Heterologous expression of cyclodextrin glycosyltransferase from Bacillus stearothermophilus in Bacillus subtilis and its application in glycosyl rutin production

Abstract

In this paper, the cgt gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus stearothermophilus was cloned into pWB980 plasmid for extracellular expression in Bacillus subtilis SCK6. Through adding a six-histidine affinity tag fused to the C-terminus, the recombinant CGTase could be purified by nickel ion affinity chromatography, and its molecular weight was approximately 76 kDa on SDS-PAGE. Then, the enzymatic properties were determined, and results were as follows: the optimum temperature and pH were identified as 40 ℃ and pH 5.0, respectively. CGTase had good tolerance to metal ions of Mn²⁺, Ca²⁺, and Mg²⁺. The enzyme activity was activated by Na⁺, Al³⁺, Fe³⁺, and Ni⁺, and it was remarkably inhibited by Cu²⁺ and Zn²⁺. To improve the aqueous solubility of rutin, CGTase was used to catalyze the transglycosylation reaction, and the conversion rate could reach as high as 80.13% under optimal conditions. Furthermore, the reaction mixture was treated with glucoamylase and microporous adsorbent resin. The yield of glycosyl-rutin was 56.1%, and its purity was 74.3%, which further improved the value of the product.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03510-5.

Introduction

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19), which can catalyze hydrolysis, cyclization, disproportionation, and coupling reactions, is an important enzyme for the food and pharmaceutical industries (Pinheiro et al. 2017; Song et al. 2021). This enzyme has been studied in detail including its catalytic mechanism, nucleotide sequence, and three-dimensional structure (the UniProt acc. No.: P31797). As a member of the α-amylase family (glycosyl hydrolase family 13), CGTase can catalyze hydrolysis reaction of different substrates to produce oligosaccharides. The cyclization reaction, which is the most widely used catalytic reaction in industry today, can catalyze the acquisition of α-, β-, and γ-cyclodextrins (CDs) consisting of six, seven, or eight glucose units, respectively, from starch or starch derivates (Wang et al. 2017; Zuo et al. 2022). Disproportionation and coupling reactions can catalyze intermolecular transglycosylation reactions. The process alters the physicochemical properties of receiver molecules such as rutin, hesperidin, steviol-glycosides, and l-ascorbic acid by rupturing the glycosidic bonds of the donor molecule and transferring glucose groups. (Mitsuzumi et al. 2011; Guo et al. 2019).

CGTase is mainly derived from microbial strains such as Bacillus, Actinobacteria, Aspergillus, and Brevibacterium (Lim et al. 2021). In general, CGTase, which is widely used in industry, comes mainly from Bacillus stearothermophilus, Bacillus subtilis, and Bacillus megaterium (Biwer et al. 2015; Tao et al. 2020). CGTase from different sources mainly use starch as substrate to catalyze the production of three different ratios of mixtures, which are classified into α-CD, β-CD, and γ-CD depending on the number of sugar groups they contain (Liu et al. 2022; Saini et al. 2022). For example, the CGTase from G.stearothermpohilus preferentially catalyzes the formation of β-CD (Leemhuis et al. 2010). Ibrahim et al. investigated the effect of the reaction conditions on the production of cyclodextrins using CGTase from B. agaradhaerens KSU-A11 (Ibrahim et al. 2011). As the reaction proceeded, β-CD was the major product and γ-CD started to be synthesized, where the CDs ratios were 0.27:0.65:0.07 for α-CD:β-CD:γ-CD, respectively. Compared with screening CGTase producing strains from nature, selecting a suitable host for the recombinant expression of CGTase and increasing production with genetic engineering technology are more promising. B. subtilis is generally assumed as an effective biological vehicle for extracellular expression of exogenous proteins with numerous valuable features such as powerful protein secretion ability, high safety, and fast growth rate (Su et al. 2021). Gimenez et al. proved that the strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, and its enzymatic activity was 16-fold higher than that of the wild strain (Gimenez et al. 2019). However, direct transformation of exogenous genes in B. subtilis was inefficient, and B. subtilis was more receptive to multimeric plasmid DNA than monomers (Canosi et al. 1978). Subsequently, Zhang et al. constructed a simple, rapid, and efficient transformation system by combining supercompetent cells and plasmid multimers. The specific method was to prepare plasmid multimers by prolonged overlap extension-PCR (POE-PCR) and then transform them into B. subtilis supercompetent cells (Zhang et al. 2011).

