Nicotinic acid immobilized enzyme conversion method

Abstract

The invention discloses a nicotinic acid immobilized enzyme conversion method, which comprises the following steps: taking wet thalli containing nitrilase, preparing a flocculation clear liquid, passing the flocculation clear liquid through an ultrafiltration membrane, carrying out embedding immobilization to obtain embedded particles not greater than 2mM, and after the embedded particles are hardened, separating and purifying the obtained conversion liquid by adopting a membrane separation method to obtain the nicotinic acid immobilized enzyme. Sequentially carrying out microfiltration, activated carbon decoloration, ultrafiltration, nanofiltration decoloration, acidification crystallization and centrifugation to finally obtain a nicotinic acid finished product. The immobilized enzyme prepared by the method disclosed by the invention can stably exist in nitrile substances, can be repeatedly used for many times, and is high in stability and low in enzyme activity loss rate, and a separated and purified product is free of impurities and high in yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immobilized enzyme, in particular to a nicotinic acid immobilized enzyme conversion method. BACKGROUND

[0002] Nicotinic acid, also known as vitamin B3, is the precursor of nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), which are essential for cellular energy metabolism (such as glycolysis, fatty acid oxidation) and redox reactions. The human body cannot synthesize enough, and must obtain it from diet (meat, fish, nuts, grains) or supplements. Nicotinic acid is widely used in the pharmaceutical industry, food and feed, additives, cosmetics, chemical industry, etc. The global market for nicotinic acid is huge, with annual production of tens of thousands of tons.

[0003] Currently, there are several methods for industrial synthesis of nicotinic acid, including 3-methylpyridine (beta-methylpyridine) oxidation, quinoline oxidation decarboxylation, ammonia oxidation, biological synthesis, and electrochemical synthesis. Among them, the 3-methylpyridine (beta-methylpyridine) oxidation method is the most mainstream and widely used method in industry. This method has relatively stable substrate sources, one-step oxidation reaction, and relatively short process. However, it has high cost, generates a lot of solid waste, produces by-products, and causes environmental pollution. Compared with biological synthesis, biological synthesis has high regional and stereoselectivity, less by-products, and is green and non-toxic, which has attracted much attention. However, biological synthesis still faces problems such as low enzyme activity, low enzyme utilization rate, and low product yield.

[0004] Using free enzymes to catalyze the production of nicotinic acid has the advantages of simple preparation, low cost, and minimal mass transfer resistance for enzyme catalysis, allowing substrates to quickly approach the active center of the enzyme. However, after the reaction, the enzyme and the product are mixed together, making it difficult to separate them effectively. Usually, the enzyme can only be used once, causing a significant waste. Currently, there are several methods for immobilizing nitrile hydrolase, including adsorption, covalent binding, cross-linked enzyme aggregates, and embedding. Adsorption is prone to enzyme loss in organic solvents (40% enzyme loss when acetonitrile is greater than 20%), and the enzyme activity decreases to 60% after 10 times of reuse. Covalent binding, such as the Fe3O4@SiO2-Epoxy nanoparticles prepared by Jiang Xiaoping et al., has an enzyme loading of 5.27 mg / g and a immobilization rate of up to 90% under optimal immobilization conditions. After immobilization, the stability of ethanol dehydrogenase is significantly improved. Moreover, the immobilized enzyme exhibits better heat resistance and good reusability. After 6 times of reuse, it still retains more than 80% of the initial enzyme activity. However, the preparation of the carrier in this method is complex and the cost is too high. Cross-linked enzyme aggregates do not require a carrier and have high enzyme loading, but the cross-linking process can lead to shielding of the active center, poor mechanical strength of the particles, and other problems. SUMMARY

[0005] The present application aims to provide a nicotinic acid immobilized enzyme conversion method, the prepared immobilized enzyme can stably exist in nitrile substances, has more repeated use times, high stability, low enzyme activity loss rate, and no impurities in the separated and purified product, and high yield.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] The nicotinic acid immobilized enzyme conversion method comprises

[0008] The wet cell containing nitrile hydrolase is taken, the flocculation supernatant is prepared to pass through the ultrafiltration membrane, embedded immobilization is carried out, and embedded particles of no more than 2 mM are obtained;

[0009] After the embedded particles are hardened, the obtained conversion liquid is separated and purified by a membrane separation method, and the nicotinic acid finished product is finally obtained by sequentially performing microfiltration, activated carbon decolorization, ultrafiltration, nanofiltration decolorization, acidification crystallization, and centrifugation.

