Optimization of oligomeric enzyme activity in ionic liquids using Rhodotorula glutinis yeast phenylalanine ammonia lyase

- Phenylalanine ammonia lyase (PAL) enzyme activity of Rhodotorula glutinis has been demonstrated in four commonly used ionic liquids.
- Enzyme activity of oligomeric PAL protein in the reaction mixture containing 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) is comparable to that obtained in aqueous buffer medium.
- Various conditions were standardized for optimal PAL forward activity in the reaction mixture containing [BMIM][PF6]; about 83% conversion of L-phenylalanine to trans-cinnamic acid was obtained.
- PAL reverse activity in ionic liquids was shown for the first time; about 59% conversion of trans-cinnamic acid to L-phenylalanine was obtained in the reaction mixture containing [BMIM][PF6].

Phenylalanine ammonia lyase (E.C.4.3.1.24, PAL) activity of Rhodotorula glutinis yeast has been demonstrated in four commonly used ionic liquids. PAL forward reaction was carried out in 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO4]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium lactate ([BMIM][lactate]). Our experiments have revealed that PAL is catalytically active in ionic liquids and the enzyme activity in ([BMIM][PF6]) is comparable to that obtained in aqueous buffer medium. Different conditions were optimized for maximal PAL forward activity including time of incubation (30.0 min) L-phenylalanine substrate concentration (30.0 mM), nature of buffer (50.0 mM Tris-HCl), pH (9.0), temperature (37 °C), and speed of agitation (100 rev min−1). Under these optimized conditions, about 83% conversion of substrate to product was obtained for the PAL forward reaction that was determined using UV spectroscopy at 290 nm. PAL reverse reaction in ([BMIM][PF6]) was determined spectrophotometrically at 520 nm; and about 59% substrate conversion was obtained. This data provides further knowledge in enzyme biocatalysis in non-aqueous media, and may be of importance when studying the function of other oligomeric/multimeric proteins and enzymes in ionic liquids.