Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

Laura Navone,Jo-Anne Blinco,Robert Speight,Pawarisa Luangthongkam,Thomas Vogl,Carlos H. Luna-Flores,Stephen Mahler,Xiaojing Chen,Juhani von Hellens

doi:10.1186/s13068-021-01936-8

Abstract

BackgroundPhytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market.ResultsIn this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme.ConclusionsOverall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

Highlights

Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid
Thermostability studies of ApV1 phytase ApV1 thermostable variant was engineered using a rational design based on the available structure of the E. coli AppA phytase [32]
ApV1 and the wild-type AppA phytase were expressed in P. pastoris and His-tag purified for biochemical characterisation

Summary

Introduction

Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. The three main desirable characteristics for phytase enzyme supplements are high specific activity in the gut, high stability during storage and feed pellet steam extrusion, and high levels of production in industrial microbial fermentations [13, 50]. Activity can be lost through thermal denaturation during feed pellet production where the enzyme may typically be exposed to temperatures approaching ~ 90 °C for up to 90 s during storage and transportation, and through degradation by proteases and the acidic conditions encountered in the gastrointestinal tract. Increasing the Tm often leads to an upward shift in optimal temperature and reduced activity at mesophilic temperatures, potentially affecting enzymatic activity at the normal body temperature of animals

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