Preparation Of Cross-linked Aggregates Of Phenylalanine Ammonia Lyase From Rhodotrula Glutinis

Phenylalanine ammonia lyase(PAL) is a key enzyme in the manufacture ofL-phenylalanine by reversing the enzyme reaction. However, the production ofL-phenylalanine from trans-cinnamic acids was of limited success, partly because of therelatively low specific activity and instability of PAL during the bioconversion. In order toimprove activity and stability, in this study, the synthesis conditions of the PAL fromRhodotorula glutinis were optimized. Moreover, the cross-linked aggregates ofphenylalanine ammonia lyase(PAL-CLEAs) were prepared. In addtion, the properties ofthe PAL-CLEAs were also investigated.1) Optimization of the synthesis conditions of the PAL from Rhodotorula glutinis.Firstly, the effects of different culture conditions were investigated on PAL activity bythe approach of ‘‘one-variable-at-a-time”, including the carbon sources, nitrogen sourcesand salts, then, the optimal region of all factors were screened with Plackett-Burmandesign and the steepest ascent method. Moreover, the optimum enzyme inducer wasscreened in shake flask. Based on the above results, the optimizing experiments werecarried out in3L fermentor, including the initial glucose concentration, the fermentationpH value and the adding time of enzyme inducer.The optimal composition of the batch medium was1g/L glucose,5g/L NaCl,35g/Lpeptone,0.25g/L KH2PO4,1.5g/L (NH4)2HPO4; the optimum cultivation conditions wereinoculum5%, initial pH value5, controlled-pH of7.0; the optimum inducer wasL-phenylalanine, PAL activity was the most maximum when L-phenylalanine was addedat8h and26h. Under these optimum conditions, the specific PAL activity could attain40.85U/g, which was7.3-fold higher than that of original condition.2) Preparation of the cross-linked aggregates of phenylalanine ammonia lyase.The effect of preparation conditions were investigated, such as collection time ofRhodotorula glutinis, precipitant concentration, cross-linking agent concentration,cross-linking time and proteic feeder. The results showed that the addition of bovine serumalbumin had no significant effect on activity of PAL-CLEAs. The crude phenylalanineammonia lyase was extracted from the Rhodotorula glutinis cell collected at25h, thenprecipitated with40%ammonium sulfate saturation， finally cross-linked with0.1%glutaraldehyde for2h. Under these optimum conditions, the activity recovery of PAL-CLEAs could attain27%. 3) Discussion of the properties of the PAL-CLEAs.The temperature, pH, storage stability, acid-base stability, denaturant stability andreusability were discussed. The results were as follows: The optimum temperature and pHof CLEAs were at50℃and8respectively. The storage stability of PAL-CLEAs wasobviously better than free enzyme in room temperature, and the PAL-CLEAs had a higheractivity for14d at25℃whereas the free enzyme had almost inactivated. The acid-baseresistance of PAL-CLEAs was a little better than free enzyme, and the relatively activitywas6.67%higher than free enzyme at pH8-10. The denaturant resistance of PAL-CLEAswas also superior to free enzyme, and the relatively activity was10%higher than freeenzyme when urae and ethanol were used as denaturing agent. Additionally, thePAL-CLEAs had a good reusability, and the PAL-CLEAs had still retained50%of theinitial activity after seven consecutive cycles in batch transformation reaction.