Heterologous expression and functional characterization of a plant alkaline phytase in Pichia pastoris.
Phytases catalyze the sequential hydrolysis of phytic acid (myo-insositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytic acid constitutes 3-5% of the dry weight of cereal grains and legumes such as corn and soybean. The high concentration of phytates in animal feed and the inability of non-ruminant animals such as swine and poultry to digest phytates leads to phosphate contamination of soil and water bodies. The supplementation of animal feed with phytases results in increased bioavailability to animals and decreased environmental contamination. Therefore, phytases are of great commercial importance. Phytases with a range of properties are needed to address the specific digestive needs of different animals. Alkaline phytase (LlALP1 and LlALP2) which possess unique catalytic properties that have the potential to be useful as feed and food supplement has been identified in lily pollen. Substantial quantities of alkaline phytase are needed for animal feed studies. In this paper, we report the heterologous expression of LlALP2 from lily pollen in Pichia pastoris. The expression of recombinant LlALP2 (rLlALP2) was optimized by varying the cDNA coding for LlALP2, host strain and growth conditions. The catalytic properties of recombinant LlALP2 were investigated extensively (substrate specificity, pH- and temperature dependence, and the effect of Ca(2+), EDTA and inhibitors) and found to be very similar to that of the native LlALP2 indicating that rLlALP2 from P. pastoris can serve as a potential source for structural and animal feed studies.
Johnson
Steven C
SC
Department of Chemistry, Michigan Technological University, Houghton, MI 49931, USA.
Yang
Mimi
M
Murthy
Pushpalatha P N
PP
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors / Q000378:metabolism; D027762:Lilium / Q000201:enzymology*; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D011058:Pollen / Q000201:enzymology; D017510:Protein Folding
D010833:Phytic Acid; D010832:6-Phytase