A glycosyltransferase and its use in the preparation of highly branched dextran

Abstract

The application discloses a glycosyltransferase and application thereof in preparation of highly branched glucan. The mutant is obtained by amino acid mutation of six sites of Y782N, W778F, V871A, S883T, T1047R and T1068F based on a glucan transferase sequence from Limosilactobulacillus reuteri. The mutant can efficiently catalyze the glycosyl transfer reaction of alpha-1,4-glucan, and introduce short-chain and high-density alpha-1,6 branched structure in the glucan chain. The mutant enzyme and amylomaltase can be used for synergistic catalysis, and highly branched alpha-glucan can be directly prepared by using sucrose as a substrate. The obtained branched alpha-glucan has high alpha-1,6 branch level, short-chain branch distribution, good water solubility and strong anti-digestion characteristics, and has good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a glycosyltransferase and its application in the preparation of highly branched dextran, and more specifically to the application of glycosyltransferase in highly branched dextran. Background Technology

[0002] Branched α-glucans are a class of polysaccharides composed of glucose residues, with α-glycosidic bonds in their backbone and branches. The branching structure can affect the molecular conformation, solubility, thermal stability, digestibility, and physiological functions of α-glucans. α-Branched glucans typically exhibit high water solubility and good rheological regulation properties, while also demonstrating good structural stability during heating, cooling, and freeze-thaw cycles. These polysaccharides also possess various physiological functions, including delaying glucose release, regulating postprandial blood glucose, improving gut microbiota, and playing an immunomodulatory role. The physicochemical properties and physiological functions of α-branched glucans are closely related to their structural characteristics, such as branching degree and chain length distribution. Studies have found that shortening the average chain length and increasing the proportion of α-1,6 glycosidic bonds generally helps improve the solubility and transparency of amylopectin and enhances its anti-retrogradation properties.

[0003] Currently, the main methods for preparing branched α-glucan include natural extraction, physicochemical modification, and enzymatic synthesis. While natural branched α-glucan is widely available and biocompatible, its structure is poorly controllable, hindering large-scale production and widespread application due to the inherent characteristics of the raw materials. Chemical modification methods involve harsh reaction conditions and produce numerous byproducts, which is detrimental to green manufacturing. In contrast, enzymatic synthesis offers advantages such as mild reaction conditions, strong regioselectivity, and controllable product structure.

[0004] Current enzymatic methods for preparing branched α-glucans typically use natural starch as a substrate, employing branching enzymes or glycosyltransferases to cleave α-1,4 glycosidic bonds and transfer the oligosaccharide chain to another segment to form α-1,6 glycosidic bonds, thereby introducing an α-1,6 branched structure. However, starch substrates have poor solubility, often requiring pretreatment steps such as gelatinization and liquefaction before the reaction, making the process complex and resulting in low yields of branched glucans. In recent years, the strategy of synthesizing branched glucans using soluble small molecules such as glucose and sucrose as substrates through a cascade catalysis of amylosucrase (AS) and branching enzymes has attracted much attention. This cascade reaction gradually extends the α-1,4 backbone and introduces α-1,6 branching points. However, traditional branching enzymes tend to transfer longer oligosaccharide fragments (DP>6), resulting in limited improvement in the degree of branching and an insufficiently refined branched structure. GH57 family glycogen branching enzymes primarily form longer branches with a degree of polymerization of 11-16 after starch treatment, while GH13 family glycogen branching enzymes primarily form branches with a degree of polymerization of 3-10 (see Gaenssle et al., The influence of amylose content on the modification of starches by glycogen branching enzymes), Food Chemistry, 2022, 393, 133294. Compared with branching enzymes, glycosyltransferases (GT) have a better ability to convert shorter α-glucan chains, and can introduce short-chain, high-density α-1,6 branched structures into glucan chains. Therefore, obtaining a novel glycosyltransferase GT that can efficiently introduce short-chain α-1,6 branches and developing a catalytic system coupled with glycosyltransferases (AS) is of great significance for preparing α-glucans with high branching degree, high water solubility, and high digestibility. Summary of the Invention

