Rennin specificity

Microbial proteinases can be classified by mechanism of action. Hartley (1960) divided them into four groups serine proteinases, thio proteinases, metalloproteinases, and acid proteinases. Morihara (1974) classified enzymes within these groups according to substrate specificity. Enzymes which split peptide substrates at the carboxyl side of specific amino acids are called carboxyendopeptidases, and those which split peptide substrates at the amino side of specific amino acids are called aminoendopeptidases. Acid proteinases, such as rennin and pepsin, split either side of specific aromatic or hydrophobic amino acid residues. The action of proteolytic enzymes on milk proteins has been reviewed by Visser (1981). 

As already described the enzyme-modified 7S protein retains a high molecular weight like the native protein it is excluded by Bio-Gel P-150 which has an exclusion limit of 150,000 daltons. The specificity of rennin is such that it is easy to control and limit the extent of digestion. When the enzyme action is monitored by ultraviolet absorption it is apparent that the rennin action is quite different from that obtained with an enzyme such as trypsin (Figure 1). Thus the UV difference spectrum for the rennin-modified protein shows an initial unfolding of the 7S protein chains as indicated by a negative peak at 236 nm. As rennin action continued this negative peak was replaced by a positive peak at about 237 nm characteristic of an ordered secondary structure. 

Hill, R.D. and Laing, R.R., Specific reaction of dansyl chloride with one lysine residue in rennin, Biochim. Biophys. Acta 132, 188-190, 1967 Chen, R.F., Huorescent protein-dye conjugates. I. Heterogeneity of sites on serum albumin labeled by dansyl chloride. Arch. Biochem. Biophys. 128, 163-175, 1968 Chen, R.F., Dansyl-labeled protein modified with dansyl chloride activity effects and fluorescence properties. Anal Biochem. 25, 412M16, 1968 Brown, C.S. and Cunningham, L.W., Reaction of reactive sulfydryl groups of creatine kinase with dansyl chloride. Biochemistry 9, 3878-3885, 1970 Hsieh, W.T. and Matthews, K.S., Lactose repressor protein modified with dansyl chloride activity effects and fluorescence properties. 

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

In the absence of calcium ions but in the presence of other casein components the normally insoluble K-casein is apparently stabilized by the calcium-sensitive caseins (157). Thus calcium ions are required to coagulate whole casein after treatment with rennin. Therefore in the native milk system the micelle-stabilizing power of K-casein is specifically destroyed by rennin, and in the presence of calcium ions in milk a coagulum is formed (2). This offers a dramatic example of how the functionality of an entire protein system can be altered by specific proteolytic action on a component of that system. 

Although the general proteolytic activities of various milk clotting enzymes may vary, their milk clotting activities are apparently predicated on the same specific cleavage of the Phe-Met bond in -casein. Apparently rennin, pepsin, chymotrypsin, a microbial protease, proteases from Endo-thia parasitica, Mucor pusillus, and Mucor miehei exert the same type of activity on -casein (2, 169). Enzymes that are currently used commercially for cheesemaking in the United States include rennin, rennin-pepsin mixtures, and microbial proteases from Endothia parasitica, Mucor pusiUus, and Mucor miehei. 

Raymond, M. N., Gamier, J., Bricas, E Cilianu, S., Blasnic, M., Chaix, A., and Lefrancier, P. (1972). Studies on the specificity of chymosin (rennin). I. Kinetic parameters of the hydrolysis of synthetic oligopeptide substrates. Biochimie 54, 145-154. 

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