Purification method and application of enzyme-catalyzed synthesized bose

Abstract

The invention provides a purification method and application of enzyme-catalyzed synthesized bose, and belongs to the technical field of bose purification. The purification method comprises the following steps: mixing beta-acetone xyloside, isopropanol and water, adding a compound enzyme system, carrying out an enzyme catalytic reaction, carrying out filtration, reduced pressure distillation, decoloration and filter pressing, carrying out electrodialysis, ultrafiltration, nanofiltration and vacuum concentration on the filtrate, adding absolute ethyl alcohol, crystallizing, and carrying out vacuum drying to obtain the bose, the electric field intensity of electrodialysis is 20-30 V / cm, the water flow velocity is 5-10 cm / s, and the current density is 20-30 mA / cm < 2 >. According to the method disclosed by the invention, the purity of the glassine is remarkably improved, the moisturizing effect of the glassine is proved to be better than that of a traditional process through a zebra fish experiment, and the area reduction inhibition rate is up to 72.3%. The vitreous pigment obtained by the invention can be widely applied to the field of cosmetics, and is especially suitable for anti-aging and skin collagen synthesis promoting products.

Description

Technical Field

[0001] The present invention belongs to the technical field of bosera purification, and in particular relates to a purification method and application of bosera synthesized by enzyme catalysis. Background Art

[0002] C-Xyloside, a glycoside compound with a wide range of bioactive properties, has attracted significant attention due to its significant applications in cosmetics, pharmaceuticals, and other fields. In particular, C-Xyloside has become a key ingredient in high-end skincare products for its anti-aging and collagen synthesis-boosting properties. Currently, C-Xyloside synthesis primarily involves chemical synthesis and enzymatic methods. Compared to traditional chemical synthesis, enzymatic methods offer advantages such as mild reaction conditions, high selectivity, and environmental friendliness, making them a research hotspot. However, the products of enzymatic synthesis are often accompanied by byproducts, unreacted substrates, residual enzyme protein, and impurity ions, which severely impact the purity and performance of C-Xyloside. Therefore, the development of efficient and low-cost purification processes has become a key bottleneck in the industrialization of enzymatic synthesis of C-Xyloside.

[0003] In the existing technology, the purification of bosine mainly relies on traditional methods such as column chromatography and solvent extraction. For example, some studies use silica gel column chromatography to separate the target product, but this method is cumbersome to operate, consumes a lot of solvent, and is difficult to apply on a large scale. While organic solvent extraction can initially remove hydrophobic impurities, it has limited effect on separating by-products with similar polarity and is prone to causing environmental pollution. In addition, residual protein or polysaccharide impurities in the enzyme-catalyzed reaction solution may further increase the difficulty of subsequent purification, resulting in reduced product yield and increased costs.

[0004] Therefore, the development of an efficient and industrially viable purification method for bosera is urgent. This method must address the unique characteristics of the enzyme-catalyzed reaction system and balance impurity removal efficiency with product yield to meet the demands of the cosmetics and pharmaceutical industries for high-purity bosera. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a purification method and application of enzyme-catalyzed synthesis of bosine. The bosine prepared by the method of the present invention has high purity and good moisturizing effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A purification method for enzyme-catalyzed synthesis of bosyltransferase comprises: mixing β-acetone xyloside, isopropyl alcohol and water, adding a composite enzyme system, carrying out an enzyme-catalyzed reaction, filtering, vacuum distilling, decolorizing, and filter pressing, subjecting the filtrate to electrodialysis, ultrafiltration, and nanofiltration, vacuum concentration, adding anhydrous ethanol, crystallizing, and vacuum drying to obtain bosyltransferase; the electric field intensity of the electrodialysis is 20-30 V / cm, the water flow rate is 5-10 cm / s, and the current density is 20-30 mA / cm 2 .

[0007] Preferably, the complex enzyme is obtained by immobilizing bosera, coenzyme NADP and isopropanol dehydrogenase on an immobilized carrier.

[0008] Preferably, the pressure of the reduced pressure distillation is 12-14 kPa, the temperature is 45-55° C., and the time is 1-2 h.

[0009] Preferably, the decolorization is carried out by adsorption using activated carbon, the pore size of the activated carbon is 0.5-2 nm, and the amount used is 3% of the weight of the β-acetone xyloside and isopropanol.

[0010] Preferably, the molecular weight cut-off of the ultrafiltration is 3000-5000Da.

[0011] Preferably, the molecular weight cut-off of the nanofiltration is 100-200 Da.

