Cloning and Expression of Phytase appA Gene from Shigella sp CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli

The phytase gene appA(S) was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppA(S)) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppA(S) contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appA(S) was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppA(P) and rAppA(E), respectively. Purified glycosylated rAppA(P) and nonglycosylated rAppA(E) had specific activity of 967 and 2982 U mg(-1), respectively. Both had pH optima of 5.5 and temperature optima of 60 degrees C. Compared with rAppA(E), rAppA(P) was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50 degrees C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppA(P) was 33% greater at 60 degrees C and 24% greater at 70 degrees C, when compared with rAppA(E). Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.