Yeast Surface Display Of Phytase And Activity Assay Of The Displayed Protein

Phytic acid and phytates are the principle storage forms of phosphorus and myo-inositol in cereal grains, soybeans and oilseeds. Due to lack of phytase, monogastric animals are incapable of hydrolyze phytic acid and phytates, resulting in the decreased usage of phosphorus in feeds. Phytase was the generic name of enzyme which catalyze the hydrolysis of phytate into myo-inositol and phosphate. On one hand phytase could enhance the mono-stomach animal absorbing phosphorus and increase its utilization rate in animal feed, on the other hand it could ease the product phytate binded by some multi-metal ions. With the development of phytase genetic engineering, the expression of recombinant phytase protein was a new hot spot by different expressional vectors.In this research, we successfully cloned the phytase gene from Aspergillus niger 424-1 by RT-PCR using first strand cDNA as template after reverse transcripting total RNA. Subsequently, a succession of processes will be following, which include the optimization of PCR reaction condition, the recovery of target fragments and the determination of the macrorestriction map. The primers were designed according to phyA gene sequence deposited in GenBank and the multiple cloning site of the plasmid pYD1, which is a vector used for protein surface display on Saccharomyces cerevisiae cells. The gene encoding phyA was amplified by PCR using the genomic RNA of Aspergillus niger. The PCR product was inserted into the plasmid pYD1. Then the construct was transformed into the yeast strain EBY100. After the target genes in the transformants were induced with 2% galactose for 36 hours, the display of the phyA protein on the yeast surface was confirmed by immnofluorescence with fluorescence microscope with yeast cells containing no vector and empty vector as controls, and the characteristics of the enzyme was analysed, and was compared with the original enzyme activity of the Aspergillus niger 424-1, respectively.Optimal pH and temperature of the displayed phytase were 7.0 and 65℃. The phytase displayed on the cell surface was found little more sensitive to temperature as compared with the native enzyme and more stable than recombinant phytase secreted into the medium. Surface displayed phytase was stable at pH 3.0-8.0, and exhibited high and similar activity at pH 5.0-7.0. The effect of cations on displayed phytase was similar to that on secreted recombinant phytase expression in EBY100 and native phytase of Aspergillus niger 424-1.