Sulfonated modified metal organic framework material, preparation method thereof and application of sulfonated modified metal organic framework material in yield increase application of aryl glycoside compounds

Abstract

The invention discloses a sulfonation modified metal organic framework material, a preparation method thereof and application of the sulfonation modified metal organic framework material in yield increase application of aryl glycoside compounds, and the sulfonation modified metal organic framework material takes a zirconium-based MOF material Zr-BTB as a precursor to react with 1, 2-ethanedisulfonic acid so as to carry out sulfonation modification. According to the sZr-BTB prepared by the invention, the material can be fixed on the surface of yarrowia lipolytica through a physical adsorption principle to form a layer of compact protective film, so that relatively macromolecular glycosidase is limited in cells and cannot be secreted, the excretion of small molecular aryl glycoside compounds is not influenced, the decomposition of a biosynthetic main product is avoided, and the yield of the yarrowia lipolytica is improved. Meanwhile, stable adsorption with yarrowia lipolytica can be maintained under a strong acidic condition, and the yield of aryl glycoside compounds produced by fermentation can be increased, so that the obstruction of non-mode microorganisms is solved, and low-cost industrialization of biosynthesis is promoted.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering, and specifically relates to a sulfonated modified metal organic framework material, a preparation method thereof, and an application thereof in increasing the production of aromatic glycoside compounds. Background Art

[0002] As the core of modern industrial biotechnology, microbial strains play an increasingly important role in the industrial field. The technology of microorganisms catalyzing reactions through their own metabolism is many times more efficient than chemical catalytic reactions. At the same time, biocatalysis can react at room temperature, and the catalytic action is specific, with advantages such as low energy consumption and high safety. Model microorganisms such as Escherichia coli and Saccharomyces cerevisiae have long been considered ideal microbial chassis. However, in recent years, due to technological breakthroughs, non-model microorganisms have become the chassis for the synthesis of many natural products. These non-model microorganisms have special physiological and metabolic characteristics, can provide rich genetic resources, and are conducive to the development of new products. Yarrowia lipolytica is an unconventional hemisaccharomyces yeast and is considered to be an excellent natural synthetic host. Yarrowia lipolytica can use glucose as a substrate for the synthesis of aromatic glycoside compounds. However, the use of Yarrowia lipolytica to ferment aromatic glycoside compounds has drawbacks that cannot be ignored. During the growth process, Yarrowia lipolytica often produces byproducts such as organic acids, which causes the pH value of the culture medium to drop to an extremely acidic level (pH 2-3). In a strong acid environment, the yeast is prone to lose activity. At the same time, during the fermentation process of Yarrowia lipolytica, hydrolases such as glycosidases and small molecule products are secreted into the extracellular space at the same time. Glycosidases can hydrolyze aromatic glycoside compounds into by-products, resulting in a decrease in the yield of the main product. These problems have hindered the development of industrial production of aromatic glycoside compounds using Yarrowia lipolytica. Summary of the invention

[0003] Purpose of the invention: In view of the shortcomings of Yarrowia lipolytica in producing aromatic glycoside compounds by biological fermentation, the present invention provides a sulfonated modified metal organic framework material. The sulfonated modified metal organic framework material prepared by the present invention is a novel zirconium-based MOF material sulfonated modified Zr-BTB (sZr-BTB) used for fixing Yarrowia lipolytica, wherein sZr-BTB can prevent the secretion of hydrolases such as glycosidases by the yeast during the fermentation process during the adsorption of Yarrowia lipolytica, thereby reducing the degradation of the product. At the same time, sZr-BTB can continuously and stably adsorb to the yeast surface under acidic conditions, and can provide long-term protection for Yarrowia lipolytica under strongly acidic conditions, effectively solving the problems of low yield of aromatic glycoside compounds produced by fermentation of Yarrowia lipolytica and low yeast activity.

[0004] The invention also provides a preparation method and application of the sulfonated modified metal organic framework material.

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

[0006] The method for preparing the sulfonated Zr-BTB material of the present invention comprises the following steps:

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

[0008] (2) adding the above solution to deionized water, mixing by ultrasonication, reacting at high temperature, cooling, washing, and centrifugally drying to obtain a 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 Zr-BTB material, which is the sulfonated metal organic framework material.

[0010] Wherein, in step (1), 1,3,5-tri(4-carboxyphenyl)benzene (H 3 BTB), benzoic acid (BA) and zirconium tetrachloride (ZrCl 4 )'s mass ratio is 10~13:440~460:9~12.

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

[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 hours, and the centrifugation speed is 8,000 to 12,000 rpm for 5 to 10 minutes.

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

[0014] (1) 1,3,5-tri(4-carboxyphenyl)benzene (H3BTB), benzoic acid (BA) and zirconium tetrachloride (ZrCl 4 ) was dissolved in dimethylformamide (DMF).

