Heterologous Expression of Histidine Acid Phytase From Pantoea sp. 3.5.1 in Methylotrophic Yeast Pichia Pastoris

Abstract

Background and Objective:The major storage form of phosphorus in plant-derived feed is presented by phytates and not digested by animals. Phytases are able to hydrolyze phytates and successfully used as feed additives. Nevertheless, nowadays, there is a constant search of new phytases and expression systems for better production of these enzymes. In this study, we describe cloning and expression of gene encoding histidine acid phytase fromPantoeasp. 3.5.1 using methylotrophic yeastPichia pastorisas the host.Methods:The phytase gene was placed under the control of the methanol-inducible AOX1 promoter and expressed inP. pastoris. Experiments of small-scale phytase expression and activity assays were used to test recombinant colonies. Four different signal peptides were screened for better secretion of phytase byP. pastoris. After 36 h of methanol induction in shake flasks, the maximum extracellular phytase activity (3.2 U/ml) was observed inP. pastorisstrain with integrated construct based on pPINK-HC vector andKluyveromyces maxianusinulinase gene signal sequence. This phytase was isolated and purified using affinity chromatography.Results:Recombinant phytase was a glycosylated protein, had a molecular weight of around 90 kDa and showed maximum activity at pH 4.0 and at 50°C. Recombinant phytase had excellent thermal stability – it retained high residual activity (100% ± 2%) after 1 hour of heat treatment at 70°C.Conclusion:The enhanced thermostability of the recombinant phytase, its expression provided by strong inducible promotor and the effectively designed expression cassette, the simple purification procedure of the secreted enzyme, and the possibility of large-scale expression make the foundation for further production of this bacterial phytase inP. pastorisat an industrial scale.