[0005] To prepare α-glucans with high branching degree, high water solubility, and high digestibility, this invention provides a glycosyltransferase mutant, obtained by amino acid mutations at six sites (Y782N, W778F, V871A, S883T, T1047R, and T1068F) based on the glucan transferase sequence from *Limosilactobacillus reuteri* shown in SEQ ID NO. 1. The mutations include changes to any one, two, three, four, or five amino acid residues at these six sites. Using this glycosyltransferase mutant, highly branched α-glucans with higher branching degree and shorter branched chains can be prepared more conveniently and efficiently using soluble small-molecule sucrose as a substrate.

[0006] In an embodiment of the present invention, the encoding genes for starch sucrase (AS) from *Deinococcus geothermalis* and glycosyltransferase (GT) from *Limosilactobacillus reuteri* were respectively ligated into the pET-21a plasmid to construct expression vectors, which were then transformed into the *E. coli* BL21(DE3) expression vector for induced expression. SDS-PAGE results showed that both enzymes could be expressed solublely heterologously in *E. coli*. Hotspot wizard 3.0 was used to predict mutation hotspot sites for GT from *Limosilactobacillus reuteri*. Based on the enzyme's three-dimensional structure, residue conservation, and mutation score, candidate mutation sites were screened for site-directed mutagenesis. Enzyme activity in wild-type and mutants was determined by iodometric titration. The results showed that the enzyme activity of single mutants Y782N, W778F, V871A, S883T, T1047R, and T1068F was higher than that of wild-type. Further, the above mutation sites were combined to obtain double mutants with different combinations. The mutants with relatively high enzyme activity were then combined with other sites in round-by-round mutations, and the relative activities were measured. The results showed that the mutant with the highest relative enzyme activity was T1047R / T1068F / W778F / Y782N, whose enzyme activity was 3.5 times that of wild-type.

[0007] According to one embodiment of the present invention, preferably, the above-mentioned glycosyltransferase mutants include Y782N, W778F, V871A, S883T, T1047R, T1068F, T1047R / Y782N, T1047R / W778F, T1047R / V871A, T1047R / S883T, T1047R / T1068F, T1047R / T1068F, T1047R / T1068F / Y782N, T1047R / T1068F / W778, T1047R / T1068F / V87 1A, T1047R / T1068F / S883T, T1047R / T1068F / W778F / Y782N, T1047R / T1068F / W778F / V871A, T1047R / T1068F / W778F / S883T, T1047R / T1068F / W778F / Y782N / V871A, T1047R / T1068F / W778F / Y782N / S883T, T1047R / T1068F / W778F / Y782N / V871A / S883T.

[0008] According to one embodiment of the present invention, preferably, the mutants include: T1047R, T1047R / T1068F, T1047R / T1068F / W778, T1047R / T1068F / W778F / Y782N, T1047R / T1068F / W778F / Y782 / V871A, and T1047R / T1068F / W778F / Y782N / V871A / S883T.

[0009] According to one embodiment of the present invention, most preferably, the mutant includes T1047R / T1068F / W778F / Y782N.

[0010] On the other hand, the present invention also relates to the coding gene of the glycosyltransferase mutant, and to genetically engineered bacteria containing the coding gene.

[0011] Preferably, the expression plasmid of the glycosyltransferase mutant is pET-21a, and the host cell is Escherichia coli BL21(DE3).

[0012] In another aspect, the present invention also provides the application of the glycosyltransferase mutant or the genetically engineered bacteria in the preparation of branched starch or branched α-glucan.

[0013] Furthermore, this invention provides a method for preparing branched starch using a dual-enzyme coupling of glycosyltransferase and starch sucrase, wherein the glycosyltransferase is the mutant described above, or a glycosyltransferase family with the amino acid sequence shown in SEQ ID No:1. Specifically, the method includes adding starch, the glycosyltransferase, and starch sucrase to a buffer solution and reacting under suitable temperature and pH conditions. The glycosyltransferase cleaves the α-1,4 glycosidic bond and transfers the oligosaccharide chain to another segment to form an α-1,6 glycosidic bond, thereby obtaining branched starch. According to an embodiment of the invention, preferably, the method includes adding starch, the glycosyltransferase, and starch sucrase to a buffer solution and then incubating at pH 7 and 40°C.