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

[0016] (3) Adding the precursor Zr-BTB material into a 1,2-ethanedisulfonic acid solution for high temperature reaction, and then collecting the sulfonated Zr-BTB material by centrifugation.

[0017] Wherein, the amount of dimethyl amide used in step (1) is 2 to 4 mL; H 3 BTB, BA, ZrCl 4 The dosages used are 10-13 mg, 440-460 mg and 9-12 mg respectively.

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

[0019] Preferably, after the material is cleaned with DMF and acetone, the material is cleaned once with hydrochloric acid having a concentration of 0.1 M, and then the hydrochloric acid is washed away with deionized water.

[0020] Among them, the amount of Zr-BTB material used in step (3) is 15-25 mg, the amount of 1,2-ethanedisulfonic acid used is 10 mg-80 mg, and 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 sulfonation modification include the following steps:

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

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

[0024] (3) Dissolve 10 mg to 200 mg of 1,2-ethanedisulfonic acid in 2 to 5 mL of DMF, 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, and then centrifuge at 8,000 to 12,000 rpm for 5 to 10 min to collect the sulfonated Zr-BTB material.

[0025] The invention discloses an application of the sulfonated modified metal organic framework material in the production of aromatic glycoside compounds by Yarrowia lipolytica.

[0026] The sulfonated metal organic framework material of the present invention is combined with Yarrowia lipolytica cells by electrostatic adsorption, and the immobilized yeast can inhibit the secretion of glycosidase during the fermentation process, thereby achieving the increase in the production of aromatic glycoside compounds produced by microbial fermentation. At the same time, sZr-BTB can stably adsorb with Yarrowia lipolytica under strong acidic conditions, thereby protecting the yeast and increasing the catalytic efficiency.

[0027] The application process is as follows:

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

[0029] (2) taking the seed liquid and inoculating it into a fermentation medium for fermentation culture;

[0030] (3) The sulfonated metal organic framework material is added to the fermentation broth to achieve the immobilization of yeast cells in the logarithmic growth phase, and the fermentation is continued to achieve the increased production of the target product aromatic glycoside compounds.

[0031] Wherein, in step (1), the Yarrowia lipolytica is cultured in a YPD seed culture medium for 24 to 36 hours; and in step (2), the Yarrowia lipolytica is cultured in a fermentation culture medium for 24 to 36 hours.

[0032] In step (3), the amount of sulfonated metal organic framework material added to the fermentation broth is 0.5-1.5 mg of sulfonated Zr-BTB material added to every 20-30 mL of fermentation broth. After addition, immobilization begins in the logarithmic growth phase, and fermentation is continued for 100-120 hours to achieve the increase in the production of the target product, aromatic glycoside compounds.

[0033] Wherein, the aromatic glycoside compound product includes one or more of gastrodin, arbutin, resveratrol, naringin, baicalin and isovitexin.

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

[0035] (1) Inoculate the glycerol tube bacteria into 3 mL of yeast extract peptone dextrose (YPD) seed culture medium and carry out seed culture at a certain temperature and rotation speed for 24 to 36 hours.

[0036] (2) Take 1.5 mL of the seed solution and inoculate it into 30 mL of fermentation medium and culture it at a certain temperature and rotation speed for 24 to 36 hours.

[0037] (3) The sulfonated metal organic framework material was added to the fermentation broth and the fermentation was continued for 120 h. Samples were taken every 24 h, and the sampled fermentation broth was frozen at -20°C to measure the gastrodin production.

[0038] The strain described in step (1) is Yarrowia lipolytica. The composition of the YPD seed culture medium is 10 g / L yeast powder, 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) comprises 10 g / L yeast powder, 20 g / L peptone, 80 g / L anhydrous glucose, 5-10 mg / L trace metal elements (4.0 g / L FeSO 4 7H 2 O,4.0g / L CaCl 2 ,1.0g / LMgCl 2 ). The culture temperature was 30°C and the rotation speed was 220-250 rpm.

[0040] Wherein, 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] The present invention proposes a method for immobilizing Yarrowia lipolytica using a sulfonated metal organic framework material (sZr-BTB). Compared with free yeast, immobilized yeast is less susceptible to external influences during growth and metabolism, and maintains yeast activity. Secondly, immobilized yeast is easy to recover during fermentation or biotransformation, and microorganisms can be reused. The sZr-BTB prepared by the present invention is a material with high specific surface area and porosity. The high specific surface area and highly ordered pore structure of sZr-BTB give it extremely strong adsorption capacity and carrier capacity. At the same time, sZr-BTB has good biocompatibility and will not adversely affect cell growth, metabolism and activity. The sZr-BTB proposed by the present invention uses physical adsorption to wrap yeast to form a protective film on its surface, so that glycosidase is restricted to the cell and cannot be secreted, and small molecule products can be excreted, avoiding the decomposition of biosynthesized aromatic glycoside compounds. At the same time, 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, thereby ensuring that the material can permanently wrap the yeast and provide protection for the yeast cells.

