The Catalytic Characteristics Of Phenylalanine Ammonialyase From Rhodotorula Mucilaginosa In Systems Containing Ionic Liquids

Ionic liquids are a liquid salt compound at room temperature. Ionic liquids have been widely developed and used in many fields because of many advantages. Especially, ionic liquids are attented in biocatalysis. Successful application of ionic liquids from a broad range of papain, lipase, cellulose, β-glucosidase and so on have been reported. However, to date, it has not been reported in catalytic application of PAL. In this study, effects of ionic liquids on PAL activity were studied in the most optimal ionic liquids screened by eight different ones.1) The partial purification of PAL in Rhodotorula mucilaginosaRhodotorula mucilaginosa cell was disrupted by glass beads, and the target protein was precipitated by ammonium sulfate with 40% saturation degree. And the salt was moved by dialysis. After concentration by PEG20 000, then the protein solution was run through Sepharose 4B gel column and DEAE-Sepharose FF ion-exchange column. The resultant protein solution was checked by SDS-PAGE. The results showed that the high purification factor was 22.71 and enzyme recovery 35.5%. SDS-PAGE revealed that PAL was patially purified.2) The screening of ionic liquidEffects of eight different concentration ionic liquids on PAL, such as 1-ethyl-3-methylimidazolium tetrafluoroborate([EMIM]BF4), 1-propyl-3-methylimidazolium tetrafluoroborate([PMIM]BF4), 1-butyl-3-methylimidazolium tetrafluoroborate([BMIM]BF4), 1-butyl-3-methylimidazolium hexafluorophosphate(BMIM]PF6), 1-allyl-3-methylimidazolium tetrafluoroborate([PtMIM]BF4), l-allyl- 3-methylimidazolium hexafluorophosphate([PtMIM]PF6), 1-hexyl-3-methylimidazolium tetrafluoroborate([HMIM]BF4), 1-hexyl-3-methylimidazolium hexafluorophosphate([HMIM]PF6) was studied. The reults showed that PAL activity was increased by 33.5% compared with that of aqueous phase.3) The effect of [HMIM]PF6 on the activity and stability of PALUsing single factor method, the optimal reaction conditions, stability and kinetic parameters of PAL was studied in [HMIM]PF6. The results showed that the optimal temperature and pH of PAL in 55% [HMIM]PF6 are 40 and 7, ℃ respectively, which have significant changes compared with that of aqueous phase. Moreover, the tolerance for high emperature and extrame pH were improved in compasion with that of aqueous phase. However, the stability against the denaturants was similar with that of aqueous phase. Furhtermore, the michaelis constant Km was lower compared to that of aqueous phase, meanwhile, the maximum reaction rate νmax was increased, which indicating apparent affinity of PAL towards the substrates was increased.