[0012] Preferably, the amount of anhydrous ethanol used is 0.8-1.2 times the weight of β-acetone xyloside.

[0013] Preferably, the crystallization temperature is 0-4°C, the rotation speed is 50-100 rpm, and the time is 50-70 min.

[0014] The present invention also provides boson obtained by the above purification method.

[0015] The present invention also provides the use of the above-mentioned bosine in preparing cosmetics.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a purification method for enzyme-catalyzed synthesis of bosyltransferase, comprising: mixing β-acetone xyloside, isopropyl alcohol and water, adding a composite enzyme system, performing an enzyme-catalyzed reaction, filtering, vacuum distilling, decolorizing, and filter pressing, subjecting the filtrate to electrodialysis, ultrafiltration, and nanofiltration, vacuum concentration, adding anhydrous ethanol, crystallizing, and vacuum drying to obtain bosyltransferase; the electric field intensity of the electrodialysis is 20-30 V / cm, the water flow velocity is 5-10 cm / s, and the current density is 20-30 mA / cm 2The method of the present invention significantly improves the purity of phosphene, and zebrafish experiments have verified that its moisturizing effect is superior to that of traditional processes, with an area shrinkage inhibition rate of up to 72.3%. The phosphene obtained by the present invention can be widely used in the field of cosmetics, especially in products for anti-aging and promoting skin collagen synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the high performance liquid chromatogram of Bose-Yin in Example 1 of Test Example 1; Figure 2 This is the high performance liquid chromatogram of Bose-Yin in Example 3 of Test Example 1; Figure 3 This is the NMR spectrum of Boson in Example 1. DETAILED DESCRIPTION

[0018] The invention provides a purification method for enzyme-catalyzed synthesis of bosyltransferase, comprising: mixing β-acetone xyloside, isopropyl alcohol and water, adding a composite enzyme system, performing an enzyme-catalyzed reaction, filtering, vacuum distilling, decolorizing, and filter pressing, subjecting the filtrate to electrodialysis, ultrafiltration, and nanofiltration, vacuum concentration, adding anhydrous ethanol, crystallizing, and vacuum drying to obtain bosyltransferase; the electric field intensity of the electrodialysis is 20-30 V / cm, the water flow velocity is 5-10 cm / s, and the current density is 20-30 mA / cm 2 .

[0019] The initial concentration of the β-acetone xyloside of the present invention is preferably 190-210 g / L, more preferably 200 g / L, the initial concentration of isopropanol is preferably 190-210 g / L, more preferably 200 g / L, the composite enzyme system is preferably obtained by immobilizing bosera, coenzyme NADP and isopropanol dehydrogenase via an immobilized carrier, and the mass ratio of the immobilized carrier, bosera, coenzyme NADP and isopropanol dehydrogenase in the composite enzyme system is preferably (50-100):(3-5):(1-2):(3-5), more preferably 75:4:1.5:4, and the dosage of the composite enzyme system is preferably 20-30 g / L, more preferably 25 g / L. The preparation of the composite enzyme system preferably includes: S1: crushing bamboo chips, soaking them in a 1-3wt% NaOH aqueous solution at 70-85°C for 1-3h, washing them with water until neutral, and drying them to a moisture content of less than 5wt% to obtain pretreated bamboo powder; S2: adding water to the pretreated bamboo powder, heating and soaking it, cooling it (for example, to 35-40°C), adding composite bacteria, aerobically fermenting it for 15-20d, filtering it, and drying it to obtain fermented bamboo powder; S3: adding water to the fermented bamboo powder, and continuously adding polyethylene glycol 4000 to a concentration of 0.1-0.2mol / L, stirring it, washing it, and drying it to obtain an enzyme immobilized carrier; S4: adding bosera, coenzyme NADP, isopropyl alcohol dehydrogenase, and the enzyme immobilized carrier to a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, reacting them in a water bath shaker, filtering it, and washing it until no protein is detected to obtain an immobilized enzyme. The composite bacteria comprises Bacillus subtilis, white rot fungus, and Phanerochaete chrysosporium in a weight ratio of 1:(1.3-1.5):(0.4-0.7); the reaction is carried out in a shaker water bath at 30-40°C for 3-8 hours. The parameters of the enzyme-catalyzed reaction are preferably: temperature 30-35°C, pH 6.8-7.2, and rotation speed 80-120 rpm, more preferably: temperature 32°C, pH 7, and rotation speed 100 rpm. The endpoint of the enzyme-catalyzed reaction is preferably a β-acetoxylase concentration of 0.5 g / L. The pH is preferably adjusted using a phosphate buffer.

