Method for preparing isothiocyanate compound

Abstract

The invention belongs to the technical field of preparation of organic compounds, and particularly relates to a preparation method of an isothiocyanate compound. According to the method, the myrosinase is fixed by using the metal organic framework, and column extraction is combined, so that the high-purity isothiocyanate compound can be obtained at high yield, the yield of the isothiocyanate compound is 90% or above, the purity is 97% or above, and the preparation efficiency is high. Compared with a traditional extraction method, the method has the advantages that immobilized myrosinase reaction and column extraction are coupled in real time, a solvent is saved, meanwhile, the product isothiocyanate compound is transferred into an organic phase in time, decomposition of the isothiocyanate compound and potential inhibition of the isothiocyanate compound on the activity of myrosinase are avoided, and the method is suitable for industrial production. The isothiocyanate compound is efficiently enriched, so that the purity and the yield of the isothiocyanate compound are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of organic compound preparation, and particularly relates to a method for preparing an isothiocyanate compound. Background Art

[0002] Isothiocyanates (ITCs), a naturally occurring active ingredient primarily derived from cruciferous plants, are believed to possess anti-cancer, anti-inflammatory, and anti-aging properties. However, ITCs are not directly present in the tissues of cruciferous plants. Instead, they are produced by myrosinase (MYR) hydrolyzing the precursor glucosinolate (GLS) after the tissues are damaged. Compared to traditional chemical synthesis, extracting ITCs from cruciferous plants offers advantages such as being environmentally friendly, inexpensive, simple, and efficient, making them more readily accepted by consumers.

[0003] However, due to the presence of impurities such as epithiospecifier protein (ESP), nitrile specifier protein (NSP) and thiocyanate-forming protein (TFP), glucosinolates are hydrolyzed into cyclic thionitrile, nitrile and thiocyanate in addition to isothiocyanate compounds, such as Figure 1 As shown, the conversion rate of glucosinolates to isothiocyanates is only 10-40%. Furthermore, isothiocyanates are prone to decomposition to form compounds such as amines and thioureas, resulting in a loss of 20-60% of the isothiocyanate compounds. The production of these byproducts and decomposition products reduces the yield and purity of isothiocyanate compounds, increases the difficulty of isothiocyanate purification, and potentially threatens the safety of isothiocyanate products. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing an isothiocyanate compound. The preparation method provided by the present invention can obtain a high-purity isothiocyanate compound with a high yield.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing an isothiocyanate compound, comprising the following steps:

[0007] (1) mixing a zinc source, concanavalin, 2-methylimidazole and water to perform a polymerization reaction to obtain a ZIF-8 core solution; the polymerization reaction temperature is 5 to 40° C., and the holding time is 5 to 60 minutes;

[0008] (2) mixing the ZIF-8 core solution with myrosinase extract, 2-methylimidazole and water to perform polymerization and immobilization reaction to obtain immobilized myrosinase;

[0009] (3) The immobilized myrosinase is loaded into a chromatography column to obtain an immobilized enzyme chromatography column, and the glucosinolate aqueous solution is sequentially passed through the immobilized enzyme chromatography column and the extraction column and then circulated to obtain an isothiocyanate compound.

[0010] Preferably, in step (1), the ratio of the amount of the zinc source to the mass of concanavalin is (0.1-5) mol: (1-500) g; and the ratio of the amount of the zinc source to the first 2-methylimidazole is 10-500:40-2000.

[0011] Preferably, in step (2), the ZIF-8 mother core solution is mixed with the myrosinase extract, 2-methylimidazole and water by stirring and premixing the ZIF-8 mother core solution and the myrosinase extract to obtain a premixed solution, and then adding a 2-methylimidazole aqueous solution dropwise to the premixed solution.

[0012] Preferably, the total protein concentration of the myrosinase extract is 2-80 mg / mL; the mass ratio of concanavalin to the total protein in the myrosinase extract is 0.1-50:2-80; the concentration of the 2-methylimidazole aqueous solution is 40-2000 mM; and the molar ratio of the zinc source to the second 2-methylimidazole in the 2-methylimidazole aqueous solution is 10-500:40-2000.

