Modification Of Enzyme Activity,optimal PH And Stability Of Phenylalanine Ammonia-lyase From Anabaena Variabilis

Phenylalanine ammonia-lyase(PAL,EC: 4.3.1.5)catalyzes the non-oxidative liberation of ammonia from L-phenylalanine to generate trans-cinnamic acid and a small amount of ammonia;at the meantime,it catalyzes the reverse reaction under a certain condition.Comparing with PALs from other organisms,Anabaena variabilis derived PAL(AvPAL)has better stability and less body immunogenicity,so it has more therapeutic prospect;meanwhile,having a broad substrate spectrum indicates AvPAL has prospect in producting non-natural chiral amino acids.However,the optimal reaction pH of AvPAL is 8.5(the pH of human plasma is around 7.3-7.4),and its limited enzyme activity and body stability obstract its application.In this study,enzyme activity had been improved by modulating the Tyr78-loop,and the optimal reaction pH had been shifted to acidic direction by 1-1.5 unit by modifying surface amino acids which were near the active center.Furthermore,after sol-gel encapsulation,the body stability of AvPAL had been enhanced.The research results were reported as follows:(1)The enzymatic property of the MIO-depended(methylidene-imidazol-5-one)enzyme is significantly affected by the flexibility of the special loop which includes the catalysis Tyr,increasing flexibility of the loop could improve the deaminase activity of the MIO-depended enzyme.In order to improve the activity of AvPAL,Tyr78-loop was modulated according to the sequence alignment with the PALs from eukaryotes.The results revealed that the specific activities of S73 N,E95V,E95 K and S73N/E95 K were improved by 34%,30%,18% and 35% respectively,compared with that of the WT at 37?,pH 8.5.(2)Based on the online program GETAREA and the crystal structure,several sites which are near the general base Tyr78 were selected: Asn69,Glu72,Glu75,Asn89 and Val90.Removing negative charges or introducing positive ones near Tyr78 by mutation,the pH optima were successfully shifted.The pH optima of E75 A,E75L and E75 Q were shifted to 7.5 with 35%,30%,24% higher specific activities than that of the wild,respectively.Meanwhile,the half-lives of E75 L and E75 Q at 70? prolonged to 190 min and 180 min(from 130 min)respectively.In addition,when kept in pH 3.5 solution,WT almost lost its all activity in half an hour,but E75 L kept 55% of its initial activity even after 3 h incubation;after 1 h incubation in 300 ?g/m L trypsin solution,WT only held 44% initial actvity,while E75 L kept more than 70% after 3 h incubation.(3)An oral formulation of therapeutic for PKU would be highly desirable because of its convenience,however,free PAL has poor tolerance to the extreme environment in the intestine.After silica sol-gel encapsulated,PAL particles had sinificantly increased stability,which might be used in the oral treatment.In the pH 3.5 solution,the free AvPAL would lost its all activity in 30 minutes,however,the particle could still hold 80% of initial activity after 3 h incubation;when kept in 300 ?g/m L trypsin solution,the half-live of the free PAL was 45 min,while after 3 h incubation,the particle still hold more than 60% of the initial activity.The mutant enzyme particles had better stabiliy.After kept in pH 3.5 solution and 300 ?g/m L tripsin solution for 3 h,E75 Q nearly kept 100% and 90% of the initial activity,espectively.