[0020] The present invention immobilizes the enzyme, and the enzyme molecule conformation is more stable after immobilization, and the tolerance to temperature, pH value and environment is significantly improved. The immobilized enzyme can be separated and recovered from the reaction solution by filtration, and can be reused, which significantly reduces industrial production costs.

[0021] The vacuum distillation of the present invention preferably has a pressure of 12-14 kPa, more preferably 13 kPa, a temperature of 45-55°C, more preferably 50°C, and a duration of 1-2 hours, more preferably 1.5 hours. Vacuum distillation can remove acetone and isopropanol from the solution after the enzyme-catalyzed reaction, thereby improving the purity of bosylamine. Furthermore, vacuum distillation can prevent the structural damage of bosylamine caused by excessive temperatures, which could affect its efficacy.

[0022] The decolorization of the present invention is preferably carried out by activated carbon adsorption. The pore size of the activated carbon is preferably 0.5-2 nm, more preferably 1 nm, and the amount used is preferably 2%-5% by weight of the β-acetone xyloside and isopropanol, more preferably 3%.

[0023] The electric field strength of the electrodialysis of the present invention is preferably 20-30 V / cm, more preferably 25 V / cm, the water flow rate is preferably 5-10 cm / s, more preferably 8 cm / s, and the current density is preferably 20-30 mA / cm 2 , more preferably 25 mA / cm 2 Electrodialysis can be used to remove components such as phosphate and sodium acetate from the solution after the enzyme-catalyzed reaction.

[0024] The molecular weight cutoff of the ultrafiltration of the present invention is preferably 3000-5000 Da, more preferably 4000 Da. Ultrafiltration can remove macromolecular impurities such as bosera, coenzyme NADP and isopropyl alcohol dehydrogenase in the complex enzyme system.

[0025] The molecular weight cutoff of the nanofiltration of the present invention is preferably 100-200 Da, more preferably 150 Da. Nanofiltration can remove small molecules (such as xylose and acetylacetone) that are difficult to separate from the solution.

[0026] The amount of anhydrous ethanol used in the present invention is preferably 0.8-1.2 times the weight of the β-acetone xyloside, more preferably 1 time; the crystallization temperature is preferably 0-4°C, more preferably 2°C; the rotation speed is preferably 50-100 rpm, more preferably 80 rpm, and the time is preferably 50-70 minutes, more preferably 60 minutes. β-acetone xyloside can be removed by crystallization.

[0027] The present invention also provides boson obtained by the above purification method.

[0028] The present invention also provides the use of the above-mentioned bosine in preparing cosmetics.

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] In the following examples, unless otherwise specified, all methods are conventional.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Boxerase S was purchased from Huzhou Yihui Biotechnology Co., Ltd., and boxerase R was purchased from Huzhou Yihui Biotechnology Co., Ltd.; coenzyme NADP was purchased from Shanxi Lanyuan Biotechnology Co., Ltd.; isopropanol dehydrogenase was purchased from Huaian Meiling Bioenzyme Technology Co., Ltd.; Bacillus subtilis was purchased from Wuhan Huizao Biotechnology Co., Ltd. Hangzhou Branch, with the product number: HZB119024; white rot fungi were purchased from Wuhan Huizao Biotechnology Co., Ltd. Hangzhou Branch, with the product number: HZB128891; Trichoderma reesei was purchased from Wuhan Huizao Biotechnology Co., Ltd. Hangzhou Branch, with the product number: HZB120157; Bacillus licheniformis was purchased from Ningbo Testo Biotechnology Co., Ltd., with the product number TS278075; Bacillus amyloliquefaciens was purchased from Ningbo Testo Biotechnology Co., Ltd., with the product number TS277468; and Phanerochaete chrysosporium was purchased from Wuhan Huizao Biotechnology Co., Ltd. Hangzhou Branch, with the product number HZB229528. Example 1

