Proteinases EC

The presence of an indigenous proteinase in milk was suggested by Babcock and Russel in 1897 but because it occurs at a low concentration or has low activity in milk, it was felt until the 1960s that the proteinase in milk may be of microbial origin. Recent changes in the dairy industry, e.g. improved hygiene in milk production, extended storage of milk at a low temperature 

Lipase Triglycerides + H jO - fatty acids+partial glycerides + glycerol Off flavours in milk flavour development in Blue cheese 

Proteinase (plasmin) Hydrolysis of peptide bonds, particularly in -casein Reduced storage stability of UHT products cheese ripening 

Catalase 2H2OJ-O2 + 2HjO Index of mastitis pro-oxidant 

Xanthine oxidase Aldehyde + H20 + 02- Acid + H 2O2 Pro-oxidant cheese ripening 

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The NC-IUBMB classifies peptidases (EC 3.4) into exopeptidases (EC 3.4.11-19), which remove one or a few amino acids, and endopeptidases (proteinases, EC 3.4.21-99), which catalyze the cleavage of peptide bonds away from either end of the polypeptide chain (Fig. 2.1). Exopeptidases are further subdivided into enzymes that carry out hydrolysis at the N-terminus or the C-terminus (Figs. 2.1 and 2.2). Thus, aminopeptidases (EC 3.4.11) cleave a single amino acid from the N-terminus [3] those removing a dipep-... 

Peptide hydrolases (peptidases or proteases, i.e., enzymes hydrolyzing peptide bonds in peptides and proteins, see Chapt. 2) have received particular attention among hydrolases. As already described in Chapt. 2, peptidases are divided into exopeptidases (EC 3.4.11 -19), which cleave one or a few amino acids from the N- or C-terminus, and endopeptidas-es (proteinases, EC 3.4.21-99), which act internally in polypeptide chains [2], The presentation of enzymatic mechanisms of hydrolysis in the following sections will begin with peptidases and continue with other hydrolases such as esterases. 

This aspartic proteinase [EC 3.4.23.22], from the ascomy-cete Endothia parasitica, catalyzes the hydrolysis of proteins with broad specificity similar to that of pepsin A, with preferential action on substrates containing hydrophobic residues at PI and PI. ... 

The group of cysteine endopeptidases (also called sulfhydryl proteases or thiol proteases) include the higher plant enzymes papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), but also numerous microbial proteolytic enzymes such as Streptococcus cysteine proteinase (EC 3.4.22.10). The enzymes have a rather broad substrate specificity, and specifically recognise aromatic substituents. The specificity is for the second amino acid from the peptide bond to be cleaved. 

Aspartic endopeptidases (EC 3.4.23) are the best-known aspartic hydrolases and the only ones to be presented here. These enzymes were formerly called acid proteinases because most of them are active at low pH. In con-... 

This enzyme [EC 3.4.23.34], also known as slow-moving proteinase and erythrocyte membrane aspartic proteinase, is similar to cathepsin D, albeit with a slightly broader specificity. 

This enzyme [EC 3.4.22.6], also known as papaya proteinase II, is a member of the peptidase family Cl. It is the major endopeptidase of papaya (Carica papaya) latex. It has a specificity similar to that of papain. In addition, there are a number of chromatographic forms of the enzyme. 

Glutamyl endopeptidase [EC 3.4.21.19] (also known as staphylococcal serine proteinase, V8 proteinase, protease V8, and endoproteinase Glu-C), a member of the peptidase family S2B, catalyzes the hydrolysis of Asp-Xaa and Glu-Xaa peptide bonds. In appropriate buffers, the specificity of the bond cleavage is restricted to Glu-Xaa. Peptide bonds involving bulky side chains of hydrophobic aminoacyl residues are hydrolyzed at a lower rate. 

This enzyme [EC 3.4.24.14], also known as procollagen A-proteinase, catalyzes the hydrolysis of the A-propep-tide of the collagen chain a-l(l) at Pro—Gin and of a-2(11) chain at Ala—Gin. As a result, A-terminal propeptides of type I and II collagens are released prior to fibril assembly. However, it does not act on type III procollagen. 

This enzyme [EC 3.4.21.53], also known as endopepti-dase La, ATP-dependent serine proteinase, and ATP-dependent protease La, catalyzes the hydrolysis of peptide bonds in large proteins (for example, globin, casein, and denaturated serum albumin) in the presence of ATP (which is hydrolyzed to ADP and orthophosphate). Vanadate ion inhibits both reactions. A similar enzyme occurs in animal mitochondria. Protease La belongs to the peptidase family S16. 

This enzyme [EC 3.4.23.19], also known as aspergillopep-sin II, proctase A, and Aspergillus niger var. macrosporus aspartic proteinase, catalyzes the hydrolysis of peptide bonds in proteins. It has been isolated from Aspergillus niger var. macrosporus and is distinct from aspergillopep-sin I in specificity and in insensitivity to pepstatin. 

This enzyme [EC 3.4.24.27], also known as Bacillus ther-moproteolyticus neutral proteinase, is a thermostable extracellular metalloendopeptidase containing zinc and four calcium ions. A member of the peptidase family M4, this enzyme catalyzes the hydrolysis of peptide bonds with a preference for Xaa—Leu > Xaa—Phe. 

