A beta-1, 6-glycosidic bond hydrolytic enzyme and application thereof in traditional Chinese medicine resources

Abstract

The application discloses a beta-1, 6-glycosidic bond hydrolytic enzyme and application thereof in traditional Chinese medicine resources. The method screens a key gene sequence with a function of hydrolyzing beta-1, 6-glycosidic bond from a Candida parapsilosis L7 genome, and names the key gene sequence as CoExg1. A gene engineering bacterium capable of efficiently heterologously expressing CoExg1 is constructed by using a molecular cloning technology. After fermentation and enzyme production, an enzyme catalytic reaction system is established, and a traditional Chinese medicine active molecule in mogroside V is directionally converted, beta-1, 6-glycosidic bonds in different regions of a substrate structure are selectively hydrolyzed, and therefore, efficient and phased synthesis of different products such as sianoside I and mogroside IIIE is realized. In addition, CoExg1 can also hydrolyze beta-glycosidic bonds in the structures of icariin and lupeol, and directionally synthesize icariin II and lupeol, thereby providing important technical support for high-value utilization of traditional Chinese medicine resources and quality improvement and efficiency enhancement of traditional Chinese medicine glycosides.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to the gene sequence mining, heterologous expression, and application of a β-1,6-glycosidic bond hydrolase in traditional Chinese medicine resources. In particular, it relates to the enzyme-catalyzed directed and phased synthesis of mogroside V into sarcosine I and mogroside IIIE, the catalytic synthesis of icariin from icariin into icariin II, and the catalytic synthesis of styracin from styracin. Background Technology

[0002] Glycosides are an important class of active ingredients in traditional Chinese medicine, widely found in plants, animals, and microorganisms. These compounds are formed by the condensation of one or more sugar groups with a non-sugar moiety via hydroxyl groups on the sugar group. The varying numbers and bond types of glucose groups attached to the molecular backbone result in a rich diversity of glycosides with the same parent nucleus. [1] Furthermore, the different chemical structures of these glycosides lead to variations in their physicochemical properties and physiological activities, each possessing its own unique characteristics. For example, mogroside V exhibits neuroprotective effects, symmonoside I has the highest sweetness, and mogroside IIIE exhibits anti-pulmonary fibrosis activity. However, during plant metabolism, the glycoside with the largest cumulative content within the same parent nucleus is usually the one linked to the largest number of sugar groups (e.g., mogroside V). [2] Other glycosides (such as symmancoside I and mogroside IIIE) are present in trace amounts, making it difficult to achieve high purity and enrichment through traditional physical extraction methods alone, which greatly limits their application. [3 , 4] .

[0003] β-glucosidase is a hydrolase capable of breaking down β-D-glucosidic bonds. Belonging to the cellulase class, its main function is to catalyze the decomposition of β-glucosides, and it has important applications in food processing and pharmaceutical research. Obtaining other glycosides from the same parent nucleus by hydrolyzing naturally abundant polysaccharide glycosides using β-glucosidase is a potentially efficient pathway. However, β-glucosidase typically hydrolyzes glycosidic bonds with specific bonding patterns (e.g., β-1,6-glycosidic, β-1,2-glycosidic, or β-1,3-glycosidic bonds) but lacks the ability to selectively hydrolyze glycosidic bonds with the same bonding pattern at different structural sites (i.e., regioselectivity). It is evident that compared to obtaining mogroside IIIE by completely hydrolyzing the two β-1,6-glycosidic bonds in the structure of mogroside V using β-glucosidase, obtaining sermonoside I by selectively hydrolyzing / preferentially hydrolyzing the β-1,6-glycosidic bond at the C3 site (excluding / later hydrolyzing the β-1,6-glycosidic bond at the C24 site) requires a higher degree of regioselectivity in the key enzyme and is more challenging. Therefore, exploring and finding novel β-glucosidases with highly regioselective hydrolysis capabilities to efficiently and selectively hydrolyze glycosidic bonds at specific sites is an important method for achieving precise acquisition of key target glycosides, and provides new technical support for the high-value utilization of traditional Chinese medicine resources and the improvement of the quality and efficiency of active molecules in traditional Chinese medicine.

[0004] References

[0005] [1] Tan Jiancheng, Zeng Sien. Research and progress on the medicinal functions of monk fruit. China Continuing Medical Education, 2019, 11(13):159-161.

