Expression Of A Thermostable Lysine Aminopeptidase From Pseudomonas Aeruginosa In Pichia Pastoris And Fermentation Optimization

Aminopeptidase?APs? can catalyze the cleavage of amino acid residues at the N-terminal position of peptides. It can be used as a flavourzyme especially in protein hydrolysate debittering and increasing the content of free amino acides in the hydrolysate. Besides, it was also applied to medical diagnosis and N-terminal sequenceing of protein. In this study, a thermostable lysine aminopeptidase gene?plap? from. P. aeruginosa was cloned and expressed in P. pastoris. The optimization of fermentation conditions and codon optimization of plap gene were studied. Additionally, not only the purification and properties, but also the applications of of recombinant aminopeptidase were investigated.The optimized fermentation conditions based on single factor experiment were identified as follows: the induction methanol concentration was 15g·L^-1, the induction pH was 5.0, the induction temperature was 28 °C, the medium volume was 25mL/250mL, the concentration of Co²⁺ was 50 ?mol·mL^-1, the induction sorbitol was 9.0 g·L^-1. Finally, the enzyme activity was 5.21 times as in original conditions. The plap gene optimized by codon usage bias was successfully expressed in P. pastoris. The expression level of PLAP was increased 20.53% higher than before codon optimized in optimal condition. The high cell-density culture of the recombinant P. pastoris was perfomed in a 7 L fermentor. When the cell density(OD₆₀₀) was 400 at the initial induction, the activity of lysine aminopetidase in supernatant reached to 7.8 U·m L^-1, which was 2.14 folds higher than that in flaskThe recombinant PLAP from the culture supernatant was purified with several steps, including ammonium sulfate salting?40%-60% saturation?, Hitrap Q HP anion exchange chromatography, and Superdex 75 gel filtration chromatography. The specific activity of the purified PLAP was 36.41 U·mg^-1. And the final recovery of the enzyme was 4.79%. The purification factor was 3.95.The properties of the purified recombinant aminopeptidase were characterized. The results showed that the optimal temperature was 80 °C and the optimal pH was 9.0. It was stable in pH 7.0-9.5. In addition, the aminopeptidase exhibited good thermostable, after incubated within 50-70 °C for 2 h, the residule activity was over 80%. The aminopeptidase was activated by Co²⁺ and Ca²⁺, but inhibited by Ni²⁺??-mercaptoethanol?DTT and EDTA. Whereas, Mg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, and PMSF had slight inhibition on it. When EDTA and Co²⁺ were coexisted, the inhibitor influence of EDTA was greatly weakend. The substrate sepecificity of the enzyme was investgated. And it turned out that the optimal substrate was Lys-pNA, followed by Leu-pNA and Arg-p NA. The sequence analysis of lysine aminopeptidase indicated three potential N-glycosylation site. The deglycosylation of PLAP by Endo Hf showed the aminopeptidase was partial glycosylated. The glycosylation had no influence on pH and pH stability, but it decreased the thermostability of PLAP.The hydrolysis of casein was investgated with alkaline protease enzyme, lysine aminopeptidase and both together. Compared with control group, the content of free amino acid increased 22.5 folds, 10.5 folds and 65 folds respectively in three kinds of solution. After treated with alkaline protease and lysine aminopeptidase, the content of Lys and Leu were the highest. And not only the content of oligopeptide but also the small peptides increased significantly. The compounds under 180 Da accounted for 22.76%, while the macromolecules that more than 2000 Da were almost been degraded.