GJ Davis, QM Wang, GA Cox, RB Johnson, M Wakulchik, CA Dotson, EC Villarreal. Expression and purification of recombinant rhinovirus 14 3CD proteinase and its comparison to the 3C proteinase. Arch Biochem Biophys 346 125-130,1997. 

Pepsin (EC 3.4.23.1) is a typical aspartic proteinase produced in the gastric mucosa of vertebrates as a zymogen form [10], This enzyme has been extensively characterized, and its three-dimensional structure has been determined at high resolution. Porcine pepsin, in particular, has been studied as model to analyze the structure-function relationship of the aspartic proteinases. Although the aspartic proteinases including mammalian and fungal enzymes are quite similar in their three-dimentional structures, there are drastic differences in the catalytic properties, especially in substrate specificities. 

In addition to these above described aspartic proteinases, there exist some other distinct families that do not contain the consensus sequence of Asp-Thr/Ser-Gly. These are called non-pepsin-type aspartic proteinases and include aspergillopepsin II (EC 3.3.23.19) [42], scytalidiopepsin B (EC 3.4.23.32) [43], pseudomonapepsin (EC 3.4.23.37) [44] and xanthomonapepsin (EC 3.4.23.33) [45, 46], These families of aspartic proteinases are not sensitive to pepstatin, hence they are also called pepstatin-insensitive carboxyl proteinases. 

While establishing purifying for aspartic proteinase (aspergillopepsin I, EC 3.4.23.18) [7, 8] of Aspergillus saitoi, which is a food microorganism strain, it was discovered to be a rich source of acid carboxypeptidase (EC 3.4.16.5), which removes acidic, neutral, and basic amino acids as well as proline from the carboxyterminal position at pH around 3 [79, 80], The optimum pH with Z-Tyr-Leu (Z- = bebzyloxycarbonyl-) of the acid carboxypeptidase from A. saitoi was 3.5. The optimum with Z-Glu-Tyr was 3.1, and that with Z-Gly-Pro-Leu-Gly, 3.2. 

Rowan et aL [21-23] isolated two previously unknown cysteine proteinases om pineappic stem, called whimiw (EC 3.4.22.31) and comuvuin, by active-idic directed affinity chromatography on Sephatose-Gly-Phe-glycinaldehydesenii-carbazone or Sepharosc-Fhe-glydnaldehydesemicarbazone and cation exchange... 

After the introduction of pronase E, other more or less nonsubstrate-specific proteolytic enzymes have been applied to assist Se speciation. Most of them were derived from DNA/RNA clean-up protocols. The new enzymes (subtilisin from Bacillus licheniformis, also named protease VIII, EC 3.4.21.14 proteinase K from Tritirachium album, EC 3.4.21.64 the crude Novo Nordisk product of Flavourzyme from Aspergillus oryzae) proved to be capable of extracting Se with varying yields and chromatographic recovery of Se species. It is important to highlight that the latter parameter also depends on the instrumentation available. In this regard, different recovery values for the same samples reported by independent research groups do not necessarily indicate successful or unsuccessful sample preparation. Similarly, extraction efficiency (defined as the ratio of extracted Se to total Se in the sample) cannot be used as such for comparison purposes because sample preparation may include some extra steps, for example, TCA precipitation or ultrafiltration, which may reduce this value even by 10-20 percent. 

Understanding how HLE inhibitors work and/or designing new inhibitors requires a model of HLE s active-site and an understanding of its mechanism of action. All serine proteinases share a similar catalytic region and mechanism of action but differ in several amino acids in the extended substrate-binding region. These changes are responsible for the specificity differences between HLE and other serine proteinases. In some cases analysis of the enzyme-inhibitor interactions has only been carried out with other related enzymes, and those results are referenced as appropriate. One closely related enzyme, porcine pancreatic elastase (PPE, EC 3.4.21.36) has... 

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. 

Trypsin (EC 3.4.21.4 no systematic name TRY) is a serine proteinase characterized by the presence at the active site of serine and histidine, both of which participate in the catalytic process. TRY hydrolyzes the peptide bonds formed by the carboxyl groups of lysine or arginine with other amino acids, although esters and amides involving these amino acids are actually spHt more rapidly than peptide bonds. 

Chymotrypsin (EC 3.4.21.1 no systematic name CHY) is also a serine proteinase. It hydrolyzes peptide bonds involving carboxyl groups of Trp, Leu, Tyr, or Phe, with preference for the aromatic residues. The specificity of CHY thus contrasts with that of pepsin, which sphts bonds involving amino groups of the aromatic amino acids. CHY also demonstrates hydrolytic activity for other types of bonds in the following order esters (especially AT-substituted tyrosine esters) > amide > peptides. ... 

Historically, any proteinases that hydrolyze elastin have been named elastases. The genes coding for the elastases are clustered on chromosome 19, and three main types of enzymes are known (I) pancreatic El, (2) pancreatic elas-tase-2 (EC 3.4.21.71), and (3) pancreatic endopeptidase-3 (EC 3.4.21.70), also called cholesterol-binding proteinase. ... 

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