[0042] In general, the technology of yeast immobilization using sZr-BTB in the present invention can improve some major defects of the non-model microorganism Yarrowia lipolytica. This method not only effectively avoids the loss of yeast activity, but also enhances the adsorption and carrier functions through its high specific surface area and pore structure. The sulfonated group of sZr-BTB can provide protection for yeast cells under acidic conditions, prevent the extracellular secretion of glycosidases, and protect aromatic glycoside compounds from degradation. The method of the present invention significantly improves the stability and efficiency of Yarrowia lipolytica in industrial production, and shows a broader prospect in biotechnology applications.

[0043] During the fermentation of Yarrowia lipolytica, glycosidases and products are secreted outside the cell, and glycosidases decompose part of the biosynthesized aromatic glycoside compounds into by-products, resulting in a low yield of the main product. Secondly, normal Yarrowia lipolytica is prone to produce by-products such as organic acids during the fermentation process, which leads to a significant decrease in the pH of the fermentation broth, which may eventually lead to the death of the yeast. In response to these problems, the material sZr-BTB proposed in the present invention can fix the material on the surface of Yarrowia lipolytica through the principle of physical adsorption, forming a dense protective film, so that relatively large molecular glycosidases are restricted to the cell and cannot be secreted, while the efflux of small molecular aromatic glycoside compounds is not affected, thereby avoiding the decomposition of the main product of biosynthesis. Secondly, the sulfonic acid groups on the sZr-BTB material proposed in the present invention can compete with the anionic groups on the yeast surface for active hydrogen ions in the fermentation broth, thereby enhancing the adsorption capacity of Yarrowia lipolytica and the material under acidic conditions. The protective film also improves the stability of Yarrowia lipolytica cells, increases catalytic efficiency and reusability. The sulfonated modified Zr-BTB (sZr-BTB) prepared by the present invention can maintain stable adsorption with Yarrowia lipolytica under strongly acidic conditions, and at the same time prevent the secretion of glycosidases and the like in the yeast to the extracellular space through adsorption encapsulation, thereby facilitating the increase in the yield of aromatic glycoside compounds produced by fermentation, thereby solving the obstacles of non-model microorganisms and promoting the low-cost industrialization of biosynthesis.

[0044] The present invention proposes for the first time a method for preparing a sulfonated zirconium-based material sZr-BTB, and applies it to the fermentation production of Yarrowia lipolytica to achieve increased production of aromatic glycoside compounds. The sulfonated Zr-BTB material designed by the present invention can wrap the Yarrowia lipolytica like a membrane, and can limit the secretion of yeast glycosidase while ensuring the excretion of substrates, and because the yeast is wrapped by the material, the yeast can maintain a certain activity in a highly acidic fermentation broth, thereby increasing the yield of aromatic glycoside compounds produced by Yarrowia lipolytica fermentation, and increasing the activity of the yeast in a harsh environment.

[0045] At present, the existing technology cannot solve the problem that Yarrowia lipolytica controls the secretion of glycosidase during the fermentation process, resulting in the decomposition of the main product aromatic glycoside compounds by glycosidase, and the acidity of the yeast fermentation broth is around pH 2-3, and the harsh environment leads to reduced yeast activity. The sZr-BTB material proposed in the present invention can wrap the yeast to prevent the secretion of glycosidase, and can protect the yeast in the acidic fermentation broth and maintain a high activity.

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

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

[0048] 2. The present invention uses Yarrowia lipolytica to carry out microbial fermentation to produce aromatic glycoside compounds. This method has the advantages of high yield, easy expansion of culture, short culture cycle, etc., and is more suitable for the industrial production of aromatic glycoside compounds.

[0049] 3. The present invention combines MOF material nanosheets with Yarrowia lipolytica by physical adsorption. Compared with free yeast, the fixed yeast is more stable during metabolism. In addition, the physical adsorption of yeast and materials makes it easy to separate the product from the yeast, simplifying the subsequent separation and purification process.

[0050] 4. Yarrowia lipolytica produces a lot of organic acids during the fermentation process. Compared with conventional metal-organic framework materials, Zr-BTB is more stable in acidic environments, allowing the material to maintain its structural integrity under acidic conditions for a long time without disintegration.

[0051] 5. The present invention proposes a method of using 1,2-ethanedisulfonic acid to modify Zr-BTB after synthesis, wherein one sulfonic acid group of 1,2-ethanedisulfonic acid coordinates with the zirconium metal on Zr-BTB, and the other sulfonic acid group is used to compete with the groups on the surface of yeast cells for hydrogen ions under acidic conditions, thereby ensuring that sZr-BTB can maintain stable adsorption with Yarrowia lipolytica for a long time under strong acidic conditions.