[0006] [2] Tan Jiazhong, Liao Na, Zhang Baotang, Fan Bin. Development, application and prospect of natural sweeteners. China Food Additives, 2022, 33: 32-39.

[0007] [3] Zhang Xing. Research progress on extraction, separation and detection technology of mogrosides. Anhui Chemical Industry, 2017, 43: 4-6.

[0008] [4] Mou Junfei, Wang Shaoxu, Luo Qin, Wang Pengcheng, Huang Sixin, Su Xiaojian, Chen Xu, Liang Chengqin, Zhou Xianli. Determination of six mogrosides in monk fruit by HPLC-MS. Food Research and Development, 2018, 39: 139-144. Summary of the Invention

[0009] This invention addresses the deficiency of key enzymes in the targeted biosynthesis of active molecules in traditional Chinese medicine (TCM) due to poor selective hydrolysis. It provides a gene sequence mining, heterologous expression, and application of a β-1,6-glycosidic bond hydrolase in TCM resources. Using bioinformatics analysis, a novel gene sequence, CoExg1, capable of hydrolyzing β-1,6-glycosidic bonds was obtained through targeted screening from the L7 genome of *Candida glabrata*. CoExg1 was heterologously expressed in genetically engineered bacteria. Utilizing the highly efficient regioselective hydrolysis function of CoExg1, priority hydrolysis of β-1,6-glycosidic bonds in different regions of the active TCM molecule structure is achieved, thereby enabling the efficient synthesis of the target product and the phased synthesis of other products.

[0010] To address the above problems, the present invention provides the following solution: a β-1,6-glycosidic bond hydrolase and its application in traditional Chinese medicine resources, characterized in that:

[0011] Using bioinformatics analysis, a gene sequence capable of hydrolyzing β-1,6-glycosidic bonds was obtained from the genome of *Candida orthopsilosis* L7 and named CoExg1. Using molecular cloning technology, a genetically engineered bacterium heterologously expressing CoExg1 was obtained. After inducing enzyme production, crude or pure enzyme solutions were used for catalytic reactions to achieve the targeted transformation and preparation of active molecules from traditional Chinese medicine. The *Candida orthopsilosis* L7 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, on April 9, 2025, with accession number CCTCC NO: M 2025731. Patent application number: 202510494063.5.

[0012] The aforementioned β-1,6-glycosidic bond hydrolase CoExg1 possesses regioselective hydrolysis capabilities, enabling priority sequential hydrolysis of β-1,6-glycosidic bonds in different regions of the substrate structure. This allows for the phased synthesis of different products, simplifying the separation and preparation processes of different products (with the same parent nucleus).

[0013] The genetically engineered bacteria mentioned above use Pichia pastoris and Escherichia coli as host cells.

[0014] The active molecules of the traditional Chinese medicine mentioned are: mogroside V, icariin, and styracin.

[0015] The crude enzyme solution catalytic reaction steps are as follows:

[0016] 2 g / L CoExg1 crude enzyme solution, 20 g / L mogroside extract (mogroside V content ≥50%, Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., batch number: DST230220-025) and 50 mM phosphate buffer (pH 6.0) were reacted at 200 r / min and 30℃ for 4 h. UPLC analysis showed that the yield of symmonoside I reached the highest level of 86.5%, with a yield of 7.5 g / L. After 12 h of reaction, symmonoside I was almost completely hydrolyzed to mogroside IIIE, with a yield as high as 95.4% and a yield of 7.1 g / L.

[0017] The steps of the pure enzyme catalytic reaction are as follows:

[0018] 0.1 g / L CoExg1 purified enzyme, 2 g / L mogroside extract (mogroside V content ≥50%), and 50 mM phosphate buffer (pH 6.0) were reacted at 200 r / min and 30℃ for 2.5 h. UPLC analysis showed that the yield of symmancoside I reached the highest level of 88.2%. After 10 h of reaction, symmancoside I was almost completely hydrolyzed to mogroside IIIE, with a yield as high as 98.1%.

[0019] 5 μM CoExg1 purified enzyme, 0.25 mM icariin or gentianin, 5% DMSO (v / v), 50 mM phosphate buffer pH 6.0, reaction at 200 rpm and 30℃ for 12 h. UPLC analysis showed that CoExg1 hydrolyzed icariin to produce icariin II with a conversion rate as high as 97.4%; and hydrolyzed gentianin to produce gentianin with a conversion rate of over 98%.

