Study Of The Expression And Enzymatic Properties Of A Highly Efficient PET Plastic Degrading Enzyme In Pichia Pastoris

At present,the widespread use of polyethylene terephthalate plastic(PET)has caused serious pollution to the ecological environment.Biodegradation,a more environmentally friendly degradation method has attracted widespread attention worldwide.However,the influence of low activity,low expression,heat resistance and other factors limit its large number of applications in industry.IsPETase is a PET plastic depolymerase which found from a bacterium called Ideonella sakaiensis by Kohei Oda’s research team in Japan.The wild-type IsPETase’s activity is limited and is unstable.HotPETase and FAST-PETase which were mutated from wild-type IsPETase exhibited excellent PET hydrolytic activity.Proteins were successfully expressed in the systems of Escherichia coli and Pichia pastoria and purified proteins of higher purity in this experiment.The expression of HotPETase in Pichia systems was much lower than that of FAST-PETase.In order to compare the activity of the two,the enzyme activity test was carried out under the optimal reaction conditions of HotPETase and FAST-PETase.The experimental results showed that the activity of FAST-PETase was higher than that of HotPETase.We found the protein expressed by P.pastors had higher activity by comparing the activity of E.coli and Pichia-expressed FAST-PETase.The expression of protein was increased by molecular biology in this experiment.The main used methods were: Optimizing codons;High copies of genes.We found a higher expressed strain(1000-6)at an antibiotic concentration of 1000mg/mL;With the addition of chaperone co-expression.PDI,HAC,and ERO1 were used in the experiment.and a high-expressed strain(ERO1-9)was found in the process of co-expression of chaperone and double mutation.Using unpurified protein and purified protein for enzyme activity test and compare their activity,the activity of unpurified protein is more than 80% of the activity of purified protein.The screened ERO1-9 strain was used to cultivate expandly in fermenter.The wet weight of the fungus reached 0.33 g/mL,and the yield of protein reached 1.78 mg/mL.