[0052] 6. The glycosidase produced during the fermentation of Yarrowia lipolytica will decompose the main product, aromatic glycoside compounds, and the sulfonated Zr-BTB material as a 2D-MOF material can be wrapped on the surface of Yarrowia lipolytica, thereby inhibiting the secretion of hydrolases such as glycosidases into the fermentation broth, while the efflux of small molecular aromatic glycoside compounds is not affected, thereby avoiding the decomposition of the main product of biosynthesis and preventing the enzyme from decomposing the product. This fixation effect increases the yield of the product aromatic glycoside compounds. At the same time, because the yeast is wrapped by the sulfonated Zr-BTB material, the yeast can maintain excellent activity in extremely acidic fermentation broth or other harsh fermentation environments. At the same time, the material prepared by the present invention has a good protective effect on yeast under high temperature conditions, increases the stability of yeast, and at a higher temperature such as 45°C fermentation temperature, the yield of gastrodin is much higher than that of free Yarrowia lipolytica. 7. The preparation method of the present invention is simple and convenient, the raw materials are easy to obtain, and can be effectively used for large-scale production, which solves the obstacles of non-model microorganisms and promotes the low-cost industrialization of biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The scanning electron microscope comparison diagram of the immobilized yeast and the free yeast provided by Example 1 and Comparative Example 1;

[0054] Figure 2 The high performance liquid chromatography gastrodin and p-hydroxybenzyl alcohol spectrum in Example 1 is adopted;

[0055] Figure 3 A comparison chart of the gastrodin yields in Example 1 and Comparative Example 1;

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

[0057] Figure 5 The high performance liquid chromatography gastrodin and p-hydroxybenzyl alcohol spectrum in Comparative Example 1 is adopted;

[0058] Figure 6 This is a graph showing the yield of gastrodin produced by fermentation of Yarrowia lipolytica immobilized by sZr-BTB with different zirconium tetrachloride ratios in Example 2;

[0059] Figure 7 This is a graph showing the yield of gastrodin produced by fermentation of Yarrowia lipolytica fixed with sZr-BTB containing different 1,3,5-tris(4-carboxyphenyl)benzene ratios in Example 3;

[0060] Figure 8 This is a graph showing the yield of gastrodin produced by fermentation of Yarrowia lipolytica immobilized by sZr-BTB with different benzoic acid ratios in Example 4;

[0061] Fig. 9 This is a graph showing the yield of gastrodin produced by fermentation of Yarrowia lipolytica fixed with sZr-BTB containing different 1,2-ethanedisulfonic acid ratios in Example 5;

[0062] Fig.10 This is a graph showing the yield of gastrodin produced by fermentation of Yarrowia lipolytica immobilized by sZr-BTB of different masses in Example 6;

[0063] Fig.11 This is a graph showing the gastrodin production at different temperatures using the immobilized Yarrowia lipolytica in Example 7;

[0064] Fig.12 This is a graph showing the gastrodin production using the immobilized Yarrowia lipolytica in Example 8 in different organic solvents. DETAILED DESCRIPTION

[0065] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0066] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

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

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

[0069] As used herein, the term "YPD" is yeast extract peptone dextrose 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: 10g / L yeast powder, 20g / L peptone, 80g / L anhydrous glucose, 5mg / L trace metal elements (4.0g / L FeSO 4 7H 2 O,4.0g / L CaCl 2 ,1.0g / LMgCl 2 )

[0074] The gastrodin-producing Yarrowia lipolytica used in the examples of the present invention is a YliGT6 strain provided by Nanjing Normal University. For details, see: A method for increasing the gastrodin production of recombinant Yarrowia lipolytica, CN 117844844A.

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

[0076] The arbutin-producing Yarrowia lipolytica is transformed by laboratory engineering, and the main operation steps include: S1. constructing a heterologous expression plasmid of Yarrowia lipolytica; S2. preparing seed liquid and purifying it to obtain the arbutin-producing Yarrowia lipolytica.

[0077] S1 includes the following steps:

[0078] AS gene (SEQ ID NO.1), UbiC gene (SEQ ID NO.2) and MNX1 gene (SEQ ID NO.3) were heterologously expressed. AS, UbiC and MNX1 were used as foreign genes, and these genes were used for the synthesis of arbutin. YaliBrick plasmid pYLXP' (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) was selected as the backbone, homology arms were designed, and PCR amplification of the plasmid backbone and the target fragment was performed at the same time, and then the fragments were spliced ​​by the Ginson assembly method to construct heterologous gene expression plasmids pYLXP'-AS, pYLXP'-UbiC and pYLXP'-MNX1 respectively.

