The Study On The Mechanism Of Enzymatically Synthesized Glycogen-like α-glucan With Controlled Molecular Structure

Glycogen-likeα-glucan（GnG）,an enzymatically synthesized hyperbranched glucose polymer,consists of（α1→4）and branching（α1→6）linkages（7%～10%of the total number of bonds）.It has been receiving increasing attention for generating food ingredients and platforms for delivery of proteins,labile components,and drugs.However,the functionality of GnG was strongly influenced by molecular weight and branching pattern,which could be changed by some reaction factors.It has been shown that sucrose could be phosphorylated to glucose-1-phosphate（G-1-P）by sucrose phosphorylase（SP）.Then,the glucose residue in G-1-P could be linked to the non-reducing end of the primer byα-glucan phosphorylase（GP）,until the chain could be branched by branching enzymes（BE）.Besides,the molecular structure of GnG could be changed by adjusting the ratio of enzymes or reactants.However,the mechanism behind the control of the GnG molecular structure has not been investigated in detail.SP,GP,and BE were chosen as tool enzymes to improve insights into the fine regulation of GnG synthesis in vitro and design of innovative GnG with controlled structure.The regulation mechanism of GnG synthesis was discussed by the fine structure of primers and the ratio of reaction factors,such as the ratio of enzymes and the ratio of substrate to primer.In addition,sucrose and high amylose corn starch（HCS）were used to synthesize GnG with different molecular structures.The method was established to provide the possibility of rational construction of polysaccharides with well-defined structures.The main research contents and results are as follows:1.Preparation of tool enzymes and establishment of GnG synthesis method.Firstly,the tool enzymes were cloned,expressed,purified,and characterized.The results showed that the optimum temperature of recombinant SP from Streptococcus mutans was 55℃and the optimum p H was 7.0.The optimum temperature of recombinant GP from Aquifex aeolicus was85℃and the optimum p H was 6.5.The optimum temperature of recombinant BE from A.aeolicus was 75℃and the optimum p H was 7.5.According to the results of temperature and p H stability,the reaction temperature of three enzymes for synthesizing GnG was 55℃and the reaction system was phosphate buffer（p H 7.0）.2.The effects of the ratio of reaction factors on GnG molecular structure were discussed.To uncover the effect of the ratio of enzymes on the molecular structure of GnG,a series of GnG with similar molecular weights（9.20×10⁶～1.03×10⁷ Da）and different branching patterns were synthesized by adjusting the relative activity ratio of GP and BE（GP/BE）.With the increase of GP/BE,the average chain length of GnG was enhanced from DP 7 to DP 9,and particle size was expanded from 44.06 nm to 54.12 nm.In addition,the trends of molecular weight changed from increasing continuously to increasing and then decreasing.The results of hydrolysis ofα-amylase andβ-amylase showed that with the increase of GP/BE,the degree ofα-amylase hydrolysis increased from 54.26%to 66.16%,while the degree ofβ-amylase hydrolysis decreased from 29.53%to 16.75%.To uncover the effect of the ratio of substrate and primer on the molecular structure of GnG,a series of GnG with molecular weight in the range of 7.80×10⁶～3.20×10⁷ Da were obtained by adjusting the molar ratio of sucrose and maltotriose.There is a negative linear correlation between the molar concentration of maltotriose and the logarithm of molecular weight（R²=0.986）.3.To uncover the effect of the fine structure of starch primer on the molecular structure of GnG,HCS,normal corn starch,and waxy corn starch（WCS）were pretreated by BE to synthesize GnG as primers.Structural analysis indicated that when HCS was used as primer,the molecular density of GnG reached 341.58 g·mol^-1·nm^-3,and the degree ofα-amylase hydrolysis was the lowest（40.03%）.In contrast,the GnG synthesized from WCS primer has the lowest molecular density(175.81 g·mol^-1·nm^-3),and the highest degree of hydrolysis of β-amylase（37.03%）.4.Based on the mechanism of the above reaction factors,a series of GnG with molecular weights ranging from 1.8×10⁶～1.5×10⁷ Da were obtained by adjusting the mass ratio of sucrose to high amylose corn starch（Su/HC）.The molecular weight of GnG is positively correlated with Su/HC（R²=0.998）.In addition,with the increase of Su/HC,the molecular density of GnG increased from 410.34 g·mol^-1·nm^-3 to 1189.00 g·mol^-1·nm^-3,the molecular particle size increased from 26.74 nm to 64.84 nm,which made the particles more uniform.