Synthesis Of Aspartame Precursor Research Articles

Application of membrane phase separation for enzymatic synthesis of aspartame precursor in biphasic reaction system

N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZAPM), an aspartame precursor, was synthesized enzymatically from N-(benzyloxycarbonyl)-L-aspartic acid (ZA) and L-phenylalanine methyl ester (PM) using a biphasic reaction system. Measurement of the partition coefficient in an aqueous/alcohol system showed that, 1-hexanol and 1-heptanol were proper solvents for the system. The membrane phase separation could be performed using a hydrophobic membrane, and 1-hexanol gave higher flux than 1-heptanol. Integration of the membrane phase separation into the biphasic enzyme reaction system was realized by the continuous stable ZAPM production.

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A preliminary study was made on the feasibility of using papain as a catalyst in the synthesis of aspartame precursor (Z-Asp-OH + Phe-OMe→Z-Asp-Phe-OMe). The synthetic reaction was carried out by an equilibrium-controlled process using three different systems: precipitation, dissolved-state, and biphasic. A comparative study was made with thermolysin-catalyzed aspartame precursor synthesis in each reaction system. Papain was useful only when the biphasic system was employed for synthesis, while thermolysin was efficient in all the systems tested. Thermolysin appeared to be superior to papain even in the biphasic system; the reason for this was ascribed to the absence of esterase activity. The yield of Z-Asp-Phe-OMe in the biphasic system was increased when papain was used in a reaction mixture containing high concentrations of the starting materials. The likely reason for this is discussed.

Performance of Perstractive Enzyme Reactor for Synthesis of Aspartame Precursor

A perstractive enzyme reactor was used for the synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZAPM), the precursor of the artificial sweetener, aspartame. The synthesis of ZAPM in the reactor proceeded by an enzymatic reaction between N-(benzyloxycarbonyl)-L-aspartic acid (ZA) and L-phenylalanine methyl ester (PM) in the aqueous phase. The synthesized ZAPM in the aqueous phase was mainly extracted into the organic phase, therefore, the concentration of ZAPM in the aqueous phase could be kept low. As a result, high conversion of ZAPM was obtained with this system. The partition coefficients of substances in the aqueous/butyl acetate biphasic system, the mass transfer coefficients of substances through the membrane and the enzymatic kinetics of ZAPM synthesis were determined experimentally. The reaction model which was based on the material mass balance equations was discussed to estimate the performance of the perstractive enzyme reactor system. The calculation values using the model and the experimental data showed good agreement with the concentration changes of the substances in the system.

Synthesis of aspartame precursor with an immobilized thermolysin in mixed organic solvents

N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester, a precursor of the synthetic sweetener, aspartame, was synthesized from N-(benzyloxycarbonyl)-L-aspartic acid and L-phenylalanine methyl ester with an immobilized thermolysin (EC 3.4.24.4) in the mixed organic solvent system of tert-amyl alcohol and ethyl acetate. A mixed solvent consisting of tert-amyl alcohol and ethyl acetate at a ratio of 33:67 (v/v) was found to be the most suitable with respect to synthetic rate and stability of the immobilized enzyme. The reaction continued to proceed quite successfully in a column reactor at 40 degrees C and at a space velocity of 3.6 h(-1) with a yield of 99%, using 40 mM Z-Asp and 200 mM PheOMe dissolved in the mixed solvent as the substrate. (c) 1995 John Wiley & Sons, Inc.

Hollow-Fibre Enzyme Reactor Integrated with Solvent Extraction for Synthesis of Aspartame Precursor

Hollow-Fibre Enzyme Reactor Integrated with Solvent Extraction for Synthesis of Aspartame Precursor

Synthesis of aspartame precursor with an immobilized thermolysin in tert‐amyl alcohol

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetner asparatame, was synthesized from N-(benzyloxycarbolyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) with an immobilized thermolysin in various organic solvents. We found that in tert-amyl alcohol containing a small amount of water the immobilized enzyme showed a high activity comparble to that in ethyl acetate with quite a high stability. The immobilized enzyme was fully stable up to 70 degrees C in tert-amyl alcohol in the absence of the subatrate, and up to 50 degrees C in the presence of the substrate. The high stability in the presence of the substrate was found due to the fact that the release of calcium ions, the stabilizing factor of thermolysin, is suppressed.The substrate concentration dependence of the initial synthetic rate with the immobilized enzyme was quite different from that with the free enzyme in the biphasic system, in contrast to that in ethyl acetate. Finally, Z-AspPheOMe was continuously synthesized in a column reactor using 200 mM PheOMe and 120 mM Z-Asp as the substrate for over 300 h at 45 degrees C and a space velocity of 1 h(-1) without any loss of acivity. (c) 1994 John Wiley & Sons, Inc.

Synthesis of aspartame precursor: α‐L‐aspartyl‐L‐phenylalanine methyl ester in ethyl acetate using thermolysin entrapped in polyurethane

Cross-linked polyurethane (PU) was prepared for entrapping thermolysin. Using the immobilized thermolysin (IT), Z-L-aspartic acid (ZA) was reacted with -Lphenylalanine methyl ester (L-PM) in water-saturated ethyl acetate to give only alpha-Z-L-aspartylL-phenylalanine methyl ester (alpha-ZAPM). Ninety-four percent conversion of alpha-ZAPM was obtained for 30 h of reaction at 40 degrees C when 46 mg of enzyme was entrapped. PU support prepared from polypropylene glycol (#2000) showed better properties than from polypropylene (#1000) and polyethylene (#1000). Addition of polyol could increase the gel fraction of PU. The IT PU-ll-G-3, prepared from 1/2 mole ratio of PPG (#2000)/glycerin, gave the highest gel fraction and best swelling, and 89.0% of residual activity was obtained after four times of reuse (72 h). The stability of immobilized thermolysin was good; the activity loss resulting from degradatin and leak of enzyme in each time of reuse were found only about 2%. The kinetics of immobilized thermolysin-catalyzed condensation reaction of ZA with L-PM in water-saturated ethyl acetate was found to be first order in L-PM and the Lineweaver-Burk plot of 1/V against 1/[ZA] yields a straight line, showing that the reaction involves consecutive reactions of ZA and L-PM with the immobilized enzyme and with the ZA-immobilized enzyme complex, with the second reaction being the rate determining step.

Synthesis of aspartame precursor by solid thermolysin in organic solvent

The enzymatic synthesis of a peptide compound was carried out successfully in homogeneous organic solvent.