A sulfonated modified metal-organic framework material, a preparation method thereof and application thereof in yield-increasing application of aryl glycosides

Abstract

The application discloses sulfonated modified metal organic framework material and a preparation method thereof and application of the sulfonated modified metal organic framework material in yield increase of aryl glycoside compounds, wherein the sulfonated modified metal organic framework material is prepared by taking a zirconium-based MOF material Zr-BTB as a precursor and reacting with 1,2 ethanedithoic acid. The prepared sZr-BTB can be fixed on the surface of Yarrowia lipolytica by the principle of physical adsorption, so as to form a dense protective film, so that the relatively large molecular glycosidase is limited in the cell and cannot be secreted, the efflux of the small molecular aryl glycoside compound is not affected, the decomposition of the main biosynthesis product is avoided, meanwhile, the stable adsorption with Yarrowia lipolytica can be maintained under strong acidic conditions, the yield of the fermentation production of the aryl glycoside compound is improved, and the hindering of the non-model microorganism is solved, and the low-cost industrialization of the biosynthesis is promoted.

Description

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-mentioned purpose, the sulfonated modified metal organic framework material provided by the application is prepared by reacting a zirconium (Zr) based MOF material Zr-BTB as a precursor with 1,2 ethanedithoic acid to perform sulfonated modification.

[0006] The preparation method of the sulfonated modified Zr-BTB material provided by the application comprises the following steps:

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

[0008] (2) The above solution is added to deionized water, ultrasonically mixed, and then high-temperature reaction, cooling, washing, centrifugal drying to obtain the precursor Zr-BTB material;

[0009] (3) 1,2-ethanedithoic acid is added to the prepared precursor Zr-BTB material, and after high-temperature reaction, centrifugal collection and water washing, the sulfonated modified Zr-BTB material is obtained, which is a sulfonated modified metal organic framework material.

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

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

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

[0013] As a preferred, the preparation method of the sulfonated modified Zr-BTB material comprises the following steps:

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

[0015] (2) The above solution is added to a certain amount of deionized water, ultrasonically mixed, and then reacted at high temperature in a reaction kettle. After cooling, the material is washed with DMF and acetone, centrifuged and dried to obtain the precursor Zr-BTB material.

[0016] (3) The precursor Zr-BTB material is added to a 1,2-ethanedisulfonic acid solution and reacted at high temperature. Then, the sulfonated Zr-BTB material is collected by centrifugation.

[0017] In step (1), the amount of dimethylformamide 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 washing the material with DMF and acetone, the material is also washed once with 0.1M hydrochloric acid, and then washed 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 10-80 mg. The ratio of the precursor Zr-BTB material to 1,2-ethanedisulfonic acid is 1:1-1:4.

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

[0022] (1) 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) are dissolved in 2-4 mL of dimethylformamide (DMF).

[0023] (2) After centrifuging to remove the supernatant, 0.1M hydrochloric acid solution is added, and the solvent is reacted at 100-130°C in a reaction kettle for 20-24 hours. After cooling, the material is washed with DMF 1-3 times, acetone 1-3 times, 0.1M hydrochloric acid 1 time, and deionized water 1-3 times. The material is centrifuged at 8,000-12,000 rpm for 5-10 minutes, and then dried to obtain the Zr-BTB material.

[0024] (3) 10-200 mg of 1,2-ethanedisulfonic acid is dissolved in 2-5 mL of DMF, and then added to 15-25 mg of the precursor Zr-BTB material. The mixture is reacted at 90-120°C for 15-20 hours. Then, the sulfonated Zr-BTB material is collected by centrifugation at 8,000-12,000 rpm for 5-10 minutes.

[0025] The application provides sulfonated modified metal organic framework materials and application of the sulfonated modified metal organic framework materials in production of aryl glycosides by Yarrowia lipolytica.

[0026] The sulfonated modified metal organic framework materials are combined with Yarrowia lipolytica cells by electrostatic adsorption, the immobilized yeast can inhibit secretion of glycosidase in a fermentation process, and thus the production of aryl glycosides by microbial fermentation is increased. Meanwhile, the sZr-BTB can be stably adsorbed with Yarrowia lipolytica under strong acidic conditions, so that the yeast is protected and the catalytic efficiency is increased.

[0027] The application process is as follows:

[0028] (1) inoculating Yarrowia lipolytica into a seed culture medium to perform seed culture;

[0029] (2) taking the seed liquid to inoculate into a fermentation culture medium to perform fermentation culture;

[0030] (3) adding the sulfonated modified metal organic framework material into the fermentation liquid to realize immobilization of the yeast cells in the logarithmic growth phase, and continuing fermentation to realize yield increase of the target product aryl glycosides.

