Study On Enzymatic Synthesis,structures And Properties Of Glucan Dendrimer

Benefiting from the highly branched structure,glucan dendrimer shows excellent monodispersity,moisture retention,low digestibility and biocompatibility,and has been initially applied in the fields of foods,cosmetics,biomedicine and smart materials.The glucan dendrimer with controllable structure can be synthesized by the bioenzymatic method,which makes up for the shortage of natural sources and broadens the application scope.Branching enzyme（BE）catalyzes the react of hydrolyzingα-1,4 bonds and introducingα-1,6 branching points,which is the key enzyme preparations for the remodeling of glycoside chain structure.This project screened out a BE gene derived from Thermococcus kodakaraensis KOD1,and designed a one-step synthesis of glucan dendrimer.After exploring the enzymatic properties and catalytic mechanism of branching enzymes,glucan dendrimer was prepared using maltodextrin as substrate,and the structural parameters were monitored and regulated.Finally,the physicochemical properties of glucan dendrimer were also characterized.First,T.kodakaraensis BE gene was cloned and expressed by genetic engineering.The recombinant enzyme was purified by Ni²⁺column affinity chromatography,and the SDS-PAGE band showed that the molecular weight of enzyme was about 80 k Da.The results of enzymatic properties exhibited that the optimum p H of BE was 7.0 and the optimum temperature was 70°C.The kinetic constant K_mand V_maxof BE were 1.49 mg/m L and 1183.3mg/min/m L.With amylose and amylopectin as substrates,the branching/hydrolysis activities of BE were 59.7 and 73.6,respectively.Second,BE was used to modify maltodextrins with different DE（DE6,DE12 and DE19）as substrates and the structure of the products was further analyzed.The modification of BE significantly increased the molecular weight andα-1,6 bond ratio of the products through interchain transglycosidation.The chain length distribution results indicated that BE preferred to transfer the chain segments of DP5-14 during the catalytic process.By analyzing the conversion efficiency of products,DE12 maltodextrin was selected as the most suitable substrate.Then,the synthesis system of glucan dendrimer was constructed with DE12 maltodextrin.Glucans dendrimers with different molecular weight distribution and branching degree were obtained by adjusting the reaction time.During the synthesis,the molecular weight of products increased logarithmically and the final molecular weight was 6.28×10⁶g/mol,along with the content ofα-1,6 bonds increased from 4.2%to 9.3%.The chain length distribution indicated that segments of DP>24 were continuously hydrolyzed into shorter segments as fragments for the subsequent reactions.Using the molecular rotor as the probe of the structure,the increase of branching unit and molecular density in the synthesis process were further verified by fluorescence analysis,and a double logarithmic equation between fluorescence intensity and molecular weight was established.On the basis of these structural parameters,the structural changes in glucan dendrimer synthesis were fitted by the Meléndez-Waddell model.It was deduced that a layered structure with an average chain length of 9 and a total of12 orders could be formed at the maximum catalytic limit of BE.Finally,the physicochemical properties of different dendrimers were characterized.The glucan dendrimer could be dissolved at room temperature to form a homogeneous milky white solution.It was confirmed by transmission electron microscopy and atomic force microscopy that the glucan dendrimer exhibited a spherical conformation in aqueous solution with a particle size range of 30-65 nm,which increased with the raise of molecular weight.The tests of storage stability showed that the glucan dendrimer was degraded under acid-heat conditions,but could be stored for a long time at p H 5.0-11.0.The glucan dendrimer solution was shear-thinning within a certain shear range,representing a typical pseudoplastic fluid.With the linear polysaccharide as the control,it was found that the glucan dendrimer showed better moisturizing properties,which might be related to the formation of a stable and ordered hydrogen bond network in the branched structure.