Expression,Immobilization And Application In Pesticide Degradation Of EstC From Streptomyces Lividans

Esterases,including carboxylesterases and lipases,play critical roles in catalyzing the formation and cleavage of ester bonds.As a kind of biological enzyme widely used in industrial applications,esterases are often employed in environmental treatment,pharmaceutical synthesis,preparation of biodiesel,food processing and other fields.Compared with normal enzymes,esterases that can maintain functional and structural stability in high temperature or alkaline environments will have a broader prospect on commercial applications.In this study,the Est C from Streptomyces lividans TK24 was successfully expressed heterologously,characterized,immobilized and its application potential in pesticide degradation was also investigated.The main contents and results of this research are as follows:1.Expression and characterization of EstCEstC,a novel esterase from Str.lividans TK24,was successfully expressed,purified and characterized.Amino acid sequences alignment and phylogenetic analysis showed that EstC could be assigned as the first member of a novel family XIX.Additionally,EstC had highly conserved structural features,including a Ser155-Asp248-His278 catalytic triad and a canonical Gly¹⁵³-His¹⁵⁴-Ser¹⁵⁵-Ala¹⁵⁶-Gly¹5757 pentapeptide.In terms of enzymatic properties,EstC exhibited maximal activity at pH 9.0 and 55°C.On the preferences of substrates,EstC showed higher activity towards short-chain substrates with the highest activity for p-nitrophenyl acetate(K_m=0.31±0.02 mM,k_cat/K_m=1923.35±9.62 s^-11 mM^-1).Notably,EstC showed hyper-thermostability.The activity of EstC had no significant changes when it was incubated under 55?for 100 h and reached half-life after incubation at 100?for 8 h.Beyond that,EstC also showed good alkali-stability in a pH range of 6.0-11.0 and about 90%residual activity was still reserved after treatment at pH 8.0 or 9.0 for 26 h,especially.Est C had a certain tolerance to different metal ions,but poor tolerance to organic solvents and detergents.2.Study on the immobilization of EstC and catalytic properties of EstC@ZIFEstC was immobilized with metal-organic frameworks?MOFs?as the carrier.By optimizing the proportion of 2-methylimidazole,zinc acetate and protein concentrations,EstC@ZIF was successfully synthesized and its systematic characterization and catalytic properties were further investigated.The morphology of EstC@ZIF presented in a cross shape and it possessed an average size of about 2?m.The specific surface area and pore diameter of EstC@ZIF were 943.19 m² g^-11 and 5.43 nm,respectively.Compared with free enzyme,the optimum pH of EstC@ZIF was still 9.0 while its optimum reaction temperature was increased from 55 to 60?.Furthermore,enzymatic kinetics hardly changed and the tolerance to some organic solvents and detergents was significantly improved before and after immobilization.Especially for methanol?30%?and SDS?1%?,the residual activity of EstC@ZIF increased from 8.06 and 20.52%to 73.42 and 68.31%,respectively.In addition,Est C@ZIF had satisfying reusability with 50%of its initial activity maintained after 9 continuous uses.3.Study on the pesticide degradation ability of EstC and EstC@ZIFEstC had potential application value in bioremediation of pesticide contamination,it could hydrolyze chlorpyrifos with strong substrate specificity.0.5 U mL-1 EstC had ability to hydrolyze 51.30 and 79.82%chlorpyrifos with an initial concentration of 5 mg L^-11 within 45 min and 80min at 37?,respectively.EstC@ZIF could hydrolyze 71.36%chlorpyrifos within 80 min and the degradation rate could still retain 59.32%after 6repetitive uses.Further analysis of the degradation products by gas chromatography-mass spectrometry?GC-MS?showed that chlorpyrifos might gradually generate 3,5,6-trichloro-2-pyridinol and 2,4-bis?1,1dimethylethyl?phenol under the hydrolysis of EstC.