Construction And Optimization Of The Biodegradation Pathway Of 1,2,3-trichloropropane

The anthropogenic compound 1,2,3-trichloropropane(TCP)has recently recognized as a groundwater pollutant,mainly due to the extensive use of the compound as a solvent and precursors for the synthesis of soil fumigation and other compounds.The production of TCP in the world amounts to 50,000 tons per year.The huge amount of industrial waste discharged into the environment is a threat to human health.The traditional methods often fail to meet the requirements and are likely to cause secondary pollution.Therefore,the application of biodegradation of such compounds has become a hot topic at home and abroad.The microbial degradation pathway contains various active enzymes such as haloalkane dehalogenase,haloalcohol dehalogenase and epoxide hydrolase,and the physicochemical properties of these free enzymes are unstable,they are very sensitive to environmental changes and they are easily deactivated.In this study,the immobilization technique of crosslinked enzyme aggregates(CLEAs)was applied to the construction of TCP degradation pathway of environmental pollutants,and the degradation efficiency of TCP was improved by optimizing the conditions.The prokaryotic expression vector of pBAD was used to obtain the high expression system of haloalkane dehalogenase,haloalcohol dehalogenase and epoxide hydrolase.Finally,the expression levels were 20%,50%,and 50%,respectively.It lays a foundation for immobilization of enzyme by CLEAs technology.Based on the existing immobilization technology of CLEAs,the reaction temperature,additive,precipitation agent,crosslinking time and other conditions were optimized.Finally,immobilized haloalkane dehalogenase and immobilized haloalcohol dehalogenase with enzyme activity retention of 90% or more.And the immobilized epoxide hydrolase enzyme activity also reached more than 50%,which can be used for subsequent studies.The performance of immobilized haloalkane dehalogenase under the optimum conditions showed that the immobilized enzyme had good thermal stability,storage stability,organic tolerance,recoverability and high enantioselectivity to substrate catalysis.Therefore,the immobilized enzyme has higher research value and can be further applied in the study of TCP multi-enzyme degradation pathway.During the multi-enzyme cascade reaction of TCP,the order of the enzyme addition,the ratio of addition,and the amount of enzyme added were studied.The results showed that the sequential addition was more than 14% higher than that of the simultaneous addition of TCP.The TCP degradation efficiency was the highest when the ratio of three enzymes was 1:1:1.The degradation rate of TCP is the highest when the total mass of enzyme is 30 mg,and TCP degradation efficiency can reach over 97% after two hours of reaction,and the production of glycerol was also successfully detected.After five times of repeated recovery of immobilized three enzymes,the degradation rate of TCP could still reach more than 75%.In summary,this study uses a more efficient and economical bio-immobilization enzyme method to achieve effective degradation of TCP.