Secretion Expression,Enzymatic Properties And Crystal Structure Analysis Of Dextrin Debranching Enzyme From Thermococccus Gammatolerans

Debranching enzymes（DBEs）are a class of hydrolases that specifically hydrolyzesα-1,6-glycosidic bonds in branched substrates.In the process of starch processing,DBEs could effectively hydrolyzeα-1,6-glycosidic bonds in starch substrates,and synergistically with other amylases could significantly improve starch conversion and reduce production costs.DBEs have been widely used in the industrial production of glucose syrup,maltose syrup,cyclodextrin,and resistant starch.Pullulanase and isoamylase are the two most studied DBEs,which possess different substrate specificities.The substrate specificity of pullulanase is weak,it could hydrolyze pullulan and amylopectin,and has no obvious specificity for different chain lengths in amylopectin.Isoamylase has strong substrate specificity,it could not hydrolyze pullulan.Isoamylase tends to hydrolyze the long branched chains in amylopectin but has a weak ability to hydrolyze the short and medium branched chains.In the production of different starch sugars,it may be necessary to add DBEs with different substrate specificities to significantly improve the saccharification efficiency.Therefore,this study aims to develop a novel debranching enzyme that specifically hydrolyzes medium-length branched chains and was named dextrin debranching enzyme（DDE,EC 3.2.1.33）.We successfully expressed the DDE in E.coli and studied its enzymatic properties and crystal structure,to lay a solid foundation for the industrial application of DDE.The main research contents and results were as follows:Firstly,the basic physical and chemical properties of DDE from Thermococccus gammatolerans were predicted by using bioinformatics tools to provide the theoretical basis for subsequent construction and expression.The results showed that the molecular weight of DDE was 68621.63 Da,and the isoelectric point was 6.35.DDE contained many hydrophilic amino acids according to the amino acid sequence analysis and had no transmembrane domain.The predicted results of the secondary and tertiary structures of DDE showed that the structures were mainly composed ofα-helix,β-sheet,and random coil.Secondly,the DDE was successfully expressed and secreted extracellularly.The conditions of shake flask fermentation for producing DDE were investigated.The gene of DDE was cloned into the expression vectors p ET-15b（+）/dde,p ET-28a（+）/dde and p ET-20b（+）/dde,respectively and then transformed into the E.coli BL21（DE3）.The fermentation conditions of E.coli BL21（DE3）（p ET-20b（+）/dde）were optimized to promote the soluble expression of DDE.The results showed that the genetically engineered bacteria were cultured in TB medium at 25°C.After induction with 0.01 m M（v/v）IPTG for 84 h,the enzyme activity was 326.0 U/m L,which was 121.8%higher than the initial culture condition.Next,DDE was purified by nickel column affinity chromatography,and the enzymatic properties were characterized.The results showed that the specific activity of DDE was 733.3U/mg,and the optimal temperature and p H were 70°C and p H 4.0,respectively.The enzyme activity remained above 50%after incubation for 90 min at 70°C,and it also exhibited excellent acid resistance in p H 3.5～5.0 buffer at 4°C,with 80%remnant activity after indicating for 3 h.The activity of DDE was dependent on metal ions,Pb²⁺and Ba²⁺could significantly activate the enzyme activity,increasing by 91%and 116%respectively.However,the DDE activity could be completely inhibited by Fe³⁺and Co²⁺.Different substrates were selected for the debranching reactions,the results showed that DDE had the greatest affinity toward the DE6 maltodextrin.The substrate preferences were ranked as follows:maltodextrin>glycogen>starch.Therefore,DDE had high substrate specificity for maltodextrin with low molecular weight,while having a low hydrolysis effect on starch and glycogen with higher molecular weight.Then,the selectivity of DDE to maltodextrin and starch with a typical branched-chain structure was investigated.The difference in substrate selectivity of DDE was compared with pullulanase and isoamylase.The molecular weight of debranched products was analyzed using gel permeation chromatography.The results showed that DDE could hydrolyze most of the polymerized amylopectin in DE6 maltodextrin into small molecular linear glucans and oligosaccharides,and this enzyme was difficult to hydrolyze the polymerized amylopectin in starch.The debranching rate of DE6 maltodextrin treated with DDE for 6 h was 80%,while the debranching rates of DDE on waxy corn starch and cassava starch were 24%and 30%,respectively.The chain length distribution of the debranched products was analyzed using ion chromatography.The results showed that DDE has strong substrate selectivity and strong affinity for DP 13～24 with medium-length branched chains.The optimal debranching length of DDE was DP13～24,while was more concentrated than that of pullulanase（DP>12）and shorter than that of isoamylase（DP>25）.Finally,the crystal structure of DDE was solved,and the DDE-maltoheptaose complex was simulated using molecular docking.The high-quality protein crystals were obtained through optimizing primary screening conditions and selected for X-ray diffraction.The three-dimensional structure of DDE was successfully resolved at a resolution of 2.51?using the molecular replacement and the data were deposed on the PDB database with an accession number of 7VMA.The catalytic domain of the DDE contained an（α/α）₆ barrel structure.After multiple sequence alignment and structural superposition,the Asp241,Asp366 and surrounding residues formed an active pocket to bind substrates.It was speculated that the Asp241 and Asp366 could be the catalytic residues at the catalytic center of the enzyme.To explore the binding mode of the substrate,molecular docking was performed to simulate the structural conformation of the DDE-maltoheptaose complex.The hydrogen bond interactions between maltoheptaose and residues Arg112,Met116,Gly357 and Ile358 were analyzed,and the substrate-binding mode could provide information for further understanding of the structure-activity relationships.