Rational design-based engineering of a thermostable phytase by site-directed mutagenesis

Abstract

Phytases are enzymes that hydrolysis phytic acid and makes mineral phosphorus available to animals. Phytases face relatively extreme heating during food processing, thus thermostability plays an important role in industrial applicability of this enzyme. Herein, we report the design of a thermostable phytase with favorable biochemical properties and high enzymatic activity using molecular dynamics and rational design-based molecular engineering. Based on the crystal structure of E. coli phytase, bioinformatics analysis and docking binding energy measurement, S392F mutant was introduced by site-directed mutagenesis in order to improve thermostability of phytase through strengthen the bounding interactions. Wilde type and Mutated constructs were expressed in E. coli BL 21. The WT and manipulated phytase were purified; their biochemical and kinetic was investigated. Results revealed that recombinant WT and mutant phytase have optimum temperature of 50 °C with no significance change but optimum pH of WT and mutant was respectively 5 and 6 with a pH shift. Furthermore, S392F phytase catalytic efficiency values showed significant improve of 25.6%, compared with the WT. Analysis of the retained enzymatic activity at high temperatures, indicated that despite of phytase stability reduction at high temperatures but mutant phytase showed more stable behavior in compare with WT phytase, So that at 70 °C showed twice thermo stability and at 80 °C and 90 °C display respectively 74% and 78.4% improvement of thermostability compared to the wild-type. In conclusion, our results implied that the designed phytase could be a potential candidate for phytase manipulation research and industrial applications with improved thermostability.

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases

“…Molecular dynamics simulations revealed up to 3-fold higher interactions in the active site of both enzymes with bound polyP compared to bound RNA, which may explain the higher rates of the enzymes toward polyP ( Figure 5 ). Structure-based modeling of substrate-enzyme complexes was previously used to guide rational engineering of enzymes such as phytase, 68 lipase, 69 , 70 aminotransferase, 71 and transketolase. 72 In a similar fashion, the structural insights obtained here could contribute to guiding principles toward optimizing the activity of APs for P-polymer dephosphorylation.…”

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

“…Structural comparisons between the mutant and native phytase revealed that substituting Ser392 with Phe caused two new hydrophobic interactions with Trp 347, which brought about the elevated structural stability of Escherichia coli AppA phytase, as shown in Figure 6. Along with the increase in thermostability, the catalytic efficiency of the mutant was also increased by 25.6% through introducing hydrophobic interactions [34]. Based on the highest B-values, nine substitutions (P41W, V42S, K43L, R181S, E182S, Q285D, K286Y, E384V, R385A) were made and four more substitutions (S80I, Q184A, S342T, E383A) on the surface loop were carried out for protein surface engineering.…”

“…Mutant S392F showed a 25.6% enhancement in catalytic efficiency over that of the native phytase. Structural comparison between S392F and native phytase revealed that substituting Ser-392 with Phe introduced two new hydrophobic interactions with Trp-347 [34].…”

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

“…However, some sensitive residues hidden in the rigid parts of the protein would escape discovery. Rigid regions are also essential for protein stability, and many strategies had been reported involving modifications at rigid regions with improved protein stability, including removing or pairing of unpaired H-bond donors/acceptors , and charged groups, filling cavities, − improving hydrophobic packing, − enlarging hydrophobic clusters, and resolving steric strain . If further improvement of the protein stability is desired, it may be necessary to consider sensitive residues in rigid regions in addition to hot spots in flexible regions as multiple mutations may have synergistic effects. − …”