[0033] Purification method of enzyme-catalyzed synthesis of bosine (1) Synthesis of Bose Prepare a clean 200L conversion kettle. Add 100L of distilled water, 20kg of β-xyloside, and 20kg of isopropanol to the kettle. After dissolution, adjust the pH of the system to 7.0 with phosphate buffer. Add the complex enzyme system at a solid-liquid ratio of 25g / L. Incubate the reaction at 32°C and 100 rpm until the concentration of β-xyloside reaches 0.5g / L. The complex enzyme is obtained by immobilizing bosera enzyme S, coenzyme NADP and isopropanol dehydrogenase on an immobilized carrier. The specific method is as follows: S1: Crush and sieve bamboo chips, select the 1500 mesh crushed material, soak it in a 2 wt% NaOH aqueous solution at 80°C for 2 h, filter it, wash the solid with water four times, and dry it in an oven at 60°C to a moisture content of 3 wt% to obtain pretreated bamboo powder; S2: Add 5 kg of water to 1 kg of pretreated bamboo powder, heat to 85 ° C, keep warm and soak for 10 hours, cool to 37 ° C, add composite bacteria, and aerobic fermentation culture for 17 days, with a ventilation (air) volume of 7.0 L / (min·m 3 ), filtering, and drying to obtain fermented bamboo powder; the composite bacteria include Bacillus subtilis, white rot fungi and Phanerochaete chrysosporium in a weight ratio of 1:1.4:0.6, and the amount of the composite bacteria added accounts for 0.1wt% of the pretreated bamboo powder; S3: Add water to the fermented bamboo powder, continue to add polyethylene glycol 4000 to a concentration of 0.15 mol / L, keep stirring at 50°C for 50 hours, wash twice with clean water, and dry to obtain an immobilized carrier; S4: Add 4 mg of bosera enzyme S, 1.5 mg of coenzyme NADP, 4 mg of isopropanol dehydrogenase, and 75 mg of enzyme immobilization support to 50 mL of 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, react in a 35°C water bath shaker for 4 h, filter, wash with 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer until no protein is detected, and filter to obtain the immobilized enzyme; (2) Purification of Boson The mixture after the enzyme catalytic reaction was filtered to recover the immobilized enzyme; the filtered enzyme catalytic reaction liquid was distilled under reduced pressure at a pressure of 13 kPa and a temperature of 50°C for 1.5 h, 1.2 kg of activated carbon with a pore size of 1 nm was added, and the filtrate was filtered under a temperature of 30°C, an electric field strength of 25 V / cm, a water flow rate of 8 cm / s, and a current density of 25 mA / cm 2 After electrodialysis for 3 minutes under the same conditions, the enzyme was removed by passing through a 4000Da ultrafiltration membrane to obtain a clear liquid, which was concentrated and desalted through a 150Da nanofiltration membrane under a pressure of 2MPa to obtain a concentrate. After vacuum concentration, the concentrate was transferred to a crystallization kettle, 20kg of anhydrous ethanol was added, and the mixture was stirred at a temperature of 2°C and a rotation speed of 80rpm for 60 minutes. The mixture was filtered to obtain a white solid, which was vacuum dried at 60°C for 2h to obtain bosylamine. Example 2

[0034] Purification method of enzyme-catalyzed synthesis of bosine (1) Synthesis of Bose Prepare a clean 200L conversion kettle. Add 100L of distilled water, 15kg of β-xyloside, and 15kg of isopropanol to the kettle. Dissolve the mixture. Adjust the pH to 6.8 with phosphate buffer. Add the enzyme complex and perform the enzymatic reaction at 30°C and 120 rpm until the β-xyloside concentration reaches 0.5g / L. Filter at 80°C for 20 minutes. The complex enzyme is obtained by immobilizing bosera R, coenzyme NADP and isopropanol dehydrogenase on an immobilized carrier. The specific method is as follows: S1: Crush bamboo chips, select the crushed material with a mesh size of 1000, soak them in a 2 wt% NaOH aqueous solution at 80°C for 2 h, filter, wash the solid with water three times, and dry it in an oven at 60°C to a moisture content of 3 wt% to obtain pretreated bamboo powder; S2: Add 5 kg of water to 1 kg of pretreated bamboo powder, heat to 90 ° C, keep warm and soak for 10 hours, cool to 35 ° C, add composite bacteria, and aerobic fermentation culture for 15 days, with a ventilation (air) volume of 7.0 L / (min·m 3), filtering, and drying to obtain fermented bamboo powder; the composite bacteria comprises Bacillus subtilis, white rot fungi, and Phanerochaete chrysosporium in a weight ratio of 1:1.4:0.6. The amount of the composite bacteria added accounts for 0.1wt% of the pretreated bamboo powder; S3: Add water to the fermented bamboo powder, and continue to add polyethylene glycol 4000 to make the final concentration of polyethylene glycol 4000 0.15 mol / L, keep stirring at 50 ° C for 52 hours, wash with clean water twice, and dry to obtain an immobilized carrier; S4: Add 3 mg of bosera R, 1 mg of coenzyme NADP, 3 mg of isopropanol dehydrogenase, and 50 mg of the immobilized support to 50 mL of 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and react in a 35°C shaker water bath for 4 h. Filter and wash with 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer until no protein is detected. Filter to obtain the immobilized enzyme. (2) Purification of Boson The mixture after the enzyme catalytic reaction was filtered to recover the immobilized enzyme; the filtered enzyme catalytic reaction liquid was distilled under reduced pressure at a pressure of 12 kPa and a temperature of 45°C for 2 h, 1.5 kg of activated carbon with a pore size of 0.5 nm was added, and the filtrate was filtered; the filtrate was heated at a temperature of 28°C, an electric field strength of 20 V / cm, a water flow rate of 5 cm / s, and a current density of 20 mA / cm 2 After electrodialysis for 5 minutes under the same conditions, the enzyme was removed by a 3000Da ultrafiltration membrane to obtain a clear liquid, which was concentrated and desalted by a 100Da nanofiltration membrane under a pressure of 1.5MPa to obtain a concentrate, which was then concentrated and removed by vacuum concentration to obtain a concentrate. 15kg of anhydrous ethanol was added to a crystallization kettle, and the mixture was stirred at a temperature of 0°C and a rotation speed of 100rpm for 70 minutes. The mixture was filtered to obtain a white solid, which was dried in vacuum at 60°C for 2h to obtain bosphene. Example 3

