Sulfonation-modified metal organic framework material, preparation method therefor, and use thereof in yield increase of aryl glycoside compounds

Abstract

Disclosed in the present invention are a sulfonation-modified metal organic framework material, a preparation method therefor, and the use thereof in the yield increase of aryl glycoside compounds. The sulfonation-modified metal organic framework material reacts with 1,2-ethanedisulfonic acid by taking a zirconium-based MOF material Zr-BTB as a precursor, so as to perform sulfonation modification. The sZr-BTB prepared in the present invention can enable a material to be fixed on the surface of Yarrowia lipolytica by means of a physical adsorption principle, so as to form a layer of a compact protective film, such that relatively macromolecular glycosidase is confined in cells and cannot be secreted, and the excretion of small-molecular aryl glycoside compounds is not influenced. The biosynthetic main product is prevented from being decomposed, and stable adsorption on Yarrowia lipolytica can also be maintained under strongly acidic conditions, which is beneficial for improving the yield of aryl glycoside compounds produced by means of fermentation, thereby breaking through non-pattern microorganism obstacles and advancing the low-cost industrialization of biosynthesis.

Description

A sulfonated modified metal-organic framework material, its preparation method, and its application in increasing the yield of aryl glycosides. Technical Field

[0001] This invention belongs to the field of biochemical engineering, specifically relating to a sulfonated modified metal-organic framework material, its preparation method, and its application in increasing the yield of aryl glycosides. Background Technology

[0002] Microbial strains, as the core of modern industrial biotechnology, play an increasingly important role in the industrial field. Microbial catalysis, through its own metabolism, catalyzes reactions with a catalytic efficiency many times higher than that of chemical catalysis. Furthermore, biocatalysis can occur at room temperature, exhibits specific catalytic activity, and offers advantages such as lower energy consumption and higher safety. Model microorganisms such as *Escherichia coli* and *Saccharomyces cerevisiae* have long been considered ideal microbial substrates. However, in recent years, due to technological breakthroughs, non-model microorganisms have become substrates for the synthesis of many natural products. These non-model microorganisms possess unique physiological and metabolic characteristics, providing abundant genetic resources and facilitating the development of new products. *Yersinia lipolytica*, an unconventional hemisomycete, is considered an excellent natural synthetic host. *Yersinia lipolytica* can utilize substrates such as glucose for the synthesis of aryl glycosides. However, using *Yersinia lipolytica* for fermentation to produce aryl glycosides has a significant drawback. During its growth, *Yersinia lipolytica* often produces byproducts such as organic acids, causing the pH of the culture medium to drop to extremely acidic levels (pH 2-3). In a strongly acidic environment, the yeast easily loses its activity. Meanwhile, during the fermentation process of *Yarrowia lipolytica*, hydrolytic enzymes such as glycosidases and small molecule products are secreted extracellularly. Glycosidases can hydrolyze aryl glycosides into byproducts, leading to a decrease in the yield of the main product. These problems hinder the development of industrial-scale production of aryl glycosides using *Yarrowia lipolytica*. Summary of the Invention

[0003] Objective of the Invention: To address the shortcomings in the production of aryl glycosides by *Yarrowia lipolyticis* biofermentation, this invention provides a sulfonated modified metal-organic framework (MOF) material. The sulfonated modified MOF material prepared by this invention is a novel zirconium-based MOF material for immobilizing *Yarrowia lipolyticis*: sulfonated modified Zr-BTB (sZr-BTB). During the adsorption of *Yarrowia lipolyticis*, sZr-BTB can prevent the secretion of hydrolytic enzymes such as glycosidases by the yeast during fermentation, thereby reducing product degradation. Simultaneously, sZr-BTB can continuously and stably adsorb onto the yeast surface under acidic conditions, providing long-term protection for *Yarrowia lipolyticis* under strongly acidic conditions. This effectively solves the problems of low yield and low yeast activity in the fermentation of *Yarrowia lipolyticis*.

[0004] The present invention also provides a method for preparing and applying the sulfonated modified metal-organic framework material.

[0005] Technical solution: To achieve the above objective, the present invention provides a sulfonated modified metal-organic framework material, wherein the sulfonated modified metal-organic framework material uses zirconium (Zr)-based MOF material Zr-BTB as a precursor and reacts with 1,2-ethanedisulfonic acid to undergo sulfonation modification.

[0006] The preparation method of the sulfonated modified Zr-BTB material of the present invention includes the following steps:

[0007] (1) Preparation of metal-organic framework material Zr-BTB: 1,3,5-tris(4-carboxyphenyl)benzene (H3BTB), benzoic acid (BA) and zirconium tetrachloride (ZrCl4) were dissolved in an organic solvent;

[0008] (2) The above solution was added to deionized water, ultrasonically mixed, reacted at high temperature, cooled and washed, and centrifuged and dried to obtain the precursor Zr-BTB material.

[0009] (3) Add 1,2-ethanedisulfonic acid to the prepared precursor Zr-BTB material. After the high-temperature reaction is completed, collect by centrifugation and wash with water to obtain the sulfonated modified Zr-BTB material, which is the sulfonated modified metal-organic framework material.

[0010] In step (1), the mass ratio of 1,3,5-tris(4-carboxyphenyl)benzene (H3BTB), benzoic acid (BA), and zirconium tetrachloride (ZrCl4) is 10-13:440-460:9-12.

[0011] In step (2), the high temperature is 100-130℃, the reaction time is 20-24h, and the centrifugation speed is 8,000-12,000rpm for 5-10min.

[0012] In step (3), 1,2-ethanedisulfonic acid is dissolved in an organic solvent, the mass ratio of 1,2-ethanedisulfonic acid to the precursor Zr-BTB material is 1:1 to 6:1, the reaction temperature is 90 to 120°C, the reaction time is 15 to 20 h, and the reaction is centrifuged at 8,000 to 12,000 rpm for 5 to 10 min.

