Subtilisin protein hydrolysis

Specificity of Subtilisin for Hydrolysis of Peptide Bonds in Proteins and Polypeptides... 

The authors of the studies cited above found that the enzymes used during sample preparation could not completely hydrolyze sample proteins into amino acids. This is partly understandable as both proteinase K and subtilisin are known to show hydrolyzing preference for specific residues of proteins. Accordingly, they do not necessarily arrive at total hydrolysis. At the same time, neither pronase E nor protease XIV could always provide 100 percent extraction of Se from the samples (see Table 19.1). Therefore, alternative methods should be developed in the field of sample preparation to replace enzymatic methods. 

Because of its wide specificity, papain will degrade most protein substrates more extensively than trypsin, pepsin, or chymotrypsin and its action is quite comparable to that of subtilisin (Hill and Schmidt, 1962 Nomoto et al., 1960a,b). Many free amino acids are liberated from proteins by papain, but it would not appear to produce as extensive hydrolysis as S. griseus protease (French et al., 1963). 

Procedure 1 pepsin, subtilisin, aminopeptidase M, prolidase This method may often be useful with proteins that can be maintained in a- largely denatured state at acid pH. The protein (usually about 10 mg per ml) may be denatured in solvents like 6 M guanidinium chloride or 8 M freshly deionised urea and dialyzed exhaustively against 5 % formic acid at room temperature at about pH 2. If cysteine or cystine residues are present, these should be reduced and alkylated ( 3.8.2) prior to enzymic hydrolysis. 

Procedure 3 papain, leucine aminopeptidase, prolidase This early procedure for enzymic hydrolysis of proteins was reported by Hill and Schmidt (1962) to be successful for hydrolysis of several proteins. Papain was found to be superior to subtilisin or a combination of trypsin and chymotrypsin for the initial hydrolysis. The method might be improved if aminopeptidase M (discovered after the method was developed) is used in place of the leucine aminopeptidase, but to our knowledge this has not been tested. The problem with diketo-piperazine formation from X-Pro dipeptides in aminopeptidase M hydrolysates of peptides (see above) may make this substitution less desirable than it would seem at first. 

Modification of Ultrafiltered versus Acid Precipitated Soy Protein. When the retentate obtained from the ultrafiltration of soybean extract is subjected to an enzymatic hydrolysis as described earlier (2) for acid precipitated protein, a hydrolysis curve (DH versus time) may be drawn. A comparison of such hydrolysis curves is shown in Fig. 2 for acid precipitated soy protein isolate and ultrafiltered soy protein isolate. The curves are drawn on the basis of the same hydrolysis parameters. The enzyme used is the microbial alkaline protease subtilisin Carlsberg (ALCALASE ). 

Several types of enzymes have found uses in LADD compositions [4,48], Most common are proteases, amylases, and lipases, which attack proteinaceous, starchy, and fatty soils, respectively. Proteases work by hydrolyzing peptide bonds in proteins. Proteases differ in their specificity toward peptide bonds. The typical protease used in LADD formulations, bacterial alkaline protease (subtilisin), is very nonspecific. That is, it will attack all types of peptide bonds in proteins. In contrast to proteases, amylases catalyze the hydrolysis of starch. They attack the internal ether bonds between glucose units, yielding shorter, water-soluble chains called dextrins. Lipases work by hydrolyzing the ester bonds in fats and oils. Often, combinations are used because of the specificity of each kind to one type of soil. The commercially available enzymes are listed in Table 9.6. 

Hydrolysis of keratin and of other proteins, with subtilisin-like specificity. Hydrolyses peptides amides. 

Modification of specific amino acid residues should affect the rate of proteolysis by those enzymes which have specificity for the unmodified residues. For example, modification of the lysine residues of a protein restricts the action of trypsin. Dimethylated casein undergoes a slower rate of hydrolysis by trypsin in vitro, and the extent of proteolysis is lower than that of casein (267). An unexpected finding was that the rate and extent of hydrolysis of dimethylated casein by a-chymotrypsin are also adversely affected, while hydrolysis by subtilisin is not. Peptides derived from dimethylated casein were inhibitory of a-chymotrypsin. 

Goto et al. 1998). None of them probably reflects properly the enzyme activity over the real substrate, so it will be a matter of judgment and experience to select the most pertinent assay with respect to the actual use of the enzyme. Hydrolases are currently assayed with respect to their hydrolytic activities however, the increasing use of hydrolases to perform reactions of synthesis in non-aqueous media make this type of assay not quite adequate to evaluate the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a trans-esterification reaction that produces thiophenol as one of the products (Han et al. 2004) in this case, a method based on a reaction leading to a fluorescent adduct of thiophenol is a good system to assess the transesterification potential of such proteases and is to be preferred to a conventional protease assay based on the hydrolysis of a protein (Gupta et al. 1999 Priolo et al. 2000) or a model peptide (Klein et al. 1989). 

The hydrolysis of amide bonds in protein and simpler entities by enzymes has been known for many years and explored extensively. The degradation of protein by a proteinase such as subtilisin is the basis of action of biological washing powders. Obviously the commercial importance of... 

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