Screening,Characterization And Immobilization Of SulE Mutant With Improved Sulfonylurea-Hydrolyzing Activity

Sulfonylurea herbicide is a kind of important selective herbicide with ultra-high efficiency and low toxicity to mammals,they have been widely used for weeds control in the world.Some of sulfonylurea herbicides such as metsulfuron methyl and sulfometuron-methyl are persistent in soil and are harmful to the rotation crops,while some of sulfonylurea herbicides such as tribenuron methyl and bensulfuron-methyl have a relatively short residual period,and are target herbicides in herbicide resistant transgenic engineering.Therefore,it is of great significance to obtain sulfonylurea herbicide detoxification enzyme and gene resources with high activity.In our previous work,a novel esterase gene sulE was cloned from Hansschlegelia zhih uaiae S113.SulE catalyzes the de-esterification of a variety of sulfonylurea herbicides with a methyl or ethyl ester to the corresponding parent acids,which are herbicidal-inactive.SulE exhibits high de-esterification activity to thifensulfuron methyl but very low activities to other sulfonylurea herbicides,which greatly limited its application.Therefore,it is necessary to improve the activities of SulE to various sulfonylurea herbicides.In this study,a mutant SulE3mut with significantly improved activity was screened from the random mutant library of SulE.The de-esterification activity and characteristics of SulE3mut were studied.The effect of each mutation site on SulE activity was analyzed by single point mutation.The immobilization of SulE3mut was also studied.The main results are as follows:1.Screening of SulE mutant with improved de-esterification activityA random mutation library of SulE was acquired by error prone PCR.After three rounds of screening,a mutant,SulE3mut with significantly improved sulfonylurea de-esterification activity was screened.The nucleic acid sequence of mutant SulE3mut was sequenced and compared with SulE.It was found that five bases have been altered:T241 A,A242C,T532A,C533A and G545C,respectively,which resulting of three amino acids substitutions,namely Y81T,S178N and G182A.2.Study on the enzymatic characteristics of SulE3mut mutantSulE3mut and SulE were expressed in Escherichia coli using pET29a(+)system,and were purified using Co2+affinity chromatograph.The sulfonylurea de-esterification activity of SulE3mut was significantly higher than that of the wild type SulE.The activities of SulE3mut to metsulfuron methyl was 10.2 times higher than that of SulE,and the activities of SulE3mut to sulfometuron methyl,tribenuron methyl and amisulfuron methyl were also increased by 9.6,2.3 and 4.9 times,respectively.The optimum pH and temperature of SulE3mut were 7.0 and 40℃,respectively.SulE3mut was more stable than SulE under high temperature conditions.3.Study on the enzymatic characteristics of single point mutant Y81T,S178N and G182A of SulEThree single-site mutants of SulE,namely Y81T,S178N and G182A,were constructed using site-directed mutagenesis kit.The three single-site mutants were expressed and purified.Compared with SulE,the activity of Y81T was decreased,while the activity of S178N and G182A was obviously increased,but much lower than that of SulE3mut.The results indicated that the activity increase of SulE3mut was resulted from the synergistic effects of the three site mutations.The three-dimensional structure simulation analysis indicated that the increase of SulE3mut enzyme activity might be due to the enlargement of the active pocket and the increase of interaction between the enzyme and heterocycle of metsulfuron methyl.4.Immobilization of SulE3mutThe SulE3mut crude enzyme was immobilized using PVA+SA as the carrier.The immobilized enzyme completely transformed 100 mg/L metsulfuron-methyl within 12 hours in water.The immobilized enzyme was more stable than non-immobilized enzyme,and the immobilized enzyme retained approximately 40%of its activity after 9 cycles of recycling.In brief,this study screened a mutant of SulE that displayed significantly higher deesterification activity towards various sulfonylurea herbicides.Mutant SulE3mut has application potential in the elimination of sulfonylurea residues and the herbicide-resistant transgenic engineering.