title: Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia pastoris.
abstract: A mature phytase cDNA, encoding 441 amino acids, from Eupenicillium parvum (BCC17694) was cloned into a Pichia pastoris expression vector, pPICZ alpha A, and was successfully expressed as active extracellular glycosylated protein. The recombinant phytase contained the active site RHGXRXP and HD sequence motifs, a large alpha/beta domain and a small alpha-domain that are typical of histidine acid phosphatase. Glycosylation was found to be important for enzyme activity which is most active at 50 degrees C and pH 5.5. The recombinant phytase displayed broad substrate specificity toward p-nitrophenyl phosphate, sodium-, calcium-, and potassium-phytate. The enzyme lost its activity after incubating at 50 degrees C for 5 min and is 50% inhibited by 5mM Cu(2+). However, the enzyme exhibits broad pH stability from 2.5 to 8.0 and is resistant to pepsin. In vitro digestibility test suggested that BCC17694 phytase is at least as effective as another recombinant phytase (r-A170) which is comparable to Natuphos, a commercial phytase, in releasing phosphate from corn-based animal feed, suggesting that BCC17694 phytase is suitable for use as phytase supplement in the animal diet.
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand.
authors.forename: Anusorn
authors.initials: A
authors.lastname: Fugthong
authors.forename: Katewadee
authors.initials: K
authors.lastname: Boonyapakron
authors.forename: Warasirin
authors.initials: W
authors.lastname: Sornlek
authors.forename: Sutipa
authors.initials: S
authors.lastname: Tanapongpipat
authors.forename: Lily
authors.initials: L
authors.lastname: Eurwilaichitr
authors.forename: Kusol
authors.initials: K
authors.lastname: Pootanakit
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D055324:Eupenicillium / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D011993:Recombinant Fusion Proteins; D010832:6-Phytase