Study On The Synthesis Mechanism And Application Of Isomaltomegalosaccharides Based On 4,6-α-GTase Converting Linear Dextrin

Isomaltosaccharides areα-glucan mainly formed by connecting glucosyl residues throughα1→6 linkages.Isomaltosaccharides with a degree of polymerization between 11～100 are named isomaltomegalosaccharides(IMMs)which have particular solubilizing,anti-inflammatory,and immunomodulating functions,and have good application potential.IMMs are prepared by dextran dextrinase catalyzing maltohexaose and maltoheptaose which are expensive,and the method has been patented.Furthermore,this method can only synthesize IMMs with an average degree of polymerization of 11,which correspond to a weak solubilizing effect.A novel method is required for the synthesis of higher relative molecular weight IMMs.In response to this problem,4,6-α-glucanotransferase Gtf B-ΔN from Limosilactobacillus reuteri 121 was used to catalyze linear dextrins to prepare IMMs,and the enzymatic property and solubilization property of IMMs were explored.Firstly,Gtf B-ΔN was heterologously expressed by Escherichia coli BL21(DE3)*.Gtf B-ΔN was eluted from the nickel affinity chromatography column,and SDS-PAGE showed that Gtf B-ΔN solution has reached electrophoresis purity,the molecular weight is about 100 k Da,and the enzyme activity is 5.23 U/mg.The temperature and p H suitable for Gtf B-ΔN catalysis were 40°C and 5.0,respectively.According to the corresponding relationship between the molecular weight of the substrate and the product,a suitable substrate for the preparation of IMMs was screened from a variety of linear dextrins synthesized.HPGPC-RID and HPAEC-PAD showed significant differences in the relative molecular weight and chain length distribution of amylosucrase product,debranched waxy corn starch,and supernatant dextrin.Low-temperature retrogradation is an effective method to obtain linear dextrin with lower relative molecular weight.The weight average molecular weight andα1→6 linkages content of the product synthesized by Gtf B-ΔN catalyzing supernatant dextrin were 1.23×10~4 g/mol(degree of polymerization 76)and 83.8%,respectively.Supernatant dextrin(2.1×10~3 g/mol)was a suitable substrate for the preparation of IMMs.The properties and structures of the oligosaccharide and IMMs components in the product with bimodal distribution were identified.Theα1→6 linkages contents of the oligosaccharide and IMMs components were 34.6%and 88.6%,respectively.HPAEC-PAD showed that the main components of oligosaccharide were glucose,maltose,and maltotriose,and a small amount of isomaltose and isomaltose and panose were also present.The relative molecular weight distribution and product composition of IMMs before and after hydrolysis by five enzymes showed that IMMs were identified asα1→6 chains withα1→4 fragments inlaid at the reducing,non-reducing end,and middle part,and the degree of polymerization of theα1→4 fragments is 2～5,2～4 and 2～6,respectively.The relative molecular weight distribution,chain length distribution enzyme activity of the Gtf B-ΔN products at different reaction times showed that Gtf B-ΔN mainly exerts hydrolysis at the beginning of the reaction,producing oligosaccharide that cause relative transglycolysis activity increases,thus achieving a dynamic balance between hydrolysis and transglycosylation;transglycosylation preferentially catalyzes longα1→4 chains.By controlling the amount of maltose added to the supernatant dextrin,various IMMs(3.6×10~3～1.23×10~4 g/mol)were synthesized,and their solubilizing effect and solubilization mechanism on ethyl red were determined.IMMs(1.23×10~4 g/mol)can increase the solubility of ethyl red to 4.5 times,and the solubilizing effect is positively correlated with the relative molecular weight.The proton chemical shifts of ethyl red measured by 1H NMR indicated that the solubilization of the IMMs was achieved through hydrophobic interaction.Fourier transform infrared spectroscopy and scanning electron microscopy showed the shift and disappearance of the characteristic peaks of ethyl red and paste-forming changes in particle morphology,which confirmed the formation of the IMMs-ethyl red complex and the possibility of hydrogen bonding contribution to solubilizing effect.