[0014] Specifically, the starch sucrase is the starch sucrase shown in SEQ ID NO.2;

[0015] Furthermore, the ratio of glycosyltransferase to amylose sucrase is 0.5-4:1, preferably 1-3:1, and more specifically 2:1.

[0016] Furthermore, the present invention also provides a method for preparing α-branched glucan using a dual-enzyme coupling of glycosyltransferase and amylose sucrase, wherein the glycosyltransferase includes the aforementioned SEQ ID NO.1 and its mutant, and the amylose sucrase includes amylose sucrase (AS) derived from Deinococcus geothermalis as shown in SEQ ID NO.2. Specifically, the method includes adding the reaction substrates starch, sucrose, or glucose, along with the glycosyltransferase and amylose sucrase, to a buffer solution and reacting under suitable temperature and pH conditions. The amylose sucrase catalyzes the formation of glucan chains from sucrose, while the glycosyltransferase catalyzes a glycosyltransfer reaction in the α-1,4-glucan chains, introducing an α-1,6-branched structure to obtain α-branched glucan.

[0017] According to one embodiment of the present invention, the optimal reaction temperature for the GT wild-type and the optimal mutant was determined to be 40°C, and the optimal pH was 7.0. The optimal reaction temperature for AS was 45°C, but it could still maintain more than 80% enzyme activity at temperatures below 40°C; the optimal pH was 8.0, and it maintained high catalytic efficiency under neutral conditions (pH 7.0). Therefore, the two enzymes can work synergistically at pH 7.0 and 40°C, while maintaining high conversion efficiency.

[0018] According to one embodiment of the present invention, the effect of the enzyme ratio of GT to AS on sucrose conversion and the branching degree of the dextran product was further determined. The branching degree was determined using nuclear magnetic resonance (NMR) method, based on... 1 The branching degree of the product was calculated by integrating the characteristic peaks corresponding to α-1,6 and α-1,4 glycosidic bonds in the ¹H NMR spectrum. The results showed that the branching degree of dextran gradually increased with increasing GT dosage. When the enzyme ratio of GT to AS was less than 0.1:1, the branching degree of the dextran product was less than 10%; when the enzyme ratio of GT to AS was 2:1, the branching degree of the dextran product reached its maximum of 16.12%; further increasing the amount of GT did not further increase the branching degree of dextran.

[0019] According to one embodiment of the present invention, preferably, the reaction substrate of the method is sucrose, the sucrose concentration is 0.25-0.5M, the amount of starch sucrase is 0.5-1.0U / mL, the amount of branching enzyme is 0.5-2.0U / mL, the reaction temperature is 30-50℃, the reaction pH is 6.0-8.0, and the reaction time is 12-36h.

[0020] According to one embodiment of the present invention, preferably, the reaction substrate of the method is sucrose, the sucrose concentration is 0.5M, the amount of starch sucrase is 1.0 U / mL, the amount of branching enzyme is 2.0 U / mL, the reaction temperature is 40℃, the reaction pH is pH 7.0, and the reaction time is 24h.

[0021] According to one embodiment of the present invention, branched dextran was prepared using the optimal GT mutant T1047R / T1068F / W778F / Y782N as a catalyst. The reaction conditions were: sucrose concentration 0.5M, AS enzyme dosage 1.0 U / mL, GT mutant enzyme dosage 2.0 U / mL, temperature 40℃, pH 7.0, and reaction time 24 h. Nuclear magnetic resonance (NMR) analysis showed that the branching degree of the dextran product under these conditions reached 21.88%, while the GT wild-type under the same conditions only reached 16.12%. The optimal GT mutant significantly improved the branching modification effect of dextran.

