Enzymatic chemically modified enzymes

For the purpose of synthesizing flavor peptides or proteins in large scale, we developed "protein recombination method" and "enzymatic synthesis using chemically modified enzyme". "Protein recombination method" was applied to the synthesis of C-terminal portion of p-casein and its analog. Chymotrypsin was chemically modified by Z-DSP in aqueous solution. It was stable for organic solvents. Using this modified enzyme, we succeeded in the synfiiesis of Inverted-Aspartame-Type Sweetener "Ac-Phe-Lys-OH" in one step. 

Today, it is well-known that peptides or proteins exhibit various kinds of taste. Our group has been researching on the relationship between taste and structure of peptides, BPIa (Bitter peptide la, Arg-Gly-Pro-Pro-Phe-Ile-Val) (7 as a bitter peptide, Om-p-Ala-HCl (OBA), Om-Tau-HCl as salty peptides(2j, and "Inverted-Aspartame-Type Sweetener" (Ac-Phe-Lys-OH) as a sweet peptide(5). The relationship between taste and chemical structure was partly made clear. Since commercial demand for these flavor peptides is increasing, we need to develop new synthetic methods which can prepare these peptides in large scale. We developed the following two methods (1) protein recombination method as a chemical method, (2) enzymatic synthesis using chemically modified enzyme as a biochemical method. 

Enzymatic Synthesis Using Chemically Modified Enzyme... 

Regioselective enzymatic acylation of large, insoluble polysaccharides is still a quite difficult task and therefore it is not surprising that only scant data have been reported up to now, most of them describing reaction outcomes which met with limited success. Nevertheless, enzymatic derivatization of polysaccharides has been performed in nonpolar organic solvents using insoluble polysaccharides with soluble [51] or suspended enzymes [52]. Chemically modified celluloses with either enhanced solubility or more readily accessible hydroxyl groups, like cellulose acetate or hydroxypropyl cellulose, were acylated by CalB, as reported by Sereti and coworkers [53]. However, the same authors failed to modify crystalline cellulose under the same reaction conditions. 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

All life processes are the result of enzyme activity. In fact, life itself, whether plant or animal, involves a complex network of enzymatic reactions. An enzyme is a protein that is synthesized in a living cell. It catalyzes a thermodynamically possible reaction so that the rate of the reaction is compatible with the numerous biochemical processes essential for the growth and maintenance of a cell. The synthesis of an enzyme thus is under tight metabolic regulations and controls that can be genetically or environmentally manipulated sometimes to cause the overproduction of an enzyme by the cell. An enzyme, like chemical catalysts, in no way modifies the equilibrium constant or the free energy change of a reaction. 

Chloramphenicol (9) is liable to breakdown by chloramphenicol acetyl-transferases [185]. Fluoro derivatives (57, 58) resist enzymatic attack but little has been heard of these, apparently because of their toxicity [319], Aminoglycoside antibiotics (AGACs) may be chemically modified by AMEs. Some derivatives (e.g. amikacin, 43) are more recalcitrant than others, e.g. kanamycin (42) (see Figure 4.2). Other enzyme-resistant AGACs of low toxicity are needed. 

Sol-gel matrices can also provide a chemical surrounding that favors enzymatic reactions. Lipases act on ester bonds and are able to hydrolyze fats and oils into fatty acids and glycerol. These are interphase-active enzymes with lipophilic domains and the catalytic times reaction occurs at the water-lipid interface. Entrapped lipases can be almost 100 times more active when a chemically modified silica matrix is used. The cohydrolysis of Si(OMe)4 and RSi(OMe)3 precursors provides alkyl groups that offer a lipophihc environment that can interact with the active site of Upases and increase their catalytic activity. Such entrapped lipases are now commercially available and offer new possibilities for organic syntheses, food industry, and oil processing. ... 

An illustration of this approach may be seen in the studies on streptococcal proteinase (Liu 1967). The activity of this enzyme is dependent upon the presence of a free sulfhydryl group. The active form of the enzyme was first converted to the inactive S-sulfenyl-sulfonate derivative. Treatment of this derivative with a chemically-reactive substrate "analogue, a-N-bromoacetylarginine methyl ester, resulted in the alkylation of a single histidine residue. The sulfhydryl group of the modified enzyme was regenerated by reduction, however, this did not restore enzymatic activity, thus providing presumptive evidence for the involvement of both a cysteinyl and a histidyl residue in the active site of this enzyme. 

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