title: A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris.
abstract: A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K (m) and V (max) for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe(2+), Fe(3+), and Al(3+). When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).
authors.affiliation: Department of Biotechnology, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand.
authors.forename: Patcharaporn
authors.initials: P
authors.lastname: Pandee
authors.forename: Pijug
authors.initials: P
authors.lastname: Summpunn
authors.forename: Suthep
authors.initials: S
authors.lastname: Wiyakrutta
authors.forename: Duangnate
authors.initials: D
authors.lastname: Isarangkul
authors.forename: Vithaya
authors.initials: V
authors.lastname: Meevootisom
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055326:Neosartorya / Q000201:enzymology* / Q000235:genetics*; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D010833:Phytic Acid; D010832:6-Phytase