| Starch is an inexpensive,widely distributed and renewable carbohydrate resource in the nature,and it has been an international research hotspot that rebuild the molecular structure of starch and convert starch into low-caloric products.4,6-α-Glucosyltransferase（4,6-α-GT）,a starch-converting enzyme from GH70,has been proved to be able to rearrange theα,1-4 andα,1-6 glycosidic bonds in the structure of starch,and synthesize reuteran-like polysaccharide（RLP,linked by alternatingα,1-4/α,1-6 glycosidic bonds）and isomalto/maltopolysaccharide（IMMP,linked by consecutiveα,1-6 glycosidic bonds）products.4,6-α-GT has great potential in the application of directional regulation of the starch structure.However,the member of 4,6-α-GT and the three-dimensional（3D）structural information of 4,6-α-GT are sparse,which limites the further academic research and application research about 4,6-α-GT.This study obtained three novel 4,6-α-GTs from different sources,they were Geobacillus sp.12AMOR14,6-α-GT（Gs GT）,Bacillus sporothermodurans 4,6-α-GT（Bs GT）and Limosilactobacillus fermentum NCC 3057 4,6-α-GT（Lf GT）.The enzymatic properties of three 4,6-α-GTs were comprehensively characterized here,as well as their protein 3D structure.And based on the enzymatic properties and the 3D structural information of 4,6-α-GTs,the structure-activity relationship of 4,6-α-GT between the active pocket and product specificity was further explored by site-directed mutagenesis.The main findings are described as follows:（1）Mining the novel 4,6-α-GT sequences on the basis of a comprehensive analysis of the amino acid sequences of 4,6-α-GT.First,we designed mixed degenerate primer based on the conserved motifs（i,ii,iii,iv,ⅵandⅶ）of 4,6-α-GT,and established a method to detect 4,6-α-GT gene,so as to mine 4,6-α-GT gene from the genome of lactic acid bacteria;Secondly,we searched NCBI database by BLAST search tool for the amino acid sequences which are homologous with the reported 4,6-α-GTs sequence,and screened several putative 4,6-α-GT sequences step by step from the homologous sequences,and artificially synthesized these putative 4,6-α-GT sequences.These putative 4,6-α-GTs were tried to be heterologously expressed in engineered strain after we truncating their sequence,finally,we successfully obtained three proteins with catalytic activity of 4,6-α-GT:Gs GT,Bs GT,and Lf GT.（2）Characterization of enzymatic properties of 4,6-α-GT.We obtained and characterized the purified protein of three 4,6-α-GTs.First,the optimal reaction conditions and thermal stability of 4,6-α-GT were determined.We found their optimum temperature were around 35-40℃and their optimum p H were around 6.0-7.0,and the thermal stability of Gs GT was outstanding.Secondly,the donor/acceptor substrate specificity of 4,6-α-GT were determined,in which Bs GT showed a unique donor specificity that can use panose as a donor.The products of 4,6-α-GT were synthesized by converting amylose substrate,and the structural characteristics of the products were characterized by 1H-NMR,13C-NMR,HPGFC,enzyme fingerprinting and in vitro intestinal simulation.It was found that Gs GT synthesized a branched RLP product with alternatingα,1-4/α,1-6 bonds,while Bs GT and Lf GT synthesized linear IMMP product with continuousα,1-6 bonds.The structure of Gs GT,Bs GT and Lf GT products contain 41%,79%and 72%α,1-6 bonds respectively,as well as the anti-digestion contents are35%,84%,and 64%,respectively.The chain-length transfer patterns of three 4,6-α-GTs were determined by the maltoheptaose-incubation experiment,and it was found that multiple glucoses were transferred during the reaction catalyzed by Gs GT,while only a single glucose was transferred during the reaction catalyzed by BsGT or LfGT.This study found that,among three new 4,6-α-GTs,Gs GT showed outstanding thermal stability,Bs GT had unique donor substrate specificity（acting on pantose）,while Bs GT and Lf GT synthesized products with highα,1-6 bond content and high anti digestion content.These three novel 4,6-α-GTs showed great academic research value,and showed application potential in the preparation of dietary fiber.（3）Crystal structure analysis of 4,6-α-GT.The crystallization conditions of three novel4,6-α-GT proteins were screened by using protein crystallization kits.After the crystallization conditions were preliminarily obtained,we further optimized these crystallization conditions for obtaining the high-quality protein crystal of 4,6-α-GT,finally the electron density datas of protein crystal were collected through X-ray diffraction.The electron density of Lf GT protein crystal was obtained in this study,and the protein 3D structure information of Lf GT was successfully solved（resolution is 2.4（?）,the PDB number of the structural model is 7DT1）through softwares（CCP4i,Phenix,and Win Coot）.The structure-activity relationship between the structural characteristics of Lf GT and its product specificity,like theα,1-6 bond content and molecular weight,was preliminarily analyzed.We chose to obtaine the 3D simulation structural models of Gs GT and Bs GT by homology modeling.It is found that they folded to form domains A,B and C similaring to Lf GT.By comparing their simulated structures,we preliminarily analyzed the relationship between their enzymatic properties and structures.（4）Rational design of L.fermentum NCC 3057 4,6-α-GT（LfGT）to control theα,1-6bonds content in its product structure.In this study,we selected the amino acid residues around acceptor pocket of Lf GT for rational design.By docking the substrate to the acceptor pocket of protein 3D structural model of Lf GT,three key amino acid sites（S346,S348 and L399）affecting theα,1-6 transglycosidic conformation of acceptor substrate were found.We targeted these three sites,performed mutagenesis,and verified the structure-activity relationship between amino acid residues at these three sites and theα,1-6 transglycosidation reaction of Lf GT.The hydrophobicity of amino acid residues at S346,S348 and L399 sites can influence theα,1-6 transglycosidation of Lf GT by affecting the adsorption of the acceptor substrate by the pocket,as well as the binding conformation of the acceptor substrate in the pocket.Finally,16Lf GT mutants were constrated in this study by combinatorial mutating the S346,S348 and L399 sites of Lf GT,which could synthesize IMMP products withα,1-6 glycosidic bond contant of 6-86%.This study provides a theoretical basis for controlling the synthesis of IMMP products with different dietary fiber contents by engineering 4,6-α-GT,and expandes the potential application of IMMP products.（5）Rational design of B.sporothermodurans 4,6-α-GT（BsGT）to change the arrangement of glycosidic bonds in its product structure.In this study,through the sequence comparison of4,6-α-GTs,two key amino acid sites（S345 and I347 of Bs GT）were found to affect the arrangement of glycosidic bonds in the product structure.Site-directed mutagenesis was performed to constructed the S345T/I347V mutant of Bs GT,and the continuousα,1-6 bond-linked IMMP product of Bs GT was successfully transformed into a linear ptoduct linked by alternatelyα,1-4/α,1-6 bonds（pullulan-like polysaccharide）.The chain length transfer pattern of S345T/I347V mutant was determined by maltoheptaose-incubation experiment,which proved that the change of product structure was caused by the change of chain length transfer pattern.Finally,we proposed the synthetic pathway for 4,6-α-GT synthesising the continuousα,1-6 bond linked product and theα,1-4/α,1-6 bond alternately linked product:the sugar chain moves in the direction of the acceptor pocket during the synthesis process of product,and the different distance（multiple or single glucose）of sugar chain movement results in the different arrangements of glycosidic bonds in the product structure.This study further clarifies the synthesis mechanism of 4,6-α-GT products,and provides a theoretical basis for controlling the product structure and precisely designing product with special structures. |
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