Site-Directed Mutagenesis Of Aspergillus Niger Phytase And Its Display On The Cell Surface Of Pichia Pastoris

Phytase?PHYA?derived from Aspergillus niger is currently recognized as one of the most promising phytase in livestock,but it does not meet the demand of industry when using wild-type strains to produce.In the present research,phyA full-length sequence of phytase from Aspergillus niger ZJUY was cloned and modified based on PCR directed mutagenesis,aimed at solving phytase denaturalization and deactivation during the process of pelleting.The mutation types included codon optimization for key sites,the introduction of hydrogen bond?T295S,Q296 R and V43N?and the deletion mutation of disulfide bond?Cys196-Cys446?.Ultimately,flocculation?Flo1p?was fused with improved phytase in the N-terminal.Phytases were successfully displayed on the surface of Pichia pastoris GS115 after induced with 1% methanol,verified by immunofluorescence and flow cytometry.The main results as follows:?1?The intron of phyA was removed by three rounds of PCR on the template of Aspergillus niger genome.The advantage of this approach is that a complete phyA without intron could be obtained when the poor quality of extracted RNA.?2?Twelve mutational sites?R62?H63?G64?R66?P68?R146?H342?D343?S295?R296?N43 and S446?were introuduced by PCR with high efficiency and the operation is simple.?3?The anchored-Flo1p?FS?and target-phyA were successfully inserted into the yeast expression vector pPICZ?C relied on seamless cloning,which with higher transformation efficiency compared to the traditional construction method.?4?Eight kinds of expression vector pPICZ?C-FS/phyA were integrated into the Pichia pastoris GS115 genome by homologous recombination using lithium chloride transformation method,respectively,and then successfully displayed on the surface of GS115 after induced with methanol.?5?The deletion mutation of disulfide bond?Cys196-Cys446?can make the emission wavelength of the endogenous fluorescence generate redshift for enzyme-displaying,and change molecular of enzyme conformation.In addition,it also increased the affinity of the enzyme to its substrate and substratspezifitaet,but decreased the catalytic efficiency.?6?The optimum temperature and pH would change to some extent when introduced hydrogen bond and delete Cys196-Cys446.?7?Through the study of thermostability,the recombinant A61 so slowly lost it activity after undergoing water bath at 90?,which can overcome denaturation during the process of granulation?60⁹0 C?.?8?Through the study of pH-stability,A31,A61 and A84 still maintained more than 80% of enzyme activity at a pH range of 1.6 to 4.0.?9?Metal ions Na⁺?K⁺?Fe²⁺?Zn²⁺ can activate the activity of phytase in low concentration,while Cu2+?Mn2+ and Co2+can inhibit the activity of phytase.