[0013] Preferably, the method for preparing the sulfonated modified Zr-BTB material includes the following steps:

[0014] (1) Dissolve 1,3,5-tris(4-carboxyphenyl)benzene (H3BTB), benzoic acid (BA) and zirconium tetrachloride (ZrCl4) in dimethylformamide (DMF).

[0015] (2) Add a certain amount of deionized water to the above solution, mix it with ultrasound, react it at high temperature in a reactor, cool it, wash it with DMF and acetone, and centrifuge and dry it to obtain the precursor Zr-BTB material.

[0016] (3) Add the precursor Zr-BTB material to a 1,2-ethanedisulfonic acid solution and react at high temperature. Then collect the sulfonated Zr-BTB material by centrifugation.

[0017] In step (1), the amount of dimethylamide used is 2-4 mL; the amounts of H3BTB, BA, and ZrCl4 used are 10-13 mg, 440-460 mg, and 9-12 mg, respectively.

[0018] In step (2), the volume of deionized water is 0.5-1 mL.

[0019] Preferably, after using DMF and acetone cleaning materials, a 0.1M hydrochloric acid cleaning material is used once, followed by rinsing with deionized water to remove the hydrochloric acid.

[0020] In step (3), the amount of Zr-BTB material used is 15-25 mg, and the amount of 1,2-ethanedisulfonic acid used is 10 mg-80 mg, wherein the ratio of the precursor Zr-BTB material and 1,2-ethanedisulfonic acid is 1:1-1:4.

[0021] More preferably, the preparation method of the Zr-BTB material and the sulfonation modification include the following steps:

[0022] (1) Dissolve 10-13 mg of 1,3,5-tris(4-carboxyphenyl)benzene (H3BTB), 440-460 mg of benzoic acid (BA) and 9-12 mg of zirconium tetrachloride (ZrCl4) in 2-4 mL of dimethylformamide (DMF).

[0023] (2) After centrifuging the above solution to remove the supernatant, add a 0.1M hydrochloric acid solution. The solvent is reacted in the reactor at 100-130℃ for 20-24h. After cooling, wash with DMF 1-3 times, acetone 1-3 times, 0.1M hydrochloric acid once, and deionized water 1-3 times. Centrifuge at 8,000-12,000 rpm for 5-10min, and then dry to obtain Zr-BTB material.

[0024] (3) Dissolve 10 mg to 200 mg of 1,2-ethanedisulfonic acid in 2 to 5 mL of DMF, and then add it to 15 to 25 mg of the precursor Zr-BTB material. React at 90 to 120 °C for 15 to 20 h. Then, centrifuge at 8,000 to 12,000 rpm for 5 to 10 min to collect the sulfonated Zr-BTB material.

[0025] The application of the sulfonated modified metal-organic framework material described in this invention in the production of aryl glycosides by Yersinia lipolyticis.

[0026] The sulfonated modified metal-organic framework material of this invention binds to Yarrowia lipolytica cells via electrostatic adsorption. This immobilized yeast can inhibit the secretion of glycosidases during fermentation, thereby increasing the production of aryl glycosides by microbial fermentation. Simultaneously, sZr-BTB can stably adsorb to Yarrowia lipolytica under strongly acidic conditions, thus protecting the yeast and increasing catalytic efficiency.

[0027] The application process is as follows:

[0028] (1) Inoculate the lipophilic yeast into the seed culture medium for seed culture;

[0029] (2) Take the seed liquid and inoculate it into the fermentation medium for fermentation culture;

[0030] (3) Sulfonated modified metal-organic framework materials are added to the fermentation broth to immobilize yeast cells in the logarithmic growth phase, and fermentation continues to increase the production of the target product, aryl glycosides.

[0031] In step (1), the Yersinia lipolytica yeast is cultured in YPD seed medium for 24-36 hours; in step (2), it is cultured in fermentation medium for 24-36 hours.

[0032] In step (3), the amount of sulfonated modified metal-organic framework material added to the fermentation broth is 0.5–1.5 mg of sulfonated modified Zr-BTB material per 20–30 mL of fermentation broth. After addition, immobilization begins in the logarithmic growth phase, and fermentation continues for 100–120 h to increase the yield of the target product, aryl glycosides.

[0033] The aryl glycoside compounds include one or more of the following: gastrodin, arbutin, resveratrol, naringin, baicalin, and vitexin.

[0034] Preferably, the application includes the following steps:

[0035] (1) Inoculate glycerol tube bacteria into 3 mL of yeast extract peptone glucose medium (YPD) seed culture medium and culture for 24–36 h under certain temperature and rotation speed conditions.

[0036] (2) Take 1.5 mL of seed liquid and inoculate it into 30 mL of fermentation medium and culture it for 24 to 36 h under certain temperature and rotation speed conditions.

[0037] (3) Sulfonated modified metal-organic framework material was added to the fermentation broth and fermented for 120 h. Samples were taken every 24 h, and the fermentation broth was frozen at -20℃ to measure the gastrodin yield.

[0038] The strain mentioned in step (1) is *Yarrowia lipolyticis*. The YPD seed culture medium consists of 10 g / L yeast extract, 20 g / L peptone, and 80 g / L anhydrous glucose. The culture temperature is 30°C, and the culture speed is 220–250 rpm.

[0039] The fermentation medium in step (2) consists of 10 g / L yeast extract, 20 g / L peptone, 80 g / L anhydrous glucose, and 5–10 mg / L trace metal elements (4.0 g / L FeSO4·7H2O, 4.0 g / L CaCl2, 1.0 g / L MgCl2). The culture temperature is 30℃, and the culture speed is 220–250 rpm.

[0040] In step (3), 0.5 to 1.5 mg of sulfonated modified Zr-BTB material is added to every 30 mL of fermentation broth.

