Optimization And Modification Of Plastic Hydrolases And Glycotransferase Via Enzyme Engineering Technologies

Green bio-manufacturing has wide applications in agriculture,environment and pharmaceutical researches via the utilization of biomass and industrial by-product.The application of various enzymes(e.g.hydrolases,synthases and transferases)used in green bio-manufacturing provides an ecological friendly way to overcome the disadvantages of traditional chemical methods in dealing with environmental pollution and pharmaceutical synthesis.Natural enzymes usually work in mild reaction condition,create no or less environmental pollutions and have better substrate recognition abilities.However,they usually cannot meet the industrial requirements.In this study,three enzymes were investigated for better applications in environmental protection and pharmaceutical synthesis under the direction of enzyme engineering.Environmental problems caused by PET and other plastics exert a baneful influence on ecological stability and human health.A few PET hydrolases have been discovered before,but they usually exhibit low efficiency or harsh reaction conditions.A novel PET degradation system consisting of PETase and MHETase hydrolases from Ideonella sakaiensis was reported by Science in 2016.However,the efficiency of this system is relatively low,thus we explored the molecular mechanism of the PETase and MHETase system in degradation of PET,and improved the catalytic ability of this system by protein engineering.In this study,the PETase protein were expressed and purified in E.coli expression system.Crystal structure analysis and sequences alignments were performed after acquiring the crystal structures:(1)PETase shows a typicalα/βhydrolase conformation,and the substrate binding region was narrower and shallower;(2)we prefer to classify PETase(EC 3.1.1.101)into a newα/βhydrolysis family,in order to distinguish it from other true cutinases.Site-specific mutagenesis of key amino acids were designed in order to verify the actual function of these sites in catalytic process.Based on the purification and analysis of 24 mutant proteins,we can find that:(1)for WT,the optimum reaction temperature is around 30℃and the suitable p H value is between 6.5-8.0 in BHET hydrolysis;when it comes to PET,the react condition is 30℃and the optimal p H is 9.0.(2)All amino acid mutants lead to significant decrease of activity except W130H in BHET hydrolysis experiment;while S185H shows highest improvement in catalysis ability among the beneficial mutations(Y58A,W130H,A180I and S185H)for PET degradation studies,which provide the possibility to further increase PETase activity by enzyme engineering.(3)The contradict results between BHET and PET demonstrate that these residues play key roles in catalytic process and recognize different substrates.(4)Y58A and S185H increase activity and thermostability synchronously,which provide basis on construct a more potential enzyme for industrial application in future.MHETase was analysis to further improve the catalysis rate of PETase.The activity results show that MHETase has excellent activity towards MHET and it will promote BHET degradation when co-exists with PETase.These results demonstrate that it’s a good strategy to degrade PET plastics by PETase-MHETase combination.Glycosylation is a common strategy to improve pharmacological activity and stability.However,the presence of multiple hydroxy groups in monosaccharides and substrate conformational selectivity make it difficult to implement glycosylation by chemical methods.It has been reported that the glycotransferase Bs-YjiC could recognize flavonoids,xanthenes,macrocyclics,anthraquinones,NDP and different monosaccharides as substrates,which provides a new way for prodrug glycosylation.However,the molecular mechanism of substrate recognition in Bs-YjiC remains unclear now.Hence,we designed experiments to understand its catalytic mechanism and improve its catalytic efficiency through structure analysis by enzyme engineering.The Bs-YjiC protein were expressed and purified in E.coli expression system in this study.Based on crystal structure analysis acquired from this study and sequences alignments by bioinformatics,we can find that:Bs-YjiC shows a typical GT-B glycotransferase,and the protein can be divided into two parts,the substrate binding to N domain and the UDP-G binding to C domain.Site-specific mutagenesis were designed and performed via analysis of the structure and the alignment for further increase its activity.Through the purification and analysis of 46 mutant proteins,we can find that:(1)when 2-chloro-4-nitrophenyl glycosides was used as the substrate,some mutants(e.g.H16A,T229A,H293A,E301A)show significant negative effect,while V108A shows the best activity for Kcat/K_M increased 4.5 times among positive mutants(e.g.M71A,V108A).(2)Pterostilbene was chosen to test the activity of Bs-YjiC in pharmaceutical synthesis.The results indicated that V108A improve the activity for 40%.These results demonstrate that Bs-YjiC shows prominent prospect in drug synthesis and more mutations and modifications could be done on Bs-YjiC to obtain better activity in the future.As described above,the condition for expression and purification of PETase,MHETase and Bs-YjiC were acquired.The results in PETase and MHETase studies proposed the potential mechanism for PET preference,provided data for improving the enzymatic activity and understanding the catalytic mechanism of PET biodegradation by enzyme engineering.The Bs-YjiC study structurally explains the possible reason for its broad substrate recognition ability;and some key amino acids were analyzed by enzyme engineering,which shed light on the improvement of catalytic efficiency and substrate recognition extension in further study.The crucial roles of biological enzymes in green bio-manufacturing were confirmed by the studies of these 3 proteins,and the feasibility of enzyme engineering in the improvement of enzymatic activities was developed,which is of vital importance in industrial and daily life.