Magnetic nitrilase catalyst for green cyclic synthesis of organic acid as well as preparation method and application of magnetic nitrilase catalyst

Abstract

The invention relates to the technical field of enzyme catalysis, relates to the technical field of bioengineering application, and in particular relates to a magnetic nitrilase catalyst for green cyclic synthesis of organic acid as well as a preparation method and application of the magnetic nitrilase catalyst. According to the method, nitrilase escherichia coli is fixed on a magnetic composite carrier in a covalent binding and adsorption-crosslinking manner to form a magnetic nitrilase catalyst, and the catalyst is used for catalytic synthesis of organic acid; after the reaction is finished, the magnetic nitrilase catalyst is recovered by magnetic attraction, and the reaction waste liquid is acidized to realize the purpose of green cyclic synthesis of the organic acid. The prepared magnetic nitrilase catalyst can stably exist in nitrile substances and has the advantages of being high in mechanical strength, low in enzyme activity loss rate and easy to store, the magnetic nitrilase catalyst and reaction liquid can be recycled, beneficial reference is provided for promoting clean, environment-friendly and efficient enzyme method production of organic acid, and the application prospect is wide. The method has huge application potential in continuous separation and industrial application.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and in particular to a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, a preparation method and application thereof. Background Art

[0002] As the fourth generation of new pesticides in the world, pyridine pesticides are an important non-ionic insecticide with the characteristics of high efficiency, low toxicity and broad spectrum. In recent years, with the growing demand for biological control in agricultural production, pyridine pesticides have developed rapidly in China. Nitrile compounds have become a hot spot in the research and development of pesticide intermediates due to their practical biological activity and unique chemical mechanism. So far, many nitrile compounds have been successfully developed into important pesticide varieties, such as organic acid pesticides such as clopyralid, fluclorac and amiloride. Among them, 3,4,5,6-tetrachloropyridine carboxylic acid is one of the important intermediates for the synthesis of pyridine herbicides and plant growth regulators such as clopyralid. It has the advantages of high efficiency, low toxicity, long lasting effect, good environmental compatibility with humans and organisms, and has broad market prospects. At present, the method for synthesizing the product 3,4,5,6-tetrachloropyridine carboxylic acid is to use 3,4,5,6-tetrachlorocyanopyridine as a raw material and use chemical methods to hydrolyze it to obtain 3,4,5,6-tetrachloropyridine carboxylic acid. There is also a process for re-synthesizing 3,4,5,6-tetrachloropyridine carboxylic acid from the by-products of amiloride production, but these processes are cumbersome, have many by-products, and produce a large amount of three wastes, which does not meet the requirements of green development. In addition, the wastewater in the production process of pyridine pesticides has the characteristics of complex composition, high toxicity, difficulty in degradation, high salt content and poor biodegradability. Direct discharge without purification and recycling will cause problems such as waste and depletion of water resources.

[0003] Nitrilase is an important industrial enzyme that can catalyze the production of carboxylic acids from nitrile compounds. The hydrolysis reaction catalyzed by it has the advantages of high efficiency, good specificity and mild conditions. Therefore, nitrilase can be used in the production of tetrachloropyralidin to solve the key problems in this route and explore a reliable, scalable and sustainable green approach.

[0004] However, free nitrilase is sensitive to reaction conditions and has poor reusability. It is also not stable enough to heat, high ion concentration, strong acid, strong base and some organic solvents, and is easy to reduce or even lose its catalytic ability. At present, the industrial biocatalytic process of nitrilase mostly uses whole cells as catalysts. The cells provide a suitable microenvironment for nitrilase, which can reduce the influence of the external environment on the enzyme activity to a certain extent. However, free cells are prone to breakage during the reaction process, releasing the contained nitrilase and cell components, resulting in problems such as easy inactivation of the enzyme and inability to reuse the cells. In addition, after the enzymatic reaction, the enzyme is not easy to separate from the substrate and the product, and there are more or less enzyme residues in the product, resulting in low product purity and poor enzyme recovery and reusability. These deficiencies of free enzymes greatly restrict their application in actual production. In order to solve the problem of enzyme existence, people usually fix the enzyme in a carrier material. The reported nitrilase immobilization research usually uses resin, chitosan, carrageenan gel and sodium alginate as carriers to obtain immobilized enzymes through crosslinking and adsorption. However, using the above method, both the enzyme immobilization efficiency and the stability of the immobilized enzyme need to be improved. Summary of the invention

[0005] The present invention is made to solve the above-mentioned problems, and aims to provide a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, a preparation method and application thereof. The magnetic nitrilase catalyst prepared by the preparation method can be reused, can greatly improve the catalytic efficiency and stability of the bacteria in the reaction process, can be applied to the synthesis process of organic acids such as 3,4,5,6-tetrachloropyridinecarboxylic acid, 3,5-dichlorobenzoic acid, 3-chlorobenzoic acid or 3,6-dichloropyridine-3-carboxylic acid, and acidification and neutralization treatment is performed on the reaction waste liquid to achieve the recycling of the reaction waste liquid.

[0006] The first aspect of the present invention provides a method for preparing a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, characterized in that it comprises the following steps:

[0007] (1) Preparation of magnetic diatomite by physical blending: diatomite, magnetic material and cross-linking agent are mixed and reacted to obtain magnetic diatomite;

[0008] (2) preparing an Escherichia coli-magnetic diatomaceous earth adsorption system: mixing a wet suspension of Escherichia coli containing nitrilase with magnetic diatomaceous earth, and stirring and adsorbing the mixture to obtain an Escherichia coli-magnetic diatomaceous earth adsorption system;

[0009] (3) Magnetic nitrilase catalyst: A cross-linking agent is added to the Escherichia coli-magnetic diatomaceous earth adsorption system, and the mixture is stirred for reaction and then treated to obtain the magnetic nitrilase catalyst.

