A method for preparing high purity glucosyl flavonoid products using mutant cyclodextrin glycosyltransferase

Abstract

The application discloses a method for preparing high-purity glucosyl flavonoid products by using mutant cyclodextrin glycosyltransferase, and belongs to the technical field of bioengineering and fine chemical industry. The application carries out molecular modification on CGTase from Geobacillus stearothermophilus, and obtains a high-selectivity mutant enzyme, the catalytic efficiency of which on beta-cyclodextrin is significantly improved, and the selectivity of mono-glucosyl rutin and mono-glucosyl hesperidin is increased from less than 50% to more than 85%. The feeding concentration of rutin reaches 20 g / L, the feeding concentration of hesperidin reaches 15 g / L, the total reaction time is less than or equal to 8 h, the production capacity is increased by more than 20 times, and the cycle is shortened by more than 60%. The process omits resin chromatography, the post-treatment is simplified into three steps, and is green and low in cost. The product purity is greater than or equal to 85%, no solvent residue is left, the product meets the requirements of medical and cosmetic grades, and is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to a method for preparing high-purity glucosyl flavonoid products using mutant cyclodextrin glycosyltransferase, belonging to the fields of bioengineering and fine chemical technology. Background Technology

[0002] Flavonoids are important bioactive substances from natural sources, widely used in functional foods, cosmetics, and pharmaceutical preparations. Hesperidin, the most abundant dihydroflavonoid glycoside in citrus peel, possesses physiological activities such as antioxidant, anti-inflammatory, microcirculation improvement, and lipid regulation. Rutin, a core component of vitamin P, has multiple effects including antioxidant, anti-inflammatory, cardiovascular protection, anti-obesity, and anti-photoaging, leading to continuously increasing market demand. However, both face significant application bottlenecks: hesperidin's water solubility is only about (6.32±0.12) μg / mL, with an oral bioavailability of approximately 3.51%; rutin is almost insoluble in water (solubility <0.02 mg / mL), with an oral absorption rate of less than 1%, and its poor water / lipid solubility severely restricts its industrialization and formulation.

[0003] Glucosylation is a mainstream modification pathway for improving the solubility and bioavailability of flavonoids. Glucosyl hesperidin and glucosyl rutin can significantly improve water solubility and stability while retaining the parent compound's activity, and have become core functional ingredients in high-end food, pharmaceutical, and skincare fields. Existing structural modifications are mainly divided into chemical methods and biotransformation methods: chemical methods (acid hydrolysis, methylation, sulfonation, metal complexation, etc.) have drawbacks such as high reagent consumption, complex byproducts, and environmental unfriendliness; enzymatic glycosylation, due to its mild conditions, high regioselectivity, and fewer byproducts, has become the mainstream technology for preparing glucosyl flavonoid derivatives.

[0004] Current industrial-scale preparations utilize cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) as the core catalyst, with starch, dextrin, or cyclodextrin as glycosyl donors, to perform transglycosylation reactions on hesperidin and rutin, respectively. Several institutions and companies, including Hayashihara Chemical Co., Ltd. (Japan), Zhejiang University of Technology, Shandong Benyue Biotechnology Co., Ltd., and Huiwen Biotechnology Co., Ltd., have published related patents and processes, achieving some progress in enzyme modification, substrate concentration, and conversion rate. However, existing technologies generally suffer from common key defects, making it difficult to meet the demands for large-scale, low-cost, and high-purity production. 1. The substrate has low solubility, the reaction system is heterogeneous, mass transfer is limited, the feed concentration is low, and the efficiency is poor: high-concentration feed is difficult to achieve, the unit volume production capacity is low, and the solubilization method is prone to problems such as solvent residue, product discoloration and deactivation. In addition, it has strict requirements for the purity of raw materials and high cost.

[0005] 2. Insufficient enzyme catalysis selectivity, low proportion of monoglucosyl products, and uncontrollable degree of polymerization: CGTase catalysis is reversible and produces complex products. In addition to the target monoglucosyl derivative, it is easy to generate polysubstituted isomers, resulting in unstable product quality. It is necessary to add glycosidase to hydrolyze polysubstituted products, which prolongs the process.

[0006] 3. Substrate and product easily form inclusion complexes, affecting stability and bioactivity: Unreacted substrates and glucosyl products are prone to inclusion complexes, reducing product performance and quality uniformity.

[0007] 4. The separation and purification process is cumbersome and has poor environmental and economic efficiency: The process is highly dependent on macroporous resin chromatography and activated carbon decolorization. The process is long, consumes a lot of solvents, generates a lot of solid waste, and has high regeneration costs, making it difficult to adapt to the green manufacturing of bulk raw materials.

[0008] In summary, there is an urgent need in this field for a green enzymatic preparation technology that can simultaneously and efficiently prepare glucosyl hesperidin and glucosyl rutin, with strong substrate concentration adaptability, high conversion rate, good monoglucosyl selectivity, simple post-processing, and low requirements for raw material purity, so as to break through the existing process bottlenecks and meet the needs of industrial production. Summary of the Invention

[0009] To address the problems of low substrate solubility, poor glycosylation efficiency, insufficient monoglucosyl selectivity, long reaction time, cumbersome purification process, and high cost in existing enzymatic methods for preparing glucosylrutin and glucosylhesperidin, this invention provides a highly efficient, green, and simplified preparation method. This invention uses cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) from *Geobacillus stearothermophilus* as the target for modification. Based on sequence analysis, single-point and combinatorial mutations were performed to obtain mutant enzymes with significantly improved catalytic performance. After molecular modification, this enzyme preferentially adapts to β-cyclodextrin as a glycosyl donor, significantly improving the glycosylation efficiency and regioselectivity for rutin and hesperidin, effectively overcoming the shortcomings of natural CGTase, such as low catalytic activity, poor glycosylation efficiency, low substrate conversion rate, and long reaction cycle for β-cyclodextrin.