[0020] Beneficial effects

[0021] 1. This invention provides a β-1,6-glycosidic bond hydrolase CoExg1, which can solve the problem that in the process of microbial directed biosynthesis of active molecules of traditional Chinese medicine, due to the large number and complexity of enzyme systems produced and the poor selectivity of key enzymes, all β-1,6-glycosidic bonds of the substrate are over-hydrolyzed, and ultimately the intermediate target product cannot be obtained in large quantities and accurately.

[0022] 2. This invention provides a β-1,6-glycosidic bond hydrolase CoExg1, which possesses highly regioselective hydrolysis characteristics that can preferentially hydrolyze the β-1,6-glycosidic bond in the glucose branch chain attached to the C3 hydroxyl group of the mogroside V structure (after almost complete hydrolysis, it then begins to hydrolyze the β-1,6-glycosidic bond in the glucose branch chain attached to the C24 hydroxyl group), thereby achieving efficient staged synthesis of the intermediate target product sermonoside I and the final product mogroside IIIE.

[0023] 3. This invention provides a β-1,6-glycosidic bond hydrolase CoExg1, which can directionally hydrolyze the β-glycosidic bond attached to the C7 hydroxyl group of the parent nucleus of icariin / mangosteenin, thereby removing one molecule of glucose and achieving efficient synthesis of icariin II / mangosteenin.

[0024] 4. This invention provides a genetically engineered bacterium that efficiently heterologously expresses the β-1,6-glycosidic bond hydrolase CoExg1.

[0025] 5. The method for large-scale preparation of siamenoside I and mogroside IIIE using crude enzyme solution provided by this invention: 2 g / L CoExg1 crude enzyme solution, 20 g / L mogroside extract (mogroside V content ≥50%), and 50 mM phosphate buffer (pH 6.0) were reacted at 200 r / min and 30℃ for 4 h. UPLC detection showed that the yield of siamenoside I reached a maximum of 86.5%, with a yield of 7.5 g / L. After 12 h of reaction, siamenoside I was almost completely hydrolyzed to mogroside IIIE, with a yield as high as 95.4% and a yield of 7.1 g / L. (Prior art article: Dekkera bruxellensis, a beer yeast that specifically bioconverts smogroside extracts into the intense natural sweetener siamenoside I, Figure 4 a. After 4 hours of catalytic reaction with crude enzyme solution, the yield of sympathoside I is less than 20%; after 8 hours of reaction, the yield of sympathoside I is less than 70%. The method of the present invention has more obvious staged hydrolysis characteristics and higher space-time yield of sympathoside I, and is innovative.

[0026] 6. The method for the staged synthesis of siamenoside I and mogroside IIIE using pure enzymes provided by this invention: 0.1 g / L CoExg1 pure enzyme, 2 g / L mogroside extract (mogroside V content ≥50%), and 50 mM phosphate buffer (pH 6.0) were reacted at 200 r / min and 30℃ for 2.5 h. UPLC analysis showed that the yield of siamenoside I reached a maximum of 88.2%; after 10 h of reaction, siamenoside I was almost completely hydrolyzed to mogroside IIIE, with a yield as high as 98.1%. (Prior art article: Dekkerabruxellensis, a beer yeast that specifically bioconverts mogroside extracts into the intense natural sweetener siamenoside I, Figure 4b. After 4 hours of pure enzyme-catalyzed reaction, the yield of sympathoside I is about 10%; after 10 hours of reaction, the yield of sympathoside I is about 30%. The method of the present invention has more obvious staged hydrolysis characteristics and higher space-time yield of sympathoside I, and is innovative.

[0027] 7. The method for synthesizing icariin II and gentianin by pure enzyme catalysis provided by the present invention is as follows: 5 μM CoExg1 pure enzyme, 0.25 mM icariin or gentianin, 5% DMSO (v / v), 50 mM phosphate buffer pH=6.0, reaction at 200 rpm and 30℃ for 12 h. UPLC detection showed that the hydrolysis of icariin by CoExg1 to generate icariin II had a conversion rate as high as 97.4%; the hydrolysis of gentianin to generate gentianin had a conversion rate as high as 98% or more.

