Sulfhydryl proteases Papain

Sulfhydryl proteases, see Actinidin, Papain Superoxide dismutase, Cu,Zn (Richardson et al., 1975)... 

Among the proteolytic enzymes, the plant proteases are the most widely used in the food industry. Most of the plant proteases which have been studied are characterized by a free sulfhydryl group which is essential for their activity. The most important of these so-called sulfhydryl or thiol proteases are papain, ficin, and bromelain. Since the literature on these enzymes has been the subject of several recent reviews (i, 2, 3, 4), major emphasis is placed in this presentation on the use of these enzymes in the food industry. Some of the more recent developments relating to the structure and function of the sulfhydryl proteases are discussed. 

One of the most characteristic differences between papain and the other sulfhydryl proteases, ficin and bromelain, is the absence of carbohydrate in papain. Glycopeptides have been isolated from ficin (48) and bromelain (49, 50, 51, 52). In the case of bromelain the carbohydrate moiety composed of mannose, xylose, fucose, and N-acetylglucosamine is covalently linked to an asparagine residue of the peptide chain through a glucosamine component of the sugar moiety (51,52). 

Many proteins, such as lysozyme (Figure 5.1), contain both a-helices and P-sheets, but do not have the special structures created by alternating P-a-P patterns. In the sulfhydryl proteases, such as papain (Figure 5.10F) and actinidin, the strands of sheets and the helices tend to be segregated in different regions of space. 

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. 

Sulfhydryl proteases, such as papain, ficin and bromelain, follow essentially the mechanism outlined in Figure 2. The acyl acceptor is... 

The application of papain in peptide synthesis is well established [23-25]. Papain can be used for fhe preparation of di- and tripepfides in an aqueous medium wifh cosolvent addition (up to 40%) and at high pH to promote synthetic activity. The enzyme is a sulfhydryl protease with no homology to the trypsin or subtilase families of hydrolases. Since the catalytic nucleophile is a cysteine and because thioesters are relatively more prone to aminolysis than oxo-esters, the enzyme could be very attractive for synfhesis. However, unlike the case with the thiol variants of some serine hydrolases, fhe proteolytic activity is still high, and the broad substrate range of proteolysis makes peptide substrate and product hydrolysis more problematic than trypsin or chymotrypsin. Extensive enzyme engineering studies on papain are lacking, probably due to the laborious procedure for isolation of active papain from inclusion bodies formed in E. coli. 

Pre-slaughter injection of the live animal has proved to be the most effective method of introducing proteolytic enzymes into meat so that they penetrate uniformly into the furthest interstices of the tissue (31). Oxidized papain (103) and proteases whereby the sulfhydryl group in the active site is reversibly blocked (104) have been injected into the jugular veins of animals approximately 30 min before slaughter to distribute the enzyme in the tissues. Subsequently, in the reducing environment of the meat, the protected enzymes are activated. However, the enzymes are apparently active only between 50°C and 82°C or while the meat is cooking. 

Actinidin is a cysteine protease that requires a free sulfhydryl group for activity (Arcus, 1959 McDowall, 1970). This protease is composed of 220 amino acid residues with molecular mass of 23,500 (Came and Moore, 1978). The amino acid sequence of the enzyme shows considerable homology with papain, a well-known cysteine protease in immature papaya fruit (Came and... 

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