[0010] The first technical solution provided by this invention is a cyclodextrin glycosyltransferase mutant, wherein the mutant is a cyclodextrin glycosyltransferase parent with the amino acid sequence shown in SEQ ID No. 1, mutated by any of the following methods: (a) Replace glutamic acid at position 291 with isoleucine or valine; (b) Replace tyrosine at position 218 with phenylalanine; (c) Replace proline at position 232 with serine; (d) Replace arginine at position 277 with lysine; (e) Replace leucine at position 191 with isoleucine; (f) Replace glutamic acid at position 289 with valine; (g) Replace tyrosine at position 218 with phenylalanine, and replace alanine at position 122 with threonine; (h) Replace tyrosine at position 218 with phenylalanine, and replace glutamic acid at position 291 with valine; (i) Glutamic acid at position 291 is replaced with valine, and alanine at position 122 is replaced with threonine; (j) Replace tyrosine at position 218 with phenylalanine, and replace leucine at position 191 with isoleucine; (k) Replace tyrosine at position 218 with phenylalanine, glutamic acid at position 291 with valine, and proline at position 232 with serine; (l) Alanine at position 122 is replaced with threonine, tyrosine at position 218 is replaced with phenylalanine, and glutamic acid at position 291 is replaced with valine.

[0011] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0012] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0013] In some embodiments, the recombinant plasmid uses PET series plasmids as expression vectors.

[0014] The fourth technical solution provided by the present invention is to express the mutant described in the first technical solution, or to contain the gene described in the second technical solution, or to transform a host cell with the recombinant plasmid described in the third technical solution.

[0015] In some embodiments, the host cell is a microbial cell.

[0016] In some embodiments, the microbial cells are Escherichia coli.

[0017] The fifth technical solution provided by this invention is a recombinant Escherichia coli, wherein the recombinant Escherichia coli is... E. coli Using BL21(DE3) as the host cell and plasmid pET-28a as the expression vector, the mutant described in the first technical solution was expressed.

[0018] The sixth technical solution provided by the present invention is a method for biosynthesizing glucosyl flavonoids. The method involves using the mutant described in the first technical solution as a catalyst to introduce into a catalytic system containing flavonoids and β-cyclodextrin to synthesize glucosyl flavonoids.

[0019] In some embodiments, the flavonoid compound is rutin or hesperidin.

[0020] In some embodiments, the catalyst in the catalytic system is measured as 50 g / L of crude enzyme solution.

[0021] In some embodiments, the initial concentration of rutin or hesperidin in the catalytic system is 15-25 g / L, and the initial concentration of β-cyclodextrin is 223-372 g / L; and the reaction system completes the complexation and glycosylation conversion of rutin and β-cyclodextrin within 6-8 h.

[0022] In some embodiments, the catalytic system is controlled by NaHCO3 at pH 6.5 and the reaction is carried out at 40 °C.

[0023] In some embodiments, after the reaction is complete, a ceramic membrane and nanofiltration are used to remove protein impurities and unreacted residual sugars from the system to obtain a glucosylrutin solution; the impurity removal process does not use macroporous adsorption resin column chromatography; subsequently, the glucosylrutin solution is directly spray-dried to obtain a high-purity glucosylrutin product.

[0024] In some embodiments, the catalyst is added in the form of wet cells obtained by shake-flask fermentation or high-density fermentation of the host cells described in the fourth technical solution or the recombinant Escherichia coli described in the fifth technical solution, or an enzyme solution obtained by crushing the wet cells.

[0025] In some embodiments, the high-density fermentation process involves inoculating the host cells described in the fourth technical solution or the recombinant *E. coli* described in the fifth technical solution into LB liquid medium containing 100 μg / mL kanamycin, and culturing at 37 °C and 200 rpm for 12-16 h to obtain a seed culture. The seed culture is then inoculated into the fermentation medium at a 2.0% inoculation rate (100 mL / 5 L) and cultured at 37 °C. The aeration rate is initially 1 vvm, increasing to 1.2 vvm after 3 h. The tank pressure is maintained at 0.05-0.06 MPa. The stirring speed is 500-700 rpm. Dissolved oxygen is controlled to be at least 20%, decreasing rapidly after 2 h of fermentation and then rapidly increasing again after about 5 h. At this point, fed medium is added to maintain dissolved oxygen at around 30%. The pH may rise slowly in the early stages of fermentation, decreasing after feeding. The pH can then be adjusted with ammonia to maintain it at 6.7-6.9. After feeding begins, the temperature is slowly lowered to 30-33 °C for 1-2 h. When OD 600 When the temperature reaches 20-25℃, cool it down to 25℃. Add 0.2 mM IPTG to induce for 14-16 h, then place the culture in a container and collect the wet cells.

[0026] The seventh technical solution provided by the present invention is the application of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the host cell described in the fourth technical solution, or the recombinant Escherichia coli described in the fifth technical solution in the preparation of glucosyl flavonoids or products containing glucosyl flavonoids.

[0027] In some embodiments, the glucosylflavonoid compound is glucosylrutin or glucosylhesperidin.

[0028] The eighth technical solution provided by this invention is a method for improving the conversion rate of cyclodextrin glycosyltransferase products, wherein the method involves performing any one of the following mutations on the cyclodextrin glycosyltransferase parent with the amino acid sequence shown in SEQ ID No. 1: (a) Replace glutamic acid at position 291 with isoleucine or valine; (b) Replace tyrosine at position 218 with phenylalanine; (c) Replace proline at position 232 with serine; (d) Replace arginine at position 277 with lysine; (e) Replace leucine at position 191 with isoleucine; (f) Replace glutamic acid at position 289 with valine; (g) Replace tyrosine at position 218 with phenylalanine, and replace alanine at position 122 with threonine; (h) Replace tyrosine at position 218 with phenylalanine, and replace glutamic acid at position 291 with valine; (i) Glutamic acid at position 291 is replaced with valine, and alanine at position 122 is replaced with threonine; (j) Replace tyrosine at position 218 with phenylalanine, and replace leucine at position 191 with isoleucine; (k) Replace tyrosine at position 218 with phenylalanine, glutamic acid at position 291 with valine, and proline at position 232 with serine; (j) Replace alanine at position 122 with threonine, tyrosine at position 218 with phenylalanine, and glutamic acid at position 291 with valine; The product is glucosylrutin or glucosylhesperidin.