Rutin is a 3-O-rutinoside of quercetin, also called rutinoside and purple quercetin. Rutin is one of the main components of vitamin P that has diverse pharmacological activities (Aditya et al. 2017; Calzada et al. 2018; Li et al. 2021). However, rutin is almost insoluble in water, resulting in poor bioavailability and a very limited application (Mauludin et al. 2009; Slámová et al. 2018). To improve this physicochemical property, researchers added the glucose group to rutin using CGTase to synthesize transglycosylated rutin (G1-Rutin), which increased the solubility approximately 30,000 times greater than that of rutin (Suzuki et al. 1991). Glycosyl-rutin acts as a powerful antioxidant with excellent free radical scavenging ability and it has been widely used in food, medicine, and cosmetic fields. Hashizume et al. indicated that the consumption of monoglucosyl rutin (200 and 400 mg/day) for 8 weeks reduced abdominal visceral fat (Hashizume et al. 2020). Nagasawa et al. demonstrated for the first time that dietary G-rutin consumption can provide potential health benefits related to the inhibition of tissue glycation reactions common to diabetes (Nagasawa et al. 2003).

In recent years, the enzymatic transglycosylation of rutin has become an effective tool (Aoki et al. 2017; Moon et al. 2018). The reaction products are mostly mixtures of different oligosyl-rutins (Gn-rutins with more than one glycosyl residue, where n represents the number of glycosyl residues); however, little research has been reported on the production of glycosyl-rutin (G1-rutin, in this paper containing one glycosyl residue). Sun et al. reported the transglycosylation of rutin using the novel enzyme produced by Bacillus sp. SK13.002. The conversion rate of rutin could reach 65.7% under optimized enzymatic reaction conditions, with the reaction mixture consisting of mono-, di-, tri-, tetra-, and penta-glycosylated rutins (Sun et al. 2013). Compared with Sun, González-Alfonso et al. added amyloglucosidase STA1 to the reaction mixture catalyzed by commercial CGTase enzymes from Geobacillus sp. and Bacillus macerans, which enhanced the yield of the monoglucosylation product by a factor of two (González-Alfonso et al. 2021).

In this paper, the heterologous expression of CGTase from B. stearothermophilus in B. subtilis was successfully achieved. As shown in Scheme 1, a two-step enzymatic process for the production of glycosyl-rutin was established via transglycosylation and deglycosylation, laying a foundation for the production of glycosyl-rutin.

Scheme 1.

Two-step enzymatic reaction for the synthesis of glycosyl-rutin

Materials and methods

Strains and plasmids

Cgt gene (GenBank: X59042.1), originating from B. stearothermophilus, was found in the NCBI database, synthesized by Tsingke Biotechnology Co., Ltd. (Hangzhou, China), and cloned into E. coli TOP10 (Fujiwara et al. 1992). Plasmid pWB980 was used as the expression vector of the cgt gene, and the host strain B. subtilis SCK6 was stored in the laboratory. B. subtilis strain SCK6 can be obtained from the Bacillus Genetic Stock Center (http://www.bgsc.org).

Construction of multimeric plasmids

The strain E. coli TOP10 pET-28α-cgt and the empty vector pWB980 were used as templates to amplify the cgt gene and linear vector fragment containing 3' and 5' overlapping termini, respectively. His-tag was added at the C-terminus of CGTase via primers CGTase-F (5′-gcgcaactcaagcttttgccGCTGGAAATCTTAATAAGGTAAACTTTAC-3′) and CGTase-R (5′-catgcctgcaggtcgactctTTAGTGGTGGTGGTGGTGGTGGTTCTGCCAATCCACTATAATTTTTC-3′). The lowercase letter in the primers represents homologous arm sequences. Then, QuickCut^TMDpnI (Takara Biomedical Technology Co., Ltd. Beijing, China) was applied to the PCR products and kept at 37 °C for 2 h. On the basis of pure PCR products serving as primers and templates for each other, POE-PCR (Fig. 1) was executed using PrimeSTAR®Max DNA Polymerase (Takara Biomedical Technology Co., Ltd. Beijing, China). The amplification protocol included denaturation at 98 °C for 5 min, followed by 30 cycles of 98 °C for 10 s, 55 °C for 5 s, and 72 °C for 65 s, with a final extension at 72 °C for 5 min. Subsequently, multimeric plasmid PWB980-cgt was amplified.

