Expression of Microbial Phytases in Yeast Systems and Characterization of the Recombinant Enzymes

Phytases are hexakisphosphate phosphorylases that initiate the hydrolysis of phosphate from phytate (myo-inositol hexakisphosphate), the major form of phosphorus in plant foods and feeds. These enzymes have been increasingly used worldwide for animal nutrition, environmental protection, and human health. In our laboratory, we have studied the expression of fungal and bacterial phytases in Pichia pastoris and Saccharomyces cerevisiae. Both sources of phytases can be expressed as functioning enzymes in the two yeast systems, and secreted into the media as extracellular proteins at relatively high yield and purity. This chapter describes detailed procedures for preparing phytase expression constructs, transforming the constructs, and fermenting the transformants. Partial purification, biochemical characterization, and in vitro function assay of the recombinant phytases are also discussed. Key Words: Aspergillus niger ; electroporation; environment; Escherichia coli ; food; feed; gene expression; glycosylation; Pichia pastoris , phosphorus; phytase; phytic acid; purification; pH profile; Saccharomyces cerevisiae ; thermostability; transformation.