| Rhodotorula glutinis CIBAS A1401 was isolated from a mango garden in Dianxi by researcher Yang Shunkai of Chengdu Institute of Biology, CAS. Due to its relatively high phenylalanine ammonia-lyase activity, this strain of yeast is now applied in factories to synthesize L-Phe. To make use of it more effectively and to enhance the activity and stability of PAL, we purified PAL from R. glutinis CIBAS A1401 and studied its basic properties.After initially being activated by the wort medium, R. glutinis CIBAS A1401 was cultured in the inducing medium through two steps, lasting 48 hrs and 21 hrs respectively. According to the PAL activity, the No. 37 strain was selected as the experimental strain. Then after the two-step fermentation, the cells were collected in 50mL centrifuge tubes with screw cap.In the next step, the target protein was precipitated between 40% and 65% ammonium sulfate saturation, and the salt was removed by being run through the redissolved precipitate in Sephadex G25 column. After concentration by PEG, the protein solution was run through Sepharose 4B filtration column and DEAE-Sepharose Fast Flow ion-exchange column. The final protein solution was checked on SDS-PAGE with one band, which proved that the enzyme was purified to homogeneity.The protein concentration and PAL activity of the protein solution were measured after each step of purification; the specific activity, fold and yield of each step were calculated. In the end, a 139-fold purification with a yield of 12.6% was obtained. Protein concentration was measured in terms of the Lowry method using BSA as the standard; PAL activity was measured by check the A278 of the reaction system, which is the characteristic wavelength of the product t-Ca.Incubating the purified enzyme and the substrate L-Phe at different pH level and different temperatures, we found the optimum pH and temperature for R. glutinis CIBAS A1401 PAL to convert L-Phe to t-Ca is pH8.5, 40℃. At the same time, we found R. glutinis CIBAS A1401 PAL remains stable in pH8.0~9.5 at 40℃. Furthermore, the special stability of this enzyme was demonstrated by the fact that for two weeks its activity decreased less than 40% & 20% at 4℃ & -20℃ without any protectors or preservatives.Based on the Lineweaver-Burk plot, the enzyme has a Km of 3.87 × 10-4mol/L for L-Phenylalanine at pH 8.5, 40℃. When the molecular weight of the subunit was measured through SDS-PAGE, one protein band was observed after staining with coomassie blue R-250. The molecular weight of the subunit was measured as 79.4kDa by the Rf of the subunit according to the standard line obtained from the Rf of the standard proteins. In addition, it was reported that PAL was made up of 4 subunits in the same size. So we can estimate that the molecular weight of R glutinis CIBAS A1401 PAL is about 320 kDa.When the activity was measured in the chloride systems with different metal ions, the result indicate that except Mg2+ & Na+, most of the metal ions inhibit its activity, such as Fe3+, Cu2+, Co2+and Hg2+.The homogenous PAL and its properties were obtained through this experiment, which laid a foundation for the future study of PAL amino acid sequence and the relationship of PAL structure & function. Accordingly, we can increase the output and quality of L-Phe by modifying PAL to enhance its activity and stability.
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