[0031] In the step (1), the Yarrowia lipolytica is seed cultured in a YPD seed culture medium for 24-36 h; and in the step (2), the fermentation culture is performed for 24-36 h.

[0032] In the step (3), the sulfonated modified metal organic framework material is added in an amount of 0.5-1.5 mg in 20-30 mL of the fermentation liquid, immobilization is started in the logarithmic growth phase after the addition, and the fermentation is continued for 100-120 h to realize yield increase of the target product aryl glycosides.

[0033] The aryl glycoside products include one or more of gastrodin, arbutin, resveratrol, naringin, wild baihuiside and isofraxidin.

[0034] As preferred, the application comprises the following steps:

[0035] (1) inoculating the glycerol tubercle into 3 mL of a yeast extract powder peptone glucose culture medium (YPD) seed culture medium to perform seed culture for 24-36 h under certain temperature and rotation speed conditions.

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

[0037] (3) Add sulfonated modified metal organic framework material to the fermentation broth and continue to ferment for 120 h, sample every 24 h, store the sample fermentation broth in a refrigerator at -20 DEG C, and measure the content of gastrodin.

[0038] In the step (1), the strain is Yarrowia lipolytica. The YPD seed culture medium contains 10 g / L of yeast powder, 20 g / L of peptone, and 80 g / L of anhydrous glucose. The culture temperature is 30 DEG C, and the culture is carried out at a rotation speed of 220-250 rpm.

[0039] In the step (2), the fermentation medium contains 10 g / L of yeast powder, 20 g / L of peptone, 80 g / L of anhydrous glucose, and 5-10 mg / L of trace metal elements (4.0 g / L of FeSO4·7H2O, 4.0 g / L of CaCl2, and 1.0 g / L of MgCl2). The culture temperature is 30 DEG C, and the culture is carried out at a rotation speed of 220-250 rpm.

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

[0041] The present application proposes a method for immobilizing Yarrowia lipolytica by using sulfonated modified metal organic framework material (sZr-BTB). Compared with free yeast, the immobilized yeast is less affected by the outside world during growth and metabolism, and the yeast activity is maintained. Secondly, the immobilized yeast is easy to recover in fermentation or biotransformation, and the microorganism can be reused. The sZr-BTB prepared in the present application is a material with high specific surface area and porosity. The high specific surface area and highly ordered pore structure of sZr-BTB make it have extremely strong adsorption capacity and carrier capacity. At the same time, sZr-BTB has good biocompatibility and does not have adverse effects on the growth, metabolism and activity of cells. The sZr-BTB proposed in the present application can form a protective film on the surface of the yeast by using physical adsorption, so that the glycosidase is limited in the cell and cannot be secreted, and the small molecule products can be excreted, avoiding the decomposition of the biosynthetic aryl glycosides. At the same time, the sulfonated groups on the sZr-BTB material can compete with the groups on the surface of the yeast cells for hydrogen ions in the fermentation broth with strong acidity, so that the material can persistently wrap the yeast and protect the yeast cells.

[0042] In summary, the present application employs sZr-BTB for yeast immobilization technology to improve some major defects of non-model microorganism Yarrowia lipolytica. This method not only effectively avoids the loss of yeast activity, but also enhances adsorption and carrier function through its high specific surface area and pore structure. The sulfonic group of sZr-BTB can provide protection for yeast cells under acidic conditions, prevent the extracellular secretion of glycosidases, and protect aryl glycosides from degradation. The method of the present application significantly improves the stability and efficiency of Yarrowia lipolytica in industrial production, making it show a broader prospect in biotechnology applications.

[0043] During the fermentation of Yarrowia lipolytica, glycosidases and products are secreted extracellularly, and glycosidases decompose part of the biosynthetic aryl glycosides into byproducts, resulting in low yield of main products. Secondly, normal Yarrowia lipolytica is prone to produce organic acid and other byproducts during fermentation, resulting in a significant decrease in the pH of the fermentation broth, which may eventually lead to yeast death. To solve these problems, the material sZr-BTB proposed in the present application can be fixed on the surface of Yarrowia lipolytica by physical adsorption, forming a dense protective film that restricts the secretion of relatively large molecules of glycosidases in the cell, while the efflux of small molecules of aryl glycosides is not affected, thereby avoiding the decomposition of the main product of biosynthesis. Secondly, the sulfonic groups on the sZr-BTB material can compete with the anionic groups on the surface of the yeast for active hydrogen ions in the fermentation broth, thereby enhancing the adsorption capacity of Yarrowia lipolytica and the material under acidic conditions. This layer of protective film also improves the stability of Yarrowia lipolytica cells, increases the catalytic efficiency and reusability. The sulfonated modified Zr-BTB (sZr-BTB) prepared in the present application can maintain stable adsorption with Yarrowia lipolytica under strong acidic conditions, while preventing the secretion of glycosidases and other enzymes in the yeast to the extracellular, thereby improving the yield of aryl glycosides in the fermentation production of aryl glycosides, and solving the obstacles of non-model microorganisms, promoting the low-cost industrialization of biosynthesis.