[0029] Compared with the prior art, the technical effects of the present invention are as follows: This invention involves molecularly modifying the gene encoding cyclodextrin glycosyltransferase (CGTase) from *Geobacillus stearothermophilus* to obtain a variety of mutant enzymes with significantly enhanced catalytic performance. Compared to the wild type, the mutant enzymes exhibit a substantial increase in catalytic efficiency for β-cyclodextrin, and the selectivity for monoglucosylrutin and monoglucosylhesperidin is improved from <50% to >85%.

[0030] In terms of catalytic efficiency, the initial reaction concentration of rutin can be increased to 20 g / L, and the unit reactor capacity can be increased by more than 20 times; the initial reaction concentration of hesperidin can reach 15 g / L; the complexation and total reaction time of the rutin / hesperidin system are both controlled within 8 hours, the production cycle is shortened by more than 60%, and the reaction efficiency and equipment utilization rate are significantly improved.

[0031] In terms of purification process, thanks to the high selectivity and high conversion efficiency of the mutant enzyme, the post-processing of the reaction solution is extremely simplified, completely eliminating the macroporous resin column chromatography step, avoiding the large-scale use of organic solvents and resin regeneration costs; the post-processing is reduced from the traditional 6-7 steps to less than 3 steps (enzyme inactivation → impurity removal → spray drying), making the process simple, green and efficient.

[0032] In terms of economy and quality, replacing starch / γ-cyclodextrin with β-cyclodextrin as a glycosyl donor significantly reduces raw material costs; the elimination of long column elution significantly reduces energy consumption; the final product purity can reach over 85%, with no solvent residue, meeting pharmaceutical and cosmetic application standards, combining economy and safety, and is suitable for large-scale industrial production. Attached Figure Description

[0033] Figure 1 A schematic diagram of the synthesis of glucosylrutin catalyzed by cyclodextrin glycosyltransferase.

[0034] Figure 2 This is a high-performance chromatogram of the substrate rutin standard.

[0035] Figure 3 The image shows the high-performance liquid chromatogram of the product glucosylrutin standard.

[0036] Figure 4 This is a schematic diagram of the synthesis of glucosylhesperidin catalyzed by cyclodextrin glycosyltransferase.

[0037] Figure 5 This is a high-performance chromatogram of the substrate hesperidin standard.

[0038] Figure 6 The image shows the high-performance liquid chromatogram of the product glucosyl hesperidin standard.

[0039] Figure 7This is an agarose gel electrophoresis pattern of PCR products obtained by single-point mutation at key sites in the recombinant plasmid pET28a-CGTase; lane M is the marker; lanes 1-15 are CGTase-N10, CGTase-A122T, CGTase-E291V, CGTase-Y218F, CGTase-A65S, CGTase-L191I, CGTase-A122S, CGTase-A65T, CGTase-Y218W, CGTase-E291I, CGTase-R219K, CGTase-P232S, CGTase-R277K, CGTase-S288G, and CGTase-E289V, respectively.

[0040] Figure 8 Agarose gel electrophoresis patterns of PCR products obtained by combined mutations at key sites in the recombinant plasmid pET28a-CGTase: CGTase-L191I / Y218F, CGTase-Y218F / A122T, CGTase-Y218F / E291V, CGTase-A122T / E291V, CGTase-Y218F / E291V / P232S.

[0041] Figure 9 SDS-PAGE detection of wet cells induced by CGTase and its mutants; lane M is the marker; lanes 1-10 are, in order: pre-induction sample, CGTase-N10, CGTase-A122T, CGTase-E291V, CGTase-Y218F, CGTase-A65S, CGTase-L191I, CGTase-A122S, CGTase-A65T, CGTase-Y218W.

[0042] Figure 10 SDS-PAGE assay of wet cells induced by CGTase and its mutants; lane M is the marker; lanes 1-12 are, in order: CGTase-E291I, CGTase-R219K, CGTase-P232S, CGTase-R277K, CGTase-S288G, CGTase-E289V, CGTase-L191I / Y218F, CGTase-Y218F / A122T, CGTase-Y218F / E291V, CGTase-A122T / E291V, CGTase-Y218F / E291V / P232S.

[0043] Figure 11 The image shows the high-performance liquid chromatogram of the extracted glucosylrutin product.

[0044] Figure 12 The image shows the high-performance liquid chromatogram of the extracted glucosyl hesperidin product. Detailed Implementation

[0045] refer to Figures 1-12 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0046] Example 1: Genetically engineered bacteria E. coli Transformation and culture of BL21(DE3) / pET28a-CGTase Origin Geobacillus stearothermophilus The amino acid sequence of the cyclodextrin glycosyltransferase CGTase is shown in SEQ ID NO.1, and the nucleotide sequence of the codon-optimized CGTase gene is shown in SEQ ID NO.2. Both were synthesized by Hangzhou Qingke Biotechnology Co., Ltd., with the insertion site located in the pET28a vector. Nco I and Xho Between the I sites. Its nucleotide sequence is 2133 bp long, corresponding to an amino acid sequence length of 711 aa, and the protein molecular weight is approximately 78.2 kDa.