[0013] Preferably, in step (2), the temperature of the polymerization and fixation reaction is 5-40° C., and the holding time is 5-480 minutes; the polymerization and fixation reaction is carried out under stirring conditions, and the stirring speed is 200-1000 rpm.

[0014] Preferably, in step (3), the density of the immobilized myrosinase is 5 to 5.1 mg / cm 3 .

[0015] Preferably, in step (3), the extractant of the extraction column comprises one or both of dichloromethane and ethyl acetate; and the volume ratio of the extractant of the extraction column to the glucosinolate aqueous solution is 1 to 10:100.

[0016] Preferably, in step (3), the introduction rate of the glucosinolate aqueous solution is 1 to 100 column volumes per hour.

[0017] Preferably, in step (3), the total cycle time is 0.1 to 50 hours.

[0018] Preferably, the isothiocyanate compound includes one or more of raphanin and sulforaphane.

[0019] The present invention provides a method for preparing isothiocyanate compounds. The method utilizes a metal-organic framework to immobilize myrosinase, combined with column extraction, to obtain high-purity isothiocyanate compounds with a high yield of over 90% and a purity of over 97%, achieving high preparation efficiency.

[0020] First, the immobilized myrosinase of the present invention is synthesized in one pot, which is highly efficient and simple in steps. It does not require nitrogen protection, ammonia water, or many organic reagents. The present invention uses the ZIF-8 metal-organic framework as a carrier for the immobilized myrosinase, can synthesize the carrier at room temperature in an aqueous solution, and can in situ include concanavalin, thereby having good biocompatibility and ensuring the activity and stability of the immobilized myrosinase.

[0021] Secondly, the present invention introduces concanavalin to construct a ZIF-8 mother core with myrosinase affinity, thereby achieving selective immobilization of myrosinase, removing impurities such as ESP that cause a decrease in product purity, and fundamentally avoiding the generation of some impurities, thereby greatly improving the purification and utilization efficiency of myrosinase. Figure 2 shown.

[0022] In addition, compared with traditional extraction methods, the present invention couples the immobilized myrosinase reaction with column extraction in real time, saving solvent (only less than 10% of the solvent volume of traditional extraction methods) while timely transferring the product isothiocyanate compounds to the organic phase, avoiding the decomposition of the isothiocyanate compounds and the potential inhibition of the isothiocyanate compounds on the myrosinase activity, efficiently enriching the isothiocyanate compounds, and improving the purity and yield of the isothiocyanate compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is a diagram of the hydrolysis products of glucosinolates under different conditions;

[0025] Figure 2 The diagram shows the synthesis and action mechanism of immobilized myrosinase;

[0026] Figure 3 This is a scanning electron micrograph of the immobilized myrosinase prepared in Example 1;

[0027] Figure 4 Schematic diagram of the device for immobilized myrosinase combined with column extraction (taking dichloromethane extraction as an example);

[0028] Figure 5 This is the liquid chromatogram of the isothiocyanate compound radish extract prepared in Example 1. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing an isothiocyanate compound, comprising the following steps:

[0030] (1) mixing a zinc source, concanavalin, 2-methylimidazole and water to perform a polymerization reaction to obtain a ZIF-8 mother core solution;

[0031] (2) mixing the ZIF-8 core solution with myrosinase extract, 2-methylimidazole and water to perform polymerization and immobilization reaction to obtain immobilized myrosinase;

[0032] (3) The immobilized myrosinase is loaded into a chromatography column to obtain an immobilized enzyme chromatography column, and the glucosinolate aqueous solution is sequentially passed through the immobilized enzyme chromatography column and the extraction column and then circulated to obtain an isothiocyanate compound.

[0033] In the present invention, a zinc source, concanavalin, 2-methylimidazole (denoted as the first 2-methylimidazole), and water are mixed (denoted as the first mixing) and subjected to a polymerization reaction to obtain a ZIF-8 core solution. In the present invention, the zinc source can be a soluble zinc salt; the soluble zinc salt can include one or both of zinc acetate and zinc nitrate, preferably zinc acetate.

