Properties And Degradation Capacity On PAEs Of Thermophilic Esterase From Picrophilus Torridus

Phthalates are widely used in industrial production and consumer goods because of their flexibility,durability and machinability.Over time,PAEs will be transferred from plastic products to the external environment,which will pollute the environment.They can interfere endocrine system of human and animals at an extremely low concentration.It is easy to teratogenic,carcinogenic and mutagenic under PAEs for a long time.PAEs are refractory organics,in nature,the speeds of their hydrolysis and photolysis are very slow.The existence of PAEs is potentially harmful to our society.In the previous laboratory work,to achieve the expression of Picrophilus Torridus gene in engineered E.coli and obtain a thermophilic esterase EstPt 1,which can degrade pNPB.This thesis will focus on analyzing the properties of thermophilic esterase EstPt 1.First of all,the thermophilic esterase EstPt 1 was successfully expressed and its relative molecular weight was determined to be 35 KD and enzyme activity of cell-free extract is 317 U/mL.After purified by Ni-NTA chromatography,the enzyme activity reached 202.3 U/mg,and ratio of purification was 3.90 times.The optimum temperature and pH for enzymatic reaction were 85°C and 9.0.And kept the enzyme at 60°C for 64 h,65°C for 12 h,70°C for 1 h,still had 50% activity on pNPB.It shows that EstPt 1 has good thermostability and can be completely used for degradation of PAEs in high temperature environment.The Michaelis constant of EstPt 1 was determined to be 6.6498 mM,and the Vmax was 11875.71 Mm/s·mL.It is also estimated the active site is Ser147-Asp241-His271.EstPt 1 has excellent degradation capacity on PAEs.We found that 5 mM DBP can be completely degraded and produce MBP in 24 h.Degradation efficiency at 60°C is better than at 37°C.It shows that EstPt 1 can apply to degradate PAEs at high temperature.In order to further improve the stability of the enzyme preparation and achieve recycling,EstPt 1 was studied for immobilization.In this paper,we designed a cross-linked enzyme aggregate method to immobilize EstPt 1.Then we added Pluronic F127 as stabilizer in the conventional CLAE method.The viability is 3.5 times that of the conventional cross-linked enzyme aggregate immobilized enzyme CLEA.And we name it as CLEA-EstPt 1.The immobilized enzyme has better acid-base tolerance and thermostability;its optimum pH is 9.0,it still has 90% activity when immobilized enzyme under pH 8.0 to 9.5,and under pH 6.0 still has 20%.It seems that the activity range of pH is expanded.The optimum temperature is increased from 85°C to 90°C;the immobilized enzyme activity is still 50% when incubated at 70°C for 5 hours;and at 4°C the activity still 20%.It is certain that immobilized enzyme can be used in some high temperature environments.In addition,it showed superior organic solvent tolerance when incubated at four different organic solvents for 2 hours respectively,the activity not only lost,but also had an upward trend.Continuous use of viability 10 times still maintained at more than 80%,it showing good reuse.The tolerance of immobilized enzyme to organic solvents was significantly enhanced.After degrading DBP for 24 hours,the residual substrate was below 15%,whereas the free enzyme was exceeded 50%.