[0044] The present application is the first to propose a preparation method of sulfonated modified zirconium-based material sZr-BTB, and apply it to the fermentation production of Yarrowia lipolytica to achieve the yield increase of aryl glycosides. The sulfonated modified Zr-BTB material designed in the present application can wrap Yarrowia lipolytica like a membrane, which can limit the secretion of yeast glycosidases while ensuring the efflux of substrates. And because the yeast is wrapped by the material, the yeast can maintain a certain activity in the strongly acidic fermentation broth, increase the yield of aryl glycosides in the fermentation production of Yarrowia lipolytica, and increase the activity of the yeast in harsh environments.

[0045] The prior art cannot solve the problem of controlling the secretion of glycosidase of Yarrowia lipolytica in the fermentation process, resulting in the decomposition of the main product aryl glycosides by glycosidase, and the acidity of the fermentation broth of the yeast is about pH 2-3, and the harsh environment leads to the decrease of the activity of the yeast. The sZr-BTB material provided in the present application can wrap the yeast, prevent the secretion of glycosidase, and protect the yeast in the acidic fermentation broth and maintain a high activity.

[0046] Advantages: Compared with the prior art, the present application has the following advantages:

[0047] 1. The present application selects Zr-BTB material, and zirconium metal is considered to be low or even non-toxic, compared with other metal organic framework materials, Zr-BTB is a 2D-MOF material, which has a larger planar size, a thinner thickness and a higher water stability. This means that Zr-BTB is easy to embed yeast cells, and the structure is not easy to be damaged.

[0048] 2. The present application uses Yarrowia lipolytica for microbial fermentation to produce aryl glycosides, which has the advantages of high yield, easy to expand culture, short culture period and the like, and is more suitable for industrialized production of aryl glycosides.

[0049] 3. The present application combines MOF nanosheet on Yarrowia lipolytica by physical adsorption, compared with free yeast, the fixed yeast is more stable in the metabolic process. And the physical adsorption of yeast and material makes the product can be easily separated from the yeast, simplifying the subsequent separation and purification process.

[0050] 4. Yarrowia lipolytica produces many organic acids during fermentation, compared with conventional metal organic framework materials, Zr-BTB is more stable in acidic environment, so that the material can maintain its structural integrity for a long time under acidic conditions and will not disintegrate.

[0051] 5. The present application proposes a method for modifying Zr-BTB after synthesis using 1,2-ethanedisulfonic acid, one sulfonic group of 1,2-ethanedisulfonic acid coordinates with zirconium metal on Zr-BTB, and the other sulfonic group is used to compete with hydrogen ions on the surface of yeast cells under acidic conditions, so as to ensure that sZr-BTB can maintain stable adsorption with Yarrowia lipolytica for a long time under strong 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 The images show a comparison of scanning electron microscope images of immobilized yeast and free yeast provided in Example 1 and Comparative Example 1.

[0054] Figure 2 The high-performance liquid chromatography (HPLC) spectra of gastrodin and p-hydroxybenzyl alcohol in Example 1 are used.

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

[0056] Figure 4 The ratio of gastrodin to p-hydroxybenzyl alcohol used in Example 1 and Comparative Example 1 is shown in the graph.

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

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

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

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

[0061] Figure 9 Figure 5 is a graph of ginsenoside production by Yarrowia lipolytica immobilized with sZr-BTB with different ratios of 1,2-ethanedisulfonic acid, according to Example 5;

[0062] Figure 10 Figure 6 is a graph of ginsenoside production by Yarrowia lipolytica immobilized with sZr-BTB with different masses, according to Example 6;

[0063] Figure 11 Figure 7 is a graph of ginsenoside production by immobilized Yarrowia lipolytica at different temperatures, according to Example 7;

[0064] Figure 12 Figure 8 is a graph of ginsenoside production by immobilized Yarrowia lipolytica at different organic solvents, according to Example 8. DETAILED DESCRIPTION

[0065] The present application is further described in the following examples and by reference to the accompanying drawings.

[0066] The materials, reagents and the like used in the examples of the present application are commercially available unless otherwise specified. The experimental methods in the examples not marked with specific conditions are usually according to the conventional conditions, or according to the conditions suggested 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 Macklin Reagent Co., Ltd.

[0068] As used herein, the term "MOF" refers to a metal organic framework.

[0069] As used herein, the term "YPD" is yeast extract powder peptone glucose medium.

