Rational Design To Improve The Catalytic Ability Of 1,4-α-glucan Branching Enzyme And Its Application In Modification Of Maltodextrin

1,4-α-Glucan branching enzyme（1,4-α-glucan branching enzyme,GBE,EC 2.4.1.18）is a glycosyltransferase,which can hydrolyze theα-1,4 glucosidic linkages in starch molecules,and then link the donor chain to the acceptor chain throughα-1,6 glucosidic linkages to form branches,increasing the degree of branching of starch.With this unique transglycosylation,GBE has wide application in food,medicine,chemical and other fields.At present,GBE has some defects such as low catalytic efficiency,so it is necessary to modify the GBE based on the structure of the enzyme.However,among current researches,few of them focused on the structure and function of GBEs so far,leading to lack of effective strategies to enhance their catalytic ability of GBEs.This study focused on the GBE derived from Rhodothermus obamensis STB05（Ro-GBE）,and a series of molecular modifications were rationally designed and performed based on crystal structure analysis.Finally,ideal mutants were obtained with improved catalytic ability and industrial application.（1）The hanging-drop method was used to conduct protein crystallization experiments on Ro-GBE.After the primary screening of the kit conditions and re-screening of the crystallization conditions,high-quality protein crystals were finally obtained under the following conditions:The protein solution contains 350 mg/m L Ro-GBE;while the well solution consisted of 25%（w/v）polyethylene glycol 3350（PEG 3350）,0.1 mol/L 2-[4-（2-hydroxyethyl）piperazin-1-yl]ethanesulfonic acid（HEPES p H 7.0）,0.1 mol/L（NH4）2SO4,protein solution and well solution were mixed half-and-half（0.5（?）L+0.5（?）L）and incubated at 20°C for 14 days.The crystal structure of Ro-GBE was determined through structural analysis,with a resolution of 2.39（?）and a PDB registration number of 6JOY.The structure showed that Ro-GBE contains three domains,CBM48,domain A and domain C.Because domain A contains the（α/β）₈-barrel structure which is unique to GH13 family amylase,it can be determined that Ro-GBE belongs to GH13 family.In addition,the crystal structure of Ro-GBE with the substrate was simulated by the molecular simulation docking software Autodock,in order to further explore the relationship between the crystal structure and the catalytic ability of Ro-GBE.（2）By focusing on the surface structure of the Ro-GBE,it was found that the substrate was bound at two positions,and the groove was just between the two sugar chain binding cracks.It was speculated that the combination of enzymes and sugar chains could be changed by regulating the structure of this groove.the G160 site is the main structure of this groove,therefore,single mutants G160A,G160R and G160F were constructed.These three mutants changed the surface grooves here to varying degrees.The results showed that there was no significant difference in the specific enzyme activity of mutant G160A compared with the wild type,while the specific enzyme activities of mutant G160R and G160F were improved to varying degrees,increased by 83.2%and 57.8%,respectively.（3）Further analysis of the composite structure of the Ro-GBE and the substrate obtained by the simulation revealed that there was a hydrogen bond interaction（3.2（?））between the Q489 site and the first sugar unit at the reducing end of the substrate,and it interacted with three water molecules（Wat92,Wat112 and Wat129）to format relatively stable structure.Therefore,corresponding mutants,including Q489E,Q489G and Q489R,were constructed to test this hypothesis.The analysis of enzyme activity showed that the substitution of Gln489 with Gly（Q489G）lost its interaction with the substrate because the Glycine have shorter side chain,which led to a decrease in enzyme activity;correspondingly,mutants Q489E and Q489R formed additional hydrogen bonds with the substrate,so that the interaction improved the enzyme activity compared with the wild type,increasing by 6.9%and 10.6%respectively.This improvement was mainly due to the catalytic groove deepened by the mutation,which made the substrate bound more firmly during the catalysis process.Besides,under the induction of the catalytic groove,the substrate was arranged in a direction that was more conducive to bond formation,thus accelerating the reaction rate.（4）Based on the above studies of mutants and the analysis of the mechanism of action of Ro-GBE,the influence mechanism of rational design on the catalytic capacity of Ro-GBE was explored.On the one hand,the deepening of grooves on the surface of Ro-GBE can promote the substrate to take the correct direction and arrangement,so as to facilitate the binding of enzyme and substrate,on the other hand,the enhanced interaction between the substrate and the catalytic group of the enzyme makes the substrate bond more tightly.Through the above methods,the possibility of donor chain shedding can be reduced,so as to ensure the smooth progress of the catalytic process and improve the catalytic efficiency.Both the change of groove structure and the change of the interaction between enzyme and substrate changed the catalytic capacity of Ro-GBE by affecting the binding of Ro-GBE to the donor chain,and this mechanism can provide guidance for the modification of other amylases.（5）The industrial application value of the mutants with improved catalytic ability were initially explored by modifying maltodextrin.The results showed that after modification by the mutant G160R and G160F,the transparency and stability of the products can be significantly improved compared with the wild-type-modified product.After the storage at4°C for 20 d,the wild-type-modified product became completely turbid,while the light transmittance of the products modified by the mutant G160R and G160F were greater than80%;in addition,after modification by mutants Q489E and Q489R,the freeze-thaw stability of maltodextrin had been significantly improved compared with the wild-type-modified product,and the proportion of precipitates after freezing and thawing was decreased by 53.5%and 60.8%respectively.These all results indicated that the mutation of Ro-GBE at these sites can significantly improve product performance.