| Esterases,are a class of enzymes that with their ability to catalyze the cleavage and synthesis of ester bonds.Their structures are usually composed by the N-terminal cap domain and the C-terminal ?/? hydrolase catalytic domain.Most of esterase with stereoselectivity,regioselectivity and wide spectrum of substrate,can be applied to a variety of industrial applications,and cold-adaptation esterases have the characteristics of stable products and energy saving due to their low temperature activity,thus attracted much attention in the areas of application in biological pharmaceutical,bioremediation and lowtemperature washing.Extremophiles adapt to extreme environments such as high temperature,low temperature and high pressure during natural evolution,are excellent sources of gene resources of extreme enzymes.At present,the variety of enzymes in China is relatively unitary,which unable to meet the industrial needs,so it is still necessary to develop the high-quality enzyme resources with special properties.The low-temperature activity of cold-adapted enzymes depend on the flexibility of their structure,and generally with weak thermal stability,which is not conducive to industrial application.Therefore,it is particularly important to improve the thermal stability of cold-adapted enzymes.At present,the engineering strategies of enzyme stability mainly include the introduction of disulfide bond,hydrogen bond,salt bridge,increasing hydrophobic force,the balance between the stability and flexibility of the enzyme is also the key to optimize the catalytic function of the enzyme at a specific temperature.In order to develop the esterases for industrial production,this project intends to clone the esterase genes from D.radiodurans R1,improve the properties through molecular biology methods,and test its degradation effect on pesticides and other environmental pollutants,to provide more options of esterases for industrial and environmental.The main research results include:1.Six esterase genes were cloned from the genome of D.radiodurans R1 and the expression vector of Escherichia coli was constructed.After induction and expression,the esterase activity was determined and EstDR4 with the higher activity among six genes was selected for further study.EstDR4 is a new member of the hormone sensitive lipase(HSL)family.EstDR4 contains a typical cap structure,catalytic triplet(Ser156-ASP253-His283)and GGGX type oxygen anion hole which peculiar to HSL family.EstDR4 can effectively hydrolyze short chain and medium chain p-nitrophenyl ester substrates(C2?C12),and has regioselectivity and enantioselectivity.EstDR4 also has hydrolytic effects on tertiary alcohol and phenyl ester.The optimum temperature and p H of EstDR4 were 30? and 8,respectively.EstDR4 showed high catalytic activity at 0? and high stability at 10??40?.The residual enzyme activity of EstDR4 were above 80% after 1 hour.In addition,the activity of EstDR4 was significantly increased in the presence of Tween 80 and Triton X-100.2.The cap domain of EstDR4.To removal and replacement the cap domain of EstDR4,we got mutants ?35 and Est2 N respectively,the results showed that esterase activity of ?35greatly decreased,however,the substrate preference to long chain esters were increased.The catalytic efficiency of Est2 N displayed a 63.82% deduction,but the half-life of 40? and 50?increased to 2.09 times and 1.69 times of wild type,the thermal stability of Est2 N was improved.The interaction residues between the cap domain and catalytic domain were analyzed,and we found more chemical bonds and electrostatic force generated in Est2 N,which make the cap domain more closer to the catalytic domain.However,the overexposure of the active site in the solvent led to the decrease of catalytic efficiency and substrate affinity of Est2 N.The results of molecular dynamics simulation showed that the RMSD value of the mutant Est2 N was lower than that of the wild type,and the RMSF value of the cap domain was also reduced significantly,indicating that the enhanced rigidity of the cap domain led to the increase of the thermal stability of Est2 N.3.With reference to conservative sites of thermophilic esterases from HSL family,the EstDR4 mutations with associated sites around the active center were constructed,and got mutants S132 A,T163A and Y285 F with improved thermal stability.The results showed that the half-life of S132 A in 40? and 50? increased 44.29% and 89.89% respectively,the halflife of T163 A in 40? and 50? increased 89.59% and 119.11% respectively,and the halflife of Y285 F in 40? and 50? increased 59.41% and 65.79% respectively.Lower RMSD and Rg values also confirmed that the three-dimensional structure of three mutants were more stable compared with the wild type.Interaction force analysis found that the enhancement of hydrophobic force of hydrophobic core is the main reason for the improvement of thermal stability,saturated mutations in sites132 and 163 further confirmed that the enhancement of hydrophobic force of hydrophobic core can lead to an improvement of thermal stability,and alanine effect on the stability of the helical structure also offered an important contribution in thermal stability.4.Mutants M212 V and M212 G with reduced steric hindrance were obtained by substrate channel analysis.The substrate specificity analysis showed that the substrate range of both mutants was expanded,but the catalytic efficiency of M212 G was reduced due to the weakened hydrophobic force of substrate channel.Mutations were performed at selected sites on the ?6 and connected loop region near the substrate channel,and stability promotion mutants F214 W and S238 D were identified.Saturation mutations were performed at sites214 and 238,and catalytic efficiency and thermal stability increased mutants F214 E and S238 N were identified.Interaction analysis of mutants found that the improvement of thermal stability is associated with additional formation of hydrogen bonds and ionic bonds,in addition,sites 214,238 connected to site 212 jointly maintain the stability of substrate channel and active pocket,F214 E can form an ionic bond with H191 to make the structure shift and reduce the smaller steric hindrance at 212 site,while S238 N and R170 formed several additional hydrogen bonds,which may cause the loop swing and the change of substrate channel,which may be the reason for the increase of catalytic efficiency.5.EstDR4 and its mutants M212 V,S132A,T163 A,Y285F,F214 E,S238N were quantitated by LC-MS to determine the degradation of four insecticides(carbaryl,fenpropathrin,?-cypermethrin,deltamethrin)and zearalenone.EstDR4 showed degradation effects on both insecticides and toxin,and the degradation effects on carbaryl,fenpropathrin,?-cypermethrin,deltamethrin and zearalenone decreased in turn.Compared with the wild type,the degradation rates of S238 N on four insecticides and M212 V on three insecticides except carbaryl were increased about 6%-8%,while the degradation rates were not affected of increased stability mutants S132 A,T163A,Y285 F and F214 E.These evolution provided a promotion effect for EstDR4 application in industry.In summary,this study analyzed the enzymatic properties of HSL family esterase EstDR4 from D.radiodurans.EstDR4 was found to be an alkaline esterase with cold adaptability and surfactant tolerance,with regioselectivity and enantioselectivity.EstDR4 also had degradation effects on insecticides and zearalenone,and had a good application prospect in fruit and vegetable detergents and bioremediation.In addition,according to the structural characteristics of HSL family esterases,EstDR4 was optimized based on the hydrophobic core,loop swing and channel steric resistance.The key sites related to the thermal stability and substrate preference of the enzyme were identified,and the mutants with higher stability,stronger catalytic efficiency and more extensive substrate preference were obtained,which provided strategic reference for the enginerring of similar proteins and contributed to the further industrial application of EstDR4. |
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