[0070] As used herein, the term "BA" is benzoic acid.

[0071] As used herein, the term "DMF" is dimethylformamide.

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

[0073] Sugar-containing fermentation medium: 10 g / L yeast powder, 20 g / L peptone, 80 g / L anhydrous glucose, 5 mg / L trace metal elements (4.0 g / L FeS04·7H20, 4.0 g / L CaCl2, 1.0 g / L MgCl2)

[0074] The Yarrowia lipolytica for producing gastrodin used in the embodiments of the present application is YliGT6 strain, which is provided by Nanjing Normal University, and details are shown in: a method for improving the production of gastrodin by recombinant Yarrowia lipolytica, CN 117844844A.

[0075] The starting strain of Yarrowia lipolytica for producing arbutin is Y.lipolytica PO1f (ATCC no. MYA-2613 TM )

[0076] The Yarrowia lipolytica for producing arbutin is engineered in the laboratory, and the main operation steps include: S1. Constructing a Yarrowia lipolytica heterologous expression plasmid; S2. Preparing a seed solution and purifying to obtain the Yarrowia lipolytica for producing arbutin.

[0077] S1 includes the following steps:

[0078] Heterologous expression of AS gene (SEQ ID NO. 1), UbiC gene (SEQ ID NO. 2) and MNX1 gene (SEQ ID NO. 3). The foreign genes use AS, UbiC and MNX1, which are used for the synthesis of arbutin. The YaliBrick plasmid pYLXP' (purchased from Hangzhou Baosai Biological Technology Co., Ltd.) is selected as the backbone, and the homologous arms are designed to perform PCR amplification on the plasmid backbone and the target fragment, and then the fragment splicing is performed by the Ginson assembly method to construct the heterologous gene expression plasmids pYLXP'-AS, pYLXP'-UbiC and pYLXP'-MNX1.

[0079] The pYLXP'-AS and pYLXP'-MNX1 plasmids digested by restriction endonuclease Blnl and Sall are spliced by T4 ligase to obtain the plasmid pYLXP'-AS-MNX1, and the same method is used for enzyme digestion and ligation to obtain the pYLXP'-AS-MNX1-UbiC plasmid. The constructed plasmid is transformed into the starting strain: Y.lipolytica po1f (po1gΔura3) using a YPD plate (g / L) containing 5-fluoroorotic acid (50 mg / L): yeast powder 10, peptone 20, glucose 20, agar powder 15; and the coating culture is performed, and 2x Flash KOD DyeMix is used for PCR verification, and the single clone with successful transformation is selected for the next operation.

[0080] S2. Preparation of seed solution and obtaining of the strain:

[0081] Randomly pick the successful transformants from the overnight culture plate, inoculate in 3ml YPD medium: yeast powder 10, peptone 20, glucose 25, 30°C, 220rpm, 120h.

[0082] After 120h of shake culture, centrifuge at 12000rpm for 10min to collect the supernatant, use a syringe to sample through a 0.22μm water filter, then use a Liquid Chromatograph Mass Spectrometer to determine the presence of arbutin and measure the arbutin content, with a mobile phase ratio of water (containing 0.05% trifluoroacetic acid): methanol (90:10), a flow rate of 0.7mL / min, a column temperature of 25°C, a detection wavelength of 282nm, and a sample injection volume of 20μL.

[0083] Draw a standard curve by quantitative analysis to determine that the clones can produce arbutin and continue to ferment.

[0084] Inoculate the high-yield arbutin yeast transformants obtained on a YPD plate, pick a single colony with a sterile toothpick and inoculate in YPD medium, 30°C, 220rpm, 24h.

[0085] Inoculate 500ul of seed liquid into 30ml of YPD fermentation medium: yeast powder 10, peptone 20, glucose 50, culture temperature 30°C, 220rpm, continuous fermentation for 120h.

[0086] Take samples every 24h to measure OD 600 , centrifuge the sample, 12000rpm for 10min to collect the supernatant, sample through a 0.22μm water filter for HPLC detection to accurately measure the arbutin yield, and use the strain as an engineering strain for subsequent immobilization operation.

[0087] Example 1

[0088] This example explores the ability of sZr-BTB to promote the fermentation of arbuscular yeasts to produce aryl glycosides. In this example, a ginsenoside-producing arbuscular yeast strain is used, and the preparation steps of sZr-BTB are as follows:

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

[0090] (2) 80 mg of 1,2-ethanedisulfonic acid was dissolved in 4 mL of DMF by ultrasonic, and was added to the above obtained 40 mg of Zr-BTB to mix uniformly, and was heated and stirred at 100°C for 20 h in a water bath at a rotation speed of 150 rpm. After cooling, it was washed with DMF for 3 times, washed with acetone for 3 times, and centrifuged at 8,000 rpm for 5 min, and dried to obtain the sZr-BTB material.

