Screening And Heterologous High-level Expression Of Heat-resistant Mutants Of Phytase

Phytase,also known as phytate hydrolase,can hydrolyze phytic acid or phytate into inositol and phosphoric acid or phosphate,and has a wide range of applications in industries such as feed,food,and environmental protection.In order to further improve the thermal stability and expression level of phytase,this experiment uses error-prone PCR technology to construct a library of E.coli phytase mutants,and through high-throughput screening,three strains of E.coli phytase with significantly improved thermal stability are obtained.The APPA mutant uses SWISS-MODEL and Swiss-Pdb Viewer to perform three-dimensional modeling and mutation site structural analysis based on its amino acid sequence,and constructs multiple gene dosage strain of the high-temperature resistant phytase P.pastoris engineering strain through genetic engineering technology,and it was inoculated in a 3.6 L fermenter for amplification and fermentation.The main results are as follows:（1）The phytase gene APPA from E.coli was mutated by molecular evolution technology based on error-prone PCR random mutation,ligated to p ET22b（+）vector,and transformed into E.coli BL21（DE3）to construct a phytase mutant library.The molybdenum blue method was used for high-throughput screening of mutants with significantly improved thermal stability compared to the wild type.The results showed that the enzyme activities of the three mutants APPA1,APPA2,and APPA3 after being treated at 90°C for 5 min were 1.923,3.354 and 3.746U·m L^-1.The wild-type strain was not detected after high temperature treatment,indicating that the thermal stability of the three mutants was greatly improved compared with the wild-type strain.（2）The nucleic acid sequence of the mutant was obtained by sequencing,and after codon optimization,it was connected to the op SP2 signal peptide and p PIC9K vector,and P.pastoris X33 was used as the host for secretion expression.After purification by anion exchange chromatography,the enzymatic properties of the thermostable phytase mutant were studied from the optimum p H,p H stability,optimum temperature,thermal stability and kinetic parameters.Three-dimensional modeling and mutation site structure were analyzed using SWISS-MODEL and Swiss-Pdb Viewer,respectively.According to the analysis results,it is speculated that the introduction of new hydrogen bonds in the secondary structure of the mutant may be the main factor in improving the thermal stability.（3）P.pastoris expression plasmid p PIC9K-op SP2-op APPA3 and modified plasmid p PIC9K-（Amp-3’AOX-Xba I-del）were used as templates for PCR amplification to obtain single gene dose expression cassette fragments and vector fragments.The single gene dose expression vector is constructed using one-step cloning technology.The multiple gene dosage strain was constructed by repeatedly integrating the expression plasmid with gene dosage.High-yield phytase strains were obtained through G418 resistant plate and shake flask screening.The flask shake results showed that with the increase of the phytase mutant gene dosage,the enzyme activity and protein content in the fermentation broth of the recombinant strain increased.The enzyme activities of single gene dose strain N1,double gene dosage strain N2,three gene dosage strain N3,four gene dosage strain N4 and five gene dosage strain N5were 24.996,45.374,74.921,106.845 and 117.829 U·m L^-1,respectively.The enzyme activity of the multiple gene dosage strains N2,N3,N4 and N5 were 81.5%,199.7%,327.4%,371.4%higher than that of the single gene dose strain N1,respectively.The specific enzyme activity of the four gene dosage strain N4 reached 116.010 U·mg^-1,which was 2.75 times that of the single gene dose strain N1.（4）A five gene dosage strain capable of high-producing phytase was inoculated into a 3.6 L fermentor for scale-up fermentation.The fermentation process was controlled at an inoculation time of 24 h,an inoculum amount of 10%,an induced cell concentration of OD₆₀₀ of 100,an induction temperature of 28°C,a methanol concentration of 0.5%（v/v）,and an induced expression time of 96 h.The enzyme activity and protein content of the final fermentation broth reached 1095.051 U·m L^-1 and 1.232 mg·m L^-1,respectively.The specific enzyme activity was the highest at 72 h of induction,reaching 1037.974 U·mg^-1.