[0079] The pYLXP'-AS and pYLXP'-MNX1 plasmids digested with restriction endonucleases Blnl and Sall were spliced ​​by T4 ligase to obtain the plasmid pYLXP'-AS-MNX1, and the pYLXP'-AS-MNX1-UbiC plasmid was obtained by restriction digestion and ligation in the same way. The constructed plasmid was transformed into the starting strain: Y.lipolyticapo1f (po1gΔura3) using YPD plates (g / L) containing 5-fluoroorotic acid (50 mg / L): yeast powder 10, peptone 20, glucose 20, agar powder 15; for coating culture, and 2× Flash KOD DyeMix was used for PCR verification, and the successfully transformed single clones were screened for the next step.

[0080] S2. Prepare seed solution and obtain strains:

[0081] Successful transformants were randomly selected from the overnight culture plates and inoculated into 3 mL YPD culture medium: 10% yeast powder, 20% peptone, and 25% glucose. The culture was shaken at 220 rpm and 30°C for 120 h.

[0082] After 120 hours of shaking culture, the supernatant was collected by centrifugation at 12000 rpm for 10 minutes. The sample was prepared by passing through a 0.22 μm water filter membrane using a syringe, and then the presence of arbutin and the content of arbutin were determined by liquid chromatography-mass spectrometry (LC-MS), wherein 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 set at 25°C, the detection wavelength was 282 nm, and the injection volume was set to 20 μL.

[0083] A standard curve was drawn through quantitative analysis to determine whether the clone could produce high arbutin and continue the subsequent fermentation.

[0084] The high-yield arbutin yeast transformants were streaked on YPD plates, and single colonies were picked with a sterilized toothpick and inoculated into YPD medium, and cultured in a shaking incubator at 220 rpm and 30°C for 24 h.

[0085] 500ul of seed solution was inoculated into 30ml YPD fermentation solution: 10% yeast powder, 20% peptone, 50% glucose, culture temperature was 30℃, and fermentation was continued at 220rpm for 120h.

[0086] Samples were taken every 24 hours to measure OD 600 The sample was centrifuged at 12000rpm for 10min to collect the supernatant, and the sample was filtered through a 0.22μm water filter membrane for high performance liquid chromatography (HPLC) detection to accurately determine the arbutin production. The strain was used as an engineered bacterium for subsequent immobilization operations.

[0087] Example 1

[0088] This example explores the ability of sZr-BTB to promote the fermentation of Yarrowia lipolytica to produce aromatic glycoside compounds. This example uses a Yarrowia lipolytica strain that produces gastrodin. The preparation steps of sZr-BTB are as follows:

[0089] (1) 10 mg of zirconium tetrachloride, 12 mg of 1,3,5-tris(4-carboxyphenyl)benzene and 450 mg of BA were dissolved in 3 mL of DMF, and then 0.5 mL of deionized water was added for ultrasonic dissolution. Then, the mixture was heated and stirred in a water bath at 120°C for 24 h. After cooling, the mixture was washed with DMF 3 times, acetone 3 times, 0.1 M hydrochloric acid 1 time, deionized water 2 times, centrifuged at 8,000 rpm for 5 min, and dried to obtain solid Zr-BTB.

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

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

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

[0093] (2) Take 1.5 mL of the activated Yarrowia 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°C and 250 rpm for 24 h. 600 About 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 shaker at 30 °C and 250 rpm. Samples were taken every 24 h and the sampled fermentation broth was frozen at -20 °C. The scanning electron microscopy image of Yarrowia lipolytica after loading is shown in the figure. Figure 1 shown.

[0095] (4) Use liquid chromatography to detect the sample in step (3). The high performance liquid chromatography uses a SinoChrom ODS-BP column, the mobile phase is 10% (methanol / water), and the flow rate is 0.7-1 mL / min. The wavelength of the ultraviolet detector is set to 225 nm. The peaks of the main product gastrodin and the by-product p-hydroxybenzyl alcohol detected by the liquid chromatography are as follows: Figure 2 As shown in Figure 2, the peak of gastrodin appears at around 13.8 min with a peak area of ​​13.624, while the peak of p-hydroxybenzyl alcohol appears at around 19 min with a peak area of ​​19.085 min. The gastrodin yield inferred from the peak values ​​is as follows: Figure 3 The ratio of gastrodin to p-hydroxybenzyl alcohol is shown in Figure 4 As shown in the results, it can be seen that the production of gastrodin gradually increased with the increase of time, but after 48 hours, the production of gastrodin decreased due to the production of glycosidase.

[0096] Comparative Example 1

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

[0098] Example 2

[0099] This example investigates the role of ZrCl in the synthesis of sZr-BTB. 4 The effect of dosage on the morphology of the material affects the encapsulation of the material on Yarrowia lipolytica and the yield of aromatic glycoside compounds.