[0035] Purification method of enzyme-catalyzed synthesis of bosine (1) Synthesis of Bose Prepare a clean 200L conversion kettle. Add 100L of distilled water, 25kg of β-xyloside, and 25kg of isopropanol to the kettle. Dissolve the mixture. Adjust the pH to 7.2 with phosphate buffer. Add the enzyme complex and perform the enzymatic reaction at 35°C and 80 rpm until the β-xyloside concentration reaches 0.5g / L. Filter at 90°C for 10 minutes. The complex enzyme is obtained by immobilizing bosera enzyme S, bosera enzyme R, coenzyme NADP and isopropanol dehydrogenase on an immobilized carrier. The specific method is as follows: S1: Crush and sieve bamboo chips, select the 1500 mesh crushed material, soak it in a 2 wt% NaOH aqueous solution at 80°C for 2 h, filter it, wash the solid with water five times, and dry it in an oven at 60°C to a moisture content of 3 wt% to obtain pretreated bamboo powder; S2: Add 5 kg of water to 1 kg of pretreated bamboo powder, heat to 85 ° C, keep warm and soak for 10 hours, cool to 37 ° C, add composite bacteria, and aerobic fermentation culture for 17 days, with a ventilation (air) volume of 7.0 L / (min·m 3 ), filtering, and drying to obtain fermented bamboo powder; the composite bacteria include Bacillus subtilis, white rot fungi and Phanerochaete chrysosporium in a weight ratio of 1:1.4:0.6, and the amount of the composite bacteria added accounts for 0.1wt% of the pretreated bamboo powder; S3: Add water to the fermented bamboo powder, continue to add polyethylene glycol 4000 to a concentration of 0.15 mol / L, keep stirring at 50°C for 50 hours, wash twice with clean water, and dry to obtain an immobilized carrier; S4: Add 2.5 mg of boserainase S, 2.5 mg of boserainase R, 25 mg of coenzyme NADP, 5 mg of isopropanol dehydrogenase, and 100 mg of enzyme immobilization carrier to 50 mL of 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and react in a 35°C shaker water bath for 4 h. Filter and wash with 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer until no protein is detected. Filter to obtain the immobilized enzyme. (2) Purification of Boson The mixture after the enzyme catalytic reaction was filtered to recover the immobilized enzyme; the filtered enzyme catalytic reaction liquid was distilled under reduced pressure at a pressure of 15 kPa and a temperature of 55°C for 1 hour, 1 kg of activated carbon with a pore size of 2 nm was added, and the filtrate was filtered; the filtrate was heated at a temperature of 30°C, an electric field strength of 30 V / cm, a water flow rate of 10 cm / s, and a current density of 30 mA / cm 2 After electrodialysis for 2 minutes under the same conditions, the enzyme was removed by passing through a 5000Da ultrafiltration membrane to obtain a clear liquid, which was concentrated and desalted through a 200Da nanofiltration membrane at a pressure of 2.5MPa to obtain a concentrate, which was then transferred to a crystallization kettle after vacuum concentration, and 25kg of anhydrous ethanol was added. The product was stirred at a temperature of 4°C and a rotation speed of 80rpm for 50 minutes, filtered, and a white solid was obtained. The product was vacuum dried at 60°C for 2h to obtain bosylamine.