Fig. 1.

Schematic diagram of DNA polymer preparation by POE-PCR

Expression of recombinant CGTase in B. subtilis

The B. subtilis SCK6 strain was inoculated into shake flasks containing 50 ml of LB medium (1 μg/mL erythromycin) and cultivated at 37 °C for 8–12 h with shaking at 200 rpm. The seed solution was then obtained and diluted to OD₆₀₀ = 1.0 with fresh LB medium containing 2% (w/v) xylose. Supercompetent B. subtilis SCK6 cells were prepared by incubating at 200 rpm for another 2 h. POE-PCR products were mixed with supercompetent B. subtilis SCK6 cells and cultured together at 37 ℃ for 1.5 h. The transformed competent cells were then spread on kanamycin and erythromycin LB agar plates for further characterization and DNA sequencing.

After validation, a single colony was selected and inoculated into 250 mL shake flasks containing 50 mL LB medium (1 μg/mL erythromycin and 50 μg/mL kanamycin). The seed solution (obtained by incubating at 37 °C for 8–12 h) was transferred to shake flasks with 40 mL of terrific broth medium at 4% inoculum and fermented for 96 h at 200 rpm. The fermentation medium contained 5 g/L maltodextrin of DE 15–20, 12 g/L of peptone, 24 g/L of yeast powder, 2.31 g/L of KH₂PO₄, 16.43 g/L of K₂HPO₄, and 2 g/L of NaCl.

Purification and SDS-PAGE

The culture broth produced by shaking flask fermentation was centrifuged at 8000 rpm (5724 × g, Sigma 1–16 K, Sigma Laborzentrifugen GmbH) and 4 °C for 10 min. The supernatant was concentrated by ultrafiltration membrane (with a molecular weight of 10 kDa) to obtain the crude enzyme solution. Then, the recombinant CGTase was purified by Ni²⁺-NTA resin column. Concentrations of 5, 100, 150, 200, and 500 mM of imidazole were set for elution, and the eluate was picked up with a pre-cooled centrifuge tube when a protein peak appeared. Each concentration was eluted until the UV baseline no longer changed. Finally, 12% (w/v) SDS-PAGE was used to examine recombinant proteins.

Characterization of purified CGTase

The cyclization activity of CGTase was determined based on the discoloration of phenolphthalein solution at 550 nm and measured by spectrophotometric analysis of the concentration of β-CD. First, 1.0 mL of 2% soluble starch solution was taken and preheated in 40℃ for 10 min. Then, 0.2 mL of crude enzyme solution was added and mixed well. After 10 min, 1.8 mL of NaOH solution (0.03 mol/L) was added to terminate the reaction, followed by 1 mL of 0.02% phenolphthalein. In addition, the absorbance was measured with distilled water as a blank and no fermentation solution as a control. One unit of enzyme activity corresponded to the amount of CGTase that produced 1 mM β-CD/min under the above enzyme-catalyzed conditions.

The optimal temperature for CGTase was obtained by recording the enzyme activity at temperatures between 20 and 80 °C. The effect of temperatures on CGTase stability was investigated by catalyzing the cyclization of soluble starch at 40 °C, 50 °C, 60 °C, 70 °C for 2 h, with samples taken every 30 min to determine the enzyme activity. The relative enzyme activity was defined as 100% at the optimum temperature for 0 h. The optimum pH of recombinant CGTase was determined at 40 °C using 0.1 M citric acid buffer and 0.2 M dibasic sodium phosphate buffer with pH values ranging from 3.0 to 8.0.

To evaluate the influence of organic solvents on CGTase activity, soluble starch was used as substrate to determine the CGTase activity after the addition of different organic solvents with a volume ratio of 10% (ethyl acetate, n-hexane, tert-butanol, ethanol, methanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, and acetone). The effect of metal ions on CGTase activity was studied in 4 mL of the reaction solution system supplemented with a final concentration of 10 mM metal ion sulfates (Mn²⁺, Ca²⁺,Mg²⁺, Cu²⁺, and Fe²⁺) and chloride (Na⁺, Al³⁺, Ni⁺, and Zn²⁺). The activity was measured without the addition of organic solvents or metal ions as a control (100%).