[0022] In another aspect, the present invention also provides an α-branched dextran prepared by the above-described α-branched dextran preparation method, wherein the branching degree exceeds 16% and the product branching is mainly composed of short chain structures with a polymerization degree of 2 to 7.

[0023] According to one embodiment of the present invention, the branched α-glucan product was degraded by isoamylase and pullulanase, and its branched structure was analyzed by high-performance anion exchange chromatography. The results showed that the branches of the branched glucan were mainly short-chain oligosaccharide structures with a degree of polymerization of 2-7.

[0024] According to one embodiment of the present invention, the structure of the branched α-glucan prepared by the two enzymes was studied using scanning electron microscopy (SEM) and X-ray diffraction (XRD). No regular crystal structure was observed in the SEM image, indicating that the sample mainly exists in an amorphous form; no obvious characteristic crystal diffraction peaks were observed in the XRD results, indicating that its crystallinity is low and it mainly exhibits an amorphous structure.

[0025] According to one embodiment of the present invention, solubility determination results show that at a concentration of 50 mg / mL, the solubility of the branched dextran sample reaches 98.60% at room temperature. This excellent water solubility helps expand the application potential of branched dextran products in food, pharmaceuticals, and functional materials. Thermogravimetric analysis (TGA) results indicate that no significant degradation of the glycan backbone was observed in the branched dextran product below 200°C, demonstrating good thermal stability, which is beneficial for its development and utilization in food processing and other applications involving heat treatment.

[0026] According to one embodiment of the present invention, the digestibility of the branched α-glucan prepared by the dual-enzyme synergistic method was evaluated using the Englyst in vitro simulated digestion method. α-Amylase and amyloglucosidase were used to simulate the enzymatic hydrolysis environment of the human digestive tract, and the degree of hydrolysis of the glucan within a set time was measured, allowing for rapid evaluation of its digestibility and glycemic potential. The results showed that the 3-hour digestibility of the highly branched glucan prepared from the GT wild-type was 67.16%, with rapidly digestible starch, slowly digestible starch, and resistant starch contents of 20.02%, 36.50%, and 43.71%, respectively. The 3-hour digestibility of the highly branched glucan prepared from the optimal GT mutant was 65.36%, with rapidly digestible starch, slowly digestible starch, and resistant starch contents of 18.05%, 32.74%, and 49.21%, respectively, and the resistant starch content was increased by 5.5% compared to the wild-type.

[0027] Therefore, the present invention further provides the application of the above-mentioned α-branched dextran in functional foods, including the production of functional foods used as low glycemic functional carbohydrates, resistant polysaccharides and food functional ingredients to delay glucose release, regulate postprandial blood glucose, improve intestinal microecology and exert immunomodulatory effects.

[0028] In summary, the glycosyltransferase mutant provided by this invention can efficiently catalyze the glycosyltransferase reaction of α-1,4-glucan, introducing short-chain, high-density α-1,6 branching structures into the glucan chain. Using this mutant enzyme in synergistic catalysis with amylase, highly branched α-glucans can be directly prepared from sucrose as a substrate. The dual-enzyme coupling strategy of this invention can prepare branched α-glucans at higher sucrose substrate concentrations, lower branching enzyme dosages, and shorter reaction times. The resulting branched α-glucans exhibit high α-1,6 branching levels, short branch distribution, good water solubility, and strong anti-digestion properties, making them promising candidates for development as low-glycemic functional carbohydrates, resistant polysaccharides, and functional food ingredients, with excellent industrial application prospects. Attached Figure Description

[0029] Figure 1 This is an SDS-PAGE gel electrophoresis pattern of the target protein obtained by isolation and purification after heterologous expression of glycosyltransferase and amylose sucrase in Escherichia coli, where M is the protein molecular weight standard.

[0030] Figure 2 High-performance anion exchange chromatograms were used to show the branch chain length distribution of the hyperbranched α-glucan product prepared by the synergistic use of glycosyltransferase and starch sucrase after degradation by isoamylase and pullulanase.

[0031] Figure 3 SEM images showing the structure of branched α-glucan products prepared by the synergistic use of glycosyltransferase and starch sucrase.

