PEG-chymotrypsin

Matsushima et al. [26] succeeded in the formation of acid-amide bonds by the catalytic action of PEG-chymotrypsin in benzene, such as benzoyl tyrosine butylamide and benzoyl tyrosine-oligophenylalanine ethyl ester. 

PEG-proteases used were abbreviated as follows CHY, PEG-chymotrypsin PAP, PEG-papain PEP, PEG-pepsin THR, PEG-thermolysin TRY, PEG-tr5 sin. 

Gaertner et al. [48] investigated peptide bond formation catalyzed by PEG-proteases in benzene. The substrate specificity of PEG-chymotrypsin for ester hydrolytic activity in aqueous solution was almost the same as that of unmodified enzyme [51] both PEG-modified and unmodified chymotrypsins catalyzed the hydrolysis of aromatic amino acid esters but these enzymes did not hydrolyze the basic amino acid ester. In the case of dipeptide synthesis in benzene, however, not only Bz-iyr-Phe-NHa but also Bz-Lys-Phe-NH2 was synthesized from corresponding amino acid derivatives by PEG-chymotrypsin. The change in the substrate specificity, which has been reported in a few cases, might be due to a surrounding environment on the enzyme molecule. 

Sinisterra et al. [85] reported the stereoselective hydrolysis of racemic A-benzoyl-phenylalanine methyl ester catalyzed by PEG-chymotrypsin in aqueous methanolic solution. The hydrolyzed products, A -benzoylphenylalanine, obtained by PEG-chymotrypsin as well as unmodified enzyme were extracted from the reaction mixture and analyzed by both polarimetry and proton nuclear magnetic resonance spectrophotometry. The enantiomeric ratios of the products were 98 2 and 50 50 (S R) for PEG-enzyme and unmodified enzyme, respectively. In this case, chymotrypsin acquired enantioselectivity by the chemical modification witii PEG. 

The influence of the structure of the main chain on the chymotrypsin catalyzed release of p-nitroaniline is shown in Fig. 15. Oligopeptide p-nitroanili-des were attached to HPMA copolymers (as side-chains) and to polyethylene glycol (end-point attachment). From the values of kcat/KM it is evident that all oligopeptide p-nitroanilides attached to PEG were cleaved faster than those attached to HPMA copolymers. It appeared that the PEG substrates fit better into the active site of chymotrypsin due to the linearity and flexibility of the PEG molecule, and the type of spacer attachments [255]. 

The lyophilization of enzymes from solutions containing salts or amphiphilic compounds is known to increase the activity in organic media by up to several orders of magnitude. Thus, the transesterification activity of a-chymotrypsin was increased 82-fold by co-lyophilization with pentaglyme [75]. The colyophilization of lipases and (poly ethylene)glycol (PEG) led to an enhanced transesterification activity in various ionic liquids [76, 77]. 

Figure 2. Predicted protein partition coefficient versus PEG molecular weight for lysozyme, chymotrypsin, albumin and catalase. Dextran molecular weight is 23,000 and polymer composition is PEG 6% Dx 8%.

Amino Acids and Peptides. - Wasserman s method of one-carbon homologation of carboxylic acids to give a-ketocarboxylates involves reaction with cyanomethylenetriphenyl-phosphorane followed by ozone (Scheme 24) and has been used as a key step in a chemo-enzymatic synthesis of isotopically labelled L-valine, L-isoleucine, and o/fo-isoleucine. Alkylation of the carbanion derived from the imino-substituted methylphosphonate diphenyl ester (186) with indol-3-ylmethyl bromide followed by appropriate deprotection has been used to prepare the phosphonate analogue (187) of tryptophan (Scheme 25). The deprotected analogue (188) and derived peptides show activity as inhibitors of chymotrypsin. Two approaches to solid phase Wadsworth-Enunons reactions which have applications in combinatorial chemistry have been reported. In one diethylphosphonoacetamide is bound to PEG-PAL resin via a peptide link, while... 

Fig. 9 Separations of five standard protein samples by mixer-settler HSCCC. Experimental conditions are as follows apparatus type-J coil planet centrifuge with 10 cm revolution radius column a mixer-settler spiral disk assembly consisting of eight barricaded disks with a 160 ml capacity A, solvent system 12.5% (w/w) PEG-1000 and 12.5% (w/w) K2HPO4 mobile phase lower phase sample five proteins each 5-6 mg in 1 ml of each phase, cytochrome c (K = 0.02), myoglobin (K = 0.59), ovalbumin (K = 1.26), lysozyme (K = 1.69), and bovine serum albumin (K = 1.95) flow rate 0.25 ml/min rpm 800 detection 280 nm stationary phase retention 52% B, solvent system PEGIOOO/K2HPO4/KH2PO4/H2O (16 8.3 4.2 71.5, w/w) sample cytochrome c (5 mg, K = 0.035), human serum albumin (20 mg, K = 0.4), 3-lactoglobulin (20 mg, K = 0.69), a-chymotrypsin (20 mg, K = 1.2), and trypsinogen (20 mg, = 2.1) in 2 ml of each phase flow rate 0.5 ml/min rpm 1000 stationary phase retention 53.6%.

As far as the protein extraction is concerned, this field has received increased attention in recent years. Dahurun and Cussler [35] studied protein extraction in membrane contactors under various experimental conditions. Solutions of cytochrome-c, myoglobin, a-chymotrypsin, catalase, and urease in phosphate buffer were extracted using an immiscible aqueous phase, polyethylene glycol (PEG). Also, membrane-based adsorption processes have been commercialized for the separation and recovery of proteins as a replacement for a packed column of adsorbent beads. Often such processes are called membrane chromatography [36a] or adsorptive membrane... 

Various proteases such as chymotrypsin [26,48], papain [49], subtilisin [50,51], ther-molysin [52], and trypsin [51,53] have been coupled with PEG derivatives for the purpose of peptide synthesis in organic solvents. 

Parallel approaches have been described for the preparation of polyacrylate-protease conjugates [396-400]. Acryloylation of subtilisin and a-chymotrypsin, followed by mixed polymerization with methyl methacrylate, vinyl acetate, styrene, or ethylvinyl ether, provides insoluble, doped polymethyl methacrylate, polyvinyl acetate, polystyrene, and polyethyl vinyl ether polymers [396]. These biocatalytic plastics perform especially well in hydrophilic and hydrophobic solvents, and have been used for peptide synthesis and the regioselective acylation of sugars and nucleosides. Similarly, modification of subtilisin and thermolysin with PEG monomethacrylate, then copolymerization with methyl methacrylate and trimethylolpropane trimethacrylate furnishes protease-polymethyl methacrylate plastics, which show good activities and stabilities in aqueous, mixed, and low-water and anhydrous organic media [397-400]. The protein-acrylate composites are unique in that they enable catalytic densities as high as 50% w/w. 

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