[0041] This invention proposes a method for immobilizing *Yersinia lipolyticis* using a sulfonated metal-organic framework material (sZr-BTB). Compared to free yeast, immobilized yeast is less susceptible to external influences during growth and metabolism, thus maintaining yeast activity. Furthermore, immobilized yeast is easily recovered during fermentation or biotransformation, allowing for the reuse of the microorganism. The sZr-BTB prepared in this invention is a material with high specific surface area and porosity. Its high specific surface area and highly ordered pore structure give it extremely strong adsorption capacity and carrier capacity. Simultaneously, sZr-BTB exhibits good biocompatibility and does not adversely affect cell growth, metabolism, or activity. The sZr-BTB proposed in this invention utilizes physical adsorption to encapsulate yeast, forming a protective film on its surface. This restricts glycosidases to intracellular secretion, while allowing small molecule products to efflux, thus preventing the decomposition of biosynthesized aryl glycosides. Meanwhile, the sulfonated groups on the sZr-BTB material can compete with the groups on the surface of yeast cells for hydrogen ions in the highly acidic fermentation broth, thus ensuring that the material can permanently encapsulate the yeast and provide protection for the yeast cells.

[0042] In summary, the yeast immobilization technology using sZr-BTB in this invention can improve some of the major defects of the non-model microorganism *Yersinia lipophila*. This method not only effectively avoids the loss of yeast activity but also enhances adsorption and carrier functions through its high specific surface area and porous structure. The sulfonated groups of sZr-BTB can provide protection for yeast cells under acidic conditions, preventing the extracellular secretion of glycosidases and protecting aryl glycosides from degradation. The method of this invention significantly improves the stability and efficiency of *Yersinia lipophila* in industrial production, giving it broader prospects in biotechnology applications.

[0043] During the fermentation of *Yarrowia lipolytica*, glycosidases and products are secreted extracellularly. Glycosidases decompose some biosynthesized aryl glycosides into byproducts, resulting in low yields of the main product. Secondly, *Yarrowia lipolytica* typically produces organic acids and other byproducts during fermentation, leading to a significant decrease in the pH of the fermentation broth and potentially causing yeast death. To address these issues, the material sZr-BTB proposed in this invention can be immobilized on the surface of *Yarrowia lipolytica* through physical adsorption, forming a dense protective film. This confines relatively large glycosidase molecules intracellularly, preventing secretion, while the efflux of small aryl glycosides remains unaffected, thus avoiding the decomposition of the biosynthesized main product. Furthermore, the sulfonic acid groups on the sZr-BTB material compete with the anionic groups on the yeast surface for active hydrogen ions in the fermentation broth, enhancing the adsorption capacity of *Yarrowia lipolytica* to the material under acidic conditions. This protective film also improves the stability of *Yarrowia lipolytica* cells, increasing catalytic efficiency and reusability. The sulfonated modified Zr-BTB (sZr-BTB) prepared in this invention can maintain stable adsorption with Yersinia lipolyticis under strongly acidic conditions. At the same time, it prevents the secretion of glycosidases and other enzymes in yeast to the extracellular space through adsorption and encapsulation, thereby improving the yield of aryl glycosides produced by fermentation. This solves the obstacle of non-model microorganisms and promotes the low-cost industrialization of biosynthesis.

[0044] This invention is the first to propose a method for preparing sulfonated modified zirconium-based material sZr-BTB, and applies it to the fermentation production of *Yarrowia lipolytica* to increase the yield of aryl glycosides. The sulfonated modified Zr-BTB material designed in this invention can encapsulate *Yarrowia lipolytica* like a membrane, restricting the secretion of yeast glycosidases while ensuring substrate efflux. Furthermore, because the yeast is encapsulated by the material, it can maintain a certain level of activity in highly acidic fermentation broths, increasing the yield of aryl glycosides produced by *Yarrowia lipolytica* fermentation and enhancing the yeast's activity in harsh environments.

[0045] Current technologies cannot address the issue of controlling glycosidase secretion during the fermentation process of *Yarrowia lipolytica*, leading to the decomposition of the main product, aryl glycosides, by glycosidases. Furthermore, the fermentation broth's acidity, typically around pH 2-3, creates a harsh environment that reduces yeast activity. The sZr-BTB material proposed in this invention encapsulates the yeast, preventing glycosidase secretion and protecting it in acidic fermentation broths while maintaining high activity.

[0046] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0047] 1. This invention selects Zr-BTB material. Zirconium metal is considered to be low-toxicity or even non-toxic. Compared with other metal-organic framework materials, Zr-BTB is a 2D-MOF material with larger planar dimensions, thinner thickness, and higher water stability. This means that Zr-BTB is easy to embed yeast cells, and its structure is not easily destroyed.

[0048] 2. This invention uses Yersinia lipolyticis for microbial fermentation to produce aryl glycosides. This method has the advantages of high yield, easy scale-up cultivation, and short cultivation cycle, and is more suitable for the industrial production of aryl glycosides.

[0049] 3. This invention binds MOF nanosheets to Yersinia lipolyticis via physical adsorption. Compared to free yeast, the immobilized yeast is more stable during metabolism. Furthermore, the physical adsorption between the yeast and the material allows for easy separation of the product from the yeast, simplifying subsequent separation and purification processes.

[0050] 4. During fermentation, Yersinia lipolyticis produces many organic acids. Compared with conventional metal-organic framework materials, Zr-BTB is more stable in acidic environments, allowing the material to maintain its structural integrity and not disintegrate for a long time under acidic conditions.

[0051] 5. This invention proposes a method of modifying Zr-BTB with 1,2-ethanedisulfonic acid after synthesis. One sulfonic acid group of 1,2-ethanedisulfonic acid coordinates with zirconium metal on Zr-BTB, while the other sulfonic acid group competes with groups on the surface of yeast cells for hydrogen ions under acidic conditions, thereby ensuring that sZr-BTB can maintain stable adsorption with Yersinia lipolyticis for a long time under strongly acidic conditions.