[0028] 8. Existing patent application number 202510494063.5 only reported the directed synthesis of sarcodactyl I from mogroside V via fermentation of Candida glabrata L7, but it did not possess the ability to further convert it into mogroside IIIE. The present invention provides a β-1,6-glycosidic bond hydrolase encoding gene, CoExg1, which can functionally achieve staged hydrolysis of β-1,6-glycosidic bonds, and can achieve the preparation of sarcodactyl I and mogroside IIIE at different time points in the same fermentation system, demonstrating significant innovation. Attached Figure Description

[0029] Figure 1 Recombinant plasmid maps of β-1,6-glycosidic bond hydrolase CoExg1 gene amplification and heterologous expression: a: CoExg1 gene amplification product; b: CoExg1-pPIC9k-his map.

[0030] Figure 2 SDS-PAGE results of screening and purification of Pichia pastoris engineered strains expressing β-1,6-glycosidic bond hydrolase CoExg1 heterologously: a: Fermentation supernatant of transformants obtained from screening with different concentrations of G418 after 72 h of induction (lanes 1,2: 0.5 mg / ml; 3,4: 1 mg / ml; 5,6: 1.5 mg / ml; 7,8: 2 mg / ml; 9: pPIC9k-his empty vector); b: SDS-PAGE image of purified enzyme CoExg1.

[0031] Figure 3 UPLC detection of mogroside V (12h) catalyzed by β-1,6-glycosidic bond hydrolase CoExg1. a: Mogroside series standards (V: mogroside V; SI: symmancoside I; IVA: mogroside IVA; IV: mogroside IV; IIIE: mogroside IIIE); be: Products of mogroside extract (mogroside V content ≥50%) after hydrolysis by CoExg1 at 0h, 3h, 4h, and 12h.

[0032] Figure 4 Pathway diagram of the staged hydrolysis of mogroside V by the β-1,6-glycosidic bond hydrolase CoExg1

[0033] Figure 5 UPLC detection of icariin catalyzed by β-1,6-glycosidic bond hydrolase CoExg1. Figure a: Series of icariin standards (1: icariin; 2: icariin I; 3: icariin II; 4: icariin); bd: Products of icariin after hydrolysis by CoExg1 at 0h, 2h, and 12h.

[0034] Figure 6 UPLC detection of β-1,6-glycosidic bond hydrolase CoExg1 catalyzing the conversion of monganin into amoeboidin. Figure a: Monganin series standards (1: monganin; 2: monganin); bd: Products of monganin after hydrolysis by CoExg1 at 0h, 6h, and 12h. Detailed Implementation

[0035] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the scope of the invention as described in detail herein.

[0036] Example 1: Mining of the gene sequence of β-1,6-glycosidic bond hydrolase CoExg1

[0037] Candida orthopsilosis L7 (accession number: CCTCC NO: M 2025731) possesses the specific biotransformation ability to hydrolyze mogroside V. Its main metabolite is symmonoside I, which has a sweet taste, and its minor product is mogroside IIIE. Its extracellular enzyme system exhibits unique substrate selectivity. In this study, the whole genome of strain L7 was sequenced using the Illumina NovaSeq 6000 sequencing platform. Functional annotation was performed using the GO, KEGG, and COG databases, focusing on screening gene clusters related to β-glucosidase (EC 3.2.1.21). Sequence alignment was performed using the BLASTP algorithm, with an E-value threshold of ≤1e. -5 Sequences with ≥30% homology to the protein sequence encoded by the exg1 gene (exonuclease-β-1,3-glucanase gene, known to have the function of hydrolyzing mogrosides) were screened, and CoExg1 was finally selected as a candidate gene.

[0038] Example 2: Construction of a plasmid vector for heterologous expression of β-1,6-glycosidic bond hydrolase CoExg1

[0039] Based on the amino acid sequence obtained from the above sequence alignment, the amino acid sequence of the signal peptide predicted using SingalP6.0 is MRSFAVFSILISCIYALA. After removing the signal, the following primers were designed: forward primer F(5'-AGGCTGAAGCTTACGTA). GA ATTC CTTACACCACAGAAAAGAG-3') and reverse primer R(5'-ATTCGCGGCCGCCCTAGG GAATTC The genome of Candida orthopsilosis L7 was extracted using the Solarbio Fungal Genome Extraction Kit (D2300). Molecular biological methods were employed, using forward primer F and reverse primer R, to amplify the β-1,6-glycosidic acid hydrolase (CoExg1) gene of Candida orthopsilosis L7 via PCR using a high-fidelity enzyme (Vazyme, P521) according to the procedure in Table 1. The cloning results are shown below. Figure 1 As shown in a.