[0010] In recent years, magnetic materials have shown unique advantages as carriers in immobilization. Due to their magnetic attraction characteristics, the catalyst preparation can be separated simply and quickly with the help of an external magnetic field. In addition, the movement direction of the immobilized cells and the regulation of the enzymatic reaction can also be achieved by adjusting the external magnetic field. Therefore, the present invention uses magnetic diatomite to immobilize Escherichia coli cells containing nitrilase to prepare a magnetic nitrilase catalyst. The Escherichia coli containing nitrilase is immobilized on Fe by covalent bonding and adsorption-crosslinking methods. 3 O 4 / SiO 2 A magnetic nitrilase catalyst is formed on the magnetic composite carrier, and tetrachloropyridine nitrile can be converted into tetrachloropyridine carboxylic acid by using the magnetic nitrilase catalyst. After the reaction is completed, the magnetic nitrilase catalyst is recovered by magnetic attraction; the obtained reaction solution is acidified and then centrifuged to separate the product, and then can be used for the next round of reaction after neutralization.

[0011] The magnetic nitrilase catalyst prepared by the invention can stably exist in nitrile substances, has the characteristics of high mechanical strength, low enzyme activity loss rate and easy storage, has the characteristics of less by-products and high product yield when catalyzing tetrachloropyridine nitrile to produce tetrachloropyridine carboxylic acid, has low cost, convenient operation, mild reaction, low pollution, high number of repeated uses, and has certain industrial use potential.

[0012] The invention adopts a magnetic diatomite composite carrier to immobilize Escherichia coli cells containing nitrilase, thereby improving the enzyme activity recovery rate, protein immobilization rate and stability of the magnetic nitrilase catalyst. The prepared magnetic nitrilase catalyst has good biocompatibility and has the advantages of easy separation, reusability and high enzyme loading, and is an ideal immobilized enzyme carrier.

[0013] In some embodiments, in step (1), the mass ratio of diatomaceous earth to magnetic material is (1-2):1.

[0014] In the embodiment, in the preparation of magnetic diatomite, too little diatomite content may affect the adsorption performance, resulting in incomplete adsorption of bacteria, resulting in reduced catalytic activity. When the diatomite content is increased and the ratio of the two is 2:1, compared with the optimal enzyme activity of 78%, the excessively high magnetite ratio may cause the displacement of the enzyme active center and cause a decrease in activity. Therefore, the mass ratio of diatomite to magnetic material preparation is limited to (1-2):1.

[0015] In some embodiments, in step (1), the magnetic material includes at least one of aluminum powder, iron powder, ferroferric oxide, and ferric oxide; the cross-linking agent includes a glutaraldehyde aqueous solution, the mass fraction of glutaraldehyde in the glutaraldehyde aqueous solution is 20% to 25%, and the volume usage of the glutaraldehyde aqueous solution is 0.3 to 0.7 mL / g based on the total mass of the magnetic material and diatomaceous earth.

[0016] In some embodiments, in step (2), the amount of magnetic diatomaceous earth added is 0.04-0.1 g / g wet bacteria.

[0017] In the examples, after determining the ratio of diatomaceous earth to magnetic material in the magnetic diatomaceous earth, the effect of the amount of magnetic diatomaceous earth added on the enzyme activity was further investigated. The results showed that excessive magnetic diatomaceous earth would also lead to a decrease in enzyme activity. This may be because when the enzyme is loaded, the reaction specific surface area is reduced, affecting the reaction mass transfer rate.

[0018] In some embodiments, in step (2), the stirring adsorption time is 2 to 5 hours.

[0019] In some embodiments, in step (3), the cross-linking agent includes an aqueous solution of polyethyleneimine, and the polyethyleneimine includes at least one of polyethyleneimine with MW1800, polyethyleneimine with MW4000, polyethyleneimine with MW10000, and polyethyleneimine with MW70000. In the aqueous solution of polyethyleneimine, the mass fraction of polyethyleneimine is 4-6%, and the amount of the aqueous solution of polyethyleneimine added is 0.4-0.8 mL / g wet bacteria.

[0020] In some embodiments, in step (3), the cross-linking agent includes an aqueous solution of glutaraldehyde, the mass fraction of glutaraldehyde in the aqueous solution of glutaraldehyde is 20% to 25%, and the amount of the aqueous solution of glutaraldehyde added is 0.3 to 0.6 mL / g wet bacteria.

[0021] The invention uses a magnetic diatomite composite material to immobilize Escherichia coli cells containing nitrilase, uses a PEI-GA crosslinking agent, and uses an amine-aldehyde condensation reaction to immobilize the cells on a carrier with a primary amino group to form a secondary aldehyde amine in a stable form, thereby preparing a magnetic nitrilase catalyst. While improving the cell stability, the addition of magnetic materials simplifies the separation step of the magnetic nitrilase catalyst in the production reaction, thereby reducing the production cost.

[0022] The second aspect of the present invention provides a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, wherein the magnetic nitrilase catalyst is prepared according to any preparation method described in the first aspect.

[0023] The third aspect of the present invention provides an application of a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, characterized in that the magnetic nitrilase catalyst described in the second aspect is used as a catalyst and 3,4,5,6-tetrachloropyridinecarbonitrile is used as a substrate to prepare 3,4,5,6-tetrachloropyridinecarboxylic acid.

