| Phytase is currently used primarily as an agricultural feed additive to promote animal’s growth and development and reduce phosphorus emissions. Protein engineering technology has become an effective strategy to engineer phytase for practical values.In this study, the mutant acid phytase (phyA6A) gene was modified by random mutagenesis to improve enzymatic activity and thermostability by using the error-prone PCR and DNA shuffling strategy. After one-round error-prone PCR, a single transformant, PP-NPep-11C, showing the strongest phytase activity and thermostability from among the4000transformants, was selected for detailed analyses. Southern Blotting analysis to this mutant showed that phyA11C gene was integrated into the chromosome genome through single-crossover with one copy of phyA. This mutant phytase was successively purified by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography and then visualized by SDS-PAGE. The molecular weight was70.15kDa, which was the same as that from the control strain PP-NPep-6A. The results of characterization studies showed that:compared with PP-NPep-6A, the optimum temperature of this mutant was61℃,1℃higher than that of PP-NPep-6A; the optimum pH was5.5, which was the same as PP-NPep-6A; there was a20%,26%,19%improvement of thermostability over that of the PP-NPep-6A enzyme after being heated at70℃,80℃,90℃for10min, respectively. Besides, the half-life of this mutant (9min) was extend by3min; the specific activity was58%higher than that of PP-NPep-6A phytase, the km value for sodium phytate was44%lower than that of PP-NPep-6A phytase and the overall catalytic efficiency (kcat/km) of this mutant one was78%higher than that of PP-NPep-6A. Nucleotide sequence alignment of the mutant phytase gene (phyA11C) with the control one(phyA A) revealed twelve mutation sites. These mutations caused three amino acid changes, that is, Thr195Leu, Gln368Glu, Phe376Tyr.Then, the phyA11C gene, combining with the mutant phytase genes of PP-NPep-6A, PP-NPm-8and PP-NPm-44-252, was sequentially modified by DNA shuffling. After high-throughput screening based on96-well plates, three genetically-engineered strains with desirable properties were obtained, named PP-NPds-8F, PP-NPds-12D and PP-NPds-12G. Southern Blotting analysis to the mutant yeast transformants showed that all mutant phyA gene (phyA8F,phyA12D, phyA12G) was integrated into the chromosome genome through single-crossover with one copy of phyA. These three mutant phytases were successively purified by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography and then visualized by SDS-PAGE. The molecular weight of these mutants were70.15kDa, which was the same as that from the control strain PP-NPep-6A. The results of characterization studies showed that compared with PP-NPep-6A,(1) the optimum temperature of PP-NPds-8F and PP-NPds-12G was60℃, which were the same as that of PP-NPep-6A; the optimum temperature of PP-NPds-12D was6TC,1℃higher than that of PP-NPep-6A;(2) the optimum pH of these three mutants was5.5;(3) PP-NPds-8F had a15%,23%,13%improvement of thermostability over that of the PP-NPep-6A enzyme after being heated at70℃,80℃,90℃for10min, respectively, the half-life of this mutant (7.9min) was extend by1.9min; PP-NPds-12D had a35%,40%,31%improvement of thermostability over that of the PP-NPep-6A enzyme after being heated at70℃,80℃,90℃for10min, respectively, the half-life of this mutant (12.8min) was extend by6.8min; PP-NPds-12G had a17%,21%,15%improvement of thermostability over that of the PP-NPep-6A enzyme after being heated at70℃,80℃,90℃for10min, respectively, the half-life of this mutant (8.4min) was extend by2.4min;(4) the specific activity of PP-NPds-8F was54%higher than that of PP-NPep-6A phytase, the km values for sodium phytate were35%lower than that of PP-NPep-6A phytase and the overall catalytic efficiency (kcat/km) of this mutant one was60%higher than that of PP-NPep-6A; the specific activity of PP-NPds-12D was44%higher than that of PP-NPep-6A phytase, the km value for sodium phytate were21%lower than that of PP-NPep-6A phytase and the overall catalytic efficiency (kcat/km) of this mutant one was26%higher than that of PP-NPep-6A; the specific activity of PP-NPds-12G was66%higher than that of PP-NPep-6A phytase, the km value for sodium phytate were61%lower than that of PP-NPep-6A phytase and the overall catalytic efficiency (kcat/km) of this mutant one was97%higher than that of PP-NPep-6A;(5) nucleotide sequence alignment of the mutant phytase gene (phyA8F) with the control one (phyA6A) revealed five mutation sites. These mutations caused one amino acid change, that is, Gln172Arg; the mutant phytase gene (phyA12D) with the control one (phyA A) revealed eight mutation sites. These mutations caused two amino acid change, that is, Gln172Arg, Lys432Arg; the mutant phytase gene (phyA) with the control one (phyA6A) revealed ten mutation sites. These mutations caused two amino acid change, that is, Gln368Glu, Lys432Arg.In order to investigate how the mutant sites (Q172R, T195L, Q368E, F376Y, K432R) influence the properties of phytase and identify whether individual mutations could be sequentially added to produce cumulative or synergistic improvements in thermostability and catalytic efficiency of phytase, phyA6A gene was modified through site-directed mutagenesis, three mutants PP-NPsm-M4(Q172R/K432R/Q368E), PP-NPsm-M5(Q172R/K432R/Q368E/F376Y), PP-NPsm-M6(Q172R/K432R/Q368E/F376Y/T195L) were constructed. These three mutant phytases were successively purified by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography and then visualized by SDS-PAGE. The molecular weight of these mutants were70.15kDa, which was the same as that from the control strain PP-NPep-6A. The results of characterization studies showed that:compared with PP-NPep-6A,(1) the optimum temperature and pH of the three mutants were the same as those of PP-NPep-6A;(2) PP-NPsm-M4showed a20%,25%,18%improvement of thermostability over that of the PP-NPep-6A enzyme after being heated at70℃,80℃,90℃for10min, respectively, the half-life of this mutant (8.8min) was extend by2.8min; PP-NPsm-M5showed a22%,29%,21%improvement of thermostability over that of the PP-NPep-6A enzyme after being heated at70℃,80℃,90℃for10min, respectively, the half-life of this mutant (10min) was extend by4min; the thermostability of PP-NPsm-M6was almost the same as that of PP-NPep-6A;(3) the specific activity of PP-NPsm-M4was54%higher than that of PP-NPep-6A phytase, the km value for sodium phytate was30%lower than that of PP-NPep-6A phytase and the overall catalytic efficiency(kcat/km) of this mutant one was23%higher than that of PP-NPep-6A; the specific activity of PP-NPsm-M5was62%higher than that of PP-NPep-6A phytase, the km value for sodium phytate was25%lower than that of PP-NPep-6A phytase and the overall catalytic efficiency (kcat/km) of this mutant one was71%higher than that of PP-NPep-6A; the specific activity of PP-NPsm-M6was5.5%lower than that of PP-NPep-6A phytase, the km value for sodium phytate was11%higher than that of PP-NPep-6A phytase and the overall catalytic efficiency (kcat/km) of this mutant one was20%lower than that of PP-NPep-6A.In summary, the mutant phytases with better catalytic efficiency and thermal stability were generated. What is more, structure analysis of mutant and natural phytase explored the relationship between structure and function and the synergy effect of mutant sites for phytase properties, which could provide the experimental basis for the further application of protein engineering techniques for improving the physico-chemical properties of the phytase. Also, the development of these phytase variants will enhance the industrialization application. |
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