[0032] Figure 4The in vitro digestion and hydrolysis rate curves of branched α-glucan products prepared by the synergistic use of glycosyltransferase and starch sucrase are shown. The products with the gray curve are prepared using glycosyltransferase mutants, while the products with the black curve are prepared using wild-type glycosyltransferase. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Example 1: Heterologous expression of glycosyltransferase and amylose sucrase

[0037] The encoding genes for starch sucrase (AS) from *Deinococcus geothermalis* and glycosyltransferase (GT) from *Limosilactobacillus reuteri* were ligated into the pET-21a plasmid to construct expression vectors, which were then transformed into the *E. coli* BL21(DE3) expression vector for induced expression. *E. coli* were inoculated into LB broth containing 100 mg / mL ampicillin and cultured overnight at 37°C and 200 rpm to obtain a seed culture. A 1% inoculum was then inoculated into 50 mL of fresh LB broth and cultured at 37°C and 200 rpm for approximately 3 hours. When OD... 600When the pH reaches approximately 0.8, add IPTG to the culture medium at a final concentration of 0.2 mM and induce induction at 16°C and 200 rpm for 16 to 24 hours. Collect the strain by centrifugation, resuspend the cells in Tris-HCl buffer (pH=7.0), then sonicate to disrupt the protein. Centrifuge to obtain the supernatant, and purify using Ni column affinity chromatography to obtain the purified wild-type and mutant target proteins. Figure 1 As shown, SDS-PAGE results indicate that both enzymes can be expressed heterologously in Escherichia coli.

[0038] .

[0039] Amino acid sequence of amylosucrase AS (SEQ ID No: 2): MLKDVLTSELAAQVRDAFDDDRDAETFLLRLERYGEDLWESLRAVYGDQVRALPGRLLEVMLHAYHARPAELRRLDEARLLRPDWLQRPEMVGYVAYTDRFAGTLKGVEERLDYLEGLGVKYLHLMPLLRPREGENDGGYAVQDYRAVRPDLGTMDDLSALARALRGRGISLVLDLVLNHVAREHAWAQKARAGDPKYRAYFHLFPDRRGPDAFEATLPEIFPDFAPGNFSWDEEIGEGEGGWVWTTFNSYQWDLNWANPDVFLEFVDIILYLANRGVEVFRLDAIAFIWKRLGTDCQNQPEVHHLTRALRAAARIVAPAVAFKAEAIVAPADLIHYLGTRAHHGKVSDMAYHNSLMVQLWSSLASRNTRLFEEALRAFPPKPTSTTWGLYVRCHDDIGWAISDEDAARAGLNGAAHRHFLSDFYSGQFPGSFARGLVFQYNPVNGDRRISGSAASLAGLEAALETGDPGRIEDAVRRLLLLHTVILGFGGVPLLYMGDELALLNDYAFEDVPEHAPDNRWVHRPQMDWALAERVRQEPSSPAGRVNTGLRHLLRVRRDTPQLHASIESQVLPSPDSRALLLRRDHPLGGMVQVYNFSEETVMLPSHVLRDVLGDHVQDRLSGSAFRLDRPTVRLEGYRALWLTAGEAPALEHHHHHH .

[0040] Example 2 Molecular modification of glycosyltransferase

[0041] Hotspot wizard 3.0 was used to predict GT mutation hotspot sites from *Limosilactobacillus reuteri*. Based on the enzyme's three-dimensional structure, residue conservation, and mutation score, candidate mutation sites were screened for site-directed mutagenesis. Enzyme activity in wild-type and mutant strains was determined using iodometric titration. The procedure was as follows: 1.25 mg / mL potato amylose was mixed with 10 μg / mL GT in 50 mM phosphate buffer (pH 7.0), and the mixture was incubated at 40 °C for 15 min. Subsequently, 10 μL of the mixture was transferred to a 96-well plate and mixed with 150 μL of iodine solution. The absorbance was measured at 660 nm. The control group was treated with phosphate buffer instead of the enzyme. The concentration of amylose was determined according to a standard curve. One unit of enzyme activity (U) was defined as the amount of enzyme required to consume 1 mg of amylose per minute.