[0024] According to the application of the third aspect, in some embodiments, the method of using the magnetic nitrilase catalyst to green-cycle synthesize 3,4,5,6-tetrachloropyridine carboxylic acid comprises:

[0025] The magnetic nitrilase catalyst is used as a catalyst, 3,4,5,6-tetrachloropyridinecarbonitrile is used as a substrate, and a pH 6.5-8 buffer solution is used as a reaction medium to form a conversion system, and the reaction is carried out at 25-50° C. and 400-800 rpm to prepare 3,4,5,6-tetrachloropyridinecarboxylic acid.

[0026] According to the application described in the third aspect, in some embodiments, the buffer is pH 7.0, 200mM PB buffer; the reaction temperature is 37-38°C; and the substrate concentration is 10-200mM.

[0027] According to the application described in the third aspect, in some embodiments, the following steps are included:

[0028] (1) using a magnetic nitrilase catalyst as a catalyst and 3,4,5,6-tetrachloropyridinecarbonitrile as a substrate, continuously catalyzing 3,4,5,6-tetrachloropyridinecarbonitrile to synthesize 3,4,5,6-tetrachloropyridinecarboxylic acid to obtain a reaction solution 1;

[0029] (2) using a magnet to absorb the magnetic nitrilase catalyst in the reaction solution 1 to obtain a reaction solution 2, performing solid-liquid separation on the reaction solution 2 to obtain a precipitate and a reaction waste liquid, adding ethanol to the precipitate, removing the precipitate by centrifugation, and rotary evaporating the supernatant liquid until the solid has a constant weight, thereby obtaining the product 3,4,5,6-tetrachloropyridine carboxylic acid;

[0030] (3) adjusting the pH of the reaction waste liquid to 2.4-3, and collecting the supernatant liquid by centrifugation;

[0031] (4) adjusting the pH of the supernatant liquid in step (3) to 6.5-7, using the adjusted supernatant liquid as a solvent, adding 3,4,5,6-tetrachloropyridine carbonitrile and a magnetic nitrilase catalyst, and repeating steps (1) to (4) to prepare 3,4,5,6-tetrachloropyridine carboxylic acid.

[0032] The present invention uses a neutralization method to treat the reaction waste liquid of tetrachloropyridine carboxylic acid synthesis, and uses the treated reaction waste liquid as a solvent for the next round of reaction, so that all materials used in the catalytic process except the raw materials are recycled for more than 30 batches. The magnetic nitrilase catalyst and the reaction liquid prepared by the method of the present invention can be recycled, and the separation of the catalytic preparation and the product can be quickly achieved. The process not only simplifies downstream separation and purification, but is also very suitable for large-scale production and continuous separation, which is of great significance for improving the use efficiency of the enzyme and reducing the use cost, and provides a useful reference for promoting clean, environmentally friendly and efficient enzymatic production of tetrachloropyridine carboxylic acid, and has great application potential in continuous separation and industrial applications.

[0033] The fourth aspect of the present invention provides an application of a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, characterized in that the magnetic nitrilase catalyst described in the second aspect is used as a catalyst for cyclic synthesis of 3,5-dichlorobenzoic acid, 3-chlorobenzoic acid or 3,6-dichloropyridine-3-carboxylic acid.

[0034] By implementing the above technical solution, the present invention has the following beneficial effects:

[0035] The magnetic nitrilase catalyst prepared by immobilizing Escherichia coli cells containing nitrilase on a magnetic diatomite composite carrier has excellent biocompatibility and stability, and also improves the enzyme activity recovery rate and protein immobilization rate of the magnetic nitrilase catalyst. It is easy to separate and reuse in the reaction, and is an ideal immobilized enzyme carrier.

[0036] The magnetic nitrilase catalyst prepared by the invention can stably exist in nitrile substances, has the characteristics of high mechanical strength, low enzyme activity loss rate and easy storage, has the characteristics of less by-products and high product yield when catalyzing tetrachloropyridine nitrile to produce tetrachloropyridine carboxylic acid, has low cost, convenient operation, mild reaction, low pollution, high number of repeated uses, and has certain industrial use potential.

[0037] The preparation method of the magnetic nitrilase catalyst constructed by the invention is simple and easy to recycle.

[0038] The present invention uses a neutralization method to treat the reaction waste liquid of synthesizing tetrachloropyridine carboxylic acid, and uses the treated reaction waste liquid as a solvent for the next round of reaction, so that all materials used in the catalytic process except the raw materials are recycled for more than 30 batches. The magnetic nitrilase catalyst and the reaction liquid prepared by the method of the present invention can be recycled, and the separation of the catalytic preparation and the product can be quickly realized. The process not only simplifies the downstream separation and purification, but is also very suitable for large-scale production and continuous separation, not only reduces the production cost, but also realizes the recycling of water, consumes less energy and protects the environment, and achieves the purpose of zero discharge of chemical wastewater and green production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a process flow chart of the present invention;

[0040] Figure 2 is a catalytic efficiency curve diagram of the magnetic nitrilase catalyst cell and the free enzyme under different pH conditions in Example 3 of the present invention;

[0041] Figure 3 is a catalytic efficiency curve diagram of magnetic nitrilase catalyst cells and free cells under different temperature conditions in Example 4 of the present invention;

[0042] Figure 4 is a diagram of the catalytic reaction process of the magnetic nitrilase catalyst cell at different substrate concentrations in Example 5 of the present invention;

[0043] Figure 5 This is a graph showing the determination of the reuse of the magnetic nitrilase catalyst cells in Example 6 of the present invention;

[0044] Figure 6 This is a reaction process diagram of recycling the waste liquid from the acid neutralization reaction to synthesize tetrachloropyridine carboxylic acid in Example 7 of the present invention;

[0045] Figure 7 This is a reaction process diagram of synthesizing tetrachloropyridine carboxylic acid by recycling magnetic nitrilase catalyst cells and waste liquid in Example 8 of the present invention. DETAILED DESCRIPTION

[0046] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is described in detail below in conjunction with embodiments and drawings.

