Blue proteolysis

Figure 6 Sequence alignment of lantibiotic and nonlantibiotic bacteriocin prepeptides. The residues in red indicate those positions that are fully conserved within that class, and those in blue are highly conserved. For the nonlantibiotic bacteriocins, only the leader sequences are shown. The site of proteolysis is indicated by the arrow. For cytolysin, the additional six residues removed by CylA are indicated in green.

The level of proteolysis in cheese varies from limited (e.g. Mozzarella) through moderate (e.g. Cheddar and Gouda) to very extensive (e.g. Blue cheeses). The products of proteolysis range from very large polypeptides, only a little smaller than the parent caseins, to amino acids which may, in turn, be catabolized to a very diverse range of sapid compounds, including amines, acids and sulphur compounds. 

Femandez-Salguero, J., Andreas Marcos Alcala, M., and Esteban, M. A. (1989). Proteolysis of Cabrales cheese and other European blue vein cheese varieties. /. Dairy Res. 56,141-145. 

Figure 10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of products of limited proteolysis of the debranching enzyme with trypsin. The molecular weights shown of the various bands were determined by the methodology described previously (26). The ratio of debrancher to trypsin was 100 to 1. The incubation was conducted for 60 minutes at 25°. The gel stain was Coomassie Brilliant Blue and the absorbance was measured at 600 nm using a Gilford gel scanner with a 0-1 O.D. chart scale (36).

Proteolysis during maturation is essential in most cheese varieties. The extent of proteolysis varies from very limited (e.g.. Mozzarella) to very extensive (e.g.. Blue mould varieties) and the products range in size from large polypeptides, comparable in size to intact caseins, through a range of medium and small peptides to free amino acids. Clearly, no one proteolytic agent is responsible for such a wide range of products. 

As mentioned in Section IV El, the extent of proteolysis varies from very limited, e.g.. Mozzarella, to very extensive, e.g., blue-mould varieties. The use of PAGE showed that the proteolytic pattern, as well as its extent, exhibit marked intervarietal differences (Ledford et al., 1966 Marcos et al., 1979). The PAGE patterns of both the water-insoluble and water-soluble fractions are, in fact, quite characteristic of the variety, as shown in Figs. 11 and 12 for a number of Cheddar, Dutch, and Swiss-type cheeses. RP-HPLC of the water-soluble fraction or subfractions thereof also shows varietal characteristics (Fig. 13). Both the PAGE and HPLC patterns vary and become more complex as the cheese matures and are in fact very useful indices of cheese maturity and to a lesser extent of its quality (O Shea, 1993). Therefore, they have potential in the objective assessment of cheese quality. 

Undoubtedly, the products of these primary biochemical events, i.e., fatty and other acids, peptides, and amino adds, contribute to cheese flavor, perhaps very significantly in many varieties and proteolysis certainly has a major influence on the various rheological properties of cheese, e.g., texture, meltability, and stretchability. However, the finer points of cheese flavor are almost certainly due to further modification of the products of the primary reactions. The most clear-cut example of this is the oxidation of fatty acids to methyl ketones in blue cheeses. Catabolism of amino acids leads to the production of numerous sapid compounds, including amines, carbonyls, acids, thiols, and alcohols. Many of these compounds may interact chemically with each other and the compounds of other reactions via the Maillard and Strecker reactions. At present, relatively little is known concerning the enzymology of amino acid catabolism in most cheeses and even less is known about the chemical reactions. It is very likely that research attention will focus on these secondary and tertiary reactions in the short-term future. 

The amino acid sequence of Ps. fluorescens azurin, Fig. 2, determined by Ambler and Brown (6) reveals information which is fundamental to any understanding of the physical properties of the blue center. During the course of this work a number of observations suggested that azurin possessed a very compact structure and that the metal played some role in maintaining this structure (6). For example, the native protein was found to be completely resistant to proteolysis by trypsin, chymotrypsin, or subtilysin B while, in contrast, the apoprotein was readily digested by these enzymes. Another manifestation of the compactness of the protein structure was the failure to detect significant amounts of tryptophan... 

Traditionally fermented dairy products have been used as beverages, meal components, and ingredients for many new products [60], The formation of flavor in fermented dairy products is a result of reactions of milk components lactose, fat, and casein. Particularly, the enzymatic degradation of proteins leads to the formation of key-flavor components that contribute to the sensory perception of the products [55], Methyl ketones are responsible for the fruity, musty, and blue cheese flavors of cheese and other dairy products. Aromatic amino acids, branched-chain amino acids, and methionine are the most relevant substrates for cheese flavor development [55]. Volatile sulfur compounds derived from methionine, such as methanethiol, dimethylsulflde, and dimethyltrisul-fide, are regarded as essential components in many cheese varieties [61], Conversion of tryptophan or phenylalanine can also lead to benzaldehyde formation. This compound, which is found in various hard- and soft-type cheeses, contributes positively to the overall flavor [57,62]. The conversion of caseins is undoubtedly the most important biochemical pathway for flavor formation in several cheese types [62,63]. A good balance between proteolysis and peptidolysis prevents the formation of bitterness in cheese [64,65],... 

Fig. 3.5 Schematic structure of aggrecan and the sugar components in it. The blue horizontal line in the middle represents the protein eomponents from the A/-terminus to the C-terminus. Aggrecan is sensitive to break-up (proteolysis) where the dotted arrow indicate, this is responsible for the degradation of cartilage tissue. (Authors own work and http //glycoforum.gr.jp/science/word/pro-teoglycan/PGAOOE.html, permission obtained from copyright-owner)...

To determine which proteins contained (ADP-ribose) , fraction V was analyzed by acetic acid/urea polyacrylamide gel electrophoresis. As shown in Fig. 2A, the major portion of radioactivity migrated with histone HI and radioactivity was also found with staining bands of the four HMG proteins. Following 3-ABm treatment for 16 h, ADP-ribosylation of HMG 14 and 17 was almost completely inhibited, while that of HMG 1 and 2 and histone HI decreased less. The reduced ADP-ribosylation cannot be attributed to differences in protein extraction or proteolysis since Coomassie blue staining patterns were very similar and 3-ABm treatment did not affect the incorporation of labeled lysine into HMG proteins and histone HI (Fig. 2B). ADP-ribosylation... 

Reaction termination. The reactions are terminated by the addition of SDS-PAGE sample buffer (10 mM Tris-HCl, pH 6.8, 1 mM EDTA, 1% DTT, 2.5-5% SDS, 0 01% bromphenol blue, final) followed immediately by boiling. Prepare a 4X stock of SDS-PAGE sample buffer and store in aliquots at -20°C. Quickly boiling the samples is essential to minimize artifactual proteolysis owing to bindmg-protein denaturation... 

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