Enzymatic solvent-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. 

Kamat, S. Critchley, G. Beckman, E. J. Russell, A. J. Biocatalytic Synthesis of Acrylates in Organic Solvents and Supercritical Fluids HI. Does Carbon Dioxide Covalently Modify Enzymes Biotechnol. Bioeng. 1995, 46, 610-620. Kamihira, M. Taniguchi, M. Kobayashi, T. Synthesis of Aspartame Precursors by Enzymatic Reaction in Supercritical Carbon Dioxide. Agric. Biol. Chem. 1987, 51, 3427-3428. 

Free or immobilized enzymes have been exploited already in a number of systems. Here, biocatalysis may take place in reversed micelles or in an aqueous phase in contact with an organic solvent. In a powdered state some enzymes are able to function in pure organic solvents. Furthermore, modified enzymes such as polymer bound enzymes or surfactant-coated enzymes have been developed so that they can solubilize in organic solvents to overcome diffusion limitation. The advantages of enzymatic reactions using organic solvents can be briefly summarized as follows ... 

Chapters are also included on yeast-mediated stereoselective biocatalysis, stereoselective synthesis of steroids, chemo-enzymatic synthesis of enantiopure arylpropionic acids, supercritical carbon dioxide as a solvent in enzyme catalysis, state-of-the-art techniques in enzyme immobilization, biocatalysis by polyethylene glycol-modified enzymes, and enzymatic deprotection techniques in organic synthesis. 

The experimental evidences that medium engineering might represent an efficient method to modify or improve enzyme selectivity (alternative to protein engineering and to the time-consuming search for new catalysts) were immediately matched by the search for a sound rationale of this phenomenon. The different hypotheses formulated to try to rationalize the effects of the solvent on enzymatic enantioselectivity can be grouped into three different classes. The first hypothesis suggests that... 

Lipase has been used in organic solvents to produce useful compounds. For example, Zark and Klibanov (8) reported wide applications of enzymes to esterification in preparing optically active alcohols and acids. Inada et al (9) synthesized polyethylene glycol-modified lipase, which was soluble in organic solvent and active for ester formation. These data reveal that lipases are very useful enzymes for the catalysis different types of reactions with rather wide substrate specificities. In this study, it was found that moditied lipase could also synthesize esters and various lipids in organic solvents. Chemically moditied lipases can help to solve today s problems in esteritication and hopefully make broader use of enzymatic reactions that are attractive to the industry. 

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. 

Recent research has demonstrated that enzymes can function in various low-water environments (1-4), and that the properties of enzymes can be favorably modified by nonaqueous solvents (5-7). Increased substrate solubility, reversal of hydrolytic reactions, and improved thermostability are among the practical advantages afforded by nonaqueous solvents. However, the role of water in enzymatic reactions at low water concentrations is not generally understood, and there is some uncertainty regarding how much the conformation of a protein changes as solvent water is removed (8.9). Nonaqueous solvent systems therefore represent important media in which to examine enzyme structure and function at the molecular level. Such studies will prove valuable for the application of enzymes in low-... 

Polyethylene glycol is used to make the enzymes soluble in organic solvents [88], and high reaction yields have been obtained with polyethylene-glycol-modified chymotrypsin [89], and papain in benzene [90], Enzymatic modification reactions with deacylation, via aminolysis, of an intermediate covalent acyl-enzyme also support the mechanism of transpeptidation in kinetically controlled peptide syn-... 

The activity of the native and modified SC was also studied for the reaction methanolysis of APEE, using the substrate, methanol, itself as a solvent. This was done for three different salt-enzyme preparations, consisting of 99% salt, 50% salt, and no added salt (except that present in buffer). It was found that the PEG modification resulted in a six- to sevenfold increase in the initial rates of conversion for the cases with no surfactant and with T 20 (Table 2). AOT was found to reduce the activity of the enzyme preparations in all experiments conducted in the solvent methanol. Previous reports on enzymatic conversion in organic solvents have shown the effect of the solvent dielectric constant on enzyme activity for salt-free enzymes [22], Relationship between activity of AOT and PEG-modified SC to the hydrophobicity coefficient of various solvents has also been studied [20], however, only for the enzymes without salt-lyophilization. The decrease in activity of enzymes in organic solvents is attributed to the decreased water availability in organic media. Additionally, as the dielectric constant increases, the potential for removal of the... 

Tlie usefnlness of enzymes is being enhanced by deploying them in organic solvents ratlier tlian aqneons media. In such solvent or solvent-water mixtiu es enzymes can catalyze reactions impossible in water, display greater stability and show behavior such as molecular memory. Enzymatic selectivity, and substrate-, stereo-, regio- and chemo-selectivity are affected and occasionally modified by the solvent. Enzyme reactions of industrial applicability have been demonstrated in organic solvents, supercritical fluids and the gaseous phase.[4 l... 

Zeolite membranes are amenable by surface modification with a variety of chemical functional groups using simple silane chemistry that may provide alternative surface chemistry pathways for enzyme immobilization. In this context, Shukla et al. [338] have recently used a chemically modified zeolite-clay composite membrane for the immobilization of porcine lipase using glutaraldehyde to provide a chemical linkage between the enzyme and the membrane. The effects of pH, temperature, and solvent on the performance of such biphasic zeolite-membrane reactors have been evaluated in the hydrolysis of olive oil to fatty acids. Similarly, Algieri et al. [339] have immobilized tyrosinase on FAU membranes for the enzymatic conversion of the 1-tyrosine to 1-DOPA as an effective drug for Parkinson s disease treatment. This approach combines the active role of zeolite membrane as enzyme support and inhibitor suppressor. Moreover,... 

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