Evolution of E. coli Phytase for Increased Thermostability Guided by Rational Parameters

Abstract

Phytases are enzymes that can hydrolyze phytate and its salts into inositol and phosphoric acid, and have been utilized to increase the availability of nutrients in animal feed and mitigate environmental pollution. However, the enzymes' low thermostability has limited their application during the feed palletization process. In this study, a combination of B-value calculation and protein surface engineering was applied to rationally evolve the heat stability of Escherichia coli phytase. After systematic alignment and mining for homologs of the original phytase from the histidine acid phosphatase family, the two models 1DKL and 1DKQ were chosen and used to identify the B-values and spatial distribution of key amino acid residues. Consequently, thirteen potential amino acid mutation sites were obtained and categorized into six domains to construct mutant libraries. After five rounds of iterative mutation screening, the thermophilic phytase mutant P56214 was finally yielded. Compared with the wild-type, the residual enzyme activity of the mutant increased from 20% to 75% after incubation at 90°C for 5 min. Compared with traditional methods, the rational engineering approach used in this study reduces the screening workload and provides a reference for future applications of phytases as green catalysts.

Sequence diversity and catalytic properties of phytases

“…The number of hydrogen, ionic and disulfide bond interactions is believed to have a strong correlation with thermostability (Zhang 2007;Liao, et al, 2013;Fei et al, 2013;Hesampour et al, 2015;Kumar et al, 2015;Tan et al, 2016;Han et al, 2018;Li et al, 2019;Zhang et al, 2020). However, here we show that, for these enzymes, this is not true.…”

“…Other studies also describe a relation between the number of hydrogen bonds and the thermostability of individual phytases (Zhang 2007;Hesampour et al, 2015). The thermostability of a E. coli phytase increased from 20 to 75% due to increased electrostatic interactions related to the formation of hydrogen bonds resulting from the replacement of charged residues by hydrophobic and aromatic ones (Li et al, 2019). However, those studies describe isolated analysis and could not be used for general considerations.…”

“…The association between the structure and the biochemical properties of enzymes can provide relevant information about factors that affect stability and catalytic activity. Several methods have been used for this purpose, including comparative sequence, structure analysis, molecular dynamics studies, or even the evaluation of random or rational mutations on enzyme activity (Zhang 2007;Liao, et al, 2013;Fei et al, 2013;Hesampour et al, 2015;Kumar et al, 2015;Tan et al, 2016;Han et al, 2018;Li et al, 2019;Zhang et al, 2020;Acquistapace et al, 2022). In this study, we report a comparative approach between sequences, threedimensional structures and biochemical characteristics of phytases already characterized and described in the literature, adopting factors such as: hydrogen, ionic and Van der Waals (VdW) interactions and disulfide bonds.…”

Integrative Structural and Computational Biology of Phytases for the Animal Feed Industry

“…Mutated residues were mainly aromatic and hydrophobic amino acids. Substituting hydrophobic and low B-value amino acids in the protein surface and highly flexible areas gave two benefits to thermostability: firstly, aromatic and hydrophobic amino acids could elevate the hydrogen bonding network and enhanced electrostatic interactions; secondly, they could reduce the flexibility of the protein structure [35]. The role of residue P257 in the thermostability of phytase PHY-US417 derived from Bacillus subtilis was examined by employing two mutations-P257R and P257A.…”