Alkaline phytase from Lilium longiflorum:: Purification and structural characterization
Alkaline phytase from Lilium longiflorum:: Purification and structural characterization
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DOI：
10.1016/j.pep.2005.07.031
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发表时间：
2006-04-01
影响因子：
1.6
通讯作者：
Murthy, PPN
中科院分区：
文献类型：
--
作者：
Garchow, BG;Jog, SP;Murthy, PPN
植酸酶催化植酸(肌醇六磷酸)的水解，植酸是细胞中含量最丰富的肌醇磷酸。植酸酶具有很大的商业价值，因为它们作为食品和动物饲料补充剂已被许多国家批准，以缓解环境和营养问题。虽然酸性植酸酶已被广泛研究，但有关碱性植酸酶的信息有限。植物中的碱性植酸酶具有独特的催化性质，但其纯化和结构性质尚未见报道。本文介绍了从植物组织中分离纯化碱性植酸酶的方法。由于受到非特异性磷酸酶和酸性植酸酶的污染以及内源浓度较低，纯化过程具有挑战性。从长花百合花粉中分离纯化碱性植酸酶的方法包括加热选择性沉淀、硫酸铵沉淀、阴离子交换层析、聚焦层析和凝胶电泳法。碱性植酸酶的纯化倍数接近3000倍，总回收率为4.2%。弗格森图法测得该酶的天然相对分子质量为118+/-7 kDa，SDS-PAGE测得变性蛋白的M-r为52-55 kDa，表明该酶为同源二聚体。二维凝胶分离和基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱分析表明，分离的蛋白质存在多种质量和电荷异构体，等电点在7.3到8.3之间。据我们所知，这是第一个从植物中提纯的碱性植酸酶。这种独特的性质表明，该酶有可能用作饲料和食品补充剂。(C)2005 Elsevier Inc.保留所有权利。
Phytases catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytases are of great commercial importance because their use as food and animal feed supplement has been approved by many countries to alleviate environmental and nutritional problems. Although acid phytases have been extensively studied, information regarding alkaline phytases is limited. Alkaline phytases with unique catalytic properties have been identified in plants, however, there is no report on the purification or structural properties. In this paper, we describe the purification of alkaline phytase from plant tissue. The purification was challenging because of contamination from non-specific phosphatases and acid phytases and low endogenous concentration. The purification of alkaline phytase from pollen grains of Lilium longiflorum involved selective precipitation by heat and ammonium sulfate followed by anion exchange and chromatofocusing chromatography and, finally, gel electrophoresis. Alkaline phytase was purified similar to 3000-fold with an overall recovery of 4.2%. The native molecular mass was estimated to be in the range of 118 +/- 7 kDa by Ferguson plot analysis and M-r of denatured protein in the range of 52-55 kDa by SDS-PAGE suggesting that the enzyme is a homodimer. Separation by 2-D gel and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis of separated proteins indicates the presence of multiple mass and charge isoforms with pI values between 7.3 and 8.3. To our knowledge, this is the first alkaline phytase to be purified from plant sources. The unique properties suggest that the enzyme has the potential to be useful as a feed and food supplement. (c) 2005 Elsevier Inc. All rights reserved.