Papain stability

Many enzymes need a certain ionic strength to maintain an optimum stabiHty and solubiHty, eg, bacterial a-amylases show optimal stabiHty in the presence of 1—2% NaCl. Some enzymes may need certain cations in low amounts for stabilization, eg, Ca " is known to stabilize subtiHsins and many bacterial a-amylases. Antioxidants (qv) such as sodium sulfite can stabilize cysteine-containing enzymes which, like papain, are often easily oxidized. 

Next we evaluate the PDLD + EVB surface for the enzymatic reaction using eq. (5.17). The resulting surface is shown in Fig. 5.6. As seen from the ligure, the protein can reduce Aby stabilizing the ionic state more than water. In fact, in the specific case of papain the protein inverts the stabilization of the covalent and ionic states relative to their order in solution. 

The liquid-liquid interface has been identified as the major factor responsible for papain deactivation in a biphasic system [66]. If the interfacial tension can be decreased to a small value using surfactant, the biocatalyst stability will be expected to increase. 

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

Resistance to peptidases was also reported for the octapeptoid 6.106 when incubated with papain, chymotrypsin, or thermolysin [229], However, resistance to peptidases may not be synonymous with a long half-life in vivo, since many factors beside peptidases can be expected to contribute to the elimination of peptoids. An indirect indication of this effect can be found for antimicrobial peptoids and particularly compound CHIR29498 (6.107) [232], In mice infected with Staphyllococus aureus, this peptoid was less active when injected 2 h post-infection compared to 0 h or 0.5 h. The conclusion drawn by the authors was that the compound requires optimization for improved absorption or stability within the body. 

Plant Proteases. These include the well known proteases papain, bromelain and ficin. Most plant enzymes are available as comparatively unpurified powder extracts, although papain is notable for being available in a stabilized and purified liquid form. Prospects for increased supply of plant enzymes, in response to greater use in traditional applications or for new processes, depend on several factors. Tlie influence of cultivation conditions, growth cycle and climate requirements make new supplies long term projects. 

Pepsin and papain hydrolysates of rapeseed protein concentrate increased foam volumes and decreased drainage compared to the untreated control (36). Foaming properties could be further enhanced by adding a stabilizer such as carboxymethylcellulose. 

Figure 12-15 Schematic drawing of the active site of a cysteine protease of the papain family with a partial structure of an acyl-enzyme intermediate in green. The thiolate-imidazolium pair of Cys 25 His 159 lies deep in the substrate-binding cleft and bridges an interface between two major structural domains, just as the Ser His pair does in serine proteases (Fig. 12-10). This may facilitate small conformational changes during the catalytic cycle. Asn 175 provides a polarizable acceptor for positive charge, helping to stabilize the preformed ion pair, and allows easy transfer of an imidazolium proton to the product of substrate cleavage. The peptide NH of Cys 25 and the side chain of Gin 19 form an oxyanion hole.

Due to the relative stability of the niacin vitamers, either acid or alkaline hydrolysis can be used to convert nicotinamide to nicotinic acid for quantitation of both vitamers as nicotinic acid (9,44). Acid hydrolysis is used to quantitate biologically available niacin. Alkaline hydrolysis releases both the biologically available and the unavailable vitamers and provides an estimate of the total niacin content. Because alkaline hydrolysis is much faster than acid hydrolysis, the latter is usually supplemented with enzymatic hydrolysis. The most common enzymes are takadiastase, papain, and clarase. On occasion, organic solvents such as methanol have been used to extract free nicotinic acid. 

It was shown <1999BML3255> that a crystalline ozonide obtained by ozonolysis of the A-allylamidc of Cbz-L-phenylalanine inhibits papain, a cysteine protease. Reduction of that ozonide in excess dimethyl sulfoxide (DMSO) generates in situ a peptide aldehyde, as proved by coupling with a stabilized Wittig ylide forming thereby an unsaturated ester. 

The gelatin sample before the papain treatment gave the following data whippability, 1.70 foam stability, 0.90 and emulsifying activity, 0.00. 

More recently, covalent chemical modification has been used as a powerful tool to enhance the functionality and stability of enzymes, for example, the covalent link of flavin to papain turned a protease into an oxido-reductase [107]. The use of this methodology was rekindled as a result of the explosion in the interest in commercial and synthetic applications of enzymes [108]. As a consequence, enzymes with new properties such as stability at extreme pH conditions, temperature, or solubility in organic solvents are being generated. 

Figure 10-25 Effect of Immobilizing of the Thermal Stability of Papain. Source From H.H. Weetall, Immobilized Enzymes and Their Application in the Food and Beverage Industry, Process Biochem., Vol. 10, pp. 3-6, 1975.

The dyed proteins, though also not very cheap, are much more suitable from an economical point of view. The use of insoluble dyed proteins (e.g. hide powder azure) is very simple and handy. After the chosen reaction time the insoluble protein is removed by filtration or centrifugation and the absorbance of the filtrate is immediately measured. For instance, Rinderknecht proved that it was possible to determine trypsin activity with HPA (hide powder azure) in ng quantities per ml and proteolytic activities in biological materials, tissue extracts, serum, urine, faeces, etc. Other authors applied this substrate for assaying proteolytic activity in beer stabilized with chillproofing preparations containing proteases (mainly papain)... 

Some of the basic information on stabilizing sulfhydryl enzymes, has been responsible for their commercialization. Without the judicious use of reducing compounds throughout the processing of the papaya latex, it would not have been possible to maximize the proteolytic activity of commercial papain preparations. Examples of other studies of enzymes which have contributed to commercialization are the determination of calcium ion as a requirement for amylase stability at high temperature, the difference in properties of catalases derived from bacterial, fungal, or... 

---