Citrobacter amalonaticus phytase on the cell surface of Pichia pastoris exhibits high pH stability as a promising potential feed supplement.

Cheng Li,Ying Lin,Yuanyuan Huang,Shuli Liang,Xiaoxiao Liu,Claude Wicker-Thomas

doi:10.1371/journal.pone.0114728

Cheng Li, Ying Lin + Show 4 more

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https://doi.org/10.1371/journal.pone.0114728

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Abstract

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

Highlights

Phosphorus is stored in many cereals and oilseeds as phytic acid [1]
Escherichia coli TOP10F9 (Invitrogen, Carlsbad, CA, USA) cells were used for DNA manipulations; these cells were cultivated in low-salt LB medium
The phytase gene phy was displayed on the cell surface of P. pastoris as a fusion with the GCW61 gene product

Summary

Introduction

Phosphorus is stored in many cereals and oilseeds (approximately 1–3%) as phytic acid [1]. Phytic acid can be degraded by phytase (myo-inositol hexakisphosphate 3- or 6phosphohydrolases; EC 3.1.3.8 or EC 3.1.3.26) This enzymatic degradation can be facilitated by the addition of phytase isolated from natural sources or by supplementation with recombinant enzymes produced in various hosts, such as fungi and bacteria. Undigested phytic acid is excreted by monogastric animals due to the absence of a sufficient level of phytase in their digestive tracts [3]. The phytases from several microorganisms and plants have been purified and characterized [8] Among these phytases, phytase from Citrobacter amalonaticus CGMCC 1696 exhibited high enzymatic activity in Pichia pastoris [9]

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