[0047] Example 1: Cultivation of engineered bacteria

[0048] Take E. coli BL21 (DE3) / pET28a (+) -NIT-T134G / W164Y / L201W (the preparation process refers to patent application number 202311747860.7, and the amino acid sequence of the nitrilase mutant NIT-T134G / W164Y / L201W is shown in SEQ ID No. 1) and inoculate it into a LB liquid culture medium test tube containing a final concentration of 100 μg / mL kanamycin resistance, culture at 37 ° C, 180 rpm for 8 to 12 hours, and then inoculate it into a fresh LB liquid culture medium shake flask containing a final concentration of 100 ug / ml kanamycin resistance at an inoculum concentration of 1% by volume, and place it in a shaker at 37 ° C, 180 rpm for 2 to 3 hours until the bacterial OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.2 mM, induce and culture at 28°C for 8-12 hours, centrifuge at 4°C and 8000 rpm for 10 minutes, discard the supernatant, collect the precipitate, and obtain the recombinant Escherichia coli wet cells containing nitrilase.

[0049] LB medium composition: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast powder, and the solvent is deionized water.

[0050] Example 2: Preparation of magnetic nitrilase catalyst

[0051] (1) Take 5g of diatomaceous earth (particle size 36.8μm) and 5g of ferroferric oxide powder, place them in 20mL of deionized water, physically blend them in a mechanical stirring device for 1h, and add 4mL of 25% glutaraldehyde as a stabilizer to cross-link the composite material. After cross-linking for 1h, the composite material is filtered and the filter cake is rinsed 2 to 3 times with deionized water to obtain a filter cake. The collected filter cake is dried at high temperature until there is no moisture, and then the magnetic diatomaceous earth is collected for use.

[0052] (2) 10 g of wet Escherichia coli cells containing nitrilase were taken and 100 ml of deionized water were added to dissolve them into a wet Escherichia coli cell suspension containing nitrilase. The suspension was stirred at 650 rpm for 10 min until it was uniform and there were no obvious large particles. Then, 1 g of magnetic diatomaceous earth was added to the wet Escherichia coli cell suspension containing nitrilase, and the suspension was stirred at 750 rpm for 1 h to adsorb the Escherichia coli cells, thereby obtaining an Escherichia coli-magnetic diatomaceous earth adsorption system.

[0053] (3) 6 ml of a cross-linking agent I polyethyleneimine (PEI) aqueous solution with a MW of 70,000 was added to the Escherichia coli-magnetic diatomaceous earth adsorption system, wherein the mass fraction of polyethyleneimine was 5%, and the mixture was stirred at 850 rpm for 1 hour. Then, 2 ml of a cross-linking agent II glutaraldehyde (GA) with a mass fraction of 25% was added to the system, and the mixture was stirred at 950 rpm for 30 minutes. The resulting suspension was separated by suction filtration, and the resulting filter cake was rinsed with deionized water for 2 to 3 times. The filter cake was collected and stored in a refrigerator at -20°C to obtain a magnetic nitrilase catalyst.

[0054] Comparative Example 1: Effect of the mass ratio of diatomaceous earth to magnetic material

[0055] The difference between this comparative example and step (1) of Example 2 is that the influence of different mass ratios of diatomite to ferroferric oxide on the catalytic efficiency of the reaction in the preparation of magnetic diatomite is explored. The mass ratios of diatomite to magnetic material are 1:1, 1:2, and 2:1 respectively, and the other steps are the same as Example 2.

[0056] The cell viability of the magnetic nitrilase catalyst prepared in this comparative example was determined by the following test method:

[0057] Cell viability assay using magnetic nitrilase catalyst: Take an amount of magnetic nitrilase catalyst cells equal to 10 g / L resting cells, resuspend in 10 mL 200 mM phosphate buffer, pH = 7.0, add 20 mM tetrachloropyridine carbonitrile as substrate to form a 10 mL system reaction solution, mix and react in a water bath shaker at 37°C and 600 rpm for 2 h, take a sample, centrifuge at 12000 rpm for 2 min, take the supernatant, filter through a 0.22 μm microfiltration membrane, and use HPLC to detect the product content and analyze.

[0058] Definition of cell activity: The amount of cells required to catalyze the production of 1 μmol of tetrachloropyralid per minute under the conditions of 37°C and pH 7.0 is 1 U.

[0059] 2h conversion rate determination: Take the magnetic nitrilase catalyst cells corresponding to the amount of free cells, resuspend them evenly in the buffer, react for 2h, take samples for liquid phase analysis, and calculate the 2h conversion rate.

[0060] Relative activity: The highest enzyme activity of magnetic nitrilase catalyst cells prepared under different conditions is set as 100%, and the ratio of other conditions to the highest enzyme activity.

[0061] Product liquid phase detection conditions: Tetrachloropyridine carboxylic acid was analyzed by high performance liquid chromatography, the chromatographic column was HypersilODSC18 column (5μm, 250nm×4.6nm), the mobile phase volume ratio of acetonitrile: water was 6:4 (v / v), the flow rate was 1mL / min, the detection wavelength was 224nm, the injection volume was 20μL, the column temperature was 40°C, and the retention time of tetrachloropyridine carboxylic acid was 6.8min.

