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HOME > J. Microbiol > Volume 49(2); 2011 > Article
Research Support, Non-U.S. Gov't
A Thermostable Phytase from Neosartorya spinosa BCC 41923 and Its Expression in Pichia pastoris
Patcharaporn Pandee 1,2, Pijug Summpunn 1,2, Suthep Wiyakrutta 3, Duangnate Isarangkul 3, Vithaya Meevootisom 2,3
Journal of Microbiology 2011;49(2):257-264.
DOI: https://doi.org/10.1007/s12275-011-0369-x
Published online: May 3, 2011
1Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand, 2Center of Excellence for Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok 10400, Thailand, 3Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
Corresponding author:  Vithaya Meevootisom , Tel: +66-2201-5671, 
Received: 15 September 2010   • Accepted: 3 December 2010
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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 Km and Vmax 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°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).

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    A Thermostable Phytase from Neosartorya spinosa BCC 41923 and Its Expression in Pichia pastoris
    J. Microbiol. 2011;49(2):257-264.   Published online May 3, 2011
    DOI: https://doi.org/10.1007/s12275-011-0369-x
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