[0034] In the present invention, the ratio of the amount of the zinc source to the mass of concanavalin can be (0.1-5) mol: (1-500) g, specifically 0.5 mol: 10 g, 0.5 mol: 50 g, 0.5 mol: 100 g, 0.5 mol: 200 g, 0.5 mol: 300 g, 0.5 mol: 400 g, 1 mol: 10 g, 1 mol: 50 g, 1 mol: 100 g, 1 mol: 200 g, 1 mol: 300 g, 1 mol: 400 g, 3 mol: 10 g, 3 mol: 50 g, 3 mol: 100 g, 3 mol: 200 g, 3 mol: 300 g or 3 mol: 400 g.

[0035] In the present invention, the molar ratio of the zinc source to the first 2-methylimidazole can be 10-500:40-2000, specifically 50:110, 50:210, 50:510, 50:1010, 50:1510, 50:1810, 100:110, 100:210, 100:510, 100:1010, 100:1510, 100:1810, 200:110, 200:210, 200:510, 200:1010, 200:1510, 200:1810, 400:110, 400:210, 400:510, 400:1010, 400:1510 or 400:1810. The invention forms a ZIF-8 mother core which wraps concanavalin by adding a first 2-methylimidazole.

[0036] In the present invention, the first mixing may be: preparing a zinc source aqueous solution, and adding the zinc source aqueous solution to a mixed aqueous solution containing concanavalin and 2-methylimidazole while stirring.

[0037] In the present invention, the concentration of the zinc source aqueous solution can be 10 to 500 mM, specifically 40 mM, 100 mM or 300 mM; the concentration of concanavalin in the mixed aqueous solution can be 0.1 to 50 mg / mL, specifically 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL or 40 mg / mL; the concentration of 2-methylimidazole can be 40 to 2000 mM, specifically 160 mM, 500 mM, 1000 mM or 1500 mM.

[0038] In the present invention, the volume ratio of the zinc source aqueous solution to the mixed aqueous solution may be 1:1.

[0039] In the present invention, the polymerization reaction temperature can be 5 to 40°C, specifically 10°C, 20°C or 30°C, and the insulation time can be 5 to 60 minutes, specifically 20 minutes or 40 minutes; the polymerization reaction can be carried out under stirring conditions, and the stirring speed can be 200 to 1000 rpm, specifically 500 rpm.

[0040] After obtaining the ZIF-8 mother core solution, the present invention mixes the ZIF-8 mother core solution with a myrosinase extract, 2-methylimidazole (denoted as the second 2-methylimidazole), and water (denoted as the second mixture) to perform polymerization and immobilization reactions to obtain immobilized myrosinase. In the present invention, the method for extracting the myrosinase extract may include the following steps: crushing a cruciferous plant or crushing a microorganism, followed by sonication and solid removal, wherein the microorganism is a microorganism genetically engineered to express myrosinase.

[0041] In the present invention, the cruciferous plant may include one or more of broccoli, radish and rapeseed, preferably radish; the part of the cruciferous plant may include one or more of seeds, leaves, stems and flowers, preferably seeds; the microorganism expressing myrosinase through genetic engineering may be the microorganism reported by Jiang Hong's team of Ocean University of China (J.Agric.Food Chem.2021,69,18,5363-5371).

[0042] In the present invention, the target particle size of the crushing can be 60-80 meshes, specifically 70 meshes; the crushing is based on cell crushing.

[0043] In the present invention, the frequency of the ultrasound can be 20 to 40 kHz, specifically 30 kHz, and the time of the ultrasound can be 10 to 30 minutes, specifically 20 minutes.

[0044] In the present invention, the solid removal may be performed by centrifugation.

[0045] In the present invention, the second mixing may be: stirring and premixing the ZIF-8 core solution and the myrosinase extract to obtain a premixed solution, and then dropwise adding a 2-methylimidazole aqueous solution to the premixed solution.

