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

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Abstract
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 α‐Z‐L‐aspartylL‐phenylalanine methyl ester (α‐ZAPM). Ninety‐four percent conversion of α‐ZAPM was obtained for 30 h of reaction at 40°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.