A Thermostable Phytase from Neosartorya spinosa BCC 41923 and Its Expression in Pichia pastoris

Patcharaporn Pandee; Pijug Summpunn; Suthep Wiyakrutta; Duangnate Isarangkul; Vithaya Meevootisom

doi:10.1007/s12275-011-0369-x

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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).

Keywords: phytase;Neosartorya spinosa;Pichia pastoris;gene cloning;enzyme characterization

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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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