[0036] Comparative Example 1 The difference between this comparative example and Example 1 is that the bosine was not purified, and the mixture after the enzyme-catalyzed reaction was filtered, concentrated under reduced pressure, and vacuum-dried at 60° C. for 12 h to obtain bosine.

[0037] Comparative Example 2 The difference between this comparative example and Example 2 is that the bosine was not purified, and the mixture after the enzyme-catalyzed reaction was filtered, concentrated under reduced pressure, and vacuum-dried at 60° C. for 12 h to obtain bosine.

[0038] Comparative Example 3 The difference between this comparative example and Example 1 is that no electrodialysis was performed.

[0039] Comparative Example 4 The difference between this comparative example and Example 1 is that no ultrafiltration membrane enzyme removal was performed.

[0040] Comparative Example 5 The difference between this comparative example and Example 1 is that no nanofiltration membrane enzyme removal was performed.

[0041] Comparative Example 6 The difference between this comparative example and Example 2 is that the composite bacteria is replaced by Bacillus subtilis.

[0042] Comparative Example 7 The difference between this comparative example and Example 2 is that the composite bacteria are replaced by white rot fungi.

[0043] Comparative Example 8 The difference between this comparative example and Example 2 is that the composite bacteria is replaced by Phanerochaete chrysosporium.

[0044] Comparative Example 9 The difference between this comparative example and Example 2 is that the composite bacteria include Bacillus subtilis and white rot fungi in a weight ratio of 1:1.4.

[0045] Comparative Example 10 The difference between this comparative example and Example 2 is that the composite bacteria includes Bacillus subtilis and Phanerochaete chrysosporium in a weight ratio of 1:0.6.

[0046] Comparative Example 11 The difference between this comparative example and Example 2 is that the composite bacteria includes Bacillus subtilis, white rot fungi and Phanerochaete chrysosporium in a weight ratio of 1.4:0.6:1.

[0047] Comparative Example 12 The difference between this comparative example and Example 2 is that the composite bacteria includes Bacillus amyloliquefaciens, Bacillus licheniformis, and Trichoderma reesei in a weight ratio of 1:1.4:0.6.

[0048] Comparative Example 13 The difference between this comparative example and Example 2 is that PEG pore formation was not performed; The preparation method of the immobilized enzyme is: S1: Crush bamboo chips to 1000-2000 mesh, soak them in 2 wt% NaOH aqueous solution at 80°C for 2 h, wash them with water until neutral, and dry them in an oven at 60°C to a moisture content of 3 wt% to obtain pretreated bamboo powder; S2: 5kg of water was added to 1kg of pretreated bamboo powder, heated to 90°C, soaked for 10h, cooled to 35°C, and a composite bacteria was added. The mixture was fermented for 15d, filtered, and dried to obtain an immobilized carrier; the composite bacteria included Bacillus subtilis, white rot fungi, and Phanerochaete chrysosporium in a weight ratio of 1:1.4:0.6; the amount of the composite bacteria added accounted for 0.1wt% of the pretreated bamboo powder; S3: Add 3 mg of bosera R, 1 mg of coenzyme NADP, 3 mg of isopropanol dehydrogenase, and 50 mg of immobilized support to 50 mL of 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, react in a 35°C water bath shaker for 4 h, filter, wash with 50 mM, pH 7.6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer until no protein is detected, and filter to obtain the immobilized enzyme.

[0049] Test Example 1 Boson purity test Liquid phase analysis was performed on the boson prepared in Examples 1-3 and Comparative Examples 1-5. The test conditions were: Column: XAmide 100A Specifications: 250 mm × 4.6 mm, 5 μm Mobile phase: acetonitrile: methanol = 92:8 (v / v); Flow rate: 1.0 mL / min Injection volume: 10 μL Column temperature: 35°C ELSD parameters: Evaporative light detector temperature: 50°C Flow rate: 2.5 mL / min.

[0050] The configuration and purity results of boson prepared in Examples 1-3 and Comparative Examples 1-5 are shown in Table 1.

[0051] The Bose liquid chromatograms of Example 1 and Example 3 are as follows: Figure 1 and Figure 2 shown.