[0040] The pPIC9k-his vector was digested with restriction endonuclease EcoRI at 37°C for 1 hour; the target gene fragments recovered from PCR and gel-recovered were ligated with homologous recombinase (Vazyme, C112) at 37°C for 1 hour; the constructed expression plasmid (as shown in the image) was then... Figure 1 (b) E. coliDH5α competent cells were introduced and cultured for 12 h on LB agar plates containing kanamycin and ampicillin. The colonies that grew on the plates were verified by PCR using universal vector primers. The plasmid containing the target gene was sequenced to verify the colony and the cloned strain with the correct target gene was selected.

[0041] Table 1. PCR amplification procedure for the CoExg1 gene

[0042]

[0043] Example 3: Construction of Pichia pastoris genetically engineered strain expressing β-1,6-glycosidic bond hydrolase CoExg1

[0044] The correctly sequenced *E. coli* clones obtained above were inoculated into LB medium and cultured at 37°C and 180 rpm for 12 h. The cells were then collected by centrifugation at 4000 rpm for 10 min. Sufficient recombinant plasmids were extracted using the Tiangen Plasmid Mini-Prep Kit (DP103), and digested with the restriction endonuclease Sac I. Histidine-deficient *Pichia pastoris* GS115 competent cells were prepared using the Beyotime (DO308) Pichia pastoris competent cell preparation and transformation kit. The single-enzyme-digested recombinant plasmids were transformed into GS115 competent cells, plated on MD solid medium, and cultured at 30°C for 3–4 days to screen for transformants carrying the His4 marker gene. Transformants on MD solid medium were washed away with 1 ml of sterile water and transferred to YPD medium. The medium was incubated at 30°C and 180 rpm until the OD value reached approximately 1.0. The medium was then diluted 10 times with sterile water. 200 μl of the diluted bacterial solution was spread onto YPD solid medium containing 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, and 2 mg / ml of genimycin G418, respectively, and incubated at 30°C for 72 h.

[0045] Two single colonies were picked from YPD solid medium containing different concentrations of G418 and inoculated into YPD medium (containing 0.2 mg / ml G418). After incubation at 30°C and 180 rpm for 24 h, they were inoculated into BMGY fermentation medium at a 1:100 ratio and incubated at 30°C and 180 rpm for 24 h. The bacterial cells were collected by centrifugation, washed twice with sterile PBS, and then inoculated into BMMY expression medium at a concentration of OD≈1.0. Fermentation was carried out at 30°C and 180 rpm for 72 h, with methanol added every 24 h to a final concentration of 0.5%.

[0046] Take 1 ml of culture medium fermented for 72 h, centrifuge at 10000 rpm for 5 min at 4 °C, collect the supernatant, add 50 μL of 0.15% sodium deoxycholate (DOC), mix well, and incubate at room temperature for 15 min. Then add 70 μL of 100% trichloroacetic acid (TCA), mix well, incubate on ice for 30 min, and centrifuge at 10000 rpm for 20 min at 4 °C. Discard the supernatant, wash the precipitate with 500 ml of pre-cooled acetone, centrifuge at 10000 rpm for 20 min at 4 °C, discard the supernatant, blot dry with paper towels, add 10 μL of loading buffer and 40 μL of PBS, heat to boiling for 5 min, and run 15 μL of gel electrophoresis. Figure 2 As shown, the strain with the best expression level was selected as the subsequent enzyme-producing strain (other strains only had slightly lower enzyme expression levels, but still had the same transformation function).

[0047] MD solid medium: 13.4 g / L YNB, 20 g / L glucose, 0.0004 g / L D-biotin, 18 g / L agar, autoclaved at 121°C for 20 min.

[0048] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose. For solid medium, an additional 18 g / L agar needs to be added. Autoclave at 121°C for 20 min.

[0049] BMGY fermentation medium: 20g peptone, 10g yeast extract, 13.4g YNB, 10ml glycerol, add water to 900ml, dispense 45ml / bottle into 250ml Erlenmeyer flasks, autoclave at 120℃ for 20min, add 5ml 1M phosphate buffer (pH 6.0), 100ul D-biotin (0.2g / L stock solution), and 50ul chloramphenicol (34mg / ml stock solution) before inoculation.