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

[0101] (1) ZrCl 4 , 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 for ultrasonic dissolution. The mixture was then heated and stirred in a water bath at 120°C for 24 h, cooled, washed with DMF 3 times, acetone 3 times, 0.1 M hydrochloric acid 1 time, deionized water 2 times, centrifuged at 8,000 rpm for 5 min, and dried to obtain solid Zr-BTB.

[0102] Among them, set ZrCl 4 The dosage is 5, 10, 15, and 20 mg.

[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 and mixed evenly. The mixture was heated in a water bath at 100° C. and stirred for 20 h at a speed of 150 rpm. After cooling, the mixture was washed with DMF three times and acetone three times, and centrifuged at 8,000 rpm for 5 min to obtain sZr-BTB material.

[0104] The subsequent preparation of sZr-BTB-immobilized Yarrowia lipolytica (YliGT6) was the same as in Example 1. The yield of gastrodin was as follows: Figure 6 As shown, according to the results, ZrCl 4 The dosage affects the morphology of the produced sZr-BTB material, which in turn affects the adsorption of Yarrowia lipolytica, resulting in changes in the yield. The experiment concluded that when 10 mg of ZrCl was added to prepare sZr-BTB, 4It is the best choice. After 120h of fermentation with Yarrowia lipolytica, the yield of gastrodin was 6g / L.

[0105] Example 3

[0106] This example explores the effect of the amount of 1,3,5-tris(4-carboxyphenyl)benzene used in the synthesis of sZr-BTB on the morphology of the material, thereby affecting the encapsulation of the material on Yarrowia lipolytica and the yield of aromatic glycoside compounds. This example uses Yarrowia lipolytica (YliGT6) that produces gastrodin, and the preparation steps of sZr-BTB are as follows:

[0107] (1) 10 mg ZrCl 4 , 1,3,5-tri(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. The mixture was then heated and stirred in a water bath at 120°C for 24 h, cooled, washed with DMF 3 times, acetone 3 times, 0.1 M hydrochloric acid 1 time, deionized water 2 times, centrifuged at 8,000 rpm for 5 min, and dried to obtain solid Zr-BTB.

[0108] The usage amounts of 1,3,5-tris(4-carboxyphenyl)benzene were 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 and mixed evenly. The mixture was heated in a water bath at 100° C. and stirred for 20 h at a speed of 150 rpm. After cooling, the mixture was washed with DMF three times and acetone three times, and centrifuged at 8,000 rpm for 5 min to obtain sZr-BTB material.

[0110] The subsequent preparation of sZr-BTB-immobilized Yarrowia lipolytica (YliGT) was the same as in Example 1. The yield of gastrodin was as follows: Figure 7 As shown in the results, the amount of 1,3,5-tri(4-carboxyphenyl)benzene affects the morphology of the produced sZr-BTB material, which in turn affects the adsorption with Yarrowia lipolytica, resulting in changes in the yield. The experiment concluded that adding 12 mg of 1,3,5-tri(4-carboxyphenyl)benzene is the best choice in the preparation of sZr-BTB. After 120 hours of fermentation, the yield of gastrodin reached 6.12 g / L.

[0111] Example 4

[0112] This example explores the effect of BA dosage on the material morphology in the synthesis of sZr-BTB, thereby affecting the material's encapsulation of Yarrowia lipolytica and the yield of aromatic glycoside compounds. This example uses Yarrowia lipolytica (YliGT) that produces gastrodin, and the preparation steps of sZr-BTB are as follows:

[0113] (1) 10 mg ZrCl 4 , 12 mg 1,3,5-tris(4-carboxyphenyl)benzene and BA were dissolved in 3 mL DMF, followed by addition of 0.5 mL deionized water for ultrasonic dissolution. Then, the mixture was heated and stirred in a water bath at 120°C for 24 h, cooled, washed with DMF 3 times, acetone 3 times, 0.1 M hydrochloric acid 1 time, deionized water 2 times, centrifuged at 8,000 rpm for 5 min, and dried to obtain solid Zr-BTB.

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

[0115] (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 and mixed evenly. The mixture was heated in a water bath at 100° C. and stirred for 20 h at a speed of 150 rpm. After cooling, the mixture was washed with DMF three times and acetone three times, and centrifuged at 8,000 rpm for 5 min to obtain sZr-BTB material.

[0116] The subsequent preparation of sZr-BTB-immobilized Yarrowia lipolytica (YliGT) was the same as in Example 1. The yield of gastrodin was as follows: Figure 8 As shown in the results, the amount of BA affects the morphology of the produced sZr-BTB material, which in turn affects the adsorption with Yarrowia lipolytica, resulting in changes in the yield. The experiment concluded that adding 450 mg of BA is the best choice in the preparation of sZr-BTB. After 120 hours of fermentation, the yield of gastrodin produced by Yarrowia lipolytica was 6.21 g / L.

