Immobilization Of Ethyl Chrysanthemate Esterase And Its Application For Efficient Production Of (+)-trans-chrysanthemic Acid

Ethyl chrysanthemate esterase (ECE) with unique stereoselectivity for (+)-trans-ethyl chrysanthemate was excreted with recombinant cells. Effects of pH and temperature on the activity and stability of free ECE and reactive conditions of the stereoselective hydrolysis of ethyl chrysanthemate to produce optically active (+)-trans-chrysanthemic acid were studied in this work.The results indicated that the ECE activity was higher within pH9.0～10.5 and the best pH was 10, and this ECE was considered as one alkaline enzyme. The ECE activity was higher at a wide range of temperature (35～55° ) and the best temperature was 45° .The optimal hydrolysis conditions were: pH 10.0, temperature 50° , 48hours. Under these conditions, the content of (+)-trans- chrysanthemic acid is 97% with a conversion rate of 100%.Immobilization of ECE was important for enzyme application in industry. The results showed that immobilized ECE on DEAE cellulose had higher activity and better stability in operation than other carriers.The optimial operation conditions were: 1g DEAE cellulose treated with 2% glutaraldehyde for 2 hours, then 20ml ECE immobilized on the carrier for 6 hours. The optimal temperature and pH were 50℃ and 10.5, respectively .The stability of immobilized ECE on pH, temperature and operation were improved.The reaction conditions on the stereoselective hydrolysis of ethyl chrysanthemate by immobilized ECE were examined in this work. The results showed that the ECE activity was inhibited by substrate and product to some extent. The reaction conversion yield and initial velocity decreased while substrate concentration was higher than 4% or product concentration higher than 10%. The maxmum conversion yield occurred at 5% ECE consumed.Further increase of enzyme quantity did not lead to better results.At last one enzyme reactor was used for the purification of (+)-trans-chrysanthemate acid. Conversion yield and enantiomer excess of product can fulfil the need of industrialization in packed-bed reactor. Content of (+)-trans-chrysanthemate acid is 97.2% after purification.