Enzymatic synthesis using chemically

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... 

To make these substrates suitable for biological assays, the introduction of functional groups that can be traced with the proper analytical techniques is essential. The use of radio-, fluorescent-, and biotin-labeled lipidated peptides has been reported. The synthesis of fluorescent substrates is chemically straightforward and allows for production of larger quantities than the enzymatic synthesis used for radiolabeled peptides and is thus preferred over the use of radioactive compounds. [1 21] Common fluorescent probes can be introduced by conjugation to a free functional group present in the peptide. The fluorescent moiety is... 

There are several methods commonly used throughout the carbohydrate field for the production of complex carbohydrates. The three most utilized methods for accessing carbohydrates for biological studies are purification from natural sources, enzymatic synthesis, and chemical synthesis. Purification from natural sources is extremely difficult and time consuming due to the... 

Four methods are known for porphobilinogen (PBG) production (i) isolation from human or animal urine (ii) chemical synthesis from 5-aminolevulinic acid (ALA) (iii) enzymatic synthesis using ALA dehydratase (the enzyme is isolated from erythrocytes, P. shermanii or Rhodopseudomonas sphaeroides) and (iv) microbiological production using heat-treated cells of P. shermanii, incubated in a medium containing ALA. 

In addition to their in vivo function, these cofactors have important in vitro functions as co-factors in enzymatic synthesis. Because these co-factors are too expensive to be used in equimolar amounts, many methods have been developed for their regeneration. These include chemical (66), biological (67) and electrochemical (68) methods. En2ymatic regeneration has found particular utihty in this appHcation (69). 

T. Furiuke, R. Sadamoto, K. Niikura, K. Monde, N. Sakairi, and S.-I. Nishimura, Chemical and enzymatic synthesis of glycocluster having seven sialyl lewis X arrays using /i-cyclodextrin as a key scaffold material, Tetrahedron, 61 (2005) 1737-1742. 

This section describes the synthesis of oxazolidine esters used as polymer hardeners that cannot be synthesized using chemical catalysis, the synthesis of polyurethane polymers with methods that avoid the use of isocyanates and the enzymatic synthesis of polyesters with low molecular weight dispersity. 

One of the best examples of the utility of enzymatic synthesis in catalyzing reactions that cannot be accomplished by any other route is the synthesis of substituted oxazolidine diesters. The oxazolidine ring is extremely water sensitive, the oxazolidine rapidly reverting back to the alkanolamine and aldehyde in the presence of water. Bis-oxazolidines have been used as hardeners for polymer coatings but the diester based on the hydroxyethyl oxazolidine and adipic acid cannot be synthesized directly with chemical catalysis because of the rapid rate of reaction of the oxazolidine ring with either the water from the esterification or the alcohol from transesterification. ... 

Initial reports on chemoenzymatic block copolymer synthesis focus on the enzymatic macroinitiation from chemically obtained hydroxy-functional polymers (route A in Fig. 4). The first report on enzymatic macroinitiation was published by Kumar et ah, who used anionically synthesized hydroxy-functional polybutadiene of various molecular weights ranging from 2600 to 19,000Da (Fig. 5) [16]. In a systematic study, the authors investigated the efficiency of the macroinitiation of CL and PDF by Novozym 435 as a function of the polybutadiene macroinitiator. The reaction profile showed that polybutadiene consumption steadily increased with the reaction... 

Esters are common components in cosmetics and skin-care products. They can be synthesized from fatty acids and alcohols using either chemical or enzymatic reactions. The chemical reactions are normally catalysed by acid catalysts. Enzymatic synthesis is carried out under milder conditions and therefore it provides products of very high purity. A range of esters such as isopropyl palmitate and isopropyl myristate are now produced industrially using enzymatic synthesis. The reactions are carried out in solvent-free systems using an immobilised lipase as catalyst. In order to get high yields in the reactions, water is removed continuously. 

Enzymatic synthesis relying on the use of aldolases offers several advantages. As opposed to chemical aldolization, aldolases usually catalyze a stereoselective aldol reaction under mild conditions there is no need for protection of functional groups and no cofactors are required. Moreover, whereas high specificity is reported for the donor substrate, broad flexibility toward the acceptor is generally observed. Finally, aldolases herein discussed do not use phosphorylated substrates, contrary to phosphoenolpyruvate-dependent aldolases involved in vivo in the biosynthetic pathway, such as KDO synthetase or DAHP synthetase [18,19]. 

Initial preparative work with oxynitrilases in neutral aqueous solution [517, 518] was hampered by the fact that under these reaction conditions the enzymatic addition has to compete with a spontaneous chemical reaction which limits enantioselectivity. Major improvements in optical purity of cyanohydrins were achieved by conducting the addition under acidic conditions to suppress the uncatalyzed side reaction [519], or by switching to a water immiscible organic solvent as the reaction medium [520], preferably diisopropyl ether. For the latter case, the enzymes are readily immobilized by physical adsorption onto cellulose. A continuous process has been developed for chiral cyanohydrin synthesis using an enzyme membrane reactor [61]. Acetone cyanhydrin can replace the highly toxic hydrocyanic acid as the cyanide source [521], Inexpensive defatted almond meal has been found to be a convenient substitute for the purified (R)-oxynitrilase without sacrificing enantioselectivity [522-524], Similarly, lyophilized and powered Sorghum bicolor shoots have been successfully tested as an alternative source for the purified (S)-oxynitrilase [525],... 

In order to try to overcome some of the problems associated with chemical synthesis of oligosaccharides containing N-acetylneuraminic acid, Sabesan and Paulson [277] have used a combination of chemical and enzymatic methods using purified sialyl-transferases in the presence of CMP-iV-acetylneuraminic acid and synthetic acceptor molecules to give sialyl derivatives of oligosaccharides which were characterised by NMR. Thus, methyl P-D-galactopyranoside, methyl P-D-lactoside and iV-acetyl-... 

GDP-ot-D-mannose (23) is the donor substrate for mannosyltransferases [139, 146, 338-340] and the precursor of GDP-(3-L-fucose (13) [173,197, 243, 341], Based on the work of Munch-Petersen [342, 343], only crude extracts from yeast have been used for the enzymatic synthesis of labeled and unlabeled 23 and GDP-deoxymannose derivatives (Table 4) [303-305, 307, 308, 344-346] as well as for the in situ regeneration of 23 (Table 4). Common to all these approaches is the use of chemically synthesized sugar-1-phosphates as substrates for GDP-Man PP. An obvious disadvantage of using crude yeast enzyme preparations is the poor quality of the enzyme source since only fresh cells or certain batches of baker s yeast are suitable for synthesis [304, 307], GDP-Man PP was purified from pig liver and used for the synthesis of 8-Azido-GDP-Man however, the enzyme lacks absolute specificity for GDP-Man in the pyrophos-phorylysis reaction [309]. 

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