[0117] Example 5

[0118] This example explores the effect of the amount of 1,2-ethanedisulfonic acid that provides sulfonic acid groups on the ability to adsorb Yarrowia lipolytica in the synthesis of sZr-BTB. Insufficient sulfonic acid groups may result in the material being unable to adsorb to the yeast in a long-lasting and stable manner, while too many groups added will result in the material not having enough sites to adsorb to the yeast. This example uses Yarrowia lipolytica (YliGT) that produces gastrodin, and the preparation steps of sZr-BTB are as follows:

[0119] (1) 10 mg ZrCl 4, 12 mg 1,3,5-tri(4-carboxyphenyl)benzene and 450 mg BA were dissolved in 3 mL DMF, followed by the addition of 0.5 mL deionized water. The reaction was transferred to a 15 mL pressure tube for ultrasonic dissolution. Then, the mixture was heated and stirred in a water bath at 120 °C for 24 h. After cooling, it was washed with DMF 3 times, acetone 3 times, 0.1 M hydrochloric acid 1 time, deionized water 2 times, centrifuged at 8,000 rpm for 5 min, and dried to obtain solid Zr-BTB.

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

[0121] The usage amounts of 1,2-ethanedisulfonic acid were set to 40, 80, 120, and 240 mg.

[0122] The subsequent preparation of sZr-BTB-immobilized Yarrowia lipolytica (YliGT) was the same as in Example 1. The yield of gastrodin was as follows: Fig. 9 As shown in the results, the ratio of 1,2-ethanedisulfonic acid to Zr-BTB has an effect on the adsorption capacity of sZr-BTB. The experiment concluded that in the preparation of sZr-BTB, the best ratio of adding Zr-BTB to 1,2-ethanedisulfonic acid is 1:2. When the mass 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 example explores the effect of the ratio of sZr-BTB material and microorganism Yarrowia lipolytica on the final product yield when they are coupled. In this example, gastrodin-producing Yarrowia lipolytica (YliGT6) was used, and the preparation method of sZr-BTB was consistent with that of Example 1.

[0125] The preparation steps of sZr-BTB immobilized Yarrowia lipolytica (YliGT6) are as follows:

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

[0127] (2) 1.5 mL of the activated Yarrowia lipolytica was inoculated into 30 mL of fresh sugar-containing fermentation medium, mixed evenly, and placed in a constant temperature shaking fermentation shaker at 30°C and 250 rpm for shaking culture for 24 h until the OD600 was approximately 0.7.

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

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

[0130] (4) Using liquid phase to detect the sample in step (3).

[0131] The yield of gastrodin Fig.10 As shown in the figure, according to the results, the coupling ratio of sZr-BTB and Yarrowia lipolytica has an impact on the yield of the main product. Too little material may result in the inability to completely encapsulate the yeast, while too much material will encapsulate the yeast too tightly, reducing the yeast activity. After the experiment, it can be concluded that adding 1 mg of sZr-BTB to 30 mL of fermentation broth is the best. After adding sZr-BTB, Yarrowia lipolytica produced 6.31 g / L of gastrodin after 120 hours of fermentation.

[0132] Example 7

[0133] This example tests the activity and yield of Yarrowia lipolytica immobilized by 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 by sZr-BTB.

[0134] Compared with Example 1, the difference between this embodiment and Example 1 is that 1 mg of sulfonated modified Zr-BTB is added to 30 mL of fermentation broth in the step, and the fermentation is carried out for 120 hours in a constant temperature shaking fermentation shaker at 35°C and 250rpm. Samples are taken every 24 hours, and the sampled fermentation broth is frozen in a -20°C refrigerator. The other two groups are compared as follows: fermentation for 120 hours in a constant temperature shaking fermentation shaker at 40°C and 250rpm; fermentation for 120 hours in a constant temperature shaking fermentation shaker at 45°C and 250rpm. Subsequently, the free yeast without adding materials is fermented at three different temperatures of 35°C, 40°C, and 45°C, and the fermentation steps are the same as in Example 1.

[0135] After fermentation, the samples were prepared and tested by high performance liquid chromatography. The gastrodin production after 120 h of fermentation was calculated based on the peak value, so as to infer the effect of different temperatures on the fixed Yarrowia lipolytica. The results are as follows Fig.11 As shown. From the results, it can be seen that with the increase of fermentation temperature, the gastrodin production in the fermentation broth decreased after 120 hours. However, the gastrodin production of Yarrowia lipolytica fixed by the material was much higher than that of free Yarrowia lipolytica at a fermentation temperature of 45°C, which can prove that the material has a good protective effect on yeast and increases the stability of yeast.

