Enhancement of Thermostability and Kinetic Efficiency of Aspergillus niger PhyA Phytase by Site-Directed Mutagenesis

Abstract

Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P < 0.05) with 24 and 22.6 % greater retention, respectively, compared with the PhyA of the wild type at 80 °C. The K m value of the improved thermostable P9 and P12 mutant enzymes for sodium phytate were 35 and 20 % lower (P < 0.05) with respect to the wild-type enzyme. In conclusion, it is feasible to simultaneously improve the thermostability and the catalytic efficiency of phytase to be used as an animal feed supplement.

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

“…1). In others studies optimized synthetic gene was used for producing extracellular enzymes like cellulase and phytase which were succeed to express recombinant proteins (Akbarzadeh et al 2013; Hesampour et al 2014, 2015). Design of 6 histidine residues were successfully applied at C-terminal of the protein and enabled us to purify active enzyme, so probably small His-Tag in C terminus of invertase had no influence on the protein conformation for its activity (Huang et al 2003).…”

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

“…At the same time, strategies such as metagenome analysis [12] and screening of thermophilic bacteria [13,14] were also utilized to develop novel heatresistant phytases. Phytases from well-known microorganisms such as Aspergillus niger [15], Escherichia coli [16] and Bacillus subtilis [1] have been reengineered by directed evolution employing random mutagenesis and multigene DNA shuffling. Furthermore, bioinformatics analysis and identification of novel thermophilic phytases, such as those from Yersinia mollaretii [17] and Citrobacter amalonaticus [18], also greatly added to the available thermoresistant phytases.…”

Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges

“…For the acidic condition present in livestock and poultry stomach [29], phytase showing high activity and stability at low pH is favored. Moreover, the steaming and pelleting process in feed manufacturing uses high temperatures of 60-80°C [18], and thus the supplemented phytase could suffer from heat denaturation. Therefore, the resistance of phytase to high temperature is a necessary property.…”