[0052] 6. During the fermentation process of *Yersinia lipolytica*, the glycosidases produced decompose the main product, aryl glycosides. The sulfonated Zr-BTB material, as a 2D-MOF material, can coat the surface of *Yersinia lipolytica*, thereby inhibiting the secretion of hydrolytic enzymes such as glycosidases into the fermentation broth. The efflux of small aryl glycosides remains unaffected, thus preventing the decomposition of the main biosynthetic product and preventing enzymes from breaking it down. This immobilization increases the yield of aryl glycosides. Furthermore, because the yeast is coated with the sulfonated Zr-BTB material, it maintains excellent activity in highly acidic fermentation broths or other harsh fermentation environments. The material prepared in this invention also provides good protection for the yeast under high-temperature conditions, increasing its stability. At higher temperatures, such as 45°C, the yield of gastrodin is significantly higher than that of free *Yersinia lipolytica*. 7. The preparation method of this invention is simple and convenient, the raw materials are readily available, and it can be effectively used for large-scale production. It overcomes the obstacles posed by non-model microorganisms and promotes the low-cost industrialization of biosynthesis. Attached Figure Description

[0053] Figure 1 is a comparison of scanning electron microscope images of immobilized yeast and free yeast provided in Example 1 and Comparative Example 1;

[0054] Figure 2 shows the high performance liquid chromatography (HPLC) spectra of gastrodin and p-hydroxybenzyl alcohol as described in Example 1.

[0055] Figure 3 is a comparison chart of gastrodin production in Example 1 and Comparative Example 1;

[0056] Figure 4 shows the ratio of gastrodin to p-hydroxybenzyl alcohol in Example 1 and Comparative Example 1.

[0057] Figure 5 shows the high performance liquid chromatography (HPLC) spectra of gastrodin and p-hydroxybenzyl alcohol in Comparative Example 1.

[0058] Figure 6 shows the yield of gastrodin produced by fermentation of *Yersinia lipolytica* immobilized with sZr-BTB at different ratios of zirconium tetrachloride in Example 2.

[0059] Figure 7 shows the yield of gastrodin produced by fermentation of Yersinia lipolytica immobilized with sZr-BTB at different ratios of 1,3,5-tris(4-carboxyphenyl)benzene in Example 3.

[0060] Figure 8 shows the yield of gastrodin produced by fermentation of *Yersinia lipolytica* immobilized with sZr-BTB at different benzoic acid ratios as described in Example 4.

[0061] Figure 9 shows the yield of gastrodin produced by fermentation of Yersinia lipolytica in Example 5 under different ratios of 1,2-ethanedisulfonic acid and sZr-BTB.

[0062] Figure 10 shows the yield of gastrodin produced by fermentation using the Yersinia lipolytica immobilized with different masses of sZr-BTB in Example 6.

[0063] Figure 11 shows the gastrodin production at different temperatures using immobilized Yersinia lipolytica from Example 7.

[0064] Figure 12 shows the gastrodin production using immobilized and lipolytic Yersinia in different organic solvents according to Example 8. Detailed Implementation

[0065] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0066] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0067] In the following examples, 1,3,5-tris(4-carboxyphenyl)benzene, benzoic acid, zirconium tetrachloride, dimethylformamide, and 1,2-ethanedisulfonic acid were purchased from Maclean's Reagent Co., Ltd.

[0068] As used in this article, the term "MOF" refers to metal-organic frameworks.

[0069] As used in this article, the term "YPD" refers to yeast extract peptone glucose medium.

[0070] As used in this article, the term "BA" stands for benzoic acid.

[0071] As used in this article, the term "DMF" stands for dimethylformamide.

[0072] As used herein, the term "sZr-BTB" refers to sulfonated Zr-BTB.

[0073] Sugar-containing fermentation medium: 10 g / L yeast extract, 20 g / L peptone, 80 g / L anhydrous glucose, 5 mg / L trace metal elements (4.0 g / L FeSO4·7H2O, 4.0 g / L CaCl2, 1.0 g / L MgCl2)

[0074] The gastrodin-producing Yersinia lipolytica strain used in this embodiment of the invention is YliGT6, provided by Nanjing Normal University. For details, see: A method for increasing the gastrodin yield of recombinant Yersinia lipolytica, CN 117844844A.

[0075] The originating strain of the arbutin-producing Yersinia lipolytica is Y. lipolytica PO1f (ATCC no. MYA-2613). TM )

[0076] The arbutin-producing *Yersinia lipolytica* was engineered in the laboratory. The main steps included: S1. Constructing a heterologous expression plasmid for *Yersinia lipolytica*; S2. Preparing and purifying the seed culture to obtain the arbutin-producing *Yersinia lipolytica* yeast.

[0077] S1 includes the following steps:

[0078] Heterologous expression of the AS gene (SEQ ID NO.1), UbiC gene (SEQ ID NO.2), and MNX1 gene (SEQ ID NO.3) was performed. The exogenous genes used were AS, UbiC, and MNX1, which are used for arbutin synthesis. The YaliBrick plasmid pYLXP' (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) was selected as the backbone. Homologous arms were designed to simultaneously amplify the plasmid backbone and the target fragment by PCR. The fragments were then assembled using the Ginson assembly method to construct the heterologous gene expression plasmids pYLXP'-AS, pYLXP'-UbiC, and pYLXP'-MNX1, respectively.

[0079] The pYLXP'-AS and pYLXP'-MNX1 plasmids were digested with restriction endonucleases Blnl and Sal, and then spliced ​​using T4 ligase to obtain the pYLXP'-AS-MNX1 plasmid. The pYLXP'-AS-MNX1-UbiC plasmid was obtained by ligation with the same restriction endonucleases. The constructed plasmids were transformed into the starter strain: Y. lipolytica po1f (po1gΔura3) using YPD plates (g / L) containing 5-fluoroorotic acid (50 mg / L): yeast extract 10 g, peptone 20 g, glucose 20 g, agar powder 15 g; and cultured using [a specific method / applicator]. Flash KOD DyeMix was used for PCR verification, and successfully transformed single clones were screened for the next step.

[0080] S2. Prepare seed culture and obtain strain:

[0081] Successful transformants were randomly selected from overnight culture plates and inoculated into 3ml LYPD medium: yeast extract 10g, peptone 20g, glucose 25g, and cultured in a shaker at 220rpm and 30℃ for 120h.