[0091] Preparation of Y. lipolytica (YliGT6) immobilized with sZr-BTB to produce gastrodin:

[0092] (1) YliGT was inoculated into 3 mL of YPD seed culture medium in a glycerol tube, mixed uniformly, and placed in a constant temperature shaking fermentation incubator at 30°C and 250 rpm, and shaken and cultured for 24 h.

[0093] (2) After activation, 1.5 mL of Y. lipolytica was inoculated into 30 mL of fresh sugar-containing fermentation medium, mixed uniformly, and placed in a constant temperature shaking fermentation incubator at 30°C and 250 rpm, and shaken and cultured for 24 h. The OD 600 was about 0.7.

[0094] (3) 1 mg of sZr-BTB was added to 30 mL of the fermentation liquid, and was fermented in a constant temperature shaking fermentation incubator at 30°C and 250 rpm for 120 h. Every 24 h, the sample was taken and stored in a refrigerator at -20°C. The scanning electron microscope image of the Y. lipolytica after loading is shown in Figure 1 .

[0095] (4) The sample in step (3) was detected by liquid phase. High performance liquid chromatography used SinoChrom ODS-BP column, and the mobile phase was 10% (methanol / water) at a flow rate of 0.7-1 mL / min. The wavelength of the ultraviolet detector was set to 225 nm. The peak values of the main product gastrodin and the by-product p-hydroxybenzyl alcohol detected by the detected liquid chromatography are shown in Figure 2 . The peak of gastrodin appeared at about 13.8 min, and the peak area was 13.624, while the peak of p-hydroxybenzyl alcohol appeared at about 19 min, and the peak area was 19.085 min. The yield of gastrodin inferred from the peak value is shown in Figure 3 . The ratio of gastrodin and p-hydroxybenzyl alcohol is shown in Figure 4 . It can be seen from the results that the yield of gastrodin gradually increases with time, but after 48 h, the yield of gastrodin decreases due to the production of glycosidase.

[0096] Comparative Example 1

[0097] The comparative example 1 is the same as the method of example 1, the difference is that the sZr-BTB material is not added, and the fermentation liquid is directly fermented in a constant temperature shaking fermentation incubator at 30℃, 250rpm for 120h. The sample is taken every 12h, and the sample fermentation liquid is stored in a refrigerator at -20℃. The surface morphology of the unloaded cell is shown in Figure 1 . It can be seen that compared with the surface morphology of the unloaded cell, the fixed yeast surface is smooth, which proves that the material and the cell are successfully adsorbed together. The peak chart of the fermentation of ginsenoside by the unloaded Yarrowia lipolytica is shown in Figure 5 . The peak of ginsenoside appears at 13.8min, and the peak area is 13.784. The peak of the by-product p-hydroxybenzyl alcohol appears at about 19.2min, and the peak area is 19.222. The ginsenoside yield inferred from the peak values of different samples is shown in Figure 3 . The immobilized Yarrowia lipolytica has better catalytic activity, and the yield of ginsenoside is higher. After 120h of fermentation, the yield of immobilized Yarrowia lipolytica reaches 6g / L, while the yield of ginsenoside of free yeast only reaches 4g / L. The ratio of ginsenoside and p-hydroxybenzyl alcohol is shown in Figure 4 . The use of sulfonated modified Zr-BTB inhibits the secretion of glycosidase, so the yield of p-hydroxybenzyl alcohol in the fermentation broth of immobilized yeast is much lower than that in the fermentation broth of free yeast.

[0098] Example 2

[0099] This example investigates the effect of the amount of ZrCl4 used in the synthesis of sZr-BTB on the morphology of the material, thereby affecting the wrapping of Yarrowia lipolytica and the yield of aryl glycosides.

[0100] In this example, ginsenoside-producing Yarrowia lipolytica (YliGT6) is used, and the preparation steps of sZr-BTB are as follows:

[0101] (1) Dissolve ZrCl4, 12mg of 1,3,5-tris(4-carboxylphenyl)benzene and 450mg of BA in 3mL of DMF, then add 0.5mL of deionized water and ultrasonic dissolution. Then heat and stir in a 120℃ water bath for 24h, after cooling, wash with DMF for 3 times, acetone for 3 times, 0.1M hydrochloric acid for 1 time, deionized water for 2 times, then centrifuge at 8,000rpm for 5min, and dry to obtain solid Zr-BTB.

[0102] Among them, the amount of ZrCl4 used is set to 5, 10, 15 and 20mg.