[0047] After obtaining the synthesized recombinant plasmid pET28a-CGTase, the plasmid was diluted according to the requirements and then transformed into competent cells. E. coli Recombinant strains were obtained from BL21(DE3). E. coli BL21(DE3) / pET28a-CGTase. The recombinant strain was streaked onto LB solid medium containing 100 μg / mL kanamycin resistance and incubated overnight at 37 °C to obtain single colonies. Each single colony was picked and incubated overnight in 50 mL LB liquid medium containing 100 μg / mL kanamycin resistance at 37 °C and 150 rpm to obtain a seed culture. The fresh seed culture was then diluted with 40% glycerol at a 1:1 volume ratio and stored at -80 °C.

[0048] strain E. coli BL21(DE3) / pET28a-CGTase was removed from the -80 ℃ freezer and thawed on ice. 5 µL of the preserved bacterial culture was streaked onto an LB agar plate containing 100 μg / mL kanamycin resistance and incubated at 37 ℃ for 12–16 h for activation. A single colony was picked and transferred to 50 mL of LB liquid medium containing 100 μg / mL kanamycin and incubated at 37 ℃ with a 150 rpm shaking incubator for 12–16 h. The resulting seed culture is the [seeded culture / protein / protein]. E. coliSeed culture of genetically engineered bacteria BL21(DE3) / pET28a-CGTase, from E. coli Plasmid pET28a-CGTase was extracted from BL21(DE3) / pET28a-CGTase bacterial culture.

[0049] LB liquid medium consists of: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, distilled water as solvent, pH 7.0–7.5. LB solid medium is LB liquid medium with 20 g / L agar added.

[0050] Example 2: Induced expression of sucrose phosphorylase SPase Tyrosine phenol lyase E. coli BL21(DE3) / pET28a-SPase was streaked onto LB agar plates containing 100 μg / mL Kan and incubated upside down at 37 °C for 12–16 h. Single colonies were picked and incubated in 50 mL LB medium containing 100 μg / mL Kan at 37 °C and 200 rpm for 12–16 h to obtain a seed culture. The seed culture was then transferred at a 2% (v / v) inoculation rate to 150 mL LB liquid medium containing the same Kan concentration and incubated on a shaker at 37 °C and 200 rpm to achieve an OD500 concentration. 600 To a final concentration of 0.2 mM, add the prepared IPTG solution to 0.6-0.8 and induce expression at 24 °C and 150 rpm for 12-16 h. After induction, transfer the bacterial culture to a centrifuge cup and centrifuge at 4 °C and 8000 rpm for 10 min, discarding the supernatant. Resuspend the bacterial pellet in 100 mM PBS buffer (pH 7.0) and centrifuge at 4 °C and 8000 rpm for 10 min, discarding the supernatant. The obtained pellet is the wet bacterial cell, which should be stored at -20 °C for later use.

[0051] Example 3: Synthesis of glucosylrutin catalyzed by cyclodextrin glycosyltransferase (CGTase) 1. Construction of the catalytic reaction system of cyclodextrin glycosyltransferase (CGTase) The enzyme solution of cyclodextrin glycosyltransferase (CGTase) was prepared according to the method described in Example 2, and a 50 mL reaction system was constructed. The reaction was carried out in a reactor at 40 °C and pH 6.5. Rutin and β-cyclodextrin were pre-complexed in the reactor for 8 h. After the complexation was completed, the pH was adjusted to 6.5 with NaHCO3 before adding the enzyme solution for reaction. The amounts of each component added are shown in Table 1. Table 1. Reaction system for the synthesis of glucosylrutin catalyzed by cyclodextrin glycosyltransferase

[0052] 2. Liquid Chromatographic Analysis of Glucosylrutin Synthesis Catalyzed by Cyclodextrin Glycosyltransferase (CGTase) (1) Treatment of reaction solution The enzyme-catalyzed reaction system was constructed according to the method in Example 3. 1 mL of sample was taken and the enzyme was inactivated at high temperature. The saccharifying enzyme was added and treated for 15 min. The enzyme was inactivated at high temperature again and centrifuged. The supernatant was taken, diluted 20 times with the mobile phase, centrifuged at 10,000 rpm for 2 min, and then filtered through a membrane for loading.

[0053] (2) High performance liquid chromatography analysis method The substrate rutin and the product glucosylrutin were detected by high performance liquid chromatography (HPLC). The HPLC column used was an ACE Excel 5 C18-PFP, with a flow rate of 1 mL / min, a column temperature of 30℃, a holding time of 15 min, and a detection wavelength of 283 nm. Mobile phase A consisted of 0.1% formic acid aqueous solution, and mobile phase B consisted of pure acetonitrile. The gradient elution programs for mobile phases A and B are shown in Table 2.

[0054] Table 2 Gradient elution program for mobile phase A and mobile phase B

[0055] The liquid chromatography spectrum of the substrate rutin standard is as follows: Figure 2 As shown, the elution time was 8.990 min. The liquid chromatography chromatogram of the product glucosylrutin standard is shown below. Figure 3 As shown, the peak elution time is 8.783 min.

[0056] Example 4: Synthesis of glucosylhesperidin catalyzed by cyclodextrin glycosyltransferase (CGTase) 1. Construction of the catalytic reaction system of cyclodextrin glycosyltransferase CGTase and its mutants The enzyme solution of cyclodextrin glycosyltransferase CGTase and its mutant was prepared according to the method described in Example 2. A 50 mL reaction system was constructed. Hesperidin and β-cyclodextrin were added sequentially to the reactor at 40 °C, followed by the addition of the enzyme solution (final concentration 50 g / L) in a fed-batch manner. The reaction was stirred thoroughly throughout, and the pH was maintained at 8.5 with NaOH throughout. The amounts of each component added are shown in Table 3. Table 3. Reaction system for the synthesis of glucosylhesperidin catalyzed by cyclodextrin glycosyltransferase.