[0082] After culturing on a shaker for 120 h, the supernatant was collected by centrifugation at 12000 rpm for 10 min. The sample was prepared by passing it through a 0.22 μm aqueous filter membrane using a syringe. The presence and content of arbutin were determined by liquid chromatography-mass spectrometry (LC-MS). The mobile phase ratio was water (containing 0.05% trifluoroacetic acid): methanol (90:10), the flow rate was 0.7 mL / min, the column temperature was 25 ℃, the detection wavelength was 282 nm, and the injection volume was 20 μL.

[0083] By drawing a standard curve through quantitative analysis, it was determined that the clone could produce high levels of arbutin and continue subsequent fermentation.

[0084] The high-arbutin-producing yeast transformants were streaked onto YPD plates, and single colonies were picked up with sterile toothpicks and inoculated into YPD medium. The culture was carried out in a shaker at 220 rpm and 30°C for 24 h.

[0085] 500 μL of seed culture was inoculated into 30 mL of YPD fermentation broth: yeast extract 10 g, peptone 20 g, glucose 50 g. The culture temperature was 30 °C and the fermentation was carried out continuously at 220 rpm for 120 h.

[0086] OD was measured every 24 hours. 600 The sample was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane for high performance liquid chromatography (HPLC) detection to accurately determine the arbutin yield. This strain was then used as an engineered bacterium for subsequent immobilization operations.

[0087] Example 1

[0088] This embodiment investigates the ability of sZr-BTB to promote the production of aryl glycosides by *Yersinia lipolytica* fermentation. This embodiment uses a *Yersinia lipolytica* strain that produces gastrodin. The preparation steps of sZr-BTB are as follows:

[0089] (1) Dissolve 10 mg zirconium tetrachloride, 12 mg 1,3,5-tris(4-carboxyphenyl)benzene and 450 mg BA in 3 mL DMF, then add 0.5 mL deionized water and sonicate to dissolve. Then heat and stir in a water bath at 120 °C for 24 h, cool, wash 3 times with DMF, 3 times with acetone, 1 time with 0.1 M hydrochloric acid, and 2 times with deionized water, centrifuge at 8,000 rpm for 5 min, and dry to obtain solid Zr-BTB.

[0090] (2) 80 mg of 1,2-ethanedisulfonic acid was dissolved in 4 mL of DMF by sonication and then added to the 40 mg of Zr-BTB obtained above and mixed evenly. The mixture was heated and stirred in a water bath at 100°C for 20 h at a speed of 150 rpm. After cooling, the mixture was washed three times with DMF and three times with acetone. It was then centrifuged at 8,000 rpm for 5 min and dried to obtain sZr-BTB material.

[0091] Preparation of sZr-BTB immobilized gastrodin-producing Yersinia lipolytica (YliGT6):

[0092] (1) Inoculate YliGT from the glycerol tube into 3 mL of YPD seed culture medium, mix well, and place in a constant temperature shaking fermentation shaker at 30℃ and 250 rpm for shaking culture for 24 h.

[0093] (2) Take 1.5 mL of the activated Yersinia lipolytica and inoculate it into 30 mL of fresh sugar-containing fermentation medium. Mix well and place in a constant temperature shaking fermentation incubator at 30℃ and 250 rpm for 24 h. OD 600 It is approximately 0.7.

[0094] (3) Add 1 mg of sZr-BTB to 30 mL of fermentation broth and ferment for 120 h in a constant temperature shaking fermentation incubator at 30 °C and 250 rpm. Take samples every 24 h and freeze the fermentation broth samples at -20 °C. The scanning electron microscope image of the loaded Yersinia lipophila is shown in Figure 1.

[0095] (4) The sample from step (3) was detected using liquid chromatography. High-performance liquid chromatography (HPLC) was performed using a SinoChrom ODS-BP column with a mobile phase of 10% (methanol / water) and a flow rate of 0.7–1 mL / min. The UV detector wavelength was set to 225 nm. The peak values ​​of the main product gastrodin and the byproduct p-hydroxybenzyl alcohol detected by HPLC are shown in Figure 2. The peak of gastrodin appeared around 13.8 min with a peak area of ​​13.624, while the peak of p-hydroxybenzyl alcohol appeared around 19 min with a peak area of ​​19.085 min. The gastrodin yield inferred from the peak values ​​is shown in Figure 3. The ratio of gastrodin to p-hydroxybenzyl alcohol is shown in Figure 4. The results show that the yield of gastrodin gradually increased with time, but decreased after 48 h due to the production of glycosidase.

[0096] Comparative Example 1

[0097] Comparative Example 1 follows the same method as Example 1, except that sZr-BTB material is not added. The fermentation broth is directly fermented in a constant-temperature shaking fermentation incubator at 30℃ and 250rpm for 120h. Samples are taken every 12h and frozen at -20℃. The surface morphology of the unloaded cells is shown in Figure 1. It can be seen that compared with the surface morphology of the unloaded cells, the surface of the immobilized yeast is smooth, proving that the material is successfully adsorbed to the cells. The peak diagram of gastrodin production by unloaded Yersinia lipolytica fermentation is shown in Figure 5. The peak of gastrodin appears at 13.8min with a peak area of ​​13.784, and the peak of the byproduct p-hydroxybenzyl alcohol appears at around 19.2min with a peak area of ​​19.222. The gastrodin yield inferred from the peak values ​​of different samples is shown in Figure 3: the immobilized Yersinia lipolytica has better catalytic activity and a higher gastrodin yield. After 120 hours of fermentation, the immobilized *Yarrowia lipolytica* yielded 6 g / L of gastrodin, while the free yeast yielded only 4 g / L. The ratio of gastrodin to p-hydroxybenzyl alcohol is shown in Figure 4. The use of sulfonated Zr-BTB inhibited the secretion of glycosidase, so the proportion of p-hydroxybenzyl alcohol in the fermentation broth of immobilized yeast was much lower than that in the fermentation broth of free yeast.

[0098] Example 2

[0099] This embodiment investigates the effect of ZrCl4 dosage on the morphology of the material in the synthesis of sZr-BTB, thereby affecting the encapsulation of Yersinia lipolyticis by the material and the yield of aryl glycosides.

