百合鳞茎苯丙氨酸解氨酶的分离纯化及酶学性质研究

经40 %～75 %硫酸铵分级沉淀和DEAE-Sepharose离子交换层析，从兰州百合（Lilium davidii var. unicolor）鳞茎中分离纯化苯丙氨酸解氨酶（PAL），纯化倍数为13.19，酶回收率4.68%。纯化的PAL经 SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测为单一蛋白带，其亚基分子量约58.7 kD。酶学性质研究表明：百合鳞茎的PAL不耐碱，更不耐酸；随着水浴时间延长和水浴温度提高，PAL活性逐渐降低，但40 ℃水浴30 min后酶活性仍保留47%；以L-苯丙氨酸为底物，40 ℃下的Km值为4.1×10^-3 mol·L^-1；金属离子Mn²⁺、Fe²⁺、Cu²⁺、Fe³⁺促进PAL活性，1.0、1.0、2.0和1.0 mmol·L^-1分别为在供试范围内的最佳处理浓度，Ag⁺、Co²⁺、Ca²⁺抑制其活性，其中Ag+的抑制作用最强。

Separation and Purification as well as Properties of Phenylalanine Ammonia-lyase in Lily Bulbs

Phenylalanine ammonia-lyase (PAL) was isolated and purified from lily (Lilium davidii var. unicolor) bulbs via precipitating with ammonium sulphate grading from 40% to 75% and DEAE-Sepharose ion-exchange chromatography techniques. A 13.19-fold purification with a yield of 4.68% was obtained. The subunit molecular weight was estimated as 58.7 kD by SDS-PAGE. Results of studies on zymological properties indicated that PAL in the lily bulbs was not able to endure acid and alkali, especially the former. The PAL activity declined gradually along with the increasing of temperature and prolonging of bath duration, however it still remained 47% after 30 min at 40 ℃. This revealed that 40 ℃ was the optimum bath temperature. PAL had a Km of 4.1×10^-3 mol·L^-1 for L-Phenylalanine at 40 ℃. Ions such as Mn²⁺, Fe²⁺, Cu²⁺ and Fe³⁺ enhanced the PAL activity and 1.0, 1.0, 2.0 and 1.0 mmol·L^-1 were the most effective respectively within the tested concentrations. Ag⁺, Co²⁺ and Ca²⁺inhibited the PAL activity and the impact of Ag⁺ was the severest.