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).

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

“…Similar results have been reported with the P. pastoris-produced N-glycosylated Bacillus subtilis phytase that shows resistance to trypsin but not pepsin (41). In contrast, the N-glycosylated phytases derived from Aspergillus japonicus BCC18313, A. niger BCC18081, Eupenicillium parvum BCC17694, and Neosartorya spinosa BCC41923 are resistant to pepsin but not to trypsin (26)(27)(28). Our results indicated that the two functional N-glycosylation motifs, NXS/T, of Yersinia phytases contribute to the enzyme proteolytic resistance to pepsin but not trypsin ( Fig.…”

“…Site-directed mutagenesis has confirmed that the N-glycosylation of scavenger receptor SREC-I at N289 confers resistance against trypsin digestion (25). Some N-glycosylated microbial phytases also exhibit resistance to degradation by pepsin (26)(27)(28). Thus, N-glycosylation represents a stabilizing factor against proteolytic cleavage by proteases (29).…”

Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

“…Substrate hydrolysis by enzymes in this family occurs via a characteristic two-step mechanism, including a nucleophilic attack of catalytic H on a scissile phosphomonoester and the hydrolysis of a covalent phosphohistidine intermediate with the release of H 15 17 . The optimal activity is usually between pH 1.3–5.5 18 19 and 45–70 °C 19 20 21 , the substrate specificity is diverse 22 and HAP family enzymes display different tolerance to acidity, heat, and proteolytic digestion 23 24 25 26 .…”

Extracellular expression of alkaline phytase in Pichia pastoris: Influence of signal peptides, promoters and growth medium

“…LlALP2 contains -Arg-His-Gly-Thr-Arg-Ala-Pro-, the signature peptide in the active site of acid phytases (maximum activity between pH 2 and 6), yet exhibits maximum enzymatic activity at pH 8.0 (the pH in the small intestines) and shows little amino acid sequence similar to acid phytases (approximately 20%) or alkaline phytase from a Bacillus amyloliquefaciens strain (less than 25%) [41], [31]. High-level expression of acid phytases from microorganisms is well documented (for example [23], [33], however heterologous expression of an alkaline phytase from a plant source in amounts necessary for feed studies has been a challenge [45], [16].…”