[0100] This embodiment uses Yersinia lipolytica (YliGT6), which produces gastrodin. The preparation steps of sZr-BTB are as follows:

[0101] (1) ZrCl4, 12 mg of 1,3,5-tris(4-carboxyphenyl)benzene and 450 mg of BA were dissolved in 3 mL of DMF, followed by the addition of 0.5 mL of deionized water and ultrasonic dissolution. The solution was then heated and stirred in a water bath at 120 °C for 24 h. After cooling, the solution was washed three times with DMF, three times with acetone, once with 0.1 M hydrochloric acid, and twice with deionized water. The solution was then centrifuged at 8,000 rpm for 5 min and dried to obtain solid Zr-BTB.

[0102] The dosage of ZrCl4 was set to 5, 10, 15, and 20 mg.

[0103] (2) 80 mg of 1,2-ethanedisulfonic acid was dissolved in 4 mL of DMF by sonication and then added to the 40 mg of Zr-BTB obtained above and mixed evenly. The mixture was heated and stirred in a water bath at 100°C for 20 h at a speed of 150 rpm. After cooling, the mixture was washed 3 times with DMF and 3 times with acetone. It was then centrifuged at 8,000 rpm for 5 min to obtain sZr-BTB material.

[0104] The subsequent preparation of sZr-BTB immobilized *YaliGT6* was consistent with Example 1. The gastrodin yield is shown in Figure 6. The results indicate that the amount of ZrCl4 affects the morphology of the generated sZr-BTB material, thus affecting its adsorption to *YaliGT6* and consequently altering the yield. Experiments concluded that adding 10 mg of ZrCl4 is the optimal choice for preparing sZr-BTB. After 120 h of fermentation with *YaliGT6*, the gastrodin yield was 6 g / L.

[0105] Example 3

[0106] This example investigated the effect of the amount of 1,3,5-tris(4-carboxyphenyl)benzene on the morphology of the sZr-BTB material during synthesis, thereby affecting the encapsulation of *Yersinia lipolytica* and the yield of aryl glycosides. This example used *Yersinia lipolytica* (YliGT6), a gastrodin-producing yeast. The preparation steps of sZr-BTB were as follows:

[0107] (1) Dissolve 10 mg ZrCl4, 1,3,5-tris(4-carboxyphenyl)benzene and 450 mg BA in 3 mL DMF, then add 0.5 mL deionized water and sonicate to dissolve. Then heat and stir in a water bath at 120 °C for 24 h, cool, wash 3 times with DMF, 3 times with acetone, 1 time with 0.1 M hydrochloric acid, and 2 times with deionized water, then centrifuge at 8,000 rpm for 5 min and dry to obtain solid Zr-BTB.

[0108] The dosage of 1,3,5-tris(4-carboxyphenyl)benzene was set at 6, 12, 24, and 48 mg.

[0109] (2) 80 mg of 1,2-ethanedisulfonic acid was dissolved in 4 mL of DMF by sonication and then added to the 40 mg of Zr-BTB obtained above and mixed evenly. The mixture was heated and stirred in a water bath at 100°C for 20 h at a speed of 150 rpm. After cooling, the mixture was washed 3 times with DMF and 3 times with acetone. It was then centrifuged at 8,000 rpm for 5 min to obtain sZr-BTB material.

[0110] The subsequent preparation of sZr-BTB immobilized Yersinia lipolytica (YliGT) was consistent with Example 1. The gastrodin yield is shown in Figure 7. The results indicate that the amount of 1,3,5-tris(4-carboxyphenyl)benzene affects the morphology of the generated sZr-BTB material, thus affecting its adsorption to Yersinia lipolytica and consequently altering the yield. Experiments concluded that adding 12 mg of 1,3,5-tris(4-carboxyphenyl)benzene is the optimal choice for preparing sZr-BTB. After 120 h of fermentation, the gastrodin yield reached 6.12 g / L.

[0111] Example 4

[0112] This embodiment investigated the effect of BA dosage on the morphology of sZr-BTB during synthesis, thereby affecting the encapsulation of *Yali lipolyticis* and the yield of aryl glycosides. This embodiment used *Yali lipolyticis* (YliGT), a gastrodin-producing yeast. The preparation steps of sZr-BTB were as follows:

[0113] (1) Dissolve 10 mg ZrCl4, 12 mg 1,3,5-tris(4-carboxyphenyl)benzene and BA in 3 mL DMF, then add 0.5 mL deionized water and sonicate to dissolve. Then heat and stir in a water bath at 120 °C for 24 h, cool, wash 3 times with DMF, 3 times with acetone, 1 time with 0.1 M hydrochloric acid, and 2 times with deionized water, centrifuge at 8,000 rpm for 5 min, and dry to obtain solid Zr-BTB.

[0114] The dosage of BA was set at 250, 350, 450, and 550 mg.

[0115] (2) 80 mg of 1,2-ethanedisulfonic acid was dissolved in 4 mL of DMF by sonication and then added to the 40 mg of Zr-BTB obtained above and mixed evenly. The mixture was heated and stirred in a water bath at 100°C for 20 h at a speed of 150 rpm. After cooling, the mixture was washed 3 times with DMF and 3 times with acetone. It was then centrifuged at 8,000 rpm for 5 min to obtain sZr-BTB material.

[0116] The subsequent preparation of sZr-BTB immobilized *YaliGT* was consistent with Example 1. The gastrodin yield is shown in Figure 8. The results indicate that the amount of BA (baicalin) affects the morphology of the generated sZr-BTB material, thus affecting its adsorption to *YaliGT* and consequently altering the yield. Experiments concluded that adding 450 mg of BA is the optimal choice for preparing sZr-BTB. After 120 h of fermentation, the gastrodin yield from *YaliGT* was 6.21 g / L.