[0042] The results showed that the enzyme activities of single mutants Y782N, W778F, V871A, S883T, T1047R, and T1068F were higher than those of wild type, and the fold increase is shown in Figure 1, with the wild type relative enzyme activity as 1.

[0043] Table 1. Relative enzyme activities of single mutants

[0044] ;

[0045] Further combinations of the above mutation sites yielded double mutants with different combinations as shown in Table 2. Mutants with relatively high enzyme activity were then further combined with other sites in successive rounds of mutation, and their relative activities were measured. The results are shown in Table 3. The relative activity of the wild type was 1. The mutant with the highest relative enzyme activity increase was T1047R / T1068F / W778F / Y782N, whose enzyme activity was 3.5 times that of the wild type.

[0046] Table 2. Relative enzyme activities of double mutants

[0047] ;

[0048] Table 3. Relative enzyme activities of combined mutants

[0049] ;

[0050] Example 3: Preparation of highly branched α-glucan by dual enzyme synergy

[0051] The optimal reaction temperature for the GT wild-type and the optimal mutant was determined to be 40℃, and the optimal pH was 7.0. The optimal reaction temperature for AS was 45℃, but it maintained over 80% enzyme activity below 40℃; the optimal pH was 8.0, and it maintained high catalytic efficiency even under neutral conditions (pH 7.0). Therefore, the two enzymes can synergistically act at pH 7.0 and 40℃ while maintaining high conversion efficiency. Based on this, a reaction system for the synergistic catalysis of sucrose to branched dextran was established, and the substrate concentration and the amount of both enzymes were optimized by controlling the sucrose consumption.

[0052] When the sucrose concentration was 0.05-0.25M, the sucrose consumption reached equilibrium after 8 hours of reaction; when the sucrose concentration was increased to 0.5M, the reaction reached equilibrium after 24 hours; however, when the sucrose concentration was further increased to 1.0M, a large amount of unconverted sucrose still remained in the system after 24 hours of reaction. Therefore, the optimal sucrose concentration was determined to be 0.5M.

[0053] When the amount of enzyme added to AS was less than 0.5 U / mL, sucrose consumption had not reached equilibrium after 24 hours of reaction. When the amount of enzyme added was increased to 1.0 U / mL, sucrose consumption tended to stabilize after 12 hours, and further increasing the amount of AS did not significantly promote sucrose conversion. Therefore, the optimal amount of enzyme added to AS was determined to be 1.0 U / mL.

[0054] The effect of the GT to AS enzyme ratio (0.01:1–2:1) on sucrose conversion and the branching degree of the dextran product was further determined. Branching degree was measured using nuclear magnetic resonance (NMR). 1 The branching degree of the product was calculated by integrating the characteristic peaks corresponding to α-1,6 and α-1,4 glycosidic bonds in the ¹H NMR spectrum. The results showed that the branching degree of dextran gradually increased with increasing GT dosage. When the enzyme ratio of GT to AS was less than 0.1:1, the branching degree of the dextran product was less than 10%; when the enzyme ratio of GT to AS was 2:1, the branching degree of the dextran product reached its maximum of 16.12%; further increasing the amount of GT did not further increase the branching degree of dextran. Therefore, the optimal ratio of GT to AS is 2:1.

[0055] In summary, the optimal reaction system for the synergistic dual-enzyme catalysis of sucrose to prepare branched dextran was determined to be: sucrose concentration 0.5 M, AS enzyme dosage 1.0 U / mL, and GT enzyme dosage 2.0 U / mL. The optimal reaction conditions were: temperature 40℃, pH 7.0, and reaction time 24 h.

