Hindawi Publishing Corporation Enzyme Research Volume 2013, Article ID 670702, 6 pages http://dx.doi.org/10.1155/2013/670702 Research Article Purification and Characterization of Phenylalanine Ammonia Lyase from Trichosporon cutaneum Andrea Goldson-Barnaby 1,2 and Christine H. Scaman 1 1 Food, Nutrition, and Health, University of British Columbia, 2205 East Mall, Vancouver, BC, Canada V6T 1Z4 2 Department of Chemistry, University of the West Indies, Kingston, Jamaica Correspondence should be addressed to Christine H. Scaman; christine.scaman@ubc.ca Received 25 June 2013; Accepted 13 August 2013 Academic Editor: Qi-Zhuang Ye Copyright © 2013 A. Goldson-Barnaby and C. H. Scaman. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identiied an extensive intron region at the N-terminus. Five amino acid residues difering from a prior report were identiied. Highest Phe : Tyr activities (1.6 ± 0.3 : 0.4 ± 0.1 mol/h g wet weight) were induced by Tyr. he enzyme has a temperature optimum of 32 ∘ C and a pH optimum of 8–8.5 and shows no metal cofactor dependence. Michaelis- Menten kinetics (Phe,   5.0 ± 1.1 mM) and positive allostery (Tyr,   2.4 ± 0.6 mM, Hill coeicient 1.9 ± 0.5) were observed. Anion exchange chromatography gave a puriication fold of 50 with 20% yield. he His-Gln motif (substrate selectivity switch region) indicates the enzyme’s ability to act on both substrates. 1. Introduction Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) catalyzes the conversion of phenylalanine to trans-cinnamic acid, as a step in the phenylpropanoid pathway of plants and in the formation of secondary products of metabolism in some microorganisms [1, 2]. In some instances, the enzyme also converts tyrosine to para-hydroxycinnamic acid. hese dual substrate enzymes are classiied as phenylalanine/tyrosine ammonia lyases, (EC 4.3.1.25). Enzymes with a greater cat- alytic eiciency for tyrosine are known as tyrosine ammonia lyase (TAL, EC 4.3.1.23). here are no known genes that code for a lyase that has activity exclusively with tyrosine. Interest in PAL is from two perspectives. First, the structural features of the enzyme responsible for its substrate speciicity have not been fully elucidated. Second, a more selective and eicient TAL is of interest for industrial appli- cations. he speciicity of PAL for phenylalanine relative to tyrosine varies by over 10 6 between biological sources, and typically, the eiciency of Phe turnover is higher than Tyr [3, 4]. An understanding of the basis for this astounding range of substrate preference is required to rationally engineer an eicient tyrosine-speciic enzyme for use in the synthesis of p-hydroxycinnamic acid for industrial applications [5]. he microbial production of aromatic chemicals continues to increase as it allows for the use of greener technologies and renewable energy sources [6]. PAL has been extensively characterized from a wide variety of plant sources [7], but only a few microbial sources of the enzyme have been investigated [8, 9]. PAL from the yeast Trichosporon cutaneum (TcPAL), identiied as an enzyme able to metabolize both Phe and Tyr and possessing a relatively high level of activity with tyrosine, was selected as a model to further investigate the dual substrate activity of this family of enzymes. Several novel indings were noted and are reported herein. he cloned gene was found to have a single intron region near the N-terminus of the enzyme and ive amino acid residues that difered from a previous report [6]. A His-Gln motif was identiied which appears to be a characteristic feature of PAL enzymes displaying dual substrate activity with tyrosine and phenylalanine. his is the irst reported characterization of the TcPAL enzyme with regard to its temperature and pH optimum as well as metal dependence.