[0050] BMMY expression medium: 20g peptone, 10g yeast extract, 13.4g YNB, add water to 900ml, dispense 45ml / bottle into 250ml Erlenmeyer flasks, autoclave at 120℃ for 20min, add 5ml 1M phosphate buffer (pH 6.0), 100ul D-biotin (0.2g / L stock solution), 50ul chloramphenicol (34mg / ml stock solution), and 5g / L methanol before inoculation.

[0051] Example 4: Staged synthesis of sermonoside I and mogroside IIIE catalyzed by β-1,6-glycosidic bond hydrolase CoExg1 and preparation

[0052] The engineered bacteria heterologously expressing CoExg1 were induced to express for 72 h according to the method in Example 3. The supernatant was collected by centrifugation at 4000 rpm for 5 min, filtered through a 0.45 μM microporous membrane, and placed into a dialysis bag with a 1 KD cutoff. The dialysis bag containing the crude enzyme solution was placed in polyethylene glycol 6000 at pH 6.0 and a concentration of 30%–40%, and concentrated at 4 °C for 4–8 h until the crude enzyme solution concentration was 10–20 g / L. Glycerol was added to a final concentration of 20%, mixed well, and then frozen at -80 °C.

[0053] The crude enzyme solution was used for a staged catalytic reaction: 2 g / L CoExg1 crude enzyme solution, 20 g / L mogroside extract (mogroside V content ≥50%), and 50 mM phosphate buffer (pH 6.0) were used. The reaction was carried out continuously at 200 rpm and 30℃ for 12 h. Samples were taken at regular intervals, and the reaction was terminated by adding methanol at a 1:1 ratio. The conversion product was detected by UPLC. The results are as follows. Figure 3As shown, CoExg1 preferentially hydrolyzes the β-1,6-glycosidic bond in the glucose side chain attached to the C3 hydroxyl group of the mogroside V structure to generate sarcoside I. After 3 hours of reaction, the conversion rate of mogroside V to sarcoside I by CoExg1 is as high as 75%, with only a very small amount of mogroside IIIE generated. After 4 hours of reaction, CoExg1 has almost completely hydrolyzed mogroside V, and then begins to hydrolyze the β-1,6-glycosidic bond in the glucose side chain attached to the C24 hydroxyl group to generate mogroside IIIE. It is not until 12 hours that almost all of it has been hydrolyzed to IIIE. The yield of sarcoside I reaches its highest point of 85.4% at 4 hours of reaction, with a yield of 7.5 g / L; the yield of mogroside IIIE reaches its highest point of 95.4% at 12 hours, with a yield of 7.1 g / L.

[0054] The reaction solutions after 4 h and 12 h of hydrolysis were terminated by adding methanol at a 1:1 ratio. An appropriate amount of AB-8 macroporous resin was soaked in methanol for 24 h and then wet-packed into a column. The resin fragments and impurities were removed by washing with 2 column volumes (BV) of methanol, followed by washing with distilled water containing 0.1% glacial acetic acid until no alcohol odor remained. Semantic glycoside I and mogroside IIIE samples (20 mg / g wet resin) were loaded separately and incubated overnight. Elution was performed using methanol-deionized water as the mobile phase at a flow rate of 2 BV / h, followed by washing with 5 BV of 40% methanol to remove impurities and a gradient elution with 5 BV of 50%–70% methanol. UPLC analysis confirmed the acquisition of sympathetic glycoside I and mogroside IIIE fractions with a purity >90%. The eluents were combined and concentrated to dryness under reduced pressure at 40℃, then reconstituted with 5 mL of deionized water. After the reconstituted sample was stored at -80℃ overnight, it was transferred to a freeze dryer and freeze-dried for 3 days to obtain the dried pure product.

