Chymotrypsin immobilized

Solvent Freely suspended chymotrypsin Immobilized chymotrypsin Ratio1"1... 

Pallavicini et al. (16) utilized a-chymotrypsin immobilized on chitin to catalyze plastein formation from leaf protein hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by soluble enzymes. Modification of Specific Functional Properties... 

Fig. 8 Photoswitchable hydrolysis of20 bya-chymotrypsin immobilized in photoisomerizable acrylamide copolymers. (A) a-Chymotrypsin immobilized in copolymer 17 (a) and (c) hydrolysis of 20 in the presence of 17a copolymer, (b) and (d) hydrolysis of20 in the presence...

Because the local pH at the surface of the support is lower than the bulk, or measured pH, the apparent pH optimum shifts to higher pH values with this and other negatively charged support materials. Figure 4.4 shows that chymotrypsin immobilized onto an ethylene-maleic anhydride support exhibits a pH optimum of 9.4, almost one full pH unit higher than that observed for the native enzyme. 

A practical demonstration of the usefulness of calorimetric sensors for work in different media was made in a study on immobilized a-chymotrypsin which was used for hjdrolysis of peptide bonds in 0.05 M Tris-HCl, pH 7.8, containing 10% DMF and for syntheses of pepude bonds in 50% DMF + 50% 0.1 M Na borate, pH 10.0. With the a-chymotrypsin immobilized in the enzyme thermistor column both reactions could be followed, the hydrolysis giving an exothermic response while the synthetic route was endothermic [32]. 

Kato et al. [54,55] reported using the proteases papain, pronase E, and chymotrypsin for the deamidation of food proteins, and Kato et al. [56] later reported deamidation of selected plant and animal proteins with chymotrypsin immobilized on controlled pore glass. The reactions were carried out at alkaline pH (pH 10) and 20°C and resulted in 5 to 20% deamidation with up to 8% peptide bond hydrolysis. Functional properties of proteins thus deamidated showed increased solubility and emulsifying and foaming properties. Enzymatic... 

Similar behaviors were observed for the LPL and chymotrypsin immobilized onto the PAM microspheres with and without spacer. 

Conclusions and Topics for Future Research 323 Table 18.3. Activities of trypsin and a-chymotrypsin immobilized by binding to various supports... 

The proteolytic enzymes, trypsin, chymotrypsin, and chymoral [8076-22-0] in combination, have been used for the treatment of post-operative hand trauma, athletic injuries, and sciatica (214—216). Trypsin has also been used successfully in treating hyaline membrane disease of newborn babies, a condition usually fatal without treatment (217). Immobilized preparations of trypsin are useful in treating acute radiation cystitis following pelvic x-irradiation therapy (218). 

For desymmetrization of diesters 3 via their hydrolysis in water, pig Hver esterase [12], o -chymotrypsin [12, 13a], and Candida antarctica Hpase (CAL-B) [14] were successfully used. However, further studies showed that respective anhydrides 5 can be used as substrates for enzyme-catalyzed desymmetrization in organic solvents [15]. The desired monoesters 4 were obtained in high yield in this way, using immobilized enzymes Novozym 435 or Chirazyme L-2 (Scheme 5.3). After the reaction, enzymes were filtered off, organic solvents were evaporated, and the crude products were crystalHzed. This was a much simpler experimental procedure in which control of the reaction progress was not necessary, and aU problems associated with extraction of products from aqueous phase and their further purification were omitted [15]. 

A series of enzyme and proteins (met-myoglobin, lysozyme, met-hemoglobin, glucose oxidase, a-chymotrypsin) was also immobilized in a-ZrP by Kumar et al. [134]. Binding constant values clearly confirm the high affinity of the various proteins with the host structure (Table 15.4). 