[0046] In the present invention, the total protein concentration of the myrosinase extract may be 2 to 80 mg / mL, specifically 5 mg / mL, 15 mg / mL, 25 mg / mL, 45 mg / mL or 65 mg / mL.

[0047] In the present invention, the mass ratio of the concanavalin and the total protein in the myrosinase extract can be 0.1-50:2-80, specifically 1:5, 1:10, 1:20, 1:50, 1:70, 10:5, 10:10, 10:20, 10:50, 10:70, 25:5, 25:10, 25:20, 25:50, 25:70, 35:5, 35:10, 35:20, 35:50 or 35:70.

[0048] In the present invention, the concentration of the 2-methylimidazole aqueous solution may be 40 to 2000 mM, specifically 100 mM, 200 mM, 500 mM, 800 mM, 1200 mM or 1600 mM.

[0049] In the present invention, the molar ratio of the zinc source to the second 2-methylimidazole in the 2-methylimidazole aqueous solution can be 10-500:40-2000, specifically 50:110, 50:210, 50:510, 50:1010, 50:1510, 50:1810, 100:110, 100:210, 100:510, 100:1010, 100:1510, 100:1810, 200:110, 200:210, 200:510, 200:1010, 200:1510, 200:1810, 400:110, 400:210, 400:510, 400:1010, 400:1510 or 400:1810. The invention stably combines the myrosinase on the ZIF-8 mother nucleus through the second 2-methylimidazole.

[0050] In the present invention, the volume ratio of the myrosinase extract to the 2-methylimidazole aqueous solution may be 1:1.

[0051] In the present invention, the rotation speed of the stirring premix can be 200-1000 rpm, specifically 500 rpm.

[0052] In the present invention, the dropping rate may be 0.1 to 2 mL / s, specifically 0.5 mL / s, 1 mL / s or 1.5 mL / s.

[0053] In the present invention, the temperature of the polymerization and fixation reaction can be 5 to 40°C, specifically 20°C, and the insulation time can be 5 to 480 minutes, specifically 60 minutes, 120 minutes, 240 minutes or 360 minutes; the polymerization and fixation reaction can be carried out under stirring conditions, and the stirring speed can be 200 to 1000 rpm, specifically 500 rpm.

[0054] In the present invention, after the polymerization and fixation reaction, the precipitate may be collected by centrifugation and then washed with water; the number of times of washing may be more than 3 times.

[0055] After obtaining the immobilized myrosinase, the present invention loads the immobilized myrosinase into a chromatography column to obtain an immobilized enzyme chromatography column, and sequentially passes the glucosinolate aqueous solution through the immobilized enzyme chromatography column and the extraction column, and then circulates to obtain an isothiocyanate compound. In the present invention, the density of the immobilized myrosinase loading can be 5 to 5.1 mg / cm 3 ; The inner diameter of the chromatography column can be 10 mm and the length can be 200 mm.

[0056] In the present invention, the concentration of the glucosinolate aqueous solution may be 0.1 to 100 g / L, specifically 1 g / L, 5 g / L, 10 g / L, 20 g / L, 50 g / L, 70 g / L or 90 g / L.

[0057] In the present invention, the extractant of the extraction column may include one or both of dichloromethane and ethyl acetate; the volume ratio of the extractant of the extraction column to the glucosinolate aqueous solution may be 1 to 10:100, specifically 2:100, 4:100, 6:100 or 8:100; the inner diameter of the extraction column may be 10 mm, and the length may be 200 mm.

[0058] In the present invention, the introduction rate of the glucosinolate aqueous solution can be 1 to 100 times the column volume per hour, specifically 6 times, 10 times, 20 times, 30 times, 50 times, 70 times or 90 times.

[0059] In the present invention, the total cycle time may be 0.1 to 50 hours, specifically 3 hours, 8 hours, 15 hours, 25 hours, 35 hours or 45 hours.

[0060] In the present invention, after the circulation, the organic phase of the obtained product system may be separated and then evaporated to dryness; the evaporation temperature may be 20-40°C, specifically 30°C.