[0055] Example 5: Purification and catalytic activity study of β-1,6-glycosidic bond hydrolase CoExg1

[0056] The above-mentioned expression strain was induced to ferment in shake flasks for 72 hours to produce enzymes. After centrifugation at 4000 rpm for 5 minutes, the supernatant was collected. The supernatant was filtered through a 0.45 μm microporous membrane and loaded at a flow rate of 1 ml / min onto a HisTrap HP affinity chromatography column (GE) pre-equilibrated with pH 6.0 phosphate buffer. Elution was performed sequentially with 5 column volumes (BV) of pH 6.0 phosphate buffer containing 0 mM, 50 mM, and 300 mM imidazole, collecting the 300 mM imidazole eluent. Purity was verified by SDS-PAGE. Figure 2 b) The eluent was then placed in a dialysis bag with a molecular weight cutoff of 1 kDa and placed in 100 volumes of phosphate buffer at pH 6.0. The solution was dialyzed at 4°C for 12 hours to remove imidazole. The dialyzed protein solution was then concentrated to 5 mg / ml by ultrafiltration at 4°C and 3500 rpm. Glycerol was added to achieve a final concentration of 20%, and the solution was stored at -80°C.

[0057] A reaction system was prepared using 0.1 g / L CoExg1 purified enzyme, 2 g / L mogroside extract (mogroside V content ≥50%), and 50 mM phosphate buffer (pH 6.0). The reaction was carried out at 200 r / min and 30℃ for 12 hours, and the reaction was terminated by adding an equal volume of methanol. UPLC analysis showed that the yield of symmenidine I reached its highest level of 88.2% after 2.5 h of reaction; after 10 h of reaction, symmenidine I was almost completely hydrolyzed to mogroside IIIE, with a yield as high as 98.1%.

[0058] A reaction system was prepared using 5 μM CoExg1 purified enzyme, 0.25 mM icariin / mangosteenin, 5% DMSO (v / v), and 50 mM phosphate buffer (pH 6.0). The reaction was carried out at 30℃ and 200 rpm for 12 hours, and then terminated by adding an equal volume of methanol. UPLC results were obtained. Figure 5 , 6 The results indicate that CoExg1 can catalyze the hydrolysis of icariin to icariin II and the hydrolysis of gentianin to gentianin. UPLC analysis showed that CoExg1 achieved a conversion rate of 97.4% in the hydrolysis of icariin to icariin II and over 98% in the hydrolysis of gentianin to gentianin.

[0059] >SEQ.No.1

[0060]

[0061] >SEQ.No.2

[0062] MRSFAVFSILISCIYALALTPQKRGESVPWNYQKQTTRGLNLGGWLVLEAYITPSLFGSWLFGDDENNIPVDEYHFTKQLGKEAAEHVLQMHWNSWYTEADFEQISYLGINTVRIPIGYWAFQLLDDDPYVQGQVEYLDKALQWARNHNLKVWIDLHGAPGSQNGFDNSGLRDTLDWQTVNGNVQVTKDVLNTIFEKYGGDDYADVVIGIELLNEPLGPSLNVDELKQFYQDGYSALRSTGSNIPVVIHDAFEAIGYWDDFSIGNNAFNVVLDHHHYQVFSAQELERSIDDHISVACNWGWDTKKESYWTITGEWSAALTDCAKWLNGVRRGARYEGQYDNSPYIGSCLQYLELDNWPEDYKTNVRKYIEAQLDAYEYTGGWIFWNWKTEDAIEWDFQRLTAAGIFPQPLTDRQYPNQCGFPSN

Claims

1. A β-1,6-glycosidic bond hydrolase, characterized in that: Genes capable of hydrolyzing β-1,6-glycosidic bonds were obtained through targeted screening of the Candida glabrata L7 genome and named as follows: CoExg1 The nucleotide sequence is shown in SEQ. No. 1; The aforementioned Candida parapsilosis L7, preservation name: Candida parapsilosis L7 ( Candida orthopsilosis L7), depositary institution: China Center for Type Culture Collection, depositary address: Wuhan University, China, deposit date: April 9, 2025, accession number: CCTCC NO: M 2025731.

2. The β-1,6-glycosidic bond hydrolase according to claim 1, characterized in that: The amino acid sequence of the β-1,6-glycosidic bond hydrolase CoExg1 is shown in SEQ.No.

2.

3. A recombinant vector comprising the β-1,6-glycosidic bond hydrolase according to claim 1 or 2, characterized in that: The vector is a Pichia pastoris or Escherichia coli expression vector.

4. The recombinant vector according to claim 3, characterized in that: The carrier is manufactured as follows: Obtained through PCR technology CoExg1 Gene fragments, with signal peptides and stop codons removed, are then processed via homologous recombination at E. coR The I single enzyme restriction site was inserted into the pPIC9k-his plasmid vector to obtain the CoExg1-Ppic9K-his recombinant vector.