[0103] (2) 80 mg of 1,2-ethanedisulfonic acid was added to 4 mL of DMF and ultrasonically dissolved, and then added to the 40 mg of Zr-BTB obtained above to mix uniformly, heated and stirred at 100°C for 20 h with a water bath, at a rotation speed of 150 rpm, washed with DMF 3 times after cooling, washed with acetone 3 times, centrifuged at 8,000 rpm for 5 min, and the sZr-BTB material was obtained.

[0104] The preparation of the subsequent sZr-BTB immobilized Yarrowia lipolytica (YliGT6) was consistent with that of Example 1. The production of gastrodin is shown in Table 2. Figure 6

[0105] Example 3

[0106] This example investigates the effect of the amount of 1,3,5-tris(4-carboxyphenyl) benzene on the morphology of the sZr-BTB material, thereby affecting the wrapping of Yarrowia lipolytica and the production of aryl glycosides. Yarrowia lipolytica (YliGT6) producing gastrodin was used in this example, and the preparation steps of sZr-BTB were as follows:

[0107] (1) 10 mg of ZrCl4, 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 for ultrasonic dissolution. Then, heating and stirring were performed at 120°C for 24 h with a water bath, and after cooling, the solid Zr-BTB was obtained by washing with DMF 3 times, washing with acetone 3 times, washing with 0.1 M hydrochloric acid once, washing with deionized water twice, and centrifuging at 8,000 rpm for 5 min.

[0108] Among them, the amount of 1,3,5-tris(4-carboxyphenyl) benzene used was set to 6, 12, 24 and 48 mg.

[0109] (2) 80 mg of 1,2-ethanedisulfonic acid was added to 4 mL of DMF and ultrasonically dissolved, and then added to the 40 mg of Zr-BTB obtained above to mix uniformly, heated and stirred at 100°C for 20 h with a water bath, at a rotation speed of 150 rpm, washed with DMF 3 times after cooling, washed with acetone 3 times, centrifuged at 8,000 rpm for 5 min, and the sZr-BTB material was obtained.

[0110] The preparation of the subsequent sZr-BTB immobilized Yarrowia lipolytica (YliGT) was consistent with that of Example 1. The production of gastrodin is shown in Table 2. Figure 7 ​As shown, according to the results, the amount of 1,3,5-tri(4-carboxylphenyl) benzene has an effect on the morphology of the generated sZr-BTB material, thereby affecting the adsorption of Yarrowia lipolytica, resulting in a change in the yield. Through experiments, it can be concluded that the addition of 12 mg of 1,3,5-tri(4-carboxylphenyl) benzene is the optimal choice in the preparation of sZr-BTB. After 120 h of fermentation, the production of gastrodin reached 6.12 g / L.

[0111] Example 4

[0112] This example investigates the effect of the amount of BA on the morphology of the material in the synthesis of sZr-BTB, thereby affecting the wrapping of Yarrowia lipolytica by the material and affecting the yield of aryl glycosides. This example uses Yarrowia lipolytica (YliGT) to produce gastrodin, and the preparation steps of sZr-BTB are as follows:

[0113] (1) 10 mg of ZrCl4, 12 mg of 1,3,5-tri(4-carboxylphenyl) benzene and BA were dissolved in 3 mL of DMF, then 0.5 mL of deionized water was added and ultrasonic dissolution was performed. Then, 24 h of stirring was performed at 120°C water bath heating, and after cooling, 3 times of DMF washing, 3 times of acetone washing, 1 time of 0.1M hydrochloric acid washing, and 2 times of deionized water washing were performed, followed by 8,000 rpm centrifugation for 5 min, and drying to obtain solid Zr-BTB.

[0114] Among them, the amount of BA used is set to 250, 350, 450, 550 mg.

[0115] (2) 80 mg of 1,2-ethanedithioic acid was added to 4 mL of DMF and ultrasonic dissolution was performed, and then it was added to the above obtained 40 mg of Zr-BTB and uniformly mixed, 20 h of stirring was performed at 100°C water bath heating, the rotation speed was 150 rpm, after cooling, 3 times of DMF washing, 3 times of acetone washing, 8,000 rpm centrifugation for 5 min were performed, and sZr-BTB material was obtained.

[0116] The subsequent preparation of sZr-BTB immobilized Yarrowia lipolytica (YliGT) is consistent with Example 1. The yield of gastrodin is as Figure 8 As shown, according to the results, the amount of BA has an effect on the morphology of the generated sZr-BTB material, thereby affecting the adsorption of Yarrowia lipolytica, resulting in a change in the yield. Through experiments, it can be concluded that the addition of 450 mg of BA is the optimal choice in the preparation of sZr-BTB. After 120 h of fermentation, the yield of gastrodin produced by Yarrowia lipolytica was 6.21 g / L.