[0052] Table 1 Effects of different groups on the configuration and purity of Bose Group S:R configuration ratio purity(%) Example 1 100:0 100.00 Example 2 0.32:99.68 99.93 Example 3 40.15:59.85 100.00 Comparative Example 1 99.65:0.35 78.65 Comparative Example 2 0.34:99.66 82.19 Comparative Example 3 99.62:0.38 92.45 Comparative Example 4 99.55:0.45 90.52 Comparative Example 5 99.59:0.41 93.26 As shown in Table 1, the S:R configuration ratio data indicate that the bosylases in Example 1, Comparative Example 1, and Comparative Examples 3-4 are primarily S-form, while the bosylases in Example 2 and Comparative Example 2 are primarily R-form, indicating that the bosylase configuration is directly related to the type of bosylase. The purity data also indicate that the purification method of the present invention significantly improves the purity of bosylase.

[0053] And by Figure 3 The nuclear magnetic resonance spectrum of boson in Example 1 also confirms that the purity of boson obtained by the purification method of the present invention can reach 100%.

[0054] Test Example 2 Determination of the effect of enzyme immobilization The Coomassie Brilliant Blue method was used to detect the adsorption rate of the enzymes in the composite enzyme systems of Examples 1-3 and Comparative Examples 6-13.

[0055] The DNS method was used to detect the apparent activity of the enzymes in the composite enzyme systems of Examples 1-3 and Comparative Examples 6-13. After the immobilized enzymes were reused 10 times, the activity retention rates of the enzymes in the composite enzyme systems were determined. The specific results are shown in Table 2.

[0056] Table 2 Effects of different groups on adsorption rate, apparent activity and activity retention Group Adsorption rate before use mg / g Apparent activity of immobilized enzyme before use (IU / g) Apparent activity retention after 10 uses % Example 1 2043 1452 95.7 Example 2 2051 1475 96.9 Example 3 2034 1461 95.5 Comparative Example 6 1722 1036 84.6 Comparative Example 7 1795 1029 82.8 Comparative Example 8 1738 1042 87.3 Comparative Example 9 1843 1174 89.1 Comparative Example 10 1896 1139 88.7 Comparative Example 11 1957 1275 91.4 Comparative Example 12 1735 1059 83.2 Comparative Example 13 1926 1203 90.5 As can be seen from the data in Table 2, the immobilized carriers of Examples 1-3 have a higher adsorption rate for the enzyme, the apparent activity of the complex enzyme system is high, and the stability of the enzyme in the complex enzyme system is strong. By comparing the data of Example 2 and Comparative Examples 6-12, it can be seen that when Bacillus subtilis, white rot fungi and Phanerochaete chrysosporium are in a specific ratio, the adsorption and stability of the immobilized carrier have a higher adsorption capacity and adsorption stability, indicating that there is a synergistic effect between the three. By comparing the data of Example 2 and Comparative Example 13, it can be seen that after fermentation, PEG pores are formed, and by using a microbial-chemical combination strategy, the two coenzymes NADP can be improved to have a higher adsorption capacity and adsorption stability. It can be seen that Examples 1-3 of the present invention can achieve efficient synergy of the "carrier-enzyme-substrate" ternary system by using a specific ratio of bacterial strains for fermentation.

[0057] Test Example 3 Verification of the moisturizing effect of Bosin Refer to the detection method of zebrafish moisturizing effect in CN118178300A patent.

[0058] The zebrafish epidermis has an osmotic pressure tolerance range, exceeding which it loses water. Treatment of zebrafish with a hyperosmotic pressure solution causes their tails to shrink and shrink due to water loss. Therefore, an inductive model of hydration and moisturizing in zebrafish was established to evaluate the moisturizing effects of bosera.

[0059] The test method for the moisturizing effect of zebrafish is as follows: (1) Breeding: Zebrafish were paired at a male-to-female ratio of 2:1 for breeding, and the eggs were collected and incubated overnight (incubator at 28°C).

[0060] (2) Experimental grouping: The experiment set up a blank group and a sample group, with 10 2-dpf juvenile fish in each group.

