Cloning,Expression,Enzymatic Characteristics And Application Of Thermostable Aminopeptidase From Pseudomonas Aeruginosa

Aminopeptidases（EC 3.4.11）are capable of removing N-terminal amino acids from proteins or peptides,which are widely used in food processing,feed manufacture,active peptide preparation,etc.Especially,the application of thermostable aminopeptidases have a broader application prospect.Our research group obtained an extracellular leucine aminopeptidase（PLAP）with high activity from Pseudomonas aeruginosa GF31,which could also degrade pyrethroids.In this paper,the enzyme was cloned and expressed in Escherichia coli to further investigate the properties,mechanism of action and its potential for practical application.（1）In order to obtain soluble expression of PLAP in E.coli,the coding gene aps of PLAP was codon-optimized and expressed in E.coli by the way of periplasmic expression.The expression strain E.coli BL21（DE3）-p ET22b（+）-aps was constructed and successfully achieved the soluble expression of recombinant PLAP.Subsequently,the optimal expression conditions were obtained as follows:Rosseta gami2（DE3）was used as the host bacterium,the final concentration of IPTG was 1 m M,the induction temperature was 20℃,and the induction time was 12 h.As a result,the expression of the enzyme in E.coli was 42%in the periplasmic space and 49.7%in the intracellular space.The purified enzyme was obtained using Co²⁺-NTA combined with weak anion chromatography and had a specific activity of 6.8 U/mg.（2）The enzymatic properties of recombinant PLAP were determined.The optimum reaction temperature was measured to be 80℃,with good thermal stability at 50-70℃;the optimum reaction p H was 8.0,which was more stable in the range of p H 7.0-9.0.Co²⁺had a strong activating effect on the enzyme activity,and Zn²⁺,Ni²⁺and Fe²⁺had a certain inhibiting effect.The enzyme was strongly inhibited by reducing agents（DTT,β-sparse ethanol）,and strongly inhibited by metal ion complexing agents（EDTA-Na₂,1,10-o-phenanthroline）,indicating that the enzyme is a metalloaminopeptidase.Bestatin as a specific inhibitor of metalloaminopeptidase had a strong inhibitory effect on its enzyme activity,while PMSF had basically no inhibitory effect.The recombinant PLAP also remained stable after incubation in high concentration salt solution and some organic solvents.The substrate specificity showed that recombinant PLAP had the best hydrolysis effect on Leu-p NA and Met-p NA,and average hydrolysis effect on Arg-p NA and Ala-p NA;while it basically had no hydrolysis activity on Glu-p NA,Pro-p NA and Phe-p NA.The kinetic analysis showed that the K_m of the enzyme was 1.11 mmol/L and V_max was 101.01μmol/（L·min）with Leu-p NA as substrate;the K_m was 2.30 mmol/L and V_max was 92.59μmol/（L·min）with Met-p NA as substrate.The analysis of ester hydrolysis ability showed that recombinant PLAP had the best hydrolysis effect on p-nitrophenyl decanoate,and the hydrolysis effect on several other esters was average.（3）The amino acid sequence comparison analysis of PLAP showed that the enzyme has the same conserved sites as other M28 family aminopeptidases,in which Glu305,Ser387,and His260 of PLAP protein sequence are catalytic ternary,and the amino acids ligated with Zn²⁺are His260,Asp272,Glu305,Asp333,HIS431.The three-dimensional model was constructed by Alpha Fold2,and the enzyme was found to consist of a PA structural domain and a catalytic structural domain.In the analysis of the molecular docking results of the aminopeptidase inhibitor Bestatin and p-nitrophenyl decanoate,it was found that amino acids such as GLU304 and ASP272 in the active center play a key role in the binding of the enzyme to the substrate.（4）The recombinant PLAP was applied to compound hydrolyzed maize protein and pyrethroid pesticide degradation.Alkaline protease was screened for the hydrolysis of maize protein,and 20%hydrolysis was obtained under optimal conditions（60℃,p H=10.0,1%enzyme addition and 5%substrate concentration）;furthermore,the hydrolysis degree could be increased to 36%at the optimal aminopeptidase addition of 1%by compounding alkaline protease with recombinant PLAP,and the content of amino acids such as Asp,Ala,Met,Leu,Arg and Phe in the hydrolysate was significantly increased.For the degradation of permethrin pesticide,the degradation rate of recombinant PLAP was 53%for50 mg/Lβ-cypermethrin at 60℃for 48 h.It also showed some degradation effect for cis-cypermethrin,deltamethrin,flucytamethrin and metsulfuron.