Expression And Site-directed Mutagenesis Of A Thermostable Serine Protease TfpA

Short supply of high-quality protein and nitrogen pollution induced by poor digestion of low-quality proteins are two major problems in current animal husbandry. Poultry feathers contain over80%crude protein with many essential amino acids which are poorly digested by animal digestive system but could be potential used as feed additive. The aim of this study was to investigate the digestive feathers’ ability of the serine protease TfpA isolated from Thermomonospora fusca, to improve the TfpA enzymatic activity by genetically manipulation (site-directed mutagenesis), and to choose a yeast system to express the functional TfpA enzyme.Study.1Studies of TfpA degradation ability of the feathers and its mechanismTo digest the feathers with the supernatants induced by X33yeast strain and the recombinant X33-pPICTFP yeast strain in BMMY medium. The results showed the supernatant induced by the recombinant X33-pPICTFP yeast strain can digest the feathers in the bacteriostatic NaN3. To further study the degradation mechanism of feathers, the reductive activity of various r-TfpA concentrations was tested, and the results showed r-TfpA did not have any reductive activity. Fianl10mmol/L PMSF inhabited82.7%protease actvity of r-TfpA, and restrained r-TfpA to digest the feathers at the same time. In the experiment of degradation feathers by TfpA, the final10mmol/L Na2SO3was added. Determination of the soluble protein concentration and free amino acids production of the supernatants at different time intervals (8h,24h, and48h) showed all the protein concentrations at the three time intervals increased a little, and the amino acids production at8h increased to126.1%and at24h,48h decreased to96.1%,98.8%. The above study showed r-TfpA digested the feather depending on its protease activity instead of its reductive activity and the reducing agent Na2SO3was able to accelerate r-TfpA degradation of the feather meal but didn’t improve the r-TfpA degradation ability of the feather meal.Study.2Expression of the directed tfpA gene mutants in Pichia pastorisAccording to the molecular structure of TfpA,11site-directed mutants were designed by molecular technology. Used pPICTFP plasmid as templates to amplify the mutational pPICTFP plasmids by PCR and transformed into P. pastoris to induce the recombinant protease and purified to analyze by Western blot. Among11mutants, the protease activity of M14(R220Q) was118.8%higher than the wild-type (WT) in the supernatant of the fementation broth, and M9(G192P), M15(D219N) were lower specific activities than WT. The purified proteins of M9, M14and M15were at the molecular weights size of21.7kDa by12%SDS-PAGE electrophoresis same to the WT expressed in P. pastoris, and were confirmed by the western blot. The specific activies of M9, M14and M15were235%,129%and231%greater than WT. The above study showed the high expression level strain of X33-pPICTFP-M14and high specific activaty enzymes of M9, M14and M15were obtained.Study.3Expression of tfpA gene in Saccharomyces cerevisiaeInduction of S.cerevisiae INVSc1, recombinant INVSc1-pYETFP, P. pastoris X33and recombinant X33-pPICTFP respectively, analyzed the growth curvre and enzyme production trend. The results showed the protease activity of r-TfpA was246.5U/mL in S.cerevisiae and was higher than160.0U/mL in P. pastoris on the fourth day. Induced the recombinant INVSc1-pYETFP to express TfpA in YPG medium and centrifuged the fermentation broth and taked on the biological membrane enrichment, ammonium sulfate precipitation, Sephadex G-50purification, SDS-PAGE and western blot analysis. The purified protein was at the molecular weights size of21.7kDa by12%SDS-PAGE electrophoresis same to the WT expressed in P. pastoris, and was confirmed by the western blot. The specific activity of purified TfpA was5,915units/mg protein, and was2.4-fold of that expressed in P.pastorios. The above study showed, comparaed with P. pastoris system, TfpA express in the S.cerevisiae that has more advantages.In conclusion, this study was the first time to reveal TfpA enzyme degradation ability of the feathers, and further to study the degradation mechanism. In this study, the he high expression level strains and high specific activaty enzymes were obtained. Moreover, this study demonstrated that TfpA expressed in S.cerevisiae system had higher expression level and specific avtivity than in P. pastoris system.