[0061] In the present invention, the isothiocyanate compound may include one or more of raphanin and sulforaphane.

[0062] In order to further illustrate the present invention, the scheme of the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] Extracting myrosinase from radish seeds: The radish seeds were crushed (particle size 70 mesh) and then ultrasonically extracted with ultrasonic wave (30kHz, 20min). The solids were removed by centrifugation to obtain crude myrosinase solution.

[0065] Prepare 10 mL of 160 mM zinc acetate aqueous solution, and slowly add 10 mL of a mixed aqueous solution containing 2 mg / mL concanavalin and 400 mM 2-methylimidazole while stirring. After the addition is complete, continue stirring for 15 minutes to react. The reaction temperature is controlled at 20°C.

[0066] Maintain stirring and add 10 mL of crude myrosinase solution with a total protein concentration of 40 mg / mL to the above solution, then slowly add 10 mL of 400 mM 2-methylimidazole aqueous solution. After the addition is complete, continue stirring for 45 minutes to react. The reaction temperature is controlled at 20°C. After stirring, centrifuge and collect the precipitate, wash it with water three times, and obtain immobilized myrosinase. Scanning electron microscopy shows Figure 3 shown.

[0067] according to Figure 3 It can be seen that the surface of the ZIF-8 crystal's regular dodecahedron structure presents obvious uniform particle-like protrusions, confirming the successful immobilization of myrosinase.

[0068] 80 mg of the immobilized myrosinase was loaded into a chromatography column (inner diameter 10 mm, length 200 mm), and a radish-derived glucosinolate aqueous solution (concentration 2 g / L) was introduced. The glucosinolate aqueous solution passed through the immobilized myrosinase and then passed into a dichloromethane extraction column. The glucosinolate aqueous solution flowing out of the extraction column was then returned to the chromatography column containing the immobilized myrosinase, as shown in FIG. Figure 4 As shown; the amount of dichloromethane is 0.1 times that of the aqueous solution of glucosinolates, and the flow rate is 3 times the column volume per hour for 1 hour to obtain 97.8% pure radish root, the liquid chromatogram is as shown Figure 5 As shown, the yield was 93%.

[0069] according to Figure 5 It can be seen that the obtained radish extract has no obvious impurities in the liquid chromatography detection.

[0070] Example 2

[0071] Extracting myrosinase from rapeseed seeds: The seeds were crushed (particle size 70 mesh) and then ultrasonically extracted (30 kHz, 20 min). The solids were removed by centrifugation to obtain a crude myrosinase solution.

[0072] Prepare 10 mL of 40 mM zinc acetate aqueous solution, and slowly add 10 mL of a mixed aqueous solution containing 1 mg / mL concanavalin and 160 mM 2-methylimidazole while stirring. After the addition is complete, continue stirring for 20 minutes to react. The reaction temperature is controlled at 25°C.

[0073] While maintaining stirring, 10 mL of crude myrosinase solution with a total protein concentration of 20 mg / mL was added to the above solution. Subsequently, 10 mL of a 160 mM 2-methylimidazole aqueous solution was slowly added dropwise. After the addition was complete, stirring was continued for 60 minutes to allow the reaction to proceed at a temperature of 25°C. After stirring, the precipitate was collected by centrifugation and washed three times with water to obtain immobilized myrosinase.

[0074] 80 mg of the immobilized myrosinase was loaded into a chromatography column (inner diameter 10 mm, length 200 mm), and a broccoli-derived glucosinolate aqueous solution (concentration 2 g / L) was introduced. The glucosinolate aqueous solution passed through the immobilized myrosinase and then passed into a dichloromethane extraction column. The glucosinolate aqueous solution flowing out of the extraction column was then returned to the chromatography column containing the immobilized myrosinase. The amount of dichloromethane used was 0.05 times that of the glucosinolate aqueous solution. The mixture was circulated for 3 hours at a flow rate of 6 column volumes per hour to obtain sulforaphane with a purity of 97.1% and a yield of 90%.