[0057] 2. Liquid Chromatographic Analysis of the Synthesis of Glucosylhesperidin Catalyzed by Cyclodextrin Glycosyltransferase (CGTase) (1) Treatment of reaction solution The enzyme-catalyzed reaction system was constructed according to the method in Example 3. 1 mL of sample was taken and the enzyme was inactivated at high temperature. The saccharifying enzyme was added and treated for 15 min. The enzyme was inactivated at high temperature again and centrifuged. The supernatant was taken, diluted 20 times with the mobile phase, centrifuged at 10,000 rpm for 2 min, and then filtered through a membrane for loading.

[0058] (2) High performance liquid chromatography analysis method The substrate hesperidin and the product glucosylhesperidin were detected by high performance liquid chromatography (HPLC). The HPLC column used was an ACE Excel 5 C18-PFP, with a flow rate of 1 mL / min, a column temperature of 30℃, a hold time of 15 min, and a detection wavelength of 283 nm. Mobile phase A consisted of 0.1% formic acid aqueous solution, and mobile phase B consisted of pure acetonitrile. The gradient elution programs for mobile phases A and B are shown in Table 4. Table 4 Gradient elution program for mobile phase A and mobile phase B

[0059] The liquid chromatography chromatogram of the substrate hesperidin standard is as follows: Figure 4 As shown, the elution time was 11.282 min. The liquid chromatography chromatogram of the product glucosyl hesperidin standard is shown below. Figure 5 As shown, the peak elution time is 10.998 min.

[0060] Example 5: Single-point mutation of cyclodextrin glycosyltransferase CGTase Following sequence alignment, homology modeling, and molecular docking analysis, 15 amino acid sites were selected for single-point mutagenesis of the cyclodextrin glycosyltransferase (CGTase) gene. The amino acid sequences of the CGTase mutants are as follows: (1) The first 10 amino acids of the amino acid sequence shown in SEQ ID No. 1 are truncated; (2) Replace the alanine at position 65 of the amino acid sequence shown in SEQ ID No. 1 with serine; (3) Replace the alanine at position 65 of the amino acid sequence shown in SEQ ID No. 1 with threonine; (4) Replace the alanine at position 122 of the amino acid sequence shown in SEQ ID No. 1 with threonine; (5) Replace the glutamic acid at position 291 of the amino acid sequence shown in SEQ ID No. 1 with valine; (6) Replace the tyrosine at position 218 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine; (7) Replace the leucine at position 191 of the amino acid sequence shown in SEQ ID No. 1 with isoleucine; (8) Replace the alanine at position 122 of the amino acid sequence shown in SEQ ID No. 1 with threonine; (9) Replace the tyrosine residue at position 218 of the amino acid sequence shown in SEQ ID No. 1 with tryptophan; (10) Replace the glutamic acid at position 291 of the amino acid sequence shown in SEQ ID No. 1 with isoleucine; (11) Replace the arginine at position 219 of the amino acid sequence shown in SEQ ID No. 1 with lysine; (12) Replace the proline at position 232 of the amino acid sequence shown in SEQ ID No.1 with serine; (13) Replace the arginine at position 277 of the amino acid sequence shown in SEQ ID No. 1 with lysine; (14) Replace serine at position 288 of the amino acid sequence shown in SEQ ID No. 1 with glycine; (15) Replace the glutamic acid at position 289 of the amino acid sequence shown in SEQ ID No.1 with valine.

[0061] The mutant was constructed using inverse PCR with plasmid pET28a-CGTase as the amplification template. The primers used are shown in Table 5, and the PCR amplification system is shown in Table 6.

[0062] PCR reaction program: pre-denaturation: 95 ℃, 5 min; complete denaturation: 95 ℃, 15 s; annealing: 58 ℃, 15 s; extension: 72 ℃, 90 s; 30 cycles; second extension: 72 ℃, 5 min; cool to 4 ℃ and incubate.

[0063] Table 5 Primers for single-point mutation of cyclodextrin glycosyltransferase (CGTase)

[0064] Table 6. PCR system for full plasmid amplification

[0065] A suitable amount of PCR product was taken and verified by agarose gel electrophoresis. After electrophoresis, the product was observed under ultraviolet light, and bright bands were visible between 5000-8000 bp, which was consistent with the theoretical value of the plasmid. The agarose gel electrophoresis results are as follows. Figure 7 As shown. Then, add to the PCR product. Dpn Incubate with 1 µL of restriction endonuclease at 37 °C for 1 h to remove methylated template. Then, PCR products are purified using a DNA gel extraction kit. Store at -20 °C for later use.

[0066] The target fragment obtained through mutation was directly transformed into the host bacterium *Escherichia coli*. E. coli BL21(DE3): Take 50 μL E. coli BL21(DE3) competent cells were thawed on ice, and 5 μL of mutant PCR product was added. The cells were then incubated on ice for 30 min. After the ice bath, the competent cells were heat-shocked at 42 °C for 50 s, followed immediately by incubation on ice for 2-3 min. Then, 600 μL of antibiotic-free LB medium was added, and the cells were incubated at 37 °C and 200 rpm for 1 h in a shaker. After incubation, the culture was centrifuged at 4 °C and 3500 rpm for 5 min, and 550 μL of the supernatant was discarded. The remaining cells were thoroughly resuspended, and 100 μL of the bacterial culture was spread onto LB agar plates containing Kan antibiotics. The cells were incubated upside down at 37 °C for 12-16 h to obtain genetically engineered *E. coli*. E. coli The BL21(DE3) / pET28a-CGTase mutant, for example, when tyrosine at position 218 of the amino acid sequence is replaced with phenylalanine, is named as follows: E. coli BL21(DE3) / pET28a-CGTase-Y218F.

[0067] The cyclodextrin glycosyltransferase mutant was induced and expressed using the method in Example 2. The expression effect was verified by SDS-PAGE electrophoresis. The electrophoresis results are as follows: Figures 8-9 As shown, the molecular weight of CGTase and its mutant proteins is approximately 78.2 kDa. The protein bands expressed by each mutant strain are significantly thicker and located at approximately 78.2 kDa, which is the correct size.

