Enzymes Aspartame synthesis

ENZYME-CATALYZED SYNTHESIS OF Z-ASPARTAME IN IONIC LIQUID... 

A commercially viable preparation of the ester of human insulin (35) has been developed using a trypsin-mediated exchange of a threonine residue for the terminal alanyl residue of porcine insulin (Scheme 3.22). This transformation was the first example of an enzymic semi-synthesis of a protein for use in medicine. (See Section 6.9.2 for further details on the production of Aspartame and insulin.)... 

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

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. 

Invertcd-Aspartame-Type Sweetener, Ac-Phe-Lys-OH, was synthesized in one step in the organic-aqueous solvent media. Maximum yield was 10%. But considering collection of enzyme and raw materials, we think that this synthesis is able to be industrialized. We also think that this modified technique can be applied to the preparation of another modified enzyme. There are a few small problems in these two methods. But we hope that these two methods will be made full use as synthetic strategies of flavor peptides or the other functional peptides. 

The food industry is a fertile area for biocatalysis applications high-fructose corn syrup (HFCS) from glucose with glucose isomerase, the thermolysin-catalyzed synthesis of the artificial sweetener Aspartame , hydrolysis of lactose for lactose-intolerant consumers, and the synthesis of the nutraceutical i-camitine in a two-enzyme system from "ybutyrobetaine all serve as examples. 

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. 

Peptide synthesis is an extremely important area of chemistry for the pharmaceutical industry, and like any specialized area of chemistry, has its own set of unique problems associated with it. Racemization and purification of final products are two of the most difficult problems in this area. The use of enzymes has been explored as a possible answer to these problems since 1938 [29]. However, proteases needed to catalyze peptide synthesis are subject to rapid autolysis under the conditions needed to affect peptide coupling, so this has generally not been a practical approach until cross-linked enzyme crystals of proteases became available. The synthetic utility of protease-CLCs was demonstrated by the thermolysin CLC (PeptiCLEC -TR)-catalyzed preparation of the aspartame precursor Z-... 

Various commercial routes for the production of L-phenyalanine have been developed because of the utilization of this amino acid in the dipeptide sweetener Aspartame. One route that has been actively pursued is the synthesis of L-phenylalanine from trans-cinnamic acid using the enzyme phenylalanine ammonia lyase (105,106). This enzyme catalyzes the reversible, nonoxidative deamination of L-phenylalanine and can be isolated from various plant and microbial sources (107,108). 

L-aspartic acid ammonia lyase, or aspartase (E.C. 4.3.1.1) is used on a commercial scale by Kyowa Hakko, Mitsubishi, Tanabe and DSM to produce L-aspartic acid, which is used as a building block for the sweetener Aspartame, as a general acidulant and as a chiral building block for synthesis of active ingrediants[1]. The reaction is performed with enzyme preparations from E. coli, Brevibacterium jlavum or other coryneform bacteria either as permeabilized whole cells or as isolated, immobilized enzymes. The process is carried out under an excess of ammonia to drive the reaction equilibrium from fumaric acid (1) in the direction of L-aspartic acid (l-2) (see Scheme 12.6-1) and results in a product of excellent quality (over 99.9% e.e.) at a yield of practically 100%. The process is carried out on a multi-thousand ton scale by the diverse producers of L-aspartic acid. Site directed mutagenesis of aspartase from E. coli by introduction of a Cys430Trp mutation has resulted in significant activation and stabilization of the enzyme P1. 

L-Phe-OCH3 is the by-product formation of p-aspartame. This isomer is of bitter taste and has to be completely removed from the a-isomer. The advantages of the enzymatic route are (i) No P-isomer is produced, (ii) the enzyme is completely stereoselective, so that racemic mixtures of the substrate or the appropiate enantiomer of the amino acid can be used, (iii) no racemization occurs during synthesis and (iv) the reaction takes place in aqueous media under mild conditions. 

Proteases - Proteases have been applied to several synthetic purposes the most Important of which are peptide bond synthesis and protein semisynthesis. Recent extensive reviews cover this area. Prote-ases have been used in ester synthesis and resolution. Semisynthesis of human insulin has been achieved by enzymic removal and replacement of one amino acid in porcine insulin. All peptide bonds in the N-termlnal octapeptide of dynorphln [H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-OH] have been formed using proteases.A precursor of aspartame has been made by themolysin-catalyzed condensation of benzyloxycarbonyl-L-aspartic acid and L-phenylalanlne methyl ester. 

The Eprs of B. subtilis and related bacilli are also very relevant for commercial applications. In particular, these enzymes are employed in the manufacture of detergents, tanning of leather, management of industrial and household wastes, bioprocessing of X-ray or photographic films for the recovery of silver, protein hydrolysate preparation in the food industry, synthesis of aspartame, and other applications [102]. Recently, the fibrinolytic activity of Vpr was discovered. Accordingly, Vpr has the potential to work as a thrombolytic agent in medical applications [103, 104]. 

Y. Bono, K. Fukushima, G. Araya, H. Nabetani, and M. Nakajima, Performance of perstractive enzyme reactor for synthesis of aspartame precursor, J. Chem. Technol. Biotechnol. 70 (1997) 171-178. 

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