[0062] The relative enzyme activity of magnetic nitrilase catalyst cells at different mass ratios of diatomaceous earth to ferric oxide is shown in Table 1.

[0063] Table 1 Diatomaceous earth and Fe 3 O 4 Effects of different ratios on the enzyme activity of catalyst cells

[0064] <![CDATA[SiO 2 :Fe 3 O 4 ]]> 1:1 1:2 1:3 2:1 3:1 Relative enzyme activity (%) 69±0.5 60±0.9 58±0.8 100 76±0.7

[0065] It can be seen from Table 1 that in M-DE / Fe 3 O 4 / SiO 2 In the preparation of composite carrier materials, too little diatomite content may affect the adsorption performance, resulting in incomplete adsorption of bacteria and reduced catalytic activity. When the diatomite content is increased to a ratio of 2:1, the optimal enzyme activity is 78%. Too high a magnetite ratio may cause the enzyme active center to shift, resulting in reduced activity.

[0066] Comparative Example 2: Effect of the amount of magnetic diatomaceous earth added

[0067] The difference between this comparative example and step (2) of Example 2 is that the effect of different amounts of magnetic diatomite added on the catalytic efficiency of the reaction is explored. The cases of 0.03 g / g wet bacteria, 0.05 g / g wet bacteria, 0.07 g / g wet bacteria, and 0.1 g / g wet bacteria (magnetic diatomite added mass g / mass of Escherichia coli wet bacteria in the Escherichia coli wet bacteria suspension containing nitrilase) are explored respectively, and the other steps are the same as Example 2.

[0068] According to the method described in Comparative Example 1, the cell viability of the magnetic nitrilase catalyst prepared in this comparative example was measured. The results are shown in Table 2. Excessive carrier (magnetic diatomaceous earth) can also lead to a decrease in enzyme activity. This may be due to the reduction of the reaction specific surface area under enzyme loading, which affects the reaction mass transfer rate.

[0069] Table 2 Effect of the amount of magnetic diatomaceous earth added on the enzyme activity of catalyst cells

[0070]

[0071] Comparative Example 3: Effect of stirring adsorption time

[0072] The difference between this comparative example and step (2) of Example 2 is that the effect of different stirring adsorption times on the catalytic efficiency of the reaction in the preparation of the E. coli-magnetic diatomaceous earth adsorption system is explored. The stirring adsorption time is 1h, 2h, 3h, 4h, and 5h respectively, and the other steps are the same as Example 2.

[0073] According to the method described in Comparative Example 1, the cell viability of the magnetic nitrilase catalyst prepared in this comparative example was determined, and the results are shown in Table 3.

[0074] Table 3 Effect of different stirring adsorption time on catalyst cell enzyme activity

[0075] Stirring adsorption time (h) 1 2 3 4 5 Relative enzyme activity (%) 76±0.2 93±0.5 100±0.3 88±0.4 80±0.1

[0076] Comparative Example 4: Effect of the amount of glutaraldehyde aqueous solution added

[0077] The difference between this comparative example and steps (1) and (3) of Example 2 is that the effect of different amounts of glutaraldehyde (GA) added on the catalytic efficiency of the reaction is explored.

[0078] First, in step (1), the addition amount of 25% glutaraldehyde (GA) aqueous solution was 0.1 mL / g, 0.2 mL / g, 0.3 mL / g, 0.4 mL / g, 0.5 mL / g, and 0.6 mL / g (glutaraldehyde (GA) aqueous solution addition volume mL / total mass of magnetic material and diatomaceous earth g), and the other steps were the same as in Example 2.

[0079] According to the method described in Comparative Example 1, the cell viability of the magnetic nitrilase catalyst prepared in this comparative example was determined, and the results are shown in Table 4.

[0080] Table 4 Effect of different GA addition amounts on the catalytic cell enzyme activity during the preparation of magnetic composite materials in step (1)

[0081]

[0082] Because mechanical and physical methods are used to mix the two carriers, GA can act as a "glue" as a cross-linking agent to make the combination of the two closer. Too low a concentration may affect the blending effect of the two, resulting in uneven mixing and affecting the catalytic efficiency.

[0083] Secondly, in step (3), the addition amount of 25% glutaraldehyde (GA) was 0.1 mL / g wet bacteria, 0.2 mL / g wet bacteria, 0.3 mL / g wet bacteria, 0.4 mL / g wet bacteria, and 0.5 mL / g wet bacteria (volume of glutaraldehyde (GA) aqueous solution added mL / mass of Escherichia coli wet bacteria in the nitrilase-containing Escherichia coli wet bacteria suspension g), and the other steps were the same as in Example 2.

[0084] According to the method described in Comparative Example 1, the cell viability of the magnetic nitrilase catalyst prepared in this comparative example was determined, and the results are shown in Table 5.

[0085] Table 5 Effect of different GA addition amounts on the enzyme activity of catalyst cells when cells are mixed with magnetic materials in step (3)

[0086]

[0087] Comparative Example 5: Effect of different amounts of polyethyleneimine added

[0088] The difference between this comparative example and step (3) of Example 2 is that the effect of different amounts of polyethyleneimine added on the catalytic efficiency of the reaction was investigated. The addition amounts of 5% polyethyleneimine aqueous solution were 0.1 mL / g wet bacteria, 0.2 mL / g wet bacteria, 0.3 mL / g wet bacteria, 0.4 mL / g wet bacteria, 0.5 mL / g wet bacteria, 0.6 mL / g wet bacteria, 0.7 mL / g wet bacteria, and 0.8 mL / g wet bacteria.

