[Objective]To explore the phenylalanine ammonia-lyase(EC 4.3.1.24;PAL)with high activity and stability,and to lay a foundation for the subsequent preparation of special dietary foods with no(low)phenylalanine.[Method]The genes RmPAL and RdPAL were cloned from Rhodotorula mucilaginosa and Rhodotorula diobovata,respectively,and the sequence and structural features of the two enzymes were analyzed by bioinformatics.The two enzymes were heterologously expressed and purified in Escherichia coli,and the optimal reaction conditions and substrate specificity were determined.In addition,the ability of RmPAL and RdPAL to convert L-Phe in casein acid hydrolysate(CAH)was determined by high-performance liquid chromatography(HPLC)and phenylalanine assay kit.[Result]The fungal-derived RmPAL and RdPAL contained three domains:the MIO domain,the core domain,and the shielding domain,with the catalytic amino acid Tyr and the substrate-specific amino acid His in the active pocket.Both RmPAL and RdPAL existed as tetramers in solution.The optimal pH and temperature of the two enzymes were 8.9 and 50℃,respectively,and they demonstrated broad pH and temperature stability,which was superior to commercial PALs derived from Rhodotorula.Furthermore,both enzymes catalyzed L-Phe and L-Tyr,with a higher catalytic efficiency towards L-Phe,approximately five times that of L-Tyr.The conversion rates of L-Phe removal from CAH were 88%and 93%for RmPAL and RdPAL,respectively.[Conclusion]Rm PAL and Rd PAL have strong stability,L-Phe hydrolysis preference,and ability to remove L-Phe from food-derived protein.
关键词
苯丙酮尿症/苯丙氨酸解氨酶/低L-Phe蛋白/催化活性/热稳定性/L-Phe转化率/特医食品
Key words
phenylketonuria/phenylalanine ammonia-lyase/low L-Phe protein/catalytic activity/thermal stability/conversion rates of L-Phe/specialized medical foods
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