Papain immobilized

Papain, Immobilized on Euperglt C Origin Carica papaya Fluka... 

Lei H, Chen L, Li X et al. (2004) The preparation of catalytically active papain immobilized on magnetic composite microspheres. Enzyme Microb Technol 35 15-21 Lioy E, Suarez J, Guzman F et al. (2001) Synthesis, biologictil, tind immunologictil properties of cyclic peptides from Plasmodium falciparum merozoite surface protein-1. Angewtind Chem Intemat Ed 40(14) 2631-2635... 

The earhest example of a membrane-enzyme oscillator was presented by Na-parstek, Caplan, and coworkers [52]. This oscillator consists of papain immobilized in a porous collodion membrane that is permeable to water, substrate, and ions. The membrane is cast as a thin film against a pH electrode and is exposed to an alkaline (pH 10) external solution of benzyl arginine ethyl ester (BAEE), which is a substrate... 

Fig. 9. Experimental shift of the activity-external pH curve due to the feed-back action of pH-active products, (a) The product is a base immobilized urease (0-0) urease in stirred solution (x-x) substrate urea, product (NH4)2 CO3 (From Sel gny et al. [40].) (b) papain immobilized in a collodion membrane (J-J) papain in stirred solution ( ) substrate benzoyl-L-arginine ethyl ester / iC00R2->i iC00"+H++i 20H (From Goldman et al. [53] and Katchalski et al. In both cases concentrated buffer or grinding to powder of membranes strongly decreases the differences observed between soluble and insoluble enzymes. 

Wash 0.5 ml of immobilized papain (Thermo Fisher) with 4 X 2 ml of 20 mM sodium phosphate, 20mM cysteine-HCl, 10 mM EDTA, pH 6.2 (digestion buffer), and finally suspend the gel in 1.0 ml of digestion buffer. 

Several examples of the binding of enzymes to poly(vinyl alcohol) are in the literature. These could possibly be used to treat enzyme deficiency diseases. In a recent example, trypsin was immobilized on poly(vinyl alcohol) fibers using maleic dialdehyde or bromal. While the reaction was more complete with bromal, the reaction with maleic dialdehyde gave a better support which showed decreasing activity with increasing enzyme content. The activity of the bromal activated system was independant of the enzyme content (52 ). Trypsin and papain were attached to poly(vinyl alcohol) by the reaction sequence shown in Equation 13. In this case, the crosslinked poly(vinyl alcohol) is treated by the 1,3-dioxalone derivative and then converted to either the isothiocyanate or the diazonium salt for coupling with the enzyme. The bound enzymes showed significant, altho reduced, activity in each case (53). 

For the first time, the use of artificial enzyme membranes allows the study of the interaction between enzyme activity and membrane potential in a well-defined context. Before the recent progress in manufacturing artificial membrane bearing immobilized enzyme, Blumenthal et al.21 described a system in which a papain solution was sandwiched between two cation and anion exchange membranes. Under short-circuit conditions the system was able to generate a current. A nonequiLbrium thermodynamic analysis was developed by the authors. 

Dissolve 10 mg of human IgG solution in 1 ml of digestion buffer and add it to the immobilized papain gel suspension. 

Binding kinetics were determined using surface plasmon resonance. Human integrin a,/3i was immobilized on the sensor chip. Kd was calculated from koS/k0 . Clones 11, 7, 4, 24, and 2 represent five humanized LM609 versions. Fab were produced by E. coli except for mouse which was prepared from IgG by papain digestion. 

Figure 10-24 Effect of Immobilizing on the pH Optimum 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.

Coupling proteins (by covalent methods) to magnetic supports is the most common approach (105). Various classes of proteins, such as lectins (110) and antibodies (111), and a large number of enzymes including invertase (112,113), papain (106, 114) a-chymotrypsin (114, 115), P-galactosidase (114), adenylate kinase (112), acetate kinase (112), and horseradish peroxidase (112), have been immobilized. In fact, such supports are now commercially available, for example, Enzacryl FEO-M (available from Aldrich Chemictd Company, USA) and Dyna (sold by Dynal, Norway). 

Weetall HH and Mason RD. Studies on immobilized papain. Biotechnol. Bioeng. 1973 15 455M-66. 

Immobilized papain and other components for antibody fragment preparation and Iodogen are available from Pierce (Rockford, IL). 

For soluble and immobilized bromelain, temperature increase is accompanied by a decrease in residual enzyme activity. A more complex form of denaturation occurs with the immobilized enzyme, which may involve a two-phase process. Immobilization offers more resistance to denaturation at the higher temperature of 60°C where the second phase is prolonged by a factor of three [60]. Differential scanning calorimetry experiments showed that bromelain is an exceptional protease among the cysteine proteases, illustrated by the fact that its thermal denaturation is consistent with an irreversible two-state model [61]. Also, the far UV circular dichroism spectrum of bromelain differs from those of papain and chymopapain and therefore represents a third spectral class within the cysteine proteinase family [62],... 

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