[0136] Example 8

[0137] This example tests the activity and yield of Yarrowia lipolytica immobilized by sZr-BTB in Example 1 of the present invention under different environments, and compares the activity of free yeast that is not immobilized by sZr-BTB.

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

[0139] After fermentation, the samples were prepared and tested by high performance liquid chromatography. The gastrodin production after 120 h of fermentation was calculated based on the peak value, so as to infer the effect of organic solvents on the immobilization of Yarrowia lipolytica. The results are as follows Fig.12 As shown in the results, it can be seen that compared with free yeast, the Yarrowia lipolytica immobilized with sZr-BTB is more stable in organic solvents. Although the activity of the yeast is still affected, the gastrodin production after 120 hours of fermentation can still reach 60-70% of the original.

[0140] Example 9

[0141] This example explores the ability of sZr-BTB to fix Yarrowia lipolytica to produce other aromatic glycoside compounds. This example uses the arbutin-producing Yarrowia lipolytica constructed as described above. Compared with Example 1, the difference between this example and Example 1 is that the strain in the step is changed to arbutin-producing Yarrowia lipolytica. At the same time, a control group without sZr-BTB material was made. The results show that, similar to Example 1 and Comparative Example 1, the sZr-BTB material increases the catalytic activity of arbutin-producing Yarrowia lipolytica, inhibits the secretion of glycosidase, and reduces the production of hydroquinone.

[0142] In summary, the sulfonated modified material Zr-BTB (sZr-BTB) prepared by the present invention is used to fix the aromatic glycoside compounds produced by Yarrowia lipolytica, which plays an important role in achieving increased production of products. Free yeast will secrete small molecule products such as glycosidases to the extracellular space during the fermentation process, resulting in the decomposition of the main product glycoside compounds by glycosidases (Comparative Example 1). In addition, during the fermentation process, Yarrowia lipolytica will produce a large amount of organic acid, resulting in the pH of the fermentation broth being reduced to 2-3. The strong acidic environment will harm the yeast and even cause the death of the yeast. Compared with Comparative Example 1, the present invention can still maintain excellent activity in a strong acid environment. The sZr-BTB prepared by the present invention can compete with the groups on the cell surface for hydrogen ions in the fermentation broth with strong acidity, thereby causing the material to be physically adsorbed to the yeast cells for a long time, thereby protecting the yeast cells. At the same time, by adsorbing and embedding cells, yeast is prevented from secreting glycosidases to the extracellular space, inhibiting the decomposition of the main product by enzymes, and increasing the yield of microbial fermentation.

Claims

1. A sulfonated metal organic framework material, characterized in that: The sulfonation-modified metal organic framework material uses a zirconium (Zr)-based MOF material Zr-BTB as a precursor and reacts with 1,2-ethanedisulfonic acid to perform sulfonation modification.

2. A method for preparing the sulfonated metal organic framework material according to claim 1, characterized in that: The steps include: (1) Preparation of 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 to deionized water, mixing by ultrasonication, reacting at high temperature, cooling and washing, and centrifugally drying to obtain a precursor Zr-BTB material; (3) Add 1,2-ethanedisulfonic acid to the prepared precursor Zr-BTB material. After the high-temperature reaction is completed, collect the precipitate by centrifugation and wash with water to obtain the sulfonated Zr-BTB material, which is the sulfonated 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-tri(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° C., the reaction time is 20-24 hours, and the centrifugation speed is 8,000-12,000 rpm for 5-10 minutes.

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, and the centrifugation speed is 8,000 to 12,000 rpm for 5 to 10 minutes.

6. Use of the sulfonated metal organic framework material according to claim 1 in the production of aromatic glycoside compounds by Yarrowia lipolytica.

7. The use according to claim 6, characterized in that: The process of the application is: (1) inoculating Yarrowia lipolytica into a seed culture medium for seed culture; (2) taking the seed liquid and inoculating it into a fermentation medium for fermentation culture; (3) Adding the sulfonated metal organic framework material to the fermentation broth and continuing the fermentation to achieve increased production of the target product, aromatic glycoside compounds.

8. The use according to claim 7, characterized in that: In step (1), the Yarrowia lipolytica is cultured in a YPD seed medium for 24 to 36 hours; and in step (2), the Yarrowia lipolytica is cultured in a fermentation medium for 24 to 36 hours.

9. The use according to claim 7, characterized in that: In step (3), the amount of sulfonated metal organic framework material added to the fermentation broth is 0.5-1.5 mg of sulfonated metal organic framework material per 20-40 mL of fermentation broth, immobilization begins in the logarithmic growth phase, and fermentation is continued for 100-120 hours to achieve increased production of the target product, aromatic glycoside compounds.

10. The use according to claim 6, characterized in that: The aromatic glycoside compound product preferably includes one or more of gastrodin, arbutin, resveratrol, naringin, baicalin and isovitexin.

Images