[0089] (volume of polyethyleneimine aqueous solution added mL / mass of E. coli wet cells in the nitrilase-containing E. coli wet cell suspension g), the other steps are the same as those in Example 2. According to the method described in Comparative Example 1, the cell viability of the magnetic composite enzyme catalyst prepared in this comparative example was determined, and the results are shown in Table 6.

[0090] Table 6 Effect of different amounts of polyethyleneimine added on the enzyme activity of catalyst cells

[0091]

[0092] Example 3: Determination of enzyme activity of magnetic nitrilase catalyst cells under different pH conditions The magnetic nitrilase catalyst cells obtained in Example 2 were added to the substrate solution, the enzyme addition amount of the magnetic nitrilase catalyst cells was 10 g / L, the substrate was tetrachloropyridine carbonitrile, the substrate concentration was 50 mM, the reaction temperature was 37° C., the rotation speed was set to 600 rpm, the reaction time was 2 h, and the amount of TCPA generated was determined by HPLC detection method to calculate the relative enzyme activity.

[0093] The pH values ​​were: citric acid-sodium citrate buffer, pH=3.0, pH=4.0, pH=5.0; PB buffer, pH=6.0, pH=7.0; tris-HCL buffer, pH=8.0, pH=9.0; glycine-sodium hydroxide buffer, pH=10.0, pH=11.0, pH=12.0.

[0094] The catalytic efficiency results of magnetic nitrilase catalyst cells and free enzyme under different pH conditions are shown in Figure 2. Figure 2 shown.

[0095] from Figure 2 The data can be analyzed to show that the free nitrilase cells show the maximum hydrolysis activity at pH = 7.0, with a relative enzyme activity of 53%. Generally speaking, the optimal pH of the immobilized cell catalytic reaction will increase or decrease. Compared with the optimal pH of free cells, the immobilized cells are suitable for reacting in a neutral environment. The shift in the optimal pH may be caused by the addition of ferric oxide, which changes the ionic environment around the enzyme active site. The results in the figure show that compared with free cells, the magnetic nitrilase catalyst cells have higher stability to changes in pH values ​​in the range of 6.0 to 9.0, and the activity decreases when the pH is higher than 7.0.

[0096] Example 4: Determination of enzyme activity of magnetic nitrilase catalyst cells under different temperature conditions The magnetic nitrilase catalyst cells obtained in Example 2 were added to the substrate solution, the enzyme amount of the magnetic nitrilase catalyst cells was 10 g / L, 50 mM tetrachloropyridine carbonitrile, combined with the optimal pH reaction conditions obtained in Example 3, the buffer solution was tris-HCL, the initial pH was 7.0, the rotation speed was set to 600 rpm, the reaction was carried out for 2 hours, and the amount of tetrachloropyridine carboxylic acid generated was determined by HPLC to calculate the relative enzyme activity.

[0097] The reaction temperatures are: 25°C, 28°C, 37°C, 41°C, and 45°C respectively.

[0098] The results of catalytic efficiency of nitrilase immobilized cells and free cells under different temperature conditions are shown in Figure 3 shown.

[0099] Combination Figure 3 From the results, the enzyme activity of magnetic nitrilase catalyst cells in the range of 28℃ to 45℃ is different from that of free cells, especially in the high temperature range of 37℃ to 45℃, the optimum temperature of free cells is 41℃, and the enzyme activity of magnetic nitrilase catalyst cells after 37℃ decreases significantly with the increase of temperature. Under high temperature conditions, the catalytic activity of the magnetic nitrilase catalyst cells is poor. However, compared with the range of 28℃ to 41℃, the magnetic nitrilase catalyst cells show relatively stable catalytic activity in the low temperature zone.

[0100] Example 5: Effect of substrate concentration on the catalytic reaction progress of magnetic nitrilase catalyst cells

[0101] The optimal magnetic nitrilase catalyst cells prepared by the immobilization method in Examples 1 to 4 were applied to catalyze the reaction of converting tetrachloropyridine nitrile into tetrachloropyridine carboxylic acid, and the catalytic efficiency of the magnetic nitrilase catalyst cells was determined. The experimental process is as follows:

[0102] In 10 ml of phosphate buffer (initial pH = 7.0), 10 g / L nitrilase Escherichia coli immobilized cells were added as a catalyst, 50, 100, 150, 200 mM tetrachloropyridinecarbonitrile was used as a substrate to form a reaction system, the reaction was carried out at 37° C. and 600 rpm for 5 to 24 h, samples were taken at 5, 8, and 24 h to terminate the reaction, the reaction liquid was separated into solid and liquid to obtain a precipitate and a reaction waste liquid, 80% ethanol was added to the precipitate, 3,4,5,6-tetrachloropyridinecarbonitrile was soluble in ethanol, and the unreacted substrate 3,4,5,6-tetrachloropyridinecarbonitrile was insoluble, the supernatant was retained by centrifugation, the supernatant was rotary evaporated at 25° C. until the solid was constant weight, the product 3,4,5,6-tetrachloropyridinecarbonitrile was obtained, and the content of 3,4,5,6-tetrachloropyridinecarbonitrile in the sample was analyzed by HPLC.

[0103] The catalytic reaction process of magnetic nitrilase catalyst cells under different substrate concentrations is shown in Figure 2. Figure 4 shown.