[0061] The sample groups were grouped as follows: Example 1 group: Example 1 Bose; Example 2 group: Example 2 Bose; Example 3 group: Example 3 Bose; S / R1 group: the mass ratio of Bose-like factor of Example 1 to Bose-like factor of Example 2 is 1:0.3; S / R2 group: the mass ratio of Bose-like factor of Example 1 to Bose-like factor of Example 2 is 1:0.5; S / R3 group: the mass ratio of Bose-like factor of Example 1 to Bose-like factor of Example 2 was 1:1; S / R4 group: the mass ratio of Bose-like factor of Example 1 to Bose-like factor of Example 2 is 1:1.5; S / R1 group: the mass ratio of Bose-like factor of Example 1 to Bose-like factor of Example 2 is 1:2; Comparative Example 1: Comparative Example 1 Bose-like; Comparative Example 2 Group: Comparative Example 2 Group Bose; Comparative Example 3 Group: Comparative Example 3 Group Bose; Comparative Example 4: Comparative Example 4 Bose-like; Comparative Example 5: Comparative Example 5 Bose-like; For groups 1 and 2: the mass ratio of the Bose-like factor in group 1 of comparative example to that in group 2 of comparative example is 1:1.

[0062] (3) Solution Preparation: Accurately weigh 1 mg of sample and add purified water to prepare a 0.5 wt% bosin solution. Add 1 mg of sample to each well. After dosing, place the 6-well plate in a 28°C incubator in the dark for 24 h.

[0063] The body surface area of ​​each group was measured after 24 h of dark incubation, and the area reduction inhibition rate was calculated. The specific results are shown in Table 3.

[0064] Area reduction inhibition rate (%) = (area of ​​the tail of the sample zebrafish - area of ​​the tail of the blank group zebrafish) ÷ area of ​​the tail of the blank group zebrafish × 100%.

[0065] Table 3 Area reduction inhibition rate of each group Group Area reduction inhibition rate (%) Group Area reduction inhibition rate (%) Example 1 group 45.7 S / R5 group 63.7 Example 2 group 40.9 Comparative Example 1 33.4 Example 3 group 66.5 Comparative Example 2 32.6 S / R1 group 64.1 Comparative Example 3 38.1 S / R2 group 66.3 Comparative Example 4 37.0 S / R3 group 72.5 Comparative Example 5 34.2 S / R4 group 65.2 1+2 pairs 63.1 It can be seen from the data in Table 3 that the effect of a single R-type or S-type boson on inhibiting the reduction of the zebrafish face and tail area is poor. The combination of R-type and S-type boson can improve the effect of inhibiting the reduction of the zebrafish face and tail area. Overall, the embodiment group has a better moisturizing effect than the comparative group, because the concentration of boson in the embodiment group is high, thereby improving the moisturizing effect. It can be seen from the data of the embodiment group 3 and the S / R1 group to the S / R5 group that when the mass ratio of S-type boson and R-type boson is 1:1, the moisturizing effect is the best. It can be seen from the data of Example 1 and Comparative Example 1 and Comparative Examples 3-5 that the electrodialysis, ultrafiltration and nanofiltration steps can improve the purity of boson, thereby improving the moisturizing effect.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for purifying bosine synthesized by enzyme catalysis, characterized in that: include: β-acetone xyloside, isopropyl alcohol and water are mixed, a composite enzyme system is added, and an enzyme catalytic reaction is carried out. The mixture is filtered, distilled under reduced pressure, decolorized and filtered, and the filtrate is subjected to electrodialysis, ultrafiltration and nanofiltration. After vacuum concentration, anhydrous ethanol is added, crystallized and vacuum dried to obtain bosylamine. The electric field strength of the electrodialysis is 20-30 V / cm, the water flow rate is 5-10 cm / s, and the current density is 20-30 mA / cm 2 .

2. The purification method according to claim 1, wherein The complex enzyme is obtained by immobilizing bosera, coenzyme NADP and isopropanol dehydrogenase via an immobilized carrier.

3. The purification method according to claim 1, wherein The pressure of the reduced pressure distillation is 12-14 kPa, the temperature is 45-55° C., and the time is 1-2 h.

4. The purification method according to claim 1, wherein The decolorization adopts activated carbon adsorption, the pore size of the activated carbon is 0.5-2nm, and the amount used is 3% of the weight of the β-acetone xyloside and isopropyl alcohol.

5. The purification method according to claim 1, wherein The molecular weight cut-off of the ultrafiltration is 3000-5000Da.

6. The purification method according to claim 1, characterized in that The molecular weight cut-off of the nanofiltration is 100-200 Da.

7. The purification method according to claim 1, characterized in that The amount of anhydrous ethanol used is 0.8-1.2 times the weight of β-acetone xyloside.

8. The purification method according to claim 1, characterized in that The crystallization temperature is 0-4°C, the rotation speed is 50-100 rpm, and the time is 50-70 min.

9. Bosine obtained by the purification method according to any one of claims 1 to 8.

10. Use of the bosine according to claim 9 in preparing cosmetics.

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