Studies On Enzymatic Properties And Structural Feature Of Glycerol Dehydratase From Klebsiella Pneumoniae 2e

As the major by-product in the biodiesel industry,crude glycerol could be converted into the high-value chemical 1,3-propanediol(1,3-PDO)by microorganisms which is a promising alternative for effective and economic utilization.However,the yield of 1,3-PDO from crude glycerol by the microorganism was severely hindered by the existence of complex impurities.The low activity of key enzymes involved in glycerol metabolism caused by crude glycerol impurities was one of the main factors affected the yield of 1,3-PDO from crude glycerol.Glycerol dehydratase was considered as one of the key and rate-limiting enzyme for 1,3-PDO production,the activity of which plays a decisive role in the synthesis rate and yield of 1,3-propanediol.The exploration of glycerol dehydratase resources with high tolerance to crude glycerol would provide important theoretical basis for the development of engineering strains applied in crude glycerol bioconversion.Our preliminary results showed that the glycerol dehydratase activity of Klebsiella pneumoniae 2e,a strain with high yield of 1,3-propylene glycol,was almost unaffected by the glycerol environment during the fermentation process of crude glycerol as substrate,showing outstanding crude glycerol impurity tolerance characteristics,which is of great research value and application prospect.In this study,glycerol dehydrating enzyme(2eGDHt)of K.pneumoniae 2e was studied.Its coding sequence,enzymatic properties,crude glycerol tolerance and functional structure stability were investigated.The results were as follows:1.The coding sequence of 2eGDHt was analyzed by bioinformatics method and the three-dimensional structure model of its protein was constructed.The results showed that 2eGDHt was composed of α,β and γ subunits,belonging to the co-enzyme B12 dependent glycerol dehydrase sequence,which was more conserved than other glycerol dehydrases of K.pneumoniae,but different from other glycerol dehydrases at Val744,it was isoleucine.AlphaFold2 three-dimensional structure modeling analysis showed that its structure was heterohexamers,which was similar to that of K.pneumoniae glycerin dehydrase(PDB id:lIWP)crystal structure is highly similar,but 2eGDHt structure contains more Coil and lessβ-sheet.2.Recombinant 2eGDHt was expressed and purified,and its enzymological and physicochemical properties and crude glycerol tolerance were investigated.Recombinant 2eGDHt was successfully expressed in E coli.The results of enzymatic physicochemical properties showed that the optimum substrate of 2eGDHt was 1,2-propanediol,and the optimum reaction temperature and pH were 37℃ and 7.0,respectively.The temperature and pH tolerance range were 35-40℃ and 6.0-9.0,respectively.A variety of metal ions had different degrees of inhibition on enzyme activity,among which calcium ions had the strongest inhibition on enzyme activity,inhibiting 40%of enzyme activity.Different chemical inhibitors had different degrees of inhibition on its enzyme activity,among which sodium dodecyl sulfonate had the greatest inhibitory effect on its enzyme activity,which could inhibit 47.4%of its enzyme activity.The results showed that the main impurities of crude glycerol,such as sodium chloride,methanol,oleic acid and linoleic acid,had little inhibitory effect on the enzyme activity of crude glycerol.At the same time,the enzyme activity was not affected basically when the impurities were mixed to simulate the environment condition of crude glycerol.These results indicate that 2eGDHt has strong crude glycerol tolerance.3.Key functional structural sites of 2eGDHt related to crude glycerol impurity tolerance were analyzed.RMSD showed that sodium chloride and linoleic acid were the key factors affecting the stability of 2eGDHt,and the crude glycerol environment had a certain effect on the stability of 2eGDHt.The results of multiple sequence alignment and RMSF indicate that residues at 740-747 of a unique coiled structure of 2eGDHt protein structure play an important role in maintaining its flexibility in the environment of crude glycerol impurities.Single point mutation combined with mutation energy prediction showed that Ile744Val mutation had little effect on the overall stability of the enzyme.Single point mutation and enzymology analysis showed that the enzyme activity of the mutant was significantly decreased under crude glycerol substrate,but no significant change under pure glycerol substrate.RMSD and RMSF of I744V indicated that the mutant had better stability and protein rigidity.These results suggest that 1744 is a key functional structural site of 2eGDHt related to crude glycerol impurity tolerance.4.The key residues that maintain the structural stability of 2eGDHt protein and their effects were explored.PDBePISA program combined with structural biology analysis results showed that 262 amino acid sites support the maintenance of heterohexamer structure.By using statistical methods,Discovery Studio,Imutant2.0 and Mupro software were combined to predict mutation energy changes before and after Single point mutation,and relevant mutation points were screened for mutation candidates.The results of point mutation experiment and enzyme analysis further showed that Arg mutation at 315 had the greatest effect on its structure when it changed to P.Further molecular dynamics simulation analysis showed that,compared with WT,the stability,flexibility,structural compactness,solvent accessible surface area and the number of hydrogen bonding decreased after Arg mutation at site 315.These experiments showed that the 315 amino acid of 2eGDHt is the key amino acid to maintain the structural stability of 2eGDHt protein.In this study,2eGDHt with crude glycerol tolerance characteristics was studied in depth.The encoding sequence of 2eGDHt was analyzed by bioinformatics method and the three-dimensional structure model of its protein was constructed.Recombinant 2eGDHt was expressed and purified,and its enzymological and physicochemical properties and crude glycerol tolerance were investigated.The key functional structural sites of 2eGDHt related to crude glycerol tolerance were analyzed.The key residues and their effects on structural stability of 2eGDHt protein were investigated.The results of this study will provide a theoretical basis for the development of engineering strains applied in crude glycerol conversion fermentation.