Gene Mining Of Thermostable Carboxylesterase And Its Application In Pesticide Degradation

In this research paper,we mainly carried out study on new carboxylesterase through screening thermophilic strains,and finally applied the developed carboxylesterase to pesticide degradation.In the plate screening experiment,an original strain Geobacillus uzenensis 23175 can hydrolyze tributyric acid glyceride and produce a significant hydrolysis circle.By analyzing the genome of the original bacteria and PCR,the gene sequence of carboxylesterase est741 was obtained.est741 has an open reading frame of 741 bp,encodes a 246 amino acid carboxylesterase(named Est741).The gene sequence has been submitted on NCBI.Based on genetic engineering technology,est741 was cloned and expressed in Escherichia coli BL21(DE3)and Pichia pastoris KM71.The optimal pH and temperature for Est741 expression were 8.0 and 50℃.Through site-directed mutation,the optimal temperature of the mutant M160K(Est741M)was increased from 50℃ to 60℃,and showed an enhanced T1/2 of 2.5 h at 70℃ in comparison to Est741(1.3 h).Additionally,Est741M immobilized on an epoxy resin showed an enhanced T1/2 of 36.8 h at 70℃ in comparison to the free enzyme.lx-Est741M could degrade various pyrethroid pesticides.After 40 min reaction with 50 U of the purified enzyme,the malathion removal was 95.8%with a malathion concentration of 20 mg/L.Using a 10 mL packed bed column reactor,the degradation ability of the immobilized enzyme lx-Est741M on pyrethroid pesticide wastewater was determined.The results showed that when 2.5 g immobilized enzyme was added to the 10 mL column reactor with a bifenthrin concentration of 500 mg/L and pump transfer rate of 0.7 mL/min,the degradation rate of lx-Est741M to bifenthrin was 90.4%.lx-Est741M exhibited high operational stability and maintained 72%initial activity after ten batches of continuous reaction for bifenthrin pesticide biodegradation.This is the first report of Est741 obtained from G.uzenensis,with improved properties.These preliminary studies on the use of epoxy resin immobilized enzymes to treat pesticide contaminated wastewater reveal potential industrial applications.