Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients

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IntroductionPhytate is the primary form of phosphorus in grasses and legumes [1,2].It can form stable and difficult-todegrade complexes with proteins, minerals, and other substances in organisms, reducing the bioavailability of proteins and mineral elements [1,2].For this reason, phytate is considered an anti-nutritional factor.Phytase, also known as myo-inositol hexakisphosphate phosphohydrolase, can degrade phytate and generate inorganic phosphorus and phosphoinositides, eliminating the anti-nutritional effects of phytate [3,4].Owing to the lack of phytase in the human digestive system, phytate cannot be metabolized, which limits the absorption of nutrients in the intestine and reduces the nutritional value of food.Phytase can reduce the phytate level in food more effectively than cooking, boiling, fermentation, soaking, and other food processing methods.Adding phytase to plant-based food ingredients can degrade phytate and release inorganic phosphorus, which can improve the absorption and utilization of phosphorus in food, relieving the anti-nutritional effect of phytate [5][6][7][8].Thus, phytase can be used as a new type of food additive in the food industry.However, numerous processing operations in the food industry necessitate execution under high temperatures.The limited thermostability of natural phytase poses a critical constraint in its practical utilization in food processing [9,10].Hence, the pursuit and advancement of improved thermostability of phytases remain a focal point of research among scholars worldwide.Currently, protein engineering strategies used to enhance the thermostability of phytase include directed evolution (irrational design), semi-rational design, and rational design [11][12][13][14].Rational design in the context of enzyme engineering involves directed modifications of enzymes based on the structure-activity relationship between molecular structure and function.Common methods employed in rational design include ion binding design, disulfide bond design, glycosylation site design, homology modeling, and molecular dynamics simulation The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry.First, homology modeling of YiAPPA was performed.By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants.Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability.The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized.Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T 50 30 ) was increased from 55.12°C to 62.44°C, and T m value was increased from 48.36°C to 53.18°C.Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg.Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability.The enhanced activity was primarily attributed to reduced enzymesubstrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate.Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36times.This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

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