[0075] Example 3

[0076] Extracting myrosinase from broccoli seeds: The broccoli seeds were crushed (particle size 70 mesh) and myrosinase was extracted by ultrasonication (30 kHz, 20 min). The solids were removed by centrifugation to obtain a crude myrosinase solution.

[0077] Prepare 10 mL of 200 mM zinc nitrate aqueous solution, and slowly add 10 mL of a mixed aqueous solution containing 2 mg / mL concanavalin and 1600 mM 2-methylimidazole while stirring. After the addition is complete, continue stirring for 30 minutes to react. The reaction temperature is controlled at 20°C.

[0078] While maintaining stirring, 10 mL of crude myrosinase solution with a total protein concentration of 40 mg / mL was added to the above solution. Subsequently, 10 mL of a 1600 mM 2-methylimidazole aqueous solution was slowly added dropwise. After the addition was complete, stirring was continued for 240 minutes to allow the reaction to proceed at a temperature of 20°C. After stirring, the precipitate was collected by centrifugation and washed three times with water to obtain immobilized myrosinase.

[0079] 80 mg of the immobilized myrosinase was loaded into a chromatography column (inner diameter 10 mm, length 200 mm), and a radish-derived glucosinolate aqueous solution (concentration 2 g / L) was introduced. The glucosinolate aqueous solution passed through the immobilized myrosinase and then passed into an ethyl acetate extraction column. The glucosinolate aqueous solution flowing out of the extraction column was then returned to the chromatography column containing the immobilized myrosinase. The amount of ethyl acetate used was 0.1 times the amount of the glucosinolate aqueous solution. The column was circulated for 10 hours at a flow rate of 12 column volumes per hour to obtain radish extract with a purity of 97.7% and a yield of 90.8%.

[0080] Comparative Example 1 (Concanavalin replaced by bovine serum albumin)

[0081] Extracting myrosinase from radish seeds: The radish seeds were crushed (particle size 70 mesh) and then ultrasonically extracted with ultrasonic wave (30kHz, 20min). The solids were removed by centrifugation to obtain crude myrosinase solution.

[0082] Prepare 10 mL of 160 mM zinc acetate aqueous solution, and slowly add 10 mL of a mixed aqueous solution containing 2 mg / mL bovine serum albumin and 400 mM 2-methylimidazole while stirring. After the addition is complete, continue stirring for 15 minutes to react. The reaction temperature is controlled at 20°C.

[0083] While maintaining stirring, 10 mL of crude myrosinase solution with a total protein concentration of 40 mg / mL was added to the above solution. Subsequently, 10 mL of a 400 mM 2-methylimidazole aqueous solution was slowly added dropwise. After the addition was complete, stirring was continued for 45 minutes to allow the reaction to proceed at a temperature of 20°C. After stirring, the precipitate was collected by centrifugation and washed three times with water to obtain immobilized myrosinase.

[0084] 80 mg of the immobilized myrosinase was loaded into a chromatography column (inner diameter 10 mm, length 200 mm), and a radish-derived glucosinolate aqueous solution (concentration 2 g / L) was introduced. The glucosinolate aqueous solution passed through the immobilized myrosinase and then passed into a dichloromethane extraction column. The glucosinolate aqueous solution flowing out of the extraction column was then returned to the chromatography column containing the immobilized myrosinase. The amount of dichloromethane used was 0.1 times the amount of the glucosinolate aqueous solution. The solution was circulated for 1 hour at a flow rate of 3 column volumes per hour, yielding radish extract with a purity of 73.2% and a yield of 90%.

[0085] Comparative Example 2 (column extraction changed to static extraction)

[0086] Extracting myrosinase from radish seeds: The radish seeds were crushed (particle size 70 mesh) and then ultrasonically extracted with ultrasonic wave (30kHz, 20min). The solids were removed by centrifugation to obtain crude myrosinase solution.

[0087] Prepare 10 mL of 160 mM zinc acetate aqueous solution, and slowly add 10 mL of a mixed aqueous solution containing 2 mg / mL concanavalin and 400 mM 2-methylimidazole while stirring. After the addition is complete, continue stirring for 15 minutes to react. The reaction temperature is controlled at 20°C.

