title: 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.
authors.affiliation: Department of Biotechnology, University of North Bengal, Siliguri, India.
authors.forename: Moushree
authors.initials: M
authors.lastname: Pal Roy
authors.affiliation: Department of Biotechnology, University of North Bengal, Siliguri, India.
authors.forename: Deepika
authors.initials: D
authors.lastname: Mazumdar
authors.affiliation: Department of Biotechnology, University of North Bengal, Siliguri, India.
authors.forename: Subhabrata
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Department of Biotechnology, University of North Bengal, Siliguri, India.
authors.forename: Shyama Prasad
authors.initials: SP
authors.lastname: Saha
authors.affiliation: Department of Biotechnology, University of North Bengal, Siliguri, India.
authors.forename: Shilpi
authors.initials: S
authors.lastname: Ghosh
mesh_terms: D010832:6-Phytase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002954:Citrobacter / Q000201:enzymology; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D015966:Gene Expression Regulation, Fungal; D005798:Genes, Bacterial; D006031:Glycosylation; D008969:Molecular Sequence Data; D010802:Phylogeny; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012760:Shigella / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D013104:Spheroplasts; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D010833:Phytic Acid; D000135:Acid Phosphatase; D010832:6-Phytase; C406788:appA protein, E coli; D014357:Trypsin