百合鳞茎苯丙氨酸解氨酶的分离纯化及酶学性质研究

园艺学报 ›› 2008, Vol. 35 ›› Issue (11): 1653-1660.

百合鳞茎苯丙氨酸解氨酶的分离纯化及酶学性质研究

孙红梅*,赵爽,王春夏,王锦霞,赵波,陈丽静

（沈阳农业大学园艺学院，辽宁省设施园艺重点实验室，沈阳110161）

收稿日期:2008-09-01 修回日期:2008-11-03 出版日期:2008-11-25 发布日期:2008-11-25
通讯作者: 孙红梅

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

SUN Hong-mei*;ZHAO Shuang;WANG Chun-xia;WANG Jin-xia;ZHAO Bo;and CHEN Li-jing

(Key Laboratory of Protected Horticulture of Liaoning Province，College of Horticulture, Shenyang Agricultural University, Shenyang 110161, China)

Received:2008-09-01 Revised:2008-11-03 Online:2008-11-25 Published:2008-11-25
Contact: SUN Hong-mei

摘要/Abstract

摘要： 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。

关键词: 兰州百合, 鳞茎, 苯丙氨酸解氨酶, 纯化, 酶学性质

Abstract: 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.

Key words: Lilium davidii var. unicolor, bulb, PAL, purification, zymological properties

中图分类号:

S 682.2

孙红梅;赵爽;王春夏;王锦霞;赵波;陈丽静. 百合鳞茎苯丙氨酸解氨酶的分离纯化及酶学性质研究[J]. 园艺学报, 2008, 35(11): 1653-1660.

SUN Hong-mei;ZHAO Shuang;WANG Chun-xia;WANG Jin-xia;ZHAO Bo;and CHEN Li-jing. Separation and Purification as well as Properties of Phenylalanine Ammonia-lyase in Lily Bulbs[J]. ACTA HORTICULTURAE SINICA, 2008, 35(11): 1653-1660.

[1]	吕学思，张栋，闵芮涵，李世琦，李铮，任梓铭，夏宜平. 外源蔗糖对离体条件下换锦花小鳞茎发生的影响[J]. 园艺学报, 2020, 47(8): 1475-1489.
[2]	许俊旭1，李青竹1，李叶2，杨柳燕1，李心1，王桢1，张永春1,*. 石蒜鳞茎膨大过程中内源激素相关基因的差异表达研究[J]. 园艺学报, 2020, 47(6): 1126-1140.
[3]	董玉惠1，王立霞1，顾启玉1，刘中良2，孙秀东1，刘世琦1,*. 利用转录组测序分析外源GA3解除大蒜气生鳞茎休眠相关基因[J]. 园艺学报, 2019, 46(12): 2335-2346.
[4]	王春夏，尹玥，王志平，严瑞，付麟岚，孙红梅*. 多倍化兰州百合和细叶百合组培苗再生和耐非生物胁迫能力[J]. 园艺学报, 2019, 46(12): 2359-2368.
[5]	孙红梅1,2,*，付麟岚1，王志平1，盖美竹1，王春夏1,2. 基于体细胞胚发生的细叶百合和兰州百合多倍体诱导及鉴定[J]. 园艺学报, 2018, 45(6): 1136-1146.
[6]	刘芳, 王家艳, 周蕴薇. 细叶百合休眠与解除SSH文库构建及其调控基因的初步筛选[J]. 园艺学报, 2014, 41(7): 1476-1483.
[7]	闫伟明1，孟焕文1，钟杨权威2，程智慧1,*. 紫皮大蒜鳞茎外皮花青苷生物合成影响因素和相关酶的研究[J]. 园艺学报, 2014, 41(2): 285-292.
[8]	靳志勇, 王硕硕, 刘娜, 巩彪, 刘世琦, 史庆华. 不同补光时间和方式对设施大蒜鳞茎膨大和品质的影响[J]. 园艺学报, 2014, 41(12): 2411-2418.
[9]	刘芳1,2，王家艳1，王晓丽3，周蕴薇1,*. 细叶百合鳞茎在低温解除休眠过程中茎尖细胞超微结构的变化[J]. 园艺学报, 2013, 40(6): 1110-.
[10]	石庆华, 刘平, 刘孟军. 果树倍性育种研究进展[J]. 园艺学报, 2012, 39(9): 1639-1654.
[11]	孙红梅*, 周兰娟, 王文娟, 袁思施, 王春夏. 百合鳞茎淀粉磷酸化酶分离纯化及酶学性质研究[J]. 园艺学报, 2012, 39(8): 1521-.
[12]	刘金, 魏景立, 刘美艳, 宋杨, 冯守千, 王传增, 陈学森. 早熟苹果花青苷积累与其相关酶活性及乙烯生成之间的关系[J]. 园艺学报, 2012, 39(7): 1235-.
[13]	朱继英, 王健, 王相友. 双孢蘑菇多酚氧化酶的分离、纯化及特性分析[J]. 园艺学报, 2012, 39(4): 763-768.
[14]	孙红梅;何玲;王微微;王春夏;李天来 . 兰州百合鳞茎可溶性酸性转化酶活性检测体系的建立 [J]. 园艺学报, 2011, 38(6): 1197-1204.
[15]	陈昆;刘世琦;张自坤;张涛;孟凡鲁 . 钾素对水培大蒜生理和品质的影响 [J]. 园艺学报, 2011, 38(3): 556-562.