[0104] like Figure 4 As shown, the conversion rate of tetrachloropyridine carbonitrile catalyzed by magnetic nitrilase catalyst cells immobilized on magnetic diatomite decreased with the increase of substrate concentration. When the substrate concentration was 50 mM, the reaction was completely completed in 24 hours, the maximum amount of product generated was stable at about 55 mM, and the difference in product generation under different conditions was not large.

[0105] Example 6: Determination of the reuse of magnetic nitrilase catalyst cells

[0106] The magnetic nitrilase catalyst cells obtained in Example 2 were taken, and 10 g / L of the magnetic nitrilase catalyst cells were used as catalysts, and reacted in a 10 ml reaction system containing 200 mM phosphate buffer, pH 7.0, and 10 mM tetrachloropyridine carbonitrile for 2 h, and then samples were taken and the tetrachloropyridine carboxylic acid content was detected by HPLC. The magnetic nitrilase catalyst cells were recovered by applying an external magnetic field, washed 3 times with deionized water, and the relative enzyme activity was determined. This was repeated multiple times, and the enzyme activity measured for the first time was set as 100%.

[0107] from Figure 5 The data analysis showed that after 18 batches of repeated use, the activity of the magnetic nitrilase catalyst cells retained more than 80% of the initial activity. After 30 batches of repeated use, the magnetic nitrilase catalyst cells retained more than 70% of the initial activity.

[0108] Example 7: Storage stability of magnetic nitrilase catalyst

[0109] Storage stability is one of the important parameters for cell immobilization, and the effect of storage conditions on the activity of immobilized enzymes is considered to be an important aspect in ensuring a long shelf life.

[0110] The magnetic immobilized nitrilase recovered after 30 batches of reactions in Example 6 was transferred to a refrigerator at 4° C. and stored for 20 days before being taken out. The substrate conversion rate could still reach more than 50% within 48 hours.

[0111] The prepared magnetic nitrilase catalyst was placed in 200 mM pH 7.0 sodium phosphate buffer and stored at the optimum temperature (37° C.) and the optimum pH 7 for 20 days. The results are shown in Table 7. The residual enzyme activity can reach more than 90%.

[0112] The prepared magnetic nitrilase catalyst was dried in the shade, stored at 4°C (stored in an EP tube after drying at room temperature) for 3 months, and then the enzyme activity was measured. The results are shown in Table 7. The residual enzyme activity after drying and storage for 90 days reached 66.3%. It can be seen that the immobilized cells after adsorption and cross-linking treatment on the carrier have good catalytic performance and storage stability.

[0113] Table 7 Storage stability of immobilized cells

[0114] Optimal storage conditions: 20 days After drying, store for 90 days Residual enzyme activity (%) 92.1 66.3

[0115] Example 8: Method for recycling waste liquid from 3,4,5,6-tetrachloropicolinic acid reaction

[0116] The treatment method for recycling waste liquid from 3,4,5,6-tetrachloropyridine carboxylic acid reaction comprises the following steps:

[0117] (1) The reaction solution in Example 6 is collected and recorded as reaction solution 1. The magnetic nitrilase catalyst cells in reaction solution 1 are sucked out by a magnet, and then centrifuged at 12000 rpm for 5 min. The supernatant is collected as reaction waste liquid; after solid-liquid separation, a supernatant and a precipitate are obtained, and the supernatant is collected as reaction waste liquid;

[0118] Product separation and purification: add 80% ethanol to the precipitate, stir at 800 rpm for 5 min, remove unreacted 3,4,5,6-tetrachloropyridinecarbonitrile by centrifugation, and evaporate the supernatant at 25°C until the solid has a constant weight to obtain the product 3,4,5,6-tetrachloropyridinecarboxylic acid.

[0119] (2) hydrochloric acid was added dropwise to the reaction waste liquid to adjust the pH value to 3, and floccules were observed in the clear liquid. The liquid was centrifuged at 12000 rpm for 5 min to remove the precipitated impurities, and the supernatant liquid was collected;

[0120] (3) NaOH was added dropwise to the supernatant treated in step (2) to adjust the pH value to 7.0, and the supernatant was used as the solvent for the subsequent reaction. The magnetic nitrilase catalyst cells were first resuspended in the solvent prepared by the treated reaction waste liquid, and the dry weight of the cells added to the transformation system was 10 gDCW / L. The amount of tetrachloropyridine carbonitrile was 150 mM, and a 50 mL reaction system was formed with pH 7.0 and the treated reaction waste liquid as the reaction medium, and tetrachloropyridine carboxylic acid was continued to be prepared under the conditions of 37° C. and 600 rpm.

[0121] like Figure 6 As shown, after the reaction waste liquid is treated with acid neutralization, it is used as a reaction medium to participate in the synthesis of tetrachloropyridine carboxylic acid. The reaction can be the same as using PBS buffer as the reaction medium. After 48 hours of reaction, the product conversion rate can also reach 80%.

[0122] Example 9: Catalyst and reaction solution are recycled to produce tetrachloropyridine carboxylic acid

[0123] According to Example 8 and Figure 6 , add 10g / L of immobilized bacteria and 150mM of tetrachloropyridinecarbonitrile to a 50mL reaction system for conversion reaction, the reaction solution pH is 7.0, the temperature is 37°C, the reaction time is 48h, and the rotation speed is 600rpm. After 48h, take part of the reaction solution for filter membrane treatment, detect the content of tetrachloropyridine carboxylic acid with high performance liquid chromatography, and calculate the conversion rate of tetrachloropyridine carboxylic acid by magnetic nitrilase catalyst cells. After the first batch of reactions is completed, the magnetic nitrilase catalyst cells are recovered by magnetic suction, washed 3 times with deionized water, and the reaction solution is acidified and neutralized to be used as a reaction medium for subsequent reactions, and the next batch of reactions is carried out, and the reaction is repeated 30 batches. Figure 7 As shown, the magnetic nitrilase catalyst cells and the reaction solution were recycled and the reaction was repeated for 30 batches, and the substrate conversion rates of each batch were all greater than 50%.

