A Thermostable Phytase from Neosartorya spinosa BCC 41923 and Its Expression in Pichia pastoris
2011
Pandee, Patcharaporn, Mahidol University, Bangkok, Thailand | Summpunn, Pijug, Mahidol University, Bangkok, Thailand | Wiyakrutta, Suthep, Mahidol University, Bangkok, Thailand | Isarangkul, Duangnate, Mahidol University, Bangkok, Thailand | Meevootisom, Vithaya, Mahidol University, Bangkok, Thailand
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℃ and 38.62 U/mg at 42℃. 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℃. 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℃ 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 Fe²+, Fe³+, and Al³+. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37℃ 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).
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