5. A method for expressing β-1,6-glycosidic bond hydrolase, characterized in that: The expression system using Pichia pastoris includes the following steps: (1) The recombinant vector according to claim 3 or 4 is placed in S ac After linearization of the I restriction site, the cells were introduced into Pichia pastoris GS115 cells, plated on MD solid medium, and screened for successfully transformed recombinant strains. (2) Using gradient concentrations of genimycin G418 for screening, multiple single clones were randomly selected from plates containing various concentrations of G418, and after small-scale shake-flask fermentation for 72 hours, SDS-PAGE analysis was performed to screen the optimal expression strain. (3) The optimal expression recombinant strain obtained by screening was subjected to large-scale shake flask fermentation to induce the expression of β-1,6-glycosidic bond hydrolase. Crude enzyme solution was prepared by dialysis bag concentration method, and pure enzyme was further obtained by HisTrap HP affinity chromatography and ultrafiltration tube concentration. MD solid medium: 13.4 g / L YNB, 20 g / L glucose, 0.0004 g / L D-biotin, 18 g / L agar, autoclaved at 121°C for 20 min.

6. A method for expressing β-1,6-glycosidic bond hydrolase, characterized in that: The use of E. coli expression systems includes the following steps: (1) The recombinant vector described in claim 3 or 4 is directly introduced into Escherichia coli BL21(DE3) cells to obtain a recombinant strain; (2) The recombinant strain was subjected to large-scale shake-flask fermentation. The expression of β-1,6-glycosidic bond hydrolase was induced by IPTG. The crude enzyme solution was obtained by ultrasonic cell disruption and freeze centrifugation. The pure enzyme was further purified by HisTrap HP affinity chromatography and concentrated by ultrafiltration tube.

7. The application of a β-1,6-glycosidic bond hydrolase according to claim 1 in traditional Chinese medicine resources, characterized in that: Using molecular cloning technology to obtain heterologous expression CoExg1 Genetically engineered bacteria, after being induced to produce enzymes, are used to catalyze reactions with crude enzyme solutions or pure enzymes to achieve the directional transformation of active molecules in traditional Chinese medicine. The active molecules of the traditional Chinese medicine are: mogroside V, icariin, or styracin, wherein mogroside V is converted into sympathoside I and mogroside IIIE, icariin is converted into icariin II, and styracin is converted into styracin.

8. The application of the β-1,6-glycosidic bond hydrolase according to claim 7 in traditional Chinese medicine resources, characterized in that: The crude enzyme-catalyzed reaction specifically includes the following steps: a reaction system of 2 g / L CoExg1 crude enzyme solution, 20 g / L mogroside extract, and 50 mM pH=6.0 phosphate buffer, wherein the mogroside extract contains mogroside V content ≥50%, and the reaction is carried out at 200 r / min and 30℃ for 4 h. UPLC detection shows that the yield of sympathoside I reaches the highest level of 86.5%, with a yield of 7.5 g / L; after 12 h of reaction, sympathoside I is almost completely hydrolyzed to mogroside IIIE, with a yield as high as 95.4%, and a yield of 7.1 g / L.

9. The application of the β-1,6-glycosidic bond hydrolase according to claim 7 in traditional Chinese medicine resources, characterized in that: The pure enzyme-catalyzed reaction specifically includes the following steps: a reaction system of 0.1 g / L CoExg1 pure enzyme, 2 g / L mogroside extract, mogroside V content ≥50%, and 50 mM pH=6.0 phosphate buffer, reacted at 200 r / min and 30℃ for 2.5 h, UPLC detection showed that the yield of sympathoside I reached the highest level of 88.2%; after 10 h of reaction, sympathoside I was almost completely hydrolyzed to mogroside IIIE, with a yield as high as 98.1%.

10. The application of a β-1,6-glycosidic bond hydrolase according to claim 7 in traditional Chinese medicine resources, characterized in that: A reaction system consisting of 5 μM CoExg1 purified enzyme, 0.25 mM icariin or argentin, 5% DMSO (v / v), and 50 mM pH=6.0 phosphate buffer was prepared and reacted at 200 rpm and 30℃ for 12 h. UPLC analysis showed that CoExg1 hydrolyzed icariin to produce icariin II with a conversion rate as high as 97.4%; and hydrolyzed argentin to produce argentin with a conversion rate of over 98%.

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