High-performance liquid chromatography method

The detection of peaks was carried out using Waters 2695 series HPLC system with ultraviolet–visible detector (Waters 2489). Chromatographic column: Welch Materials, Inc. BX-C18 (4.6 mm × 250 mm, 5 μm); mobile phase: mobile phase A was 100% acetonitrile (V/V) and mobile phase B was 0.02% phosphoric acid (V/V), The detection method was based on a gradient elution: 13–20% A, 0–12 min; 20–50% A, 12–15 min; 50–80% A, 15–16 min; 80% A, 16–18 min, and 80–13% A, 18–24 min. The flow rate was 0.8 mL/min with an injection volume of 10 μL, the detection wave length was 254 nm, and the operating temperature was 30 °C.

Conditions of enzymatic transglycosylation reaction

The fermentation broth of recombinant CGTase was concentrated (10 kDa) to obtain the crude enzyme solution, which was subjected to glycosylation reaction containing 2% rutin. The reaction solution was diluted 50 times and filtered through a 0.22 μm microporous membrane for high-performance liquid chromatography (HPLC). The conversion of rutin (%) = glycosylated rutin/(glycosylated rutin + residual rutin) × 100.

Transglycosylation of rutin: a reaction mixture (ca. 1 mL), including crude enzyme solution after concentration (100 μL), 0.1 g of maltodextrin (pre-dissolved in 400 μL of citric acid–disodium hydrogen phosphate buffer with different pH values of 4.0–8.0), and 0.02 g of rutin (pre-dissolved in 500 μL of methanol) was incubated at different temperatures (30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 ºC) for different durations (6, 12, 18, 24, 30, 36, and 42 h). Furthermore, various organic solvents (ethanol, acetone, methanol, and dimethyl sulfoxide), methanol addition (5%-50%), glycosyl donors (glucose, maltose, sucrose, β-CD, maltodextrin, and soluble starch), and substrate mass ratios (maltodextrin /rutin) were tested to determine the best conditions for the production of transglycosylated rutins (G-Rutins). After the incubation, samples were boiled for 5 min to inactivate the enzyme and then filtered for carrying out HPLC.

Synthesis of glycosyl-rutin

Based on the above transglycosylation reaction solution, the product glycosy-rutin (G1-rutin) was obtained by adding 1000 U/mL of glucoamylase at 25 °C for 30 min at 1000 rpm. The glucoamylase was purchased from Shandong Longkete Enzyme Preparation Co., Ltd. After the reaction solution was boiled in water to inactivate the enzyme, it was diluted 50 times and HPLC was performed to determine the results.

Purification and structural characterization

The glycosyl-rutin reaction solution and an equivalent volume of ddH₂O were applied to the macroporous resin B57503 (purchased from Suzhou Bojie Resin technology co., ltd.) for the reaction. Macroporous resins are a type of polymeric adsorbent resins with a large pore structure, but no exchange groups, which can selectively adsorb organic substances from solution. The resin was removed after 5 h of adsorption at 40 °C and 550 rpm. The sugars on the surface of the resin were first washed with ddH₂O, and then the impurities in the reaction solution were removed with 10% ethanol. Next, the products adsorbed in the resin were eluted with 80% ethanol, which was then removed by a rotary evaporator. Finally, the product glycosyl-rutin with high purity was obtained by lyophilization. Then, 3 mg of the lyophilized product was added to 1 mL of 20% methanol solution, and its structure was identified by LC–MS. The molecular weight of purified glycosyl-rutin was assessed using LTQ Orbitrap XL ETD mass spectrometer (Thermo Scientific, Bremen, Germany). Samples were analyzed by direct infusion and ionized by MALDI in positive reflector mode.

Results and discussion

Construction of plasmid multimer

The PCR products were verified by 1.0% agarose gel electrophoresis. In Fig. 2, clear gene bands could be seen under UV light, and the apparent molecular mass of cgt gene and carrier pWB980 were in accordance with their theoretical molecular mass 2060 base pairs (bp) and 3787 bp. The recombinant plasmid was transformed into B. subtilis SCK6. Then, DNA sequencing (Tsingke Biotechnology Co., Ltd. Hangzhou, China) confirmed that the target gene was successfully inserted into the plasmid (the sequencing results shown in Supplementary material).

