Year-end Sale % OFF 
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.
Highlights
IntroductionA significant portion of phosphorus in feed grains is present as phytic acid and its insoluble complexes with metal ions – phytates
According to the US Department of Agriculture, the most produced type of meat in 2019 was poultry - 125.6 million metric tons
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
Summary
A significant portion of phosphorus in feed grains is present as phytic acid and its insoluble complexes with metal ions – phytates. Such complexes cannot be utilized by animals, leading to the problem of insufficient quantity of available phosphorus in animal feeds. Microbial enzymes – phytases - are able to hydrolyze insoluble phytates with the release of free phosphates together with the chelated metal ions such as calcium, iron, copper and others and are ubiquitous enzymes in the animal nutrition industry [3]. Nowadays, there is a constant search of new phytases and expression systems for better production of these enzymes. 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
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