[0056] Example 4: GT optimal mutant improves branching degree of branched dextran

[0057] Branched dextran was prepared using the optimal GT mutant as a catalyst under the following conditions: sucrose concentration 0.5 M, AS enzyme dosage 1.0 U / mL, GT mutant enzyme dosage 2.0 U / mL, temperature 40℃, pH 7.0, and reaction time 24 h. Nuclear magnetic resonance (NMR) analysis showed that the branching degree of the dextran product under these conditions reached 21.88%, compared to only 16.12% for the wild-type GT under the same conditions. The optimal GT mutant significantly enhanced the branching modification effect of dextran.

[0058] Example 5: Analysis of the branched structure of α-glucan

[0059] The branched α-glucan product was degraded by isoamylase and pullulanase, and its branched structure was analyzed by high-performance anion exchange chromatography. Figure 2 As shown, the results indicate that the branches of the branched dextran are mainly short-chain oligosaccharide structures with a degree of polymerization of 2-7. This result demonstrates that using the glycosyltransferase or mutant described in this invention in combination with starch sucrase can promote the formation of short-chain branched structures, unlike the long-chain branches obtained by traditional branching enzymes.

[0060] Example 6: Physicochemical Properties Analysis of Branched α-glucan

[0061] The structure of the branched α-glucan prepared by the two enzymes was studied using scanning electron microscopy (SEM) and X-ray diffraction (XRD). Figure 3 As shown, no regular crystal structure was observed in the SEM image, indicating that the sample mainly exists in an amorphous state; no obvious characteristic crystal diffraction peaks were observed in the XRD results, indicating that its crystallinity is low and it mainly exhibits an amorphous structure.

[0062] Solubility tests showed that the branched dextran sample achieved a solubility of 98.60% at room temperature at a concentration of 50 mg / mL. This excellent water solubility helps expand the application potential of branched dextran products in food, pharmaceuticals, and functional materials. Thermogravimetric analysis (TGA) results indicated that no significant degradation of the glycan backbone was observed below 200℃, demonstrating good thermal stability, which is beneficial for its development and utilization in food processing and other applications involving heat treatment.

[0063] Example 7: Analysis of the in vitro digestion characteristics of branched α-glucan

[0064] The digestive properties of the branched α-glucan prepared by the dual-enzyme synergistic method of this invention were evaluated using the Englyst in vitro simulated digestion method. α-Amylase and amylase were used to simulate the enzymatic hydrolysis environment of the human digestive tract, and the degree of hydrolysis of the glucan was measured within a set time, allowing for rapid evaluation of its digestive properties and glycemic potential. The specific method is as follows: 200 mg of sample was weighed and added to 15 mL of sodium acetate buffer solution (100 mM, pH 5.2). After shaking at 37°C and 600 rpm for 10 min, 10 mL of a mixed enzyme solution (containing 290 U / mL α-amylase and 100 U / mL glucoamylase) prepared with sodium acetate (100 mM, pH 5.2) was added, and the mixture was continued to be shaken at 37°C. At 0, 20, 40, 60, 90, 120, 150, and 180 min, 0.5 mL of sample solution was taken and mixed with 2 mL of anhydrous ethanol to inactivate the enzyme. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. The glucose content in the supernatant was determined using the GOPOD method. The digestion-hydrolysis rate (HD) and the relative contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were calculated using the following formulas.

[0065] ;

[0066] The results show that, Figure 4 As shown, the 3-hour digestibility of the highly branched dextran prepared from the GT wild-type was 67.16%, with fast-digesting starch, slow-digesting starch, and resistant starch contents of 20.02%, 36.33%, and 43.65%, respectively. The 3-hour digestibility of the highly branched dextran prepared from the optimal GT mutant was 58.80%, with fast-digesting starch, slow-digesting starch, and resistant starch contents of 16.28%, 33.26%, and 50.46%, respectively, and the resistant starch content was 6.81% higher than that of the wild-type.

Claims

1. A glycosyltransferase mutant, characterized in that... It is obtained by amino acid mutations at six sites, Y782N, W778F, V871A, S883T, T1047R, and T1068F, based on the dextran transferase sequence from *Limosilactobacillus reuteri* shown in SEQ ID NO.