Fig. 2.

Validation electropherogram of PCR products. Lane M: 2 K plus II marker; Lane 1: cgt gene; Lane 2: carrier pWB980; Lane 3 and Lane 4: multimer plasmid pWB980-cgt

SDS-PAGE analysis of purified CGTase

The amount of the recombinant CGTase in the fermentation culture was 1.27 mg/mL. The recombinant CGTase can be refined by Ni²⁺-NTA and eluted at 150 mM imidazole concentration when gradient elution was performed. After a 27-fold purification process, the specific activity of CGTase was 11.30 U/mg. Figure 3 (Lane 2) shows that the band associated with the crude extracts of the CGTase appeared as a much more pronounced line for recombinant B. subtilis PWB980. The purified enzyme appeared as a single, clear band on SDS-PAGE (lane 3) with a molecular mass of approximately 76 kDa, which was in general agreement with the theoretical molecular mass 78.9 kDa.

Fig. 3.

SDS-PAGE analyses of the recombinant CGTase. Lane 0: molecular weight markers; Lane 1: the fermentation broth sample without the target gene introduced; Lane 2: the fermentation broth sample with the target gene introduced; Lane 3: sample after nickel ion affinity chromatography

Biochemical properties of recombinant CGTase

According to Fig. 4A, the enzyme performed best at a temperature of 40 °C, whereas the CGTase still showed about 60% activity at 70 °C. The residual enzyme activity after 2 h of reaction under different temperatures was selected to examine the heat resistance of CGTase, because the enzyme activity was affected closely by temperatures and action time. The enzyme activity kept more than 90% of the initial enzyme activity between 40 and 50℃. The recombinant enzyme still exhibited beyond 60% enzyme activity when acted at 70 °C for 2 h (Fig. 4B), indicating that the enzyme had good heat resistance.

Fig. 4.

Characterization of recombinant CGTase. A Optimum temperature, B temperature stability, C optimum pH, D metal ions, and E organic solvents on the activity of CGTase

The recombinant CGTase retained high levels of activity at a pH range of 4.0–7.0 with an optimum temperature of 40℃ (Fig. 4C). The pH stability of recombinant CGTase is shown in Figure S1. The highest enzyme activity was obtained at a pH of 5.0, which was consistent with many reports that CGTase showed high enzyme activity at an acidic pH condition. The detection of metal ions showed that CGTase has good tolerance to metal ions of Mn²⁺, Ca²⁺, and Mg²⁺. Among the ten types of metal ions, Na⁺, Al³⁺, Fe³⁺, and Ni⁺ have some activation effect on CGTase; however, Cu²⁺ has a substantial inhibitory effect on CGTase (Fig. 4D). Cu²⁺ is a heavy metal ion that may bind to the essential group of the enzyme and affect the active center of the enzyme. CGTase has good organic solvent tolerance (10%, V/V); ethyl acetate and n-hexane have an activating effect on the enzymatic activity of CGTase (Fig. 4E).

Biosynthesis of glycosyl-rutin

Given the importance of pH in enzyme-catalyzed reactions, the effect of pH on the transglycosylation reaction of rutin at 40 °C was discussed and is illustrated in Fig. 5A. The optimum pH of the recombinant CGTase was 6.0, at which rutin conversion reached 62.16%. This enzyme had strong activity at pH 5.0–6.5. Figure 5B displays the effect of temperatures on CGTase at an optimum pH of 6.0. Compared with the previous study by Sun et al., where the optimum temperature was 35 °C, the CGTase obtained in our experiment had considerable high temperature tolerance. The optimum temperature of CGTase-catalyzed reaction was 50 ℃, and a high catalytic activity between 40 and 60 ℃ was maintained.

Fig. 5.