[0124] Example 10: Green Cycle Catalysis of Other Structurally Similar Substrates by Magnetic Nitrilase Catalyst

[0125] According to the description in Example 2, a magnetic nitrilase catalyst was obtained to explore the effect of green cyclic synthesis on the catalytic performance of other substrates with similar structures.

[0126] According to Example 9, 10 g / L of immobilized bacteria and 150 mM substrate (the substrates are 3,5-dichlorobenzonitrile, 3-chlorobenzonitrile and 3,6-dichloropyridine-2-carbonitrile, respectively) were added to a 50 mL reaction system, the reaction solution pH was 7.0, the temperature was 37 ° C, the reaction time was 48 h, and the speed was 600 rpm. After 48 h, a portion of the reaction solution was taken for filter membrane treatment, and the content of the product was detected by high performance liquid chromatography, and the conversion rate of the magnetic nitrilase catalyst cell transformation product was calculated. After the first batch of reactions was completed, the magnetic nitrilase catalyst was recovered by magnetic suction, washed 3 times with deionized water, and the reaction solution was acidified and neutralized as a reaction medium for subsequent reaction recycling, and the next batch of reactions was carried out, and the reaction was repeated for 30 batches.

[0127] Table 8 Catalytic efficiency of magnetic complex enzyme cyclic catalysis of different substrates

[0128]

[0129]

[0130] As shown in Table 8, after repeating the reaction for 20 batches, the substrate conversion rate of each batch was still above 80%, and after repeating the reaction for 30 batches, the substrate conversion rate of each batch was still above 55%.

[0131] The above-mentioned embodiments are preferred examples of the present invention and are not intended to limit the protection scope of the present invention.

Claims

1. A method for preparing a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, characterized in that: The following steps are involved: (1) Preparation of magnetic diatomite by physical blending: diatomite, magnetic material and cross-linking agent are mixed and reacted to obtain magnetic diatomite; (2) preparing an Escherichia coli-magnetic diatomaceous earth adsorption system: mixing a wet suspension of Escherichia coli containing nitrilase with magnetic diatomaceous earth, and stirring and adsorbing the mixture to obtain an Escherichia coli-magnetic diatomaceous earth adsorption system; (3) Magnetic nitrilase catalyst: A cross-linking agent is added to the Escherichia coli-magnetic diatomaceous earth adsorption system, and the mixture is stirred for reaction and then treated to obtain the magnetic nitrilase catalyst.

2. The preparation method according to claim 1, characterized in that: In the step (1), the mass ratio of diatomaceous earth to magnetic material is (1-2):

1.

3. The preparation method according to claim 2, characterized in that: In the step (1), the magnetic material comprises at least one of aluminum powder, iron powder, ferroferric oxide and ferric oxide; the cross-linking agent comprises a glutaraldehyde aqueous solution, the mass fraction of glutaraldehyde in the glutaraldehyde aqueous solution is 20% to 25%, and the volume usage of the glutaraldehyde aqueous solution is 0.3 to 0.7 mL / g based on the total mass of the magnetic material and diatomaceous earth.

4. The preparation method according to claim 1, characterized in that: In the step (2), the amount of magnetic diatomaceous earth added is 0.04-0.1 g / g wet bacteria.

5. The preparation method according to claim 2, characterized in that: In the step (2), the stirring and adsorption time is 2 to 5 hours.

6. The preparation method according to claim 1, characterized in that: In the step (3), the cross-linking agent includes an aqueous solution of polyethyleneimine, and the polyethyleneimine includes at least one of polyethyleneimine with MW1800, polyethyleneimine with MW4000, polyethyleneimine with MW10000, and polyethyleneimine with MW70000. In the aqueous solution of polyethyleneimine, the mass fraction of polyethyleneimine is 4-6%, and the amount of the aqueous solution of polyethyleneimine added is 0.4-0.8 mL / g wet bacteria.

7. The preparation method according to claim 1, characterized in that: In the step (3), the cross-linking agent comprises an aqueous solution of glutaraldehyde, the mass fraction of glutaraldehyde in the aqueous solution of glutaraldehyde is 20% to 25%, and the amount of the aqueous solution of glutaraldehyde added is 0.3 to 0.6 mL / g wet bacteria.

8. A magnetic nitrilase catalyst for green cyclic synthesis of organic acids, characterized in that: The magnetic nitrilase catalyst is prepared according to the preparation method according to any one of claims 1 to 7.

9. An application of a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, characterized in that: The magnetic nitrilase catalyst according to claim 8 is used as a catalyst and 3,4,5,6-tetrachloropyridinecarbonitrile is used as a substrate to prepare 3,4,5,6-tetrachloropyridinecarboxylic acid.

10. An application of a magnetic nitrilase catalyst for green cyclic synthesis of organic acids, characterized in that: The magnetic nitrilase catalyst according to claim 8 is used as a catalyst to cyclically synthesize 3,5-dichlorobenzoic acid, 3-chlorobenzoic acid or 3,6-dichloropyridine-3-carboxylic acid.

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