[0068] To verify the activity of different mutants, crude enzyme solutions of CGTase mutants were prepared using the method in Example 2. The CGTase mutants were then subjected to catalytic reactions using the method in Example 3, with a rutin content of 5 g / L, a β-cyclodextrin content of 74.65 g / L, and a total system volume of 10 mL. The catalytic results of the engineered bacteria with cyclodextrin glycosyltransferase mutants were analyzed by liquid chromatography using the method in Example 3. The catalytic effect was compared by comparing the conversion rate of product AA-2G in the catalytic reaction solution after 2 h. The results are shown in Table 7. Compared with cyclodextrin glycosyltransferase (CGTase), several mutations significantly increased catalytic activity, namely CGTase-A122T, CGTase-E291V, CGTase-Y218F, CGTase-L191I, CGTase-E291I, CGTase-P232S, CGTase-R277K, and CGTase-E289V.

[0069] Table 7. Catalytic results of CGTase and its mutants detected by liquid phase analysis.

[0070] Remark: Marked as beneficial mutation Crude enzyme solutions of CGTase and its mutants were prepared using the method in Example 2. The catalytic reaction of CGTase and its mutants was carried out using the method in Example 4, with a hesperidin content of 5 g / L, a β-cyclodextrin content of 25 g / L, and a total system volume of 10 mL. The catalytic results of cyclodextrin glycosyltransferase and its mutant engineered bacteria were analyzed by liquid chromatography using the method in Example 4. The catalytic effect was compared by comparing the conversion rate of glucosylhesperidin in the catalytic reaction solution after 2 h. The results are shown in Table 8. Compared with cyclodextrin glycosyltransferase (CGTase), several mutations significantly increased the catalytic activity, namely CGTase-A122T, CGTase-E291V, CGTase-Y218F, CGTase-L191I, and CGTase-E291I.

[0071] Table 8. Catalytic results of CGTase and its mutants detected by liquid phase.

[0072] Remark: Marked as beneficial mutation Example 6: Combinatorial Mutation of Cyclodextrin Glycosyltransferases Based on the characteristics of different structural domains, CGTase was subjected to combined mutations through rational design. Using reverse PCR technology, combined mutant strains were constructed according to the method described in Example 5, with the following amino acids replaced: (1) Replace the tyrosine at position 218 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine and the alanine at position 122 with threonine to obtain a mutant strain. E. coli BL21(DE3) / pET28a-CGTase-Y218F / A122T; (2) Replace the tyrosine at position 218 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine and the glutamic acid at position 291 with valine to obtain a mutant strain. E. coli BL21(DE3) / pET28a-CGTase-Y218F / E291V; (3) Replace tyrosine at position 218 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine, glutamic acid at position 291 with valine, and proline at position 232 with serine to obtain a mutant strain. E. coli BL21(DE3) / pET28a-CGTase-Y218F / E291V / P232S; (4) Replace the glutamic acid at position 291 of the amino acid sequence shown in SEQ ID No. 1 with valine and the alanine at position 122 with threonine to obtain the mutant strain. E. coli BL21(DE3) / pET28a-CGTase-E291V / A122T; (5) Replace the tyrosine at position 218 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine and the leucine at position 191 with isoleucine to obtain a mutant strain. E. coli BL21(DE3) / pET28a-CGTase-Y218F / L191I; (6) Replace the tyrosine at position 218 of the amino acid sequence shown in SEQ ID No. 1 with phenylalanine, the glutamic acid at position 291 with valine, and the alanine at position 122 with threonine to obtain a mutant strain. E. coli BL21(DE3) / pET28a-CGTase-A122T / Y218F / E291V; Wet cells of the CGTase mutant were prepared using the method in Example 2. The catalytic results of the crude enzyme solution were analyzed by liquid chromatography using the method in Example 3. The catalytic effect of each mutant was compared by comparing the conversion rate of rutin in the catalytic reaction solution at 2 h, 4 h, 6 h, and 8 h. The results are shown in Table 9. Compared with the cyclodextrin glycosyltransferase CGTase, the conversion rates of the combined mutants were all improved. Among them, the triple mutant CGTase-Y218F / E291V / P232S performed best, achieving a product conversion rate of 88% after 8 h of reaction.

[0073] Table 9. Results of liquid phase detection of CGTase and its mutant engineered bacteria.

[0074] Wet cells of CGTase and its mutants were prepared using the method in Example 2. The catalytic results of the crude enzyme solution were analyzed by liquid chromatography using the method in Example 4. The catalytic effect of each mutant was compared by comparing the conversion rate of hesperidin in the catalytic reaction solution after 8 hours. Compared with the cyclodextrin glycosyltransferase CGTase, the combined mutants CGTase-Y218F / A122T, CGTase-A122T / L191I, CGTase-E291V / A122T, and CGTase-A122T / Y218F / E291V all showed improved catalytic conversion rates. Among them, the triple mutant CGTase-A122T / Y218F / E291V performed best, achieving a product conversion rate of 85.58% after 8 hours of reaction.

[0075] Example 7: Comparison of different complexation times of rutin and β-cyclodextrin Jiangnan University patent CN 118325991 A mentions that rutin and γ-cyclodextrin need to be complexed for 12 hours under conditions of 60℃ and 500 rpm shaking to construct the γ-cyclodextrin-rutin complex system. In this invention, under the catalysis of a mutant enzyme in Example 6, the glycosyl donor was changed to β-cyclodextrin, and the phosphate buffer was changed to pure water. Simultaneously, the complexation time and temperature were optimized. Using 12 hours of complexation at 60℃ and 500 rpm shaking as a control, different complexation times of 4 hours, 8 hours, and 12 hours were designed; three temperature gradients were set at 40℃, 50℃, and 60℃; and the shaking speeds were set at 300 rpm, 400 rpm, and 500 rpm.

