Alkaline phytase from Lilium longiflorum: purification and structural characterization.
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 approximately 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 Mr 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.
Garchow
Barry G
BG
Department of Chemistry, Michigan Technological University, Houghton, MI 49931, USA.
Jog
Sonali P
SP
Mehta
Bakul Dhagat
BD
Monosso
Jodi M
JM
Murthy
Pushpalatha P N
PP
D010832:6-Phytase / Q000737:chemistry* / Q000302:isolation & purification*; D000821:Animal Feed; D002853:Chromatography, Liquid / Q000379:methods; D019587:Dietary Supplements; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D027762:Lilium / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D010940:Plant Proteins; D010832:6-Phytase