[0117] Example 5

[0118] This embodiment explores the effect of the amount of 1,2-ethanedithioic acid on the adsorption capacity of sZr-BTB for Yarrowia lipolytica. If the amount of sulfonic acid groups is not enough, the material may not be able to adsorb the yeast stably and durably. If too many groups are added, the material may not have enough sites to adsorb the yeast. This embodiment uses Yarrowia lipolytica (YliGT) to produce gastrodin. The preparation steps of sZr-BTB are as follows:

[0119] (1) 10 mg of 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. The reaction was carried out in a 15 mL pressure tube under ultrasonic dissolution. Then, it was heated and stirred in a 120°C water bath for 24 h. After cooling, it was washed with DMF 3 times, acetone 3 times, 0.1 M hydrochloric acid 1 time, and deionized water 2 times, and then centrifuged at 8,000 rpm for 5 min. The solid Zr-BTB was obtained after drying.

[0120] (2) 1,2-ethanedithioic acid was added to 4 mL of DMF and ultrasonically dissolved. 40 mg of the above-obtained Zr-BTB was uniformly mixed, heated and stirred in a 100°C water bath for 20 h at a speed of 150 rpm. After cooling, it was washed with DMF 3 times, acetone 3 times, and centrifuged at 8,000 rpm for 5 min to obtain the sZr-BTB material.

[0121] Among them, the amount of 1,2-ethanedithioic acid used is set to 40, 80, 120, and 240 mg.

[0122] The subsequent preparation of sZr-BTB immobilized Yarrowia lipolytica (YliGT) is consistent with Example 1. The gastrodin yield is shown in Figure 9 According to the results, the ratio of 1,2-ethanedithioic acid to Zr-BTB has an effect on the adsorption capacity of sZr-BTB. Through experiments, it can be concluded that the optimal ratio of Zr-BTB to 1,2-ethanedithioic acid in the preparation of sZr-BTB is 1:2. When the amount of sZr-BTB added is 80 mg, Yarrowia lipolytica produces 6.22 g / L of gastrodin after 120 h of fermentation.

[0123] Example 6

[0124] This embodiment explores the effect of the ratio of sZr-BTB material and Yarrowia lipolytica on the yield of the final product. This embodiment uses Yarrowia lipolytica (YliGT6) to produce gastrodin. The preparation method of sZr-BTB is consistent with Example 1.

[0125] The preparation steps of sZr-BTB immobilized Yarrowia lipolytica (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] Gastrodin production such as Figure 10 As shown in the results, the coupling ratio of sZr-BTB to *Yarrowia lipolytica* affects the yield of the main product. Too little material may result in incomplete encapsulation of the yeast, while too much material will encapsulate the yeast too tightly, reducing yeast activity. The experiment 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, sample preparation was performed using high performance liquid chromatography detection, and the gastrodin yield after 120 h of fermentation was calculated according to the peak value, so as to infer the influence of different temperatures on the fixed Yarrowia lipolytica, and the results are shown in Figure 11 From the results, it can be seen that with the increase of the fermentation temperature, the gastrodin yield in the fermentation broth after 120 h is reduced. However, the gastrodin yield of the material-fixed Yarrowia lipolytica at the fermentation temperature of 45 ℃ is much higher than that of the free Yarrowia lipolytica, which can prove that the material has a good protective effect on the yeast and increases the stability of the yeast.

[0136] Example 8

[0137] In this example, the activity and yield of the Yarrowia lipolytica fixed by sZr-BTB in Example 1 under different environments were tested, and the activity of the free yeast not fixed by sZr-BTB was compared.

[0138] In this example, compared with Example 1, the difference lies in that 30 mL of the fermentation broth is added with 1 mg of sZr-BTB and 30 mL of methanol, and the fermentation is performed in a constant temperature shaking fermentation incubator at 30 ℃ and 250 rpm for 120 h. The sample is taken every 24 h, and the sample fermentation broth is stored in a refrigerator at -20 ℃. Another two groups are compared, that is, 30 mL of the fermentation broth is added with 1 mg of sZr-BTB and 30 mL of ethanol, and the fermentation is performed in a constant temperature shaking fermentation incubator at 30 ℃ and 250 rpm for 120 h, and the sample is taken every 24 h, and the sample fermentation broth is stored in a refrigerator at -20 ℃; 30 mL of the fermentation broth is added with 1 mg of sZr-BTB and 30 mL of dimethyl sulfoxide (DMSO), and the fermentation is performed in a constant temperature shaking fermentation incubator at 30 ℃ and 250 rpm for 120 h. The sample is taken every 24 h, and the sample fermentation broth is stored in a refrigerator at -20 ℃. Subsequently, the free yeast without the material is fermented in the environments of methanol, ethanol and DMSO, and the fermentation steps are the same as those in Example 1.