[0117] Example 5

[0118] This embodiment investigated the effect of the amount of 1,2-ethanedisulfonic acid providing sulfonic acid groups on the adsorption capacity of *Yergium lipolyticum* in the synthesis of sZr-BTB. Insufficient sulfonic acid groups may lead to unstable and unsustainable adsorption between the material and yeast, while excessive addition of groups may result in insufficient adsorption sites for the material. This embodiment used *Yergium lipolyticum* (YliGT), a gastrodin-producing yeast. The preparation steps of sZr-BTB were as follows:

[0119] (1) Dissolve 10 mg ZrCl4, 12 mg 1,3,5-tris(4-carboxyphenyl)benzene and 450 mg BA in 3 mL DMF, then add 0.5 mL deionized water. Transfer the mixture to a 15 mL pressure-resistant tube and sonicate to dissolve. Then heat and stir in a water bath at 120 °C for 24 h. After cooling, wash three times with DMF, three times with acetone, once with 0.1 M hydrochloric acid, and twice with deionized water. Centrifuge at 8,000 rpm for 5 min and dry to obtain solid Zr-BTB.

[0120] (2) Add 1,2-ethanedisulfonic acid to 4 mL of DMF and sonicate to dissolve. Take 40 mg of the Zr-BTB obtained above and mix evenly. Heat and stir in a water bath at 100℃ for 20 h at a speed of 150 rpm. After cooling, wash with DMF 3 times and acetone 3 times. Centrifuge at 8,000 rpm for 5 min to obtain sZr-BTB material.

[0121] The dosage of 1,2-ethanedisulfonic acid was set at 40, 80, 120, and 240 mg.

[0122] The subsequent preparation of sZr-BTB immobilized Yersinia lipolytica (YliGT) was consistent with Example 1. The gastrodin yield is shown in Figure 9. The results indicate that the ratio of 1,2-ethanedisulfonic acid to Zr-BTB affects the adsorption capacity of sZr-BTB. Experiments concluded that a 1:2 ratio of Zr-BTB to 1,2-ethanedisulfonic acid is optimal for the preparation of sZr-BTB. When 80 mg of sZr-BTB was added, Yersinia lipolytica produced 6.22 g / L of gastrodin after 120 h of fermentation.

[0123] Example 6

[0124] This embodiment investigates the effect of the ratio of sZr-BTB material and the microbial Yersinia lipolyticis on the final product yield when coupled. This embodiment uses Yersinia lipolyticis (YliGT6), which produces gastrodin, and the preparation method of sZr-BTB is the same as in Example 1.

[0125] The preparation steps of sZr-BTB immobilized lipolytic Yersinia oryzae (YliGT6) are as follows:

[0126] (1) Inoculate YliGT from the glycerol tube into 3 mL of YPD seed culture medium, mix well, and place in a constant temperature shaking fermentation shaker at 30℃ and 250 rpm for shaking culture for 24 h.

[0127] (2) Take 1.5 mL of the activated Yersinia lipolytica and inoculate it into 30 mL of fresh sugar-containing fermentation medium. Mix well and place it in a constant temperature shaking fermentation shaker at 30℃ and 250 rpm for 24 h of shaking culture. The OD600 is about 0.7.

[0128] (3) Add sZr-BTB to 30 mL of fermentation broth and ferment for 120 h in a constant temperature shaking fermentation incubator at 30 °C and 250 rpm. Take samples every 24 h and freeze the sampled fermentation broth at -20 °C.

[0129] The dosage of sZr-BTB was set at 0.3, 1, 5, and 10 mg.

[0130] (4) Use liquid chromatography to detect the sample from step (3).

[0131] Figure 10 shows the gastrodin yield. The results indicate that the coupling ratio of sZr-BTB to *Yarrowia lipolytica* affects the main product yield. Too little material may prevent complete encapsulation of the yeast, while too much material will encapsulate the yeast too tightly, reducing yeast activity. Experiments concluded that adding 1 mg of sZr-BTB to 30 mL of fermentation broth is optimal. After adding sZr-BTB, *Yarrowia lipolytica* produced 6.31 g / L of gastrodin after 120 h of fermentation.

[0132] Example 7

[0133] This embodiment tests the activity and yield of Yersinia lipophila immobilized with sZr-BTB at different temperatures in Example 2 of the present invention, and compares it with the activity of free yeast that is not immobilized with sZr-BTB.

[0134] Compared to Example 1, this example differs in that 1 mg of sulfonated Zr-BTB was added to 30 mL of fermentation broth, and fermentation was carried out for 120 h in a constant-temperature shaking fermentation incubator at 35°C and 250 rpm. Samples were taken every 24 h, and the fermentation broth samples were frozen at -20°C. Two other comparison groups were also conducted: fermentation for 120 h in a constant-temperature shaking fermentation incubator at 40°C and 250 rpm; and fermentation for 120 h in a constant-temperature shaking fermentation incubator at 45°C and 250 rpm. Subsequently, free yeast without added materials was fermented at three different temperatures (35°C, 40°C, and 45°C), following the same fermentation steps as in Example 1.

[0135] After fermentation, samples were prepared and analyzed using high-performance liquid chromatography (HPLC). The gastrodin yield after 120 hours of fermentation was calculated based on the peak values, thus inferring the effect of different temperatures on immobilized *Yarrowia lipolytica*. The results are shown in Figure 11. The results show that the gastrodin yield in the fermentation broth decreased after 120 hours with increasing fermentation temperature. However, the gastrodin yield of the immobilized *Yarrowia lipolytica* was significantly higher than that of the free *Yarrowia lipolytica* at a fermentation temperature of 45℃. This demonstrates that the immobilized material provides good protection for the yeast and increases its stability.

[0136] Example 8

[0137] This embodiment tests the activity and yield of Yersinia lipophila immobilized with sZr-BTB under different environments in Example 1 of the present invention, and compares it with the activity of free yeast that is not immobilized with sZr-BTB.

