Chymotrypsin protein digestion using

More than 97% of the available soybean meal is used for feed where extensive heat treatment is necessary to maximize feed conversion efficiency by livestock. Toasting inactivates protease inhibitors (especially trypsin and chymotrypsin inhibitors) and the enzyme urease, and improves protein digestibility. None of these objectives can be obtained without protein being denatured and loss in water solubility however, depending on the method used, meals with great differences in protein solubilities or dispersibilities can be produced. The optimum amount of heat treatment in toasting soybean meal is still debated among animal nutritionists. 

Fig. 2. The use of overlapping fragments to determine the sequence of a peptide. The protein is first digested with trypsin and the resulting peptides separated and sequenced. The protein is separately digested with chymotrypsin and the resulting peptides again separated and sequenced. The order of the peptide fragments in the protein can be determined by comparing the sequences obtained.

A number of proteolytic enzymes participate in the breakdown of proteins in the digestive systems of mammals and other organisms. One such enzyme, chymotrypsin, cleaves peptide bonds selectively on the carboxylterminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine (Figure 91). Chymotry psin is a good example of the use of covalent modification as a catalytic strategy. The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate. This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis. 

The enzymatic digestion of the purified proteins was carried out as described (13). The enzymes TPCK-treated pancreatic trypsin (EC 3.4.21.4 Type XIII) and TLCK-treated pancreatic chymotrypsin (EC 3.4.21 Type VII) were from Sigma Chemical Company. The amount of proteinase used was 2% of the concentration of the purified protein. The proteins were dissolved in 200 oL of 0.1 N ammonium bicarbonate buffer (pH=7.0), followed by addition of the enzyme dissolved in deionized water. The enzymatic digestion was carried out by incubating the samples for 18 hours at 37° and the reaction was stopped by addition of 10% trifluoroacetic acid solution. The proteins were lyophilized. 

Proteolytic enzymes, such as the serine proteases, are among the best characterized of all enzymes.They are important in digestive processes because they break down proteins. They each catalyze the same type of reaction, that is. the breaking of peptide bonds by hydrolysis. The crystal structures of several serine proteases have been determined, and the mechanism of hydrolysis is similar for each. The specificity of each enzyme is, however, different and is dictated by the nature of the side chains flanking the scissile peptide bond (the bond that is broken in catalytic mechanism. Chymotrypsin is one of the best characterized of these serine proteases. The preferred substrates of chymotrypsin have bulky aromatic side chains. The crystal structure determination of the active site of chymotrypsin, illustrated in Figure 18.12, has provided much of the information used to elucidate a plausible mechanism of action of the enzyme. In the first step of any catalyzed reaction, the enzyme and substrate form a complex, ES, the Michaelis complex. The hydrolysis of the peptide bond by chymotrypsin involves three amino acid residues,... 

Enzymes are biological catalysts. The majority of the enz)rmes that have been studied are proteins. Reactions that would take days or weeks or require extremely high temperatures without enzymes are completed in an instant. For example, the digestive enzymes pepsin, trypsin, and chymotrypsin break down proteins in our diet so that subunits can be absorbed for use by our cells. 

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