Biochemical characterization and recombinant expression of alkaline phytase from Lilium longiflorum

• Main Author: Jog, Sonali P
• Format: Dissertation
• Language: English
• Subjects: biology, plant physiology; chemistry, biochemistry
• ISBN: 0542438631; 9780542438639

Phytases are the primary enzymes responsible for the hydrolysis of phytic acid. Phytic acid (InSP6, myo-inositol hexakisphosphate) is the most abundant inositol phosphate in cells; in cereal grains and legumes it constitutes 3-5% of the dry cell weight of seeds. The inability of monogastric animals such as swine and poultry to absorb complexed phytic acid has led to nutritional and environmental problems. To alleviate the detrimental affects of high concentrations of phytate, swine and poultry feed is supplemented by phytases. The efficacy of phytases in addressing the environmental and nutritional problems has led to increased use in agriculture (half a billion dollar market) and the need for phytases with a range of biochemical and biophysical properties for different target applications.
This dissertation describes the biochemical characterization, purification, and recombinant expression of alkaline phytase from Lilium longiflorum . The enzyme exhibits narrow substrate specificity, it hydrolyzed InSP6 and para-nitrophenyl phosphate (pNPP). Alkaline phytase followed Michaelis-Menten kinetics with a K m of 81 muM and Vmax of 217 nmol Pi/min/mg with InSP6 and a Km of 372 muM and Vmax of 1272 nmol Pi/min/mg with pNPP. The pH optimum was 8.0 with InSP6 as substrate and 7.0 with pNPP. Alkaline phytase was activated by calcium and inactivated by ethylenediaminetetraacetic acid; however, the enzyme retained a low level of activity even in Ca2+-free medium. Fluoride as well as myo-inositol hexasulfate did not have any inhibitory affect, whereas vanadate inhibited the enzyme. The enzyme was activated by sodium chloride and potassium chloride and inactivated by magnesium chloride; the activation by salts followed the Hofmeister series.
The purification of alkaline phytase from lily pollen involved selective precipitation by heat and ammonium sulfate followed by anion exchange and chromatofocusing chromatography and, finally, gel electrophoresis. Alkaline phytase was purified ∼3000-fold with an overall recovery of 4.2%. The native molar mass was estimated to be in the range of 118 +/- 7 kDa by Ferguson plot analysis and Mr of the denatured protein in the range of 52-55 kDa by SDS-PAGE, suggesting that the enzyme is a homodimer.
cDNAs encoding alkaline phytase isoforms, Llalp1 and Llalp2 were recently cloned in our laboratory. Recombinant alkaline phytase (LlALP2) was expressed in two E. coli host strains, BL21(DE3) and Origami(TM)2. In BL21(DE3) cells, the recombinant alkaline phytase activity accumulated in inclusion bodies, whereas, in Origamj(TM)2 the activity was present in the soluble form (18%) as well as in inclusion bodies (82%). The total yield of LlALP2 in Origami(TM)2 cells (8.8 U L-1) was a third that in BL21(DE3) (24 U L-1). Biochemical characteristics of the recombinant alkaline phytase in inclusion bodies and the soluble fraction were similar to the wild-type alkaline phytase from lily pollen. Although recombinant proteins in inclusion bodies are generally inactive, recombinant LlALP2 is a rare example of the presence of enzymatically active enzyme in inclusion bodies.