RME shows particular promise in the recovery of proteins/enzymes [12-14]. In the past two decades, the potential of RME in the separation of biological macromolecules has been demonstrated [15-20]. RMs have also been used as media for hosting enzymatic reactions [21-23]. Martinek et al. [24] were the first to demonstrate the catalytic activity of a-chymotrypsin in RMs of bis (2-ethyl-hexyl) sodium sulfosuccinate (Aerosol-OT or AOT) in octane. Since then, many enzymes have been solubilized and studied for their activity in RMs. Other important applications of RME include tertiary oil recovery [25], extraction of metals from raw ores [26], and in drug delivery [27]. Application of RMs/mi-croemulsions/surfactant emulsions were recognized as a simple and highly effective method for enzyme immobilization for carrying out several enzymatic transformations [28-31]. Recently, Scheper and coworkers have provided a detailed account on the emulsion immobiUzed enzymes in an exhaustive review [32]. 

Fadnavis, N. W. and Luisi, P. L. (1989). Immobilized enzymes in reverse micelles studies with gel-entrapped Trypsin and alpha-Chymotrypsin in AOT reverse micelles. Biotechnol. Bioeng., 33, 1277-82. 

Immobilization of enzymes to solid supports can increase activity over a wide range of solvents [78]. As seen in Table 3.2, the transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) with 1-propanol by a-chymotrypsin (Scheme 3.2) immobilized to glass is 1-2 orders of magnitude higher than that of the free, lyophilized enzyme. 

Table 3.2 Values of (kc /Km)app (M 1 min-1) x 100 for freely suspended and glass-immobilized a-chymotrypsin in various solvents. In all solvent conditions, the enzyme is 1-2 orders of magnitude more active than the free, lyophilized enzyme [78].

The separation of enantiomers can be effected either by transforming them into diastereoisomers using a chiral reagent and separating them on conventional phases or by separating the enantiomers on chiral phases. The utilization of chiral phases has not yet become routine, but studies of enantiomeric dipeptides have been carried out (115,116). Pirkle et al. (117) and Hyun et al. (118) separated enantiomeric di- and tripeptides (methyl esters of /V-3-5-dinitrobenzoyl derivatives) on chiral stationary phases (CSPs) derived from (R)-a-arylalkylamines, (S)-N-(2-naphthyl) valine, or (S)-1 -(6,7-dimethyl-1 -naphthyl) isobutylamine. These workers were able to separate four peaks for each dipeptide derivative, corresponding to the two enantiomeric pairs (R,R)/(S,S) and (R,S)/(S,R). Cyclodextrin-bonded stationary phases and chiral stationary immobilized a-chymotrypsin phases were used to separate enantiomeric peptides (118a,b). 

Wainer et al. [77] presented a CSP based on a-chymotrypsin protein, and, initially, the chiral resolution of certain amino acids and amino ester was achieved on this protein CSP [14,77]. Later, this CSP was used for the chiral resolution of dipeptides and profens [78,79]. Recently, Felix and Descorps [80,81] used immobilized a-chymotrypsin for the chiral resolution of a variety of racemic compounds. Cellobiohydrolase-I (CBH-I) immobilized to silica gel was found to... 

Immobilization in a sol-gel matrix accelerated the propanolysis of N-acetyl-i-phenylalanine ethyl ester in cyclohexane for several serine proteases compared to the non-immobilized lyophilized enzymes 31-fold for Subtilisin Carlsberg, 43-fold for a-chymotrypsin, and 437-fold for trypsin (van Unen, 2001). The activity yield upon immobilization was 90% (a-chymotrypsin). The rate enhancement effect of immobilization on the enzyme activity is highest in hydrophobic solvents. 

Scheme 6 Photoswitching of a-chymotrypsin through its immobilization in photoisomerizable polymers.

The biocatalyst a-chymotrypsin s ability to hydrolyze 20 is inhibited in the presence of copolymer 19a loaded with 0.2 mol% of the triphenyl carbinol units. 47b Photoirradiation of 19a results in heterolytic bond cleavage and the formation of the cationic copolymer 19b. In this polymer structure, the biocatalyzed hydrolysis of 20 is activated (V = 1.0 pM min-1). The polymer-induced photostimulated activation and deactivation of a-chymotrypsin in the different membrane environments correlates with the permeability and transport properties of the substrate 20 through the different structures of the polymer membranes.1471 Flow dialysis experiments showed that the polymer states 17a, 18a, and 19a are nonpermeable to 20, and hence the biocata-lytic functions of the immobilized enzyme are blocked. The polymer structures 17b,... 

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