and at a pH near neutrality (Baribo and Foster 1952). An analysis of pro-teinases present in 1-year-old Cheddar cheese indicates that micrococci may contribute to proteolytic activity (Marth 1963). Proteolytic micrococci also contribute to the ripening of surface-ripened cheeses such as brick and Camembert (Lenoir 1963 Langhus et al. 1945). Micrococcal proteases probably contribute to development of ripened cheese flavor when ripening temperatures are above 10°C (Moreno and Kosikowski 1973). This effect results from degradation of /3-casein. 

The iotai proteolytic activity of pancreas powder is determined by comparing the quantity of peptides nonprecipitabie by a 556 m/V solution of trichloroacetic acid R released per minute from a substrate of casein solution with the quantity of such peptides released by pancreas powder (protease) UK from the same substrate in the same conditions. For the test suspension and the reference suspen-sion, prepare the suspension and carry out tiie dilution at (W-°C. 

Application and Principle This procedure is used to determine proteolytic activity, expressed in spectrophotometric acid protease units (SAP), of preparations derived from Aspergillus niger var. and Aspergillus oryzae var. The test is based on a 30-min enzymatic hydrolysis of a Hammarsten Casein Substrate at pH 3.0 and at 37°. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration. The quantity of solubilized casein in the filtrate is determined spectrophotometrically. 

Table 4.3 Activity half-life of proteases in the presence of organic solvent [28, 29], Values in parentheses are the results of repetitions carried out in 2007. Enzymes were incubated at 30 C and pH 8 in the presence of 25% organic solvent. Residual hydrolytic activity was measured by a casein assay.

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