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

1988; Wiley; Volume: 32; Issue: 5 Linguagem: Inglês

10.1002/bit.260320504

1097-0290

Chin‐Pin Yang, Chein‐Shyong Su,

Biopolymer Synthesis and Applications

Abstract Cross‐linked polyurethane (PU) was prepared for entrapping thermolysin. Using the immobilized thermolysin (IT), Z‐ L ‐aspartic acid (ZA) was reacted with ‐L phenylalanine methyl ester ( L ‐PM) in water‐saturated ethyl acetate to give only α‐Z‐ L ‐aspartyl L ‐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.