[0138] Compared with Example 1, the difference in this embodiment is that 1 mg of sZr-BTB and 30 mL of methanol were added to 30 mL of fermentation broth, and fermentation was carried out for 120 h in a constant temperature shaking fermentation incubator at 30℃ and 250 rpm. Samples were taken every 24 h, and the fermentation broth samples were frozen at -20℃. The other two comparison groups are as follows: 1 mg of sZr-BTB and 30 mL of ethanol were added to 30 mL of fermentation broth, and fermentation was carried out for 120 h in a constant temperature shaking fermentation incubator at 30℃ and 250 rpm. Samples were taken every 24 h, and the fermentation broth samples were frozen at -20℃; 1 mg of sZr-BTB and 30 mL of dimethyl sulfoxide (DMSO) were added to 30 mL of fermentation broth, and fermentation was carried out for 120 h in a constant temperature shaking fermentation incubator at 30℃ and 250 rpm. Samples were taken every 24 h, and the fermentation broth samples were frozen at -20℃. Subsequently, the free yeast without added materials was fermented in three different organic solvent environments: methanol, ethanol, and DMSO. The fermentation steps were the same as in Example 1.

[0139] After fermentation, samples were prepared and analyzed using high-performance liquid chromatography (HPLC). The gastrodin yield after 120 h of fermentation was calculated based on the peak values, thus inferring the effect of organic solvents on immobilized *Yarrowia lipolytica*. The results are shown in Figure 12. The results indicate that *Yarrowia lipolytica* immobilized with sZr-BTB is more stable in organic solvents compared to free yeast. Although it still affects yeast activity, the gastrodin yield after 120 h of fermentation can still reach 60-70% of the original yield.

[0140] Example 9

[0141] This embodiment investigates the ability of *Yersinia lipolytica* immobilized with sZr-BTB to produce other aryl glycosides. This embodiment uses the arbutin-producing *Yersinia lipolytica* strain constructed above. The difference between this embodiment and Example 1 is that the strain used is changed to an arbutin-producing *Yersinia lipolytica* strain. A control group without sZr-BTB material was also prepared. The results show that, similar to Example 1 and Comparative Example 1, sZr-BTB material increases the catalytic activity of the arbutin-producing *Yersinia lipolytica* strain, inhibits glycosidase secretion, and reduces hydroquinone production.

[0142] In summary, the sulfonated modified material Zr-BTB (sZr-BTB) prepared in this invention plays a crucial role in increasing the yield of aryl glycosides produced by *Yarrowia lipolytica*. During fermentation, free yeast secretes small molecule products such as glycosidases extracellularly, leading to the decomposition of the main glycoside products by these enzymes (Comparative Example 1). Furthermore, *Yarrowia lipolytica* produces a large amount of organic acids during fermentation, causing the pH of the fermentation broth to drop to 2-3. This highly acidic environment is harmful to yeast and can even lead to yeast death. Compared to Comparative Example 1, this invention maintains excellent activity even in highly acidic environments. The sZr-BTB prepared in this invention can compete for hydrogen ions with cell surface groups in highly acidic fermentation broths, thereby achieving sustained physical adsorption of the material to yeast cells and protecting them. Simultaneously, by adsorbing and encapsulating the cells, it prevents yeast from secreting glycosidases extracellularly, inhibiting the decomposition of the main products by enzymes and increasing the yield of microbial fermentation.

Claims

1. A sulfonated modified metal-organic framework material, characterized in that, The sulfonated metal-organic framework material is sulfonated by reacting zirconium (Zr)-based MOF material Zr-BTB with 1,2-ethanedisulfonic acid.

2. A method for preparing a sulfonated modified metal-organic framework material according to claim 1, characterized in that, Includes the following steps: (1) Preparation of metal-organic framework material Zr-BTB: 1,3,5-tris(4-carboxyphenyl)benzene, benzoic acid and zirconium tetrachloride (ZrCl4) were dissolved in an organic solvent; (2) The above solution was added to deionized water, ultrasonically mixed, reacted at high temperature, cooled and washed, and centrifuged and dried to obtain the precursor Zr-BTB material. (3) Add 1,2-ethanedisulfonic acid to the prepared precursor Zr-BTB material. After the high-temperature reaction is completed, centrifuge to collect the precipitate and wash it with water to obtain the sulfonated modified Zr-BTB material, which is the sulfonated modified metal-organic framework material.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass ratio of 1,3,5-tris(4-carboxyphenyl)benzene (H3BTB), benzoic acid (BA), and zirconium tetrachloride (ZrCl4) is 10-13:440-460; 9-12.

4. The preparation method according to claim 2, characterized in that, In step (2), the high temperature is 100-130℃, the reaction time is 20-24h, and the centrifugation speed is 8,000-12,000rpm for 5-10min.

5. The preparation method according to claim 2, characterized in that, In step (3), 1,2-ethanedisulfonic acid is dissolved in an organic solvent. The mass ratio of 1,2-ethanedisulfonic acid to Zr-BTB is 1:1 to 6:

1. The reaction temperature is 90 to 120°C. The reaction time is 15 to 20 hours. The mixture is centrifuged at 8,000 to 12,000 rpm for 5 to 10 minutes.

6. The application of the sulfonated modified metal-organic framework material according to claim 1 in the production of aryl glycosides by Yersinia lipolyticis.

7. The application according to claim 6, characterized in that, The application process is as follows: (1) Inoculate the lipophilic yeast into the seed culture medium for seed culture; (2) Take the seed liquid and inoculate it into the fermentation medium for fermentation culture; (3) Sulfonated modified metal-organic framework materials are added to the fermentation broth and fermentation is continued to increase the production of the target product, aryl glycosides.

8. The application according to claim 7, characterized in that, In step (1), the lipophilic yeast was cultured in YPD seed medium for 24–36 h; in step (2), it was cultured in fermentation medium for 24–36 h.

9. [Amended according to Rule 26, 17.11.2025] The application according to claim 7 is characterized in that, In step (3), the amount of sulfonated modified metal-organic framework material added to the fermentation broth is 0.5 to 1.5 mg per 20-40 mL of fermentation broth. Immobilization begins in the logarithmic growth phase, and fermentation continues for 100-120 h to increase the production of the target product, aryl glycosides.

10. The application according to claim 6, characterized in that, The aryl glycoside compound product preferably includes one or more of the following: gastrodin, arbutin, resveratrol, naringin, baicalin, and vitexin.

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