[0088] While maintaining stirring, 10 mL of crude myrosinase solution with a total protein concentration of 40 mg / mL was added to the above solution. Subsequently, 10 mL of a 400 mM 2-methylimidazole aqueous solution was slowly added dropwise. After the addition was complete, stirring was continued for 45 minutes to allow the reaction to proceed at a temperature of 20°C. After stirring, the precipitate was collected by centrifugation and washed three times with water to obtain immobilized myrosinase.

[0089] The immobilized myrosinase was used to hydrolyze a radish-derived glucosinolate aqueous solution (concentration of 2 g / L) for 1 hour, and the glucosinolate aqueous solution was statically extracted with dichloromethane, where the amount of dichloromethane used was 0.1 times that of the glucosinolate aqueous solution, to obtain radish sucrose with a purity of 89.8% and a yield of 47%.

[0090] It can be seen from the above examples that the preparation method provided by the present invention can obtain high-purity isothiocyanate compounds with high yield. The yield of the isothiocyanate compounds is above 90%, the purity is above 97%, and the preparation efficiency is high.

[0091] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing an isothiocyanate compound, characterized in that: The following steps are involved: (1) mixing a zinc source, concanavalin, 2-methylimidazole and water to perform a polymerization reaction to obtain a ZIF-8 core solution; the polymerization reaction temperature is 5 to 40° C., and the holding time is 5 to 60 minutes; (2) mixing the ZIF-8 core solution with myrosinase extract, 2-methylimidazole and water to perform polymerization and immobilization reaction to obtain immobilized myrosinase; (3) The immobilized myrosinase is loaded into a chromatography column to obtain an immobilized enzyme chromatography column, and the glucosinolate aqueous solution is sequentially passed through the immobilized enzyme chromatography column and the extraction column and then circulated to obtain an isothiocyanate compound.

2. The preparation method according to claim 1, characterized in that In step (1), the ratio of the amount of the zinc source to the mass of concanavalin is (0.1-5) mol: (1-500) g; The molar ratio of the zinc source to 2-methylimidazole is 10-500:40-2000.

3. The preparation method according to claim 1, characterized in that In step (2), the mixing is as follows: the ZIF-8 core solution and the myrosinase extract are stirred and premixed to obtain a premixed solution, and then a 2-methylimidazole aqueous solution is added dropwise to the premixed solution.

4. The preparation method according to claim 3, characterized in that The total protein concentration of the myrosinase extract is 2 to 80 mg / mL; The mass ratio of the concanavalin to the total protein in the myrosinase extract is 0.1-50:2-80; The concentration of the 2-methylimidazole aqueous solution is 40 to 2000 mM; The molar ratio of the zinc source to the 2-methylimidazole in the 2-methylimidazole aqueous solution is 10-500:40-2000.

5. The preparation method according to any one of claims 1 to 4, characterized in that In step (2), the polymerization and fixation reaction temperature is 5 to 40° C., and the holding time is 5 to 480 minutes; The polymerization and fixation reactions are carried out under stirring conditions, and the stirring speed is 200-1000 rpm.

6. The preparation method according to claim 1, characterized in that In step (3), the density of the immobilized myrosinase is 5-5.1 mg / cm 3 .

7. The preparation method according to claim 1, characterized in that In step (3), the extraction agent of the extraction column includes one or both of dichloromethane and ethyl acetate; The volume ratio of the extractant of the extraction column to the glucosinolate aqueous solution is 1 to 10:

100.

8. The preparation method according to claim 1 or 6, characterized in that In step (3), the introduction rate of the glucosinolate aqueous solution is 1 to 100 column volumes per hour.

9. The preparation method according to claim 1, characterized in that In step (3), the total cycle time is 0.1 to 50 hours.

10. The preparation method according to claim 1 or 2, characterized in that: The isothiocyanate compound includes one or more of raphanin and sulforaphane.

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