| Pyrethroid pesticide is one of the most widely used insecticide in the current world, while its residual and accumulation in the environment pose a great threat to human health and ecosystem. Biodegradation has become an effective measure in recovering the pesticide residual due to its advantages of high efficiency, safety and non-secondary-pollution.In this work, strain GF31 was characterized using 16S rDNA gene analysis and identified as a Pseudomonas aeruginosa, which has been deposited under CGMCC 7173 in China General Microbiological Culture Collection Center. The distribution assay on the pyrethroid-degrading enzyme of GF31 indicated that this enzyme mainly shows an extracellular enzymatic activity, accounting for 81.5% of the total enzyme activity, and such an enzyme is estimated to be an extracellular enzyme, different from the current intracellular pyrethroid-degrading enzymes generally reported. The cultivation medium was optimized, and the optimized medium contains:molasses 5 g/L, peptone 10 g/L, NaH2PO4 0.12 g/L, phenylalanine 1.0 g/L. In addtion,0.1% of Tween 80 is added after 12 h of cultivation. Yield of enzyme of GF31 strain increased about 2.6 folds, laying the foundation for the subsequent studies such as the purification of the degrading enzyme and the characterization of enzymatic.A scheme for purification of enzyme was designed according to the basic property of the GF31 pyrethroid-degrading enzyme. Upon a 4-step process of ultrafiltration, ammonium sulfate precipitation, ion exchange, and gel filtration, the pyrethroid-degrading enzyme was purified up to 32.8 times with a recovery of 26.6%, and the specific activity reached 2571.9 U/mg. SDS-PAGE gel electrophoresis indicated that the purified enzyme showed a single band with a molecular weight of 53 KDa.A mass spectra molecular weight was detected to be 51 KDa for the enzyme. In combination with the results from SDS-PAGE gel electrophoresis, it is suggested that the enzyme is a mono-subunit protein, and the N-terminal amino acid thereof is in an order:NH2-Thr-Pro-Gly-Lys-Pro-Asn-Pro-Ser-Ile-Cys. Reducing and non-reducing SDS polyacrylamide gel electrophoresis indicated that there was disulfide bond within the degrading enzyme molecule; glycosylation determation indicated that the degrading enzyme contains no or little glycosyl. Database searching for the peptide mass fingerprint of the purified enzyme showed that it has a high similarity with the presumed aminopeptidase derived from Pseudomonas aeruginosa M18 and PAO1, which is in turn deemed as belonging to the same kind of protein.The gene APs (Genebank accession number KT735188) encoding the pyrethroid-degrading enzyme derived from Pseudomonas aeruginosa strain GF31 was cloned by PCR based on the sequence of aminopeptidase gene of Pseudomonas aeruginosa PAO1 as templete, and the heterologous expression of APs in E.coli was successfully achieved. Bioinformatics analysis of this pyrethroid-degrading enzyme gene APs showed that the enzyme consists of 536 amino acids, with the first 24 amino acids representing a signal peptide, amino acid 25-36 reprsenting a propeptide, followed by 500 amino acid residues as a mature protein, with a molecular weight of 53.7 KD, which is consistent with the molecular weight of the purified protein. Analysis on conserved domain and amino acid sequence showed that the pyrethroid-degrading enzyme can be classified as an aminopeptidase from the superfamily of zinc-peptidases (M28), having an aminopeptidase-type catalytic ternary-component (Glu341, Ser423 and His296), and five amino acid residues to be coordinated with Zn:His296, Asp308, Glu341, Asp369, and His467. This is the first aminopeptidase reported to have pyrethroid-degrading ability. Primary structure of the pyrethroid-degrading enzyme APs was analyzed, which is substantially consistent with the the amino acid composition of the purified enzyme, with major error only occurs in the contents of Glu, Gly and Pro. Secondary structure of the enzyme was predicted, and it showed an a-helix content of 32.8%, a (3-sheet content of 20.6% and a random coil content of 46.6%. Predicted tertiary structure of APs was obtained by homologous modeling with Streptomyces griseus aminopeptidase (sharing the highest homology based on the PDB protein database) as a templete.A relatively systematic study on the degradation characteristics of the pyrethroid-degrading enzyme was carried out, providing a theoretical foundation for the practical engineering application of the enzyme. For this enzyme, the optimal degrading pH is 7.0, and the optimal degrading temperature is 60℃; it possesses a good stability within 70℃ and a pH range of 5.0-9.0; Ag+, Hg+ and Cu2+ have significant inhibitive effect on this enzyme, and other metal ions have little effect on the activity of this enzyme. Metal ion chelating agents EDTA and phenanthroline have no inhibitive effect on the activity of this pyrethroid-degrading enzyme, suggesting that the activity of this pyrethroid-degrading enzyme is independent of metal ion. Surfactants SDS and Triton X-100 can significantly inhibit the activity of this pyrethroid-degrading enzyme. The pyrethroid-degrading enzyme can catalyze the degradation of various pyrethroids such as cypermethrin, fenpropathrin, fenvalerate and deltamethrin, wherein the degradating activity on cypermethrin is comparatively high. Determination on reaction kinetics showed that when taking cypermethrin as a substrate, Vmax= 0.0017 μM/s, Km= 47.7 μM, kcat= 0.008 s-1; when taking L-leucine-paranitroaniline (Leu-pNA) as a substrate, Vmax= 0.99 μM/s, Km 2668.8 μM,kcat= 183.3 s-1, indicating that the pyrethroid-degrading enzyme can catalyze the hydrolysis of pNA substrate with a higher efficiency, and it is further determined as an aminopeptidase. |
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