| Catecholic compounds which used as important chemical reagents are widely applied in the processing of pharmaceuticals,chemicals,cosmetics,pesticides,dyes.They are released into aquatic and terrestrial ecosystem as a result of anthropogenic activities,and leading to serious environmental issues.Extradiol dioxygenases(EDOs)have been found in Bacillus,Pseudomonas,Alcaligenes,and other bacteria.They can break the C-C bond adjacent to the hydroxyl group of catecholic pollutants.Hence,EDOs are important enzymes in the biodegradation of phenolic pollutants.Till now,most of the isolated dioxygenases are mesophilic enzymes.Compared with mesophilic enzymes,thermophilic ones show better resistance to high temperature,chemicals,detergents,or proteolysis,which are essential properties for industrial applications.Therefore,it is important to obtain high-performance thermophilic dioxygenase.In the present study,4 different thermophilic dioxygenases were cloned from different thermophilic microorganisms.After screening,highly active Tcu3516 was chosen for further characterization.Homology modeling,docking,and molecular dynamics simulation were used to understand how the enzyme interacts with the substrate.Based on this result,the loops locating around the entrance of catalytic pocket were tailored through semi-rational design to obtain valuable mutants.Then,the acyl unsaturated fatty acid as the precursor of the bioplastic was synthesized,and the catalysis of the non-natural medium-chain acyl catechol derivates by Tcu3516 was optimized.Finally,the enzyme was redesigned to carry out a ring cleavage reaction on long-chain and ultra-long-chain alcy catechol substrates by reducing steric hindrance,and the catalytic ability of Tcu3516 against catechol derivates with long chain substituents was improved.The main contents and results of this thesis are as follows:1.Based on NCBI databases and bioinformatics analysis,4 new types of thermophilic extradiol dioxygenases with research prospects were cloned from Thermomonospora curvata DSM43183,Rubrobacter xylanophilus,Sulfolobus islandicus REY15 A,Thermobispora bispora DSM 43833,and they were named Tcu3516,Rxy5255,Si0380 and Tbi2513,respectively.The high-activity Tcu3516 was selected based on the activity of these four enzymes.2.The sequence analysis results showed that Tcu3516 belongs to the VOC family/type I.Comparing with other reported dioxygenases,the identity ranged from25% to 44%.The phylogenetic tree analysis further proved that Tcu3516 belongs to a unique branch that has not been reported.We found the optimum temperature and p H were 50 °C and 9.0 based on the characterization of its properties.The enzyme kept good thermal stability at 55 °C.The kinetic data showed it has a good catalytic efficiency.3.Tcu3516 can catalysis variety of natural substrates,including catechol,3-methylcatechol,3-chlorocatechol,4-chlorocatechol,2,3-dihydroxybiphenly,and 3,4-dihydroxyphenylacetic acid.A comparison with previously studied EDOs shows that Tc3516 has a wider substrate spectrum.These are exactly the required properties for the degradation of diverse phenolic environmental pollutants.Based on this,sitedirected mutations of the larger amino acid residues on the loops locating around the entrance of catalytic pocket were carried out by computer-aided homology modeling and molecular docking analysis to obtain mutants F301 V,D285A,and H205 V.We found that the above mutants have significantly improved catalytic activity on different catechol substrates.It is worth noting that the activity of D285 A has increased 2-17 folds erent substrates.Combined with the results of the enzymatic reaction kinetics,it further showed that these loops do have a critical influence on the substrate specificity and catalytic efficiency.4.The catalytic reaction of 3-(1-ketobutyl)catechol with medium chain by Tcu3516 was optimized to obtain keto/ hydroxy unsaturated fatty acids,which are used as the precursor material of degradable bioplastics.The results showed that the optimal reaction temperature was 30 °C,the optimal solvent was sodium phosphate buffer containing 10% N,N-dimethylformamide(DMF),and the total turnover number(TTN)could reach the highest after 10 h in the air.It provides a basis for the industrial application of dioxygenase.Tcu3516 shows limited catalytic ability on long-chain acyl catechol and ultra-long-chain acyl catechol which restrict its production for more promising hydroxy unsaturated fatty acids.Based on this,mutation sites were selected through molecular docking to reduce the steric hindrance between the enzyme molecule and the acyl substituent of the substrate.After the aquired F301 V,D285A,and H205 V mutants,the R256 A,M173A,W292 V,and D262 A mutants were also successfully constructed.The catalytic experiment results showed that F301 V,locating on the lid structure,had higher catalytic ability towards medium-chain acyl catechol and ultralong-chain acyl one than the wild type.This result indicates that the modifying of the lid structure will also increase the enzyme's catalytic ability on large substrates,revealing the importance of the lid structure.In short,this research has screened and obtained a new thermophilic extradiol dioxygenase Tcu3516 and its mutants.They show promising application value in biodegrading phenolic pollutants and synthesizing high-value hydroxy unsaturated fatty acids. |
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