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

Abstract

The phytase gene appAS 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 (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS 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 appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg(-1), respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. 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.

Fig 1. Nucleotide (1–1299) and deduced amino acid sequences (432) of the putative phytase gene appA_S, from Shigella sp. CD2.

The conserved HAP family active site motifs are underlined. Stop codon is shown by asterisk.

Fig 2. Multiple alignment of homologs of the Shigella sp. CD2 phytase AppA_S.

Conserved active site motifs are boxed and conserved cysteine residues are shown by arrows. The source and GenBank Accession Nos. of proteins are: Shigella sp. CD2, CCA94903; Escherichia coli AppA, EDX38944; Dickeya paradisiaca, ABW76125; Klebsiella pneumoniae ASR1, AAM23271; Yersinia intermedia, ABI95370; Citrobacter braakii, AAS45884; Obesumbacterium proteus, AAQ90419; Pectobacterium carotovorum subsp. carotovorum, ABY76184.

Fig 3. Phylogenetic tree of homologs of the Shigella sp. CD2 phytase AppA_S.

The bar represents 2 substitutions per 10 amino acids. GenBank Accession Nos. are as in Fig 2 legend.

Fig 4. (a) SDS-PAGE analysis of rAppA_P expressed in P. pastoris GS115. Lane, M-molecular weight markers, 1-extracellular fraction of P. pastoris GS115 transformed with pPIC9-appA_S, 2- extracellular fraction of P. pastoris GS115 transformed with pPIC9. (b) SDS-PAGE analysis of glycosylated and deglycosylated rAppA_P.

Lane, 1- glycosylated rAppA_P, 2- deglycosylated rAppA_P, M- molecular weight markers.

Fig 5. (a) SDS-PAGE analysis of rAppA_E expressed in E. coli BL21(DE3). Lane, M-molecular weight markers, 1- soluble fraction of induced BL21 transformed with pET20b(+), 2-pellet fraction of induced BL21 transformed with pET20b(+), 3- soluble fraction of induced BL21 transformed with pET-appA_S, 4- pellet fraction of induced BL21 transformed with pET-appA_S. (b) Western blot analysis.

Lane, M- Molecular weight marker, 1- purified rAppA_E, 2-purified and deglycosylated rAppA_P.

Fig 6. Characterization of purified rAppA_E and rAppA_P.

(a) pH profile (b) Temperature profile and (c) Thermal stability. Results of phytase activity represent the mean of three independent values.