The Open Microbiology Journal




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RESEARCH ARTICLE

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



Aliya Suleimanova1, *, Daria Bulmakova1, Margarita Sharipova1
1 Laboratory of Microbial Biotechnology, Institute of Fundamental Medicine and Biology, Kazan (Volga region) Federal University, Kazan, Russia

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 from Pantoea sp. 3.5.1 using methylotrophic yeast Pichia pastoris as the host.

Methods:

The phytase gene was placed under the control of the methanol-inducible AOX1 promoter and expressed in P. 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 by P. pastoris. After 36 h of methanol induction in shake flasks, the maximum extracellular phytase activity (3.2 U/ml) was observed in P. pastoris strain with integrated construct based on pPINK-HC vector and Kluyveromyces maxianus inulinase 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 in P. pastoris at an industrial scale.

Keywords: Phytate , Histidine acid phytase, Heterologous expression, Pichia pastoris, Alcohol oxidase promoter (PAOX1), Fermentation.


Article Information


Identifiers and Pagination:

Year: 2020
Volume: 14
First Page: 179
Last Page: 189
Publisher Id: TOMICROJ-14-179
DOI: 10.2174/1874285802014010179

Article History:

Received Date: 07/02/2020
Revision Received Date: 24/6/2020
Acceptance Date: 26/6/2020
Electronic publication date: 30/07/2020
Collection year: 2020

© 2020 Suleimanova et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Laboratory of Microbial Biotechnology, Institute of Fundamental Medicine and Biology, Kazan (Volga region) Federal University, 420008, Kazan, Kremlyovskaya Street, 18, Russia. Tel: +7 843 233 78 14, E-mail: aliya.kzn@gmail.com



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