[0139] After fermentation, sample preparation was performed using high performance liquid chromatography detection, and the gastrodin yield after 120 h of fermentation was calculated according to the peak value, so as to infer the influence of different temperatures on the fixed Yarrowia lipolytica, and the results are shown in Figure 12 According to the results, compared with the free yeast, the Yarrowia lipolytica fixed by sZr-BTB is more stable in the organic solvent, although the activity of the yeast is still affected, but the gastrodin yield after 120 h of fermentation can still reach 60-70% of the original.

[0140] Example 9

[0141] The present example explores the ability of sZr-BTB to immobilize Yarrowia lipolytica to produce other aryl glycosides. The Yarrowia lipolytica producing arbutin used in the present example is constructed as described above. The difference between the present example and Example 1 is that the strain in the step is changed to Yarrowia lipolytica producing arbutin. At the same time, a comparative example without sZr-BTB material is prepared. The results show that, similar to Example 1 and Comparative Example 1, the sZr-BTB material increases the catalytic activity of Yarrowia lipolytica producing arbutin, inhibits the secretion of glycosidase, and reduces the production of hydroquinone.

[0142] In summary, the sulfonated modified material Zr-BTB (sZr-BTB) prepared in the present application is used to immobilize Yarrowia lipolytica to produce aryl glycosides, which plays an important role in increasing the yield of the product. Free yeast secretes small molecule products such as glycosidase into the extracellular during fermentation, which leads to the decomposition of the main product glycosides by glycosidase (Comparative Example 1). In addition, Yarrowia lipolytica produces a large amount of organic acid during fermentation, which leads to a decrease in the pH of the fermentation broth to 2-3. The strong acidic environment can harm the yeast, and even cause the yeast to die. Compared with Comparative Example 1, the present application can still maintain excellent activity in a strong acid environment. The sZr-BTB prepared in the present application can compete with the hydrogen ions on the surface of the cell in the strong acid fermentation broth, thereby persistently adsorbing the material to the yeast cell, thereby protecting the yeast cell. At the same time, by adsorbing and embedding the cell, the yeast is prevented from secreting glycosidase into the extracellular, which inhibits the decomposition of the main product by the enzyme, and increases the yield of microbial fermentation.

Claims

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

2. A method of preparing a sulfonated modified metal-organic framework material according to claim 1, characterized in that, The method comprises the following steps: (1) preparing a metal organic framework material Zr-BTB: dissolving 1,3,5-tri(4-carboxyphenyl) benzene, benzoic acid and zirconium tetrachloride (ZrCl4) in an organic solvent; (2) adding the above solution into deionized water, uniformly mixing under ultrasonic, high-temperature reaction, cooling and washing, centrifugal drying to obtain a precursor Zr-BTB material; (3) adding 1,2-ethanedithioic acid into the prepared precursor Zr-BTB material, centrifugal collecting the precipitate after high-temperature reaction, and water washing to obtain a sulfonated modified Zr-BTB material, i.e. the sulfonated modified metal organic framework material.

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

4. The production method according to claim 2, characterized by, In step (2), the high-temperature temperature is 100-130℃, the reaction time is 20-24h, and the centrifugal 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-ethanedithioic acid is dissolved in an organic solvent, the mass ratio of 1,2-ethanedithioic acid and Zr-BTB is 1:1-6:1, the reaction temperature is 90-120℃, the reaction time is 15-20h, and the centrifugal speed is 8,000-12,000rpm for 5-10min.

6. The sulfonated modified metal organic framework material of claim 1 is applied to the production of aryl glycosides compounds by Yarrowia lipolytica.

7. Use according to claim 6, characterized in that, The application process is as follows: (1) inoculating Yarrowia lipolytica into a seed culture medium for seed culture; (2) taking the seed liquid to inoculate into a fermentation culture medium for fermentation culture; (3) adding the sulfonated modified metal organic framework material into the fermentation liquid for further fermentation to realize the yield increase of the target product aryl glycosides compounds.

8. Use according to claim 7, characterized in that, In step (1), the Yarrowia lipolytica is seed cultured in a YPD seed culture medium for 24-36h; in step (2), the fermentation culture is carried out in a fermentation culture medium for 24-36h.

9. Use according to claim 7, characterized in that, In step (3), the sulfonated modified metal organic framework material is added in an amount of 0.5-1.5mg per 20-40mL of the fermentation liquid, and is immobilized in the logarithmic growth phase, and the fermentation is continued for 100-120h to realize the yield increase of the target product aryl glycosides compounds.

10. Use according to claim 6, characterized in that, The aryl glycosides compound products include one or more of gastrodin, arbutin, resveratrol, naringin, wild baicalin and isofraxidin.

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