Effect of the A pH, B temperature, reaction mixtures containing crude enzyme solution after concentration (100μL), 0.1 g maltodextrin (pre-dissolved in 400μL citric acid–Na₂HPO₄ buffer, 0.02 g rutin (pre-dissolved in 500μL methanol), C organic solvent, D methanol addition, E glycosyl donors, F maltodextrin/rutin (pH6.0, 50℃,1000 rpm,24 h), and G reaction time on the glycosylation of rutin catalyzed by CGTase

Rutin’s insolubility in water limits its industrial application and prevents it from being modified by enzymatic catalysis. It is clear that the addition of organic solvents greatly enhanced the solubility of rutin. However, results presented in Fig. 4E indicate that methanol (10%, V/V) has a 20% inhibitory effect on CGTase activity. With the increase of organic solvent addition, the initial reaction concentration of rutin increased, while the CGTase activity was probably completely inhibited. To test this hypothesis, the effect of different organic solvents (50%, V/V) on CGTase transglycosylation was examined. As shown in Fig. 5C, the addition of methanol and DMSO made a remarkable improvement in the conversion of rutin. Although DMSO is considered the universal solvent, its safety is low. Compared with this, methanol is volatile and can be easily removed from the reaction system. Furthermore, the effect of different methanol additions on the conversion rate of rutin also needs to be investigated. As a result, the maximum conversion of transglycosylation was 72.71% when the addition of methanol in the reaction system was 40% (Fig. 5D). Previous studies found that with 50% (v/v) methanol, the residual CGTase hydrolysis and cyclization activities dropped to 54.6 and 52.2% with 50% (v/v) methanol, respectively (Sun et al 2011). Therefore, the enzymatic process of rutin transglycosylation must last for many hours.

CGTase is mainly used to catalyze the production of CDs from starch in industry. This paper investigated the impact of various glycosyl donors on the transglycosylation reaction, and the results showed that soluble starch and β-CD were adopted as glycosyl donors with 20.9% and 42.3% conversion of rutin, respectively. By contrast, the conversion of rutin was as high as 72.7% when maltodextrin was used as the glycosyl donor (Fig. 5E). The increase of glycosyl donor ratio was beneficial to improving the product conversion rate. Considering the subsequent purification steps for sugar removal, the optimal mass ratio of rutin to maltodextrin substrate was determined to be 1:5 (Fig. 5F). In the first 6 h, the reaction rate was the fastest. With increasing time, the catalytic reaction rate decreased, and the rate slowed down substantially after 12 h. The reaction proceeded until 36 h, when the conversion of rutin reached the highest rate of 80.13%, and the reaction reached equilibrium (Fig. 5G).

Glucoamylase catalyzed the hydrolysis of glycosyl-rutin

HPLC analyses of enzymatic products before and after the reaction catalyzed by glucoamylase are shown in Figure S2. The HPLC method can be used to separate and obtain transglycosylated rutins effectively from rutin. The retention time of rutin and glycosyl-rutin (G1-rutin) were 17.040 and 16.657 min, respectively. The other products oligosyl-rutins (G2-rutin, G3-rutin, G4-rutin, and G5-rutin) were detected at the times of 15.840, 14.980, 14.212, and 13.409 min, respectively. It can be clearly seen from Figure S2 that the addition of glucoamylase dramatically catalyzed the conversion of oligosaccharide rutin to glycosyl rutin, and the yield of G1-rutin increased gradually.

Purification and structural characterization of enzyme-catalyzed products

Then, macroporous resin B57503 was used for adsorption and purification to obtain the product glycosyl-rutin. The yield of the product was 56.1%, and the purity was 74.3%. Thus, each released peak was analyzed using liquid chromatography/mass spectrometry to verify that the released peaks were indeed the correct G-rutins. The results of LC–MS analysis are shown in Figure S3. The relative molecular weight of rutin was 609.46, and the relative molecular weight of the product glycosyl-rutin (G1-rutin) was 771.47, indicating that the product was glycosyl-rutin.

Conclusions

In this paper, CGTase derived from B. stearothermophilus was successfully transferred into B. subtilis and achieved efficient extracellular expression. Recombinant CGTase catalyzed the production of transglycosylated rutin, and the effects on different conditions were studied. Then, the glucoamylase converted oligosaccharide rutin to glycosyl-rutin. Glycosyl-rutin was adsorbed by macroporous resin B57503 with a final yield of 56.1% and a purity of 74.3%. In summary, the purified product glycosyl-rutin further improved the value of rutin.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization: JZ and XL; data curation: WS and MZ; funding acquisition: JZ and XL; investigation: WS, MZ and YZ; supervision: JZ and XL; writing original draft: WS and MZ.

Data availability

The data are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest in this work.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

The authors confirm that the manuscript has been read and approved by all named authors. The authors further confirm that the order of authors listed in the manuscript has been approved by all of us.