Endopeptidases hydrolysis using

Although any of several combinations of proteases can be used, ideally, one or more non-specific endopeptidases should be used first to convert the protein into many small peptides. These small peptides can then be degraded to amino acids by aminopeptidases and prolidase (hydrolyzes X-Pro bonds). Sometimes, carboxypeptidases are also used. Although leucine aminopeptidase has been used as the amino-peptidase (see Hill and Schmidt 1962), it may be preferable to use aminopeptidase M (Rohm and Haas, supplied by Henley and Co. of N.Y.), since this enzyme removes most residues at acceptable rates. Leucine aminopeptidase removes hydrophobic residues most rapidly, whereas some other residues are removed very slowly. Most procedures should probably include the use of prolidase (Miles) since many aminopeptidases do not cleave X-Pro bonds at appreciable rates. If it is found that proline is not released quantitatively by these procedures, the use of citrus leaf carboxypeptidase C (Rohm and Haas) can be tried after the initial endopeptidase hydrolysis and before the addition of aminopeptidase M and prolidase. Carboxypeptidase C (also yeast carboxypeptidase Y - see Hayashi et al. 1973) hydrolyzes proline bonds (as well as all others), but if proline is at or adjacent to the NH2 terminus of a peptide, it would probably not be released. In all procedures a control consisting of the enzymes only should be run in parallel with the hydrolyzed sample, and corrections should be made for any amino acids found by analysis of the control. suhic / /< > , mi... 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

In another series of investigations, [D-Ala6]LHRH was incubated with various cell types and found to have a rate of hydrolysis 3-8 times lower than that of LHRH [174][176], The use of enzyme inhibitors showed [d-Ala6]LHRH to be resistant to the endopeptidases neprilysin and thimet olig-opeptidase, but to remain sensitive to the peptidyl-dipeptidase ACE. 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

The S -(-)-a-[(acetylthio)methyl]phenylpropionic acid (21) is a key chiral intermediate for the neutral endopeptidase inhibitor (22) [48], We [44] have demonstrated the lipase-catalyzed stereoselective hydrolysis of thioester bond of racemic a-[(acetylthio)methyl]phenylpropionic acid (21) in organic solvent to yield A-(+)-a-[(mercapto)methyl]phenylpropionic acid (23) and Y-(-)-(21). Using lipase PS-30, the Y-(-)-(21) was obtained in 40% reaction yield (theoretical max. 50%) and 98% e.e. (Fig. 9). 

Such applications appear to be more attractive for the use of bioreactors than traditional uses of endopeptidases for chillproofing beer, juice clarification, and curd formation in cheesemaking which currently use well established soluble enzyme processes. In the case of curd formation, hydrolysis of micellar k-casein by immobilized chymosin is questionable (56). 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

The initial attempts to achieve an enzyme-catalyzed deprotection of the carboxyl groups used endopeptidases such as chymotrypsin [32-34] or trypsin [33,35,36]. These transformations involve the danger of an undesired hydrolysis of the peptide bonds. This disadvantage can be overcome by the use of... 

Chymotrypsin [ban, inn] (EC 3.4.21.1 Catarase Zonulysin ) is an enzyme (MW c. 25,0(X) a-form). It is a (serine) endopeptidase stored as zymogen in granules of pancreatic P-cells of mammals. It catalyses the hydrolysis of amide and ester bonds of peptides and proteins, particularly those adjacent to the carbonyl group of hydrophobic L-amino acids. Therapeutically, it is used in ocular surgery, especially for removal of cataracts. It was used formerly as a... 

The combination of immobilized enzyme columns with HPLC/thermospray MS can be very useful in peptide identification and sequencing [6,7], There are a number of ways of combining the immobilized enzyme column, HPLC and MS detection for peptide analysis. Use of an endopeptidase column prior to HPI.C separation and MS detection will enable separation of each hydrolysis product for Identification. Figure 2 shows the trypsin column/HPLC/thermospray MS of Y-endorphin. The selected ion chromatograms show the retention time for each tryptic hydrolysis product Tj and T. Typically, this column configuration can only be used on purified samples since no separation or column clean-up is performed before hydrolysis. 

The peptide or protein can also be partially hydrolyzed using endopeptidases. An endopeptidase is an enzyme that catalyzes the hydrolysis of a peptide bond that is not at the end of a peptide chain. Trypsin, chymotrypsin, and elastase are endopeptidases... 

Enzymatic methods are most often used for determining the Asn, Gin and Trp content. Exo- and endopeptidases are enzymes that catalyse the hydrolysis of specific peptide bonds (section 7.6.1). As these enzymes exhibit a high specificity, it is essential to use a mixture of them to ensure hydrolysis of all peptide bonds. Enzymes should be used at low concentrations ( 1 %) as they are proteins themselves that can degrade and thus contaminate the reaction mixture. 

The human organism is not able to use dietary proteins as such. They must be hydrolysed into single amino acid molecules before they can be absorbed. The hydrolysis of proteins (mostly denatured proteins) is catalysed by proteolytic enzymes called proteases (proteinases or peptidases), which have relatively high substrate specificity. They catalyse the hydrolysis of interior peptide bonds to form peptides of different sizes (endopeptidases such as pepsin, trypsin and chymotrypsin) or attack the terminal amino acids (exopeptidases). Hydrolysis of the N-terminal amino acids is... 

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