[0076] The triple mutant CGTase-Y218F / E291V / P232S wet cells were prepared using the method in Example 2. The crude enzyme solution was used for catalysis using the method in Example 3. The catalytic results of the crude enzyme solution were analyzed by liquid chromatography using the method in Example 4. The catalytic effects of different complexation methods were compared by comparing the conversion rate of rutin in the catalytic reaction solution after 2 hours. The results are shown in Table 10. Complexation time, temperature, and rotation speed all affect the catalytic results. From an economic and environmental perspective, complexation at 50℃ and 400 rpm for 8 hours is preferred.

[0077] Table 10 Catalytic results under different complexation conditions

[0078] Example 8: High-density fermentation and crude enzyme solution preparation of CGTase-Y218F / E291V / P232S and CGTase-A122T / Y218F / E291V 1. Seed culture strain E. coli BL21(DE3) / pET28a-CGTase-Y218F / E291V / P232S、 E. coli Remove BL21(DE3) / pET28a-CGTase-A122T / Y218F / E291V from a -80 ℃ freezer. Streak the glycerol tube onto a slant containing 100 μg / mL kanamycin. Incubate at 37 ℃ for 13-15 h. Add 6 mL of sterile water to the incubated slant and scrape the bacterial culture from the slant into the sterile water using an inoculation loop. Inoculate 1% of the culture into LB medium and incubate at 37 ℃ and 220 rpm for 10-11 h. At this point, OD... 600 Around 5.0.

[0079] LB liquid medium consists of: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, distilled water as solvent, pH 7.0-7.5. LB solid medium is LB liquid medium with 20 g / L agar added.

[0080] 2. High-density fermentation Prepare 5 L of fermentation medium, as shown in Table 11. Prepare 1 L of fed-batch medium, as shown in Table 12. Sterilize both fermentation and fed-batch media at 121 °C for 30 min. After sterilization, when the temperature drops to 60 °C, add kanamycin at 100 μg / mL to the fermentation medium. When the temperature drops to 37 °C, inoculate the seed culture into the fermentation medium at a 2.0% inoculum rate (100 mL / 5 L) and incubate at 37 °C. Aeration should begin at 1 vvm and increase to 1.2 vvm after 3 h. Maintain tank pressure at 0.05–0.06 MPa. Stirring speed should be 500–700 rpm. Dissolved oxygen should be maintained at a minimum of 20%. Dissolved oxygen decreases rapidly after 2 h of fermentation and then rises rapidly after about 5 h; at this point, fed-batch medium should be added to maintain dissolved oxygen at approximately 30%. The pH may rise slowly in the early stages of fermentation, then decrease after feeding. The pH can then be adjusted with ammonia water, maintaining it at 6.7-6.9. After feeding begins, slowly lower the temperature to 30-33 °C for 1-2 hours. When OD... 600 When the temperature reaches 20-25°C, cool it down to 25°C. Add 0.2 mM IPTG and induce for 14-16 hours before removing from the container.

[0081] Table 11 Fermentation medium formulation (5 L)

[0082] Table 12 Feeding medium formulation (1 L)

[0083] 3. Preparation of cell wall-breaking enzyme solution Before transferring the fermentation broth to the fermentation tank, use the fermentation tank cooling system to lower the temperature to 10-15℃. Pass the fermentation broth through a ceramic membrane and wash it twice with tap water to ensure as much of the broth is washed away, concentrating it to a cell count of 200 g / L. The membrane-passing process generates heat, so the temperature needs to be lowered to below 20℃. The cooled bacterial suspension is then homogenized using a high-pressure homogenizer at 60-80 kg / m². The homogenization process will raise the temperature, which must not exceed 30℃. The homogenization cycle is repeated twice. Throughout the homogenization process, the pH of the enzyme solution must be controlled, adjusting it to approximately 7.0-7.2 using a sodium hydroxide solution. After homogenization, cool the solution to 0℃, dispense the enzyme solution, and freeze it in a cold storage for later use.

[0084] Example 9: Transglycosylation reaction and separation and extraction process of 20 g / L high concentration of rutin To further confirm the strain E. coli The activity of BL21(DE3) / pET28a-CGTase-Y218F / E291V / P232S was investigated, and the product rutin was isolated using a simple separation and extraction method. First, the method of Example 8 was used for high-density fermentation to produce the corresponding enzyme solution. Then, a 5 L catalytic reaction system was constructed, with 100 g of rutin and 1.5 kg of β-cyclodextrin complexed at 50 °C with high-speed spiral stirring for 8 h. Then, 125 mL of enzyme solution (equivalent to a crude enzyme concentration of 50 g / L) was added, and the pH was adjusted to 6.5 with NaHCO3, and the temperature was 40 °C. The method of Example 4 was used to further investigate the activity of rutin. E. coli The catalytic results of BL21(DE3) / pET28a-CGTase-Y218F / E291V / P232S were analyzed by real-time liquid phase detection. The reaction reached the threshold at about 8 h, and the conversion rate of glucosylrutin was 88%.

[0085] After the reaction was complete, industrial-grade saccharifying enzyme (final concentration 1.5 mL / L) was added, and the reaction was carried out at 50 ℃ for 15 min. The reaction was terminated when no β-cyclodextrin residue was detected in the reaction solution by high-performance liquid chromatography (HPLC). The reaction solution was directly passed through a ceramic membrane for enzyme removal, removal of bacterial / cell debris, colloids, large molecular weight proteins and pigments, undissolved solid particles, and other macromolecular substances. Glucose oxidase was then added to the permeate to oxidize the glucose from β-cyclodextrin to gluconic acid, removing excess sugars. The permeate was then passed through a ceramic membrane again to remove glucose oxidase. The permeate was then directly filtered through a nanofiltration membrane (molecular weight cutoff 300~500 Da) to retain the product glucosylrutin. Finally, the retentate was spray-dried to obtain a pale yellow glucosylrutin product with a purity of 85% as determined by HPLC. The results are as follows. Figure 11 As shown.

