Molecular Modification Of Cutinase ICCG And Its Application In The Degradation Of Poly(Butyleneadipate-co-terephthalate)

At present,the"white pollution"caused by the accumulation of plastic waste poses a huge threat to the ecological environment.Poly（butylene adipate co terephthalate）（PBAT）is a biodegradable polyester that is widely used in various fields due to its excellent biodegradability as an aliphatic polyester and excellent processability as an aromatic polyester.However,the degradation rate of PBAT under natural conditions is too slow,and its waste accumulation may become a new environmental pollutant.Therefore,biodegradable enzymes are needed to accelerate its degradation.PBAT is formed by the condensation of butylene adipate（BA）and butylene terephthalate（BT）in different proportions.This article first uses PBAT with a high BT content as the substrate,abbreviated as PBAT（H）,and compares the degradation efficiency of polyester degrading enzymes from different sources to determine that the enzyme with the best degradation effect is the cutinase ICCG;Then,the enzyme was rationally designed to construct dominant mutants;At the same time,new PBAT（H）intermediate degradation enzymes were explored and a dual enzyme synergistic system was constructed to relieve intermediate inhibition and improve the degradation efficiency of PBAT（H）.The main research results of this article are as follows:（1）Screening of PBAT（H）polyester degrading enzymes.Five enzymes reported to have good effects on polyester degradation were selected and expressed in Escherichia coli BL21（DE3）.Then they were applied to the degradation of PBAT（H）respectivelly.Cutinase ICCG was determined to be the enzyme with the best degradation effect;In order to study the effect of BT content on the biodegradability of PBAT,four materials with different BT content were selected for enzymatic hydrolysis.The results showed that the higher the BT content,the lower the degradation rate.At the same time,the optimal system for the degradation of PBAT（H）by cutinase ICCG was determined,with a reaction temperature of 75℃,a mass ratio of enzyme to substrate(E·S^-1)of 0.4%,and a substrate concentration of 1%.Under this system,the degradation rate was 77.50%after 48 hours of reaction.（2）Reasonably design the cutinase ICCG to improve the degradation rate of PBAT（H）.15mutants were constructed and expressed by selecting 11 amino acids of the binding slots and the binding pathway of cutinase ICCG and PBAT（H）.Furthermore,the dominant mutants were combined and stacked to obtain the optimal four mutants A59R/T192K/Q217K/H218Y.The degradation rate of PBAT（H）at 24 hours can reach 59.49%,which is 6 times that of the parent ICCG;Then,the degradation system of the four mutants was optimized:the optimal temperature is 75℃,the optimal E·S^-1=0.12%,and the optimal substrate concentration is 2%.In this system,the degradation rate for 24 hours is 84.54%.（3）The synergistic degradation of PBAT by cutinase ICCG and ferulic acid esterase（FAE）.To relieve the inhibitory effect of PBAT（H）degradation intermediates monohydroxybutyl terephthalate（MHBT）and dihydroxybutyl terephthalate（BHBT）on cutinase ICCG,a dual enzyme degradation system was constructed.Based on the comparison of the structure of the hydrolytic enzyme MHETase,which can degrade the intermediate product of polyethylene terephthalate（PET）degradation,a new enzyme ferulic acid esterase,which can degrade the intermediate product under high temperature conditions,was explored.Five types of ferulic acid esterases FAE1,FAE4,Tt FAE,Ts FAE,and Ct FAE were used to degrade MHET,BHET,MHBT,and BHBT,respectively.The results showed that the degradation effects of FAE4,Ct FAE,and Ts FAE were significantly improved compared to MHETase,with FAE4 having the best degradation effect.The final four mutants A59R/T192K/Q217K/H218Y were combined with FAE4 to synergistically degrade PBAT（H）.At 60℃,the degradation rate increased from 42.66%of the single enzyme effect of the four mutants to 63.85%.