1. The mutations include changes to any one, two, three, four, or five amino acid residues at the six sites.

2. The glycosyltransferase mutant as described in claim 1, wherein the mutant is selected from Y782N, W778F, V871A, S883T, T1047R, T1068F, T1047R / Y782N, T1047R / W778F, T1047R / V871A, T1047R / S883T, T1047R / T1068F, T1047R / T1068F, T1047R / T1068F / Y782N, T1047R / T1068F / W778, T1047R / T1068F / V871 A. T1047R / T1068F / S883T, T1047R / T1068F / W778F / Y782N, T1047R / T1068F / W778F / V871A, T1047R / T1068F / W778F / S883T, T 1047R / T1068F / W778F / Y782N / V871A, T1047R / T1068F / W778F / Y782N / S883T, T1047R / T1068F / W778F / Y782N / V871A / S883T; Preferably, the mutant is selected from: T1047R, T1047R / T1068F, T1047R / T1068F / W778, T1047R / T1068F / W778F / Y782N, T1047R / T1068F / W778F / Y782 / V871A, T1047R / T1068F / W778F / Y782N / V871A / S883T; Preferably, the mutant is selected from: T1047R / T1068F / W778F / Y782N.

3. The encoding gene of the glycosyltransferase mutant as described in claim 1 or 2.

4. Genetically engineered bacteria containing the encoding gene as described in claim 3.

5. The glycosyltransferase mutant as described in claim 1, preferably, has an expression plasmid of pET-21a and a host cell of Escherichia coli BL21(DE3).

6. The use of the glycosyltransferase mutant as described in claim 1 or the genetically engineered bacteria as described in claim 4 in the preparation of branched starch or branched α-glucan.

7. A method for preparing branched starch using a dual-enzyme coupling of glycosyltransferase and starch sucrase, characterized in that... The glycosyltransferase described herein is either the mutant as described in claim 1 or a glycosyltransferase with the amino acid sequence shown in SEQ ID No: 1; Specifically, the method includes adding starch, the glycosyltransferase, and starch sucrase to a buffer solution and reacting them at 35-45°C and pH 6-8 to obtain branched starch. Preferably, the method includes adding starch, the glycosyltransferase, and starch sucrase to a buffer solution, and then incubating at pH 7 and 40°C. Specifically, the starch sucrase is the starch sucrase shown in SEQ ID NO.2; Furthermore, the ratio of glycosyltransferase to amylose sucrase is 0.5-4:1, preferably 1-3:1, and more specifically 2:

1.

8. A method for preparing α-branched dextran using a dual-enzyme coupling of glycosyltransferase and starch sucrase, characterized in that... The glycosyltransferase described herein is either the mutant as described in claim 1 or a glycosyltransferase with the amino acid sequence shown in SEQ ID No: 1; Specifically, the method includes adding the reaction substrate sucrose and the glycosyltransferase and amylase to a buffer solution to react and obtain α-branched dextran; Preferably, the reaction substrate of the method is sucrose, the sucrose concentration is 0.25-0.5M, the amount of starch sucrase is 0.5-1.0U / mL, the amount of glycosyltransferase is 0.5-2.0U / mL, the reaction temperature is 30-50℃, the reaction pH is 6.0-8.0, and the reaction time is 12-36h. Preferably, the reaction substrate of the method is sucrose, the sucrose concentration is 0.5M, the amount of starch sucrase is 1.0 U / mL, the amount of glycosyltransferase is 2.0 U / mL, the reaction temperature is 40℃, the reaction pH is pH 7.0, and the reaction time is 24h. Specifically, the starch sucrase is the starch sucrase shown in SEQ ID NO.

2.

9. An α-branched dextran, characterized in that... It is prepared using the method described in claim 8, with a branching degree exceeding 16%, and the product branches are mainly short-chain structures with a polymerization degree of 2 to 7.

10. The application of α-branched dextran as described in claim 9 in functional foods, including the production of functional foods used as low-glycemic functional carbohydrates, resistant polysaccharides, and food functional ingredients to delay glucose release, regulate postprandial blood glucose, improve gut microbiota, and exert immunomodulatory effects.

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