[0086] Example 10: Transglycosylation reaction and separation and extraction process of 15 g / L high concentration of hesperidin To further confirm the strain E. coli The activity of BL21(DE3) / pET28a-CGTase-A122T / Y218F / E291V was investigated, and the product hesperidin was isolated using a simple separation and extraction method. First, the method of Example 7 was used for high-density fermentation to produce the corresponding enzyme solution. Then, a 5 L catalytic reaction system was constructed, containing 100 g of hesperidin, 500 g of β-cyclodextrin, and 125 mL of enzyme solution (equivalent to a crude enzyme concentration of 50 g / L, added to the reaction solution in a fed-batch manner). The pH was maintained at 8.5 and the temperature at 40 °C throughout the process using NaOH. The method of Example 4 was then used to further investigate the activity of hesperidin. E. coliThe catalytic results of BL21(DE3) / pET28a-CGTase-A122T / Y218F / E291V were analyzed by real-time liquid chromatography. The reaction reached the threshold at about 8 h, and the conversion rate of glucosyl hesperidin was 85%.

[0087] After the reaction, the pH was adjusted to around 7.0 with acetic acid, and then industrial-grade saccharifying enzyme (final concentration 1.5 mL / L) was added. The reaction was carried out at 50℃ for 15 min. The reaction was terminated when no β-cyclodextrin residue was detected in the reaction solution by high-performance liquid chromatography (HPLC). The reaction solution was directly passed through a ceramic membrane for enzyme removal, removal of bacterial / cell debris, colloids, large molecular weight proteins and pigments, undissolved solid particles, and other macromolecular substances. Glucose oxidase was then added to the permeate to oxidize the glucose from β-cyclodextrin to gluconic acid, removing excess sugars. The permeate was then passed through a ceramic membrane again to remove glucose oxidase. The permeate was then directly filtered through a nanofiltration membrane (molecular weight cutoff 300-500 Da) to retain the product glucosyl hesperidin. Finally, the retentate was spray-dried to obtain a pale yellow glucosyl hesperidin product with a purity of 85% as determined by HPLC. The results are as follows. Figure 12 As shown.

[0088] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A cyclodextrin glycosyltransferase mutant, characterized in that, The mutant is a cyclodextrin glycosyltransferase parent with the amino acid sequence shown in SEQ ID No. 1, mutated by any of the following methods: (a) Replace glutamic acid at position 291 with isoleucine or valine; (b) Replace tyrosine at position 218 with phenylalanine; (c) Replace proline at position 232 with serine; (d) Replace arginine at position 277 with lysine; (e) Replace leucine at position 191 with isoleucine; (f) Replace glutamic acid at position 289 with valine; (g) Replace tyrosine at position 218 with phenylalanine, and replace alanine at position 122 with threonine; (h) Replace tyrosine at position 218 with phenylalanine, and replace glutamic acid at position 291 with valine; (i) Glutamic acid at position 291 is replaced with valine, and alanine at position 122 is replaced with threonine; (j) Replace tyrosine at position 218 with phenylalanine, and replace leucine at position 191 with isoleucine; (k) Replace tyrosine at position 218 with phenylalanine, glutamic acid at position 291 with valine, and proline at position 232 with serine; (l) Alanine at position 122 is replaced with threonine, tyrosine at position 218 is replaced with phenylalanine, and glutamic acid at position 291 is replaced with valine.

2. The gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. Expressing the mutant of claim 1, or containing the gene of claim 2, or transforming a host cell with the recombinant plasmid of claim 3.

5. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli E. coli Using BL21(DE3) as the host cell and plasmid pET-28a as the expression vector, the mutant described in claim 1 is expressed.

6. A method for biosynthesizing glucosylflavonoids, characterized in that, The method involves using the mutant described in claim 1 as a catalyst in a catalytic system containing flavonoids and β-cyclodextrin to synthesize glucosyl flavonoids, wherein the flavonoids are rutin or hesperidin.

7. The method according to claim 6, characterized in that, In the catalytic system, the catalyst is measured at 50 g / L as crude enzyme solution, the initial concentration of the flavonoids is 15-25 g / L, and the initial concentration of β-cyclodextrin is 223-372 g / L.

8. The use of the mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the host cell of claim 4, or the recombinant Escherichia coli of claim 5 in the preparation of glucosyl flavonoids or products containing glucosyl flavonoids, characterized in that, The glucosyl flavonoids are glucosylrutin or glucosyl hesperidin.

9. A method for improving the conversion rate of cyclodextrin glycosyltransferase products, characterized in that, The method involves performing one of the following mutations on the cyclodextrin glycosyltransferase parent amino acid sequence as shown in SEQ ID No. 1: (a) Replace glutamic acid at position 291 with isoleucine or valine; (b) Replace tyrosine at position 218 with phenylalanine; (c) Replace proline at position 232 with serine; (d) Replace arginine at position 277 with lysine; (e) Replace leucine at position 191 with isoleucine; (f) Replace glutamic acid at position 289 with valine; (g) Replace tyrosine at position 218 with phenylalanine, and replace alanine at position 122 with threonine; (h) Replace tyrosine at position 218 with phenylalanine, and replace glutamic acid at position 291 with valine; (i) Glutamic acid at position 291 is replaced with valine, and alanine at position 122 is replaced with threonine; (j) Replace tyrosine at position 218 with phenylalanine, and replace leucine at position 191 with isoleucine; (k) Replace tyrosine at position 218 with phenylalanine, glutamic acid at position 291 with valine, and proline at position 232 with serine; (l) Replace alanine at position 122 with threonine, tyrosine at position 218 with phenylalanine, and glutamic acid at position 291 with valine. The product is glucosylrutin or glucosylhesperidin.

Images