Study On The Degradation Of Phthalate Esters By Carboxylesterase And Its Whole Cell Catalyst

Phthalic acid esters（PAEs）are important synthetic organic compounds that are used in large quantities as plasticisers in industrial production and have been detected in excessive residues in a variety of environments.The continuous accumulation of PAEs not only pollutes the environment,but also seriously threatens the health of humans and other organisms.Therefore,there is a great need to remove PAEs from the environment.In contrast to physical and chemical methods,bioremediation is an approach that allows complete degradation without secondary contamination.Carboxylesterase usually plays an important role in the cleavage of PAEs ester bonds,which indicates that it is necessary to develop new esterases to degrade PAEs.In this paper,two carboxylesterase genes,est N26 and est N3,were cloned from Bacillus subtilis DBP-1 and Bacillus licheniformis DBP-4,respectively,which had been preserved in the laboratory for screening.So far,there have been no reports on the degradation of phthalate esters by EstN26 and EstN3.Therefore,in this paper,EstN26 and EstN3 were heterologously expressed and displayed on the cell surface of Escherichia coli using Ice Nucleation Protein（INP）to make a whole-cell catalyst,and the enzymatic properties of EstN26 and EstN3and their whole cells were determined,as well as preliminary assays for the degradation of PAEs,with the following main findings:（1）Determination of the enzymatic properties of EstN26 and EstN3.The enzymatic properties of EstN26 and EstN3 were determined as follows:the optimum temperature was 40°C and the optimum p H was 8.5.EstN26 could be left at 4°C for 24 days and EstN3 at 4°C for 20 days.EstN26 was treated at p H 7.5-9.0for 1 hour and the remaining relative activity was>40%,EstN3 was treated at p H6.0-9.0 for 1 hour and the remaining relative enzymatic activity was>36%.83%-96%relative enzyme activity remaining for EstN26 in the presence of metal ions Na⁺,K⁺and Mg²⁺and 64%-100%relative enzyme activity remaining for EstN3 in the presence of Fe³⁺,K⁺,Na⁺and Ca²⁺ions.The kinetic parameters K_m of EstN26 and EstN3 for p-NPC₄ were 1.312 m M and 1.105 m M,and V_max were 7461mmol/min/m L and 1110 mmol/min/m L,respectively.（2）Determination of the enzymatic properties of EstN26 and EstN3 whole-cell catalysts.The enzymatic properties of the whole-cell catalysts were determined as follows:the optimum temperature was 40°C and the optimum p H was 8.5.After 40days of tolerance at 4°C,30°C and 37°C,48%,33%and 26%of the relative enzyme activity remained in EstN26 whole cells and 42%,22%and 14%in EstN3whole cells,respectively.The residual activity of EstN26 whole cells was>47%after 1 hour treatment at p H 7.5-9.0,and the residual activity of EstN3 whole cells was>73%after 1 hour treatment at p H 6.0-9.0.K⁺ions increased the relative enzyme activity of EstN26 whole cells by 13%and whole cells of EstN3 had78%-100%relative remaining enzyme activity in the presence of Fe³⁺,K⁺,Mn²⁺,Co²⁺and Tween 80 and ethanol.The kinetic parameters K_m for p-NPC₄ were 3.440m M and 1.312 m M and V_max were 9013 mmol/min/m L and 3762 mmol/min/m L for whole cells of EstN26 and EstN3,respectively.（3）Preliminary assay of EstN26 and EstN3 and their whole cells for degradation of phthalate esters.EstN26 and EstN3 and their whole cells showed degradation of various PAEs（DBP,DMP,DEP,BBP）and thin layer analysis showed that EstN26 and EstN3 and their whole cells reacted with these PAEs and produced new substances in addition to the remaining substrate.The results of the targeted mutation and subsequent experiments showed that the amino acids at the L362（corresponding to the I360 site of EstN3）site had an important effect on the protein-catalyzed substrate degradation.The specific activity of mutants L362F,L362W,L362Y and I360F,I360W,I360Y was higher than that of wild type,that is,the degradation efficiency of substrate was higher than that of wild type.