[0010] Further, the preparation process of the flocculation supernatant passing through the ultrafiltration membrane specifically comprises:

[0011] The wet cell containing nitrile hydrolase is taken, 200 g / L of bacterial suspension is prepared by using 50 mM pH 8.0 phosphate buffer solution, the cell is broken by a high-pressure homogenizer under a pressure of 80 MPa for 3 times to obtain broken cell liquid, 5 ml / L of polyquaternary ammonium salt-10 solution with a content of 40% is added to the broken cell liquid to flocculate cell fragment particles, the supernatant is obtained by centrifugation after stirring for 1 hour, the obtained flocculation supernatant is passed through the ultrafiltration membrane, and the salt in the supernatant is removed.

[0012] Further, the embedded immobilization process specifically comprises:

[0013] The sodium alginate solution is prepared:

[0014] An appropriate amount of calcium alginate powder is taken, sterile deionized water is added, and 3% of calcium alginate solution is prepared. Under gentle heating and continuous stirring, the solution is dissolved until a uniform, transparent, and particle-free viscous solution is formed, and the solution is cooled to room temperature;

[0015] The calcium chloride solution is prepared:

[0016] An appropriate amount of anhydrous calcium chloride is taken, dissolved in sterile deionized water, and 2% of calcium chloride solution is prepared, which is placed in a beaker for subsequent reception of the dripping calcium alginate droplets;

[0017] The embedded mixed solution is prepared:

[0018] The cooled calcium alginate solution and the enzyme liquid to be immobilized are uniformly and gently mixed under sterile conditions, and then uniformly added to the 2% calcium chloride solution. After the dripping is completed, the stirring is continued for 1 h, and the reaction is washed with pure water for 3-4 times.

[0019] Furthermore, the hardening process of the embedded particles includes:

[0020] The encapsulated particles were dissolved in 700 mL of water, the pH was adjusted to 7.5, and the temperature was controlled at 30℃. 70% 3-cyanopyridine was added at a flow rate of 27 mL / h and the stirring speed was controlled at 250 rpm. Samples were taken for testing after 8 h or 15 h. 0.05% calcium chloride was added before each batch of reaction to harden the encapsulated particles.

[0021] Microfiltration conditions:

[0022] Processing solution: NA reaction solution;

[0023] Material properties: Product content 25%, specific gravity 1.04-1.07.

[0024] Purpose of treatment: To remove suspended solids and obtain a clear filtrate.

[0025] Processing technology: Membrane element: filtration accuracy 10nm (tentative); operating conditions: temperature 50-60℃, pressure 2-3 bar.

[0026] Compression ratio 7.5 times, 3 times dialysis water.

[0027] Furthermore, the microfiltration process includes:

[0028] The resulting conversion reaction solution was microfiltered at a temperature of 50-60℃, a pressure of 2-3 bar, a compression ratio of 7.5 times, and 3 times the amount of dialysis water.

[0029] Furthermore, the activated carbon decolorization process includes:

[0030] Add 0.5% activated carbon by volume, heat to 60-80 degrees Celsius, stir for 1 hour, and obtain a decolorizing solution with a light transmittance of 30-50% for activated carbon decolorization.

[0031] Ultrafiltration conditions:

[0032] Treatment solution: NA decolorizing solution.

[0033] Material properties: Na content 20%; pH 7.7; temperature 25-30℃; conductivity 33 mS / cm.

[0034] Processing procedure: Molecular weight cutoff of 1KD, temperature 25-30℃, pressure 20 bar, compression 20 times, collect the clear liquid with a content of 200g / L; the turbid liquid is collected and returned to the activated carbon decolorization area.

[0035] Furthermore, the ultrafiltration process includes:

[0036] The molecular weight cutoff is 1 kDa. The temperature is 25-30℃ and the pressure is 20 bar, which is compressed 20 times. The clear liquid is collected with a content of 200 g / L. The turbid liquid is collected and then decolorized with activated carbon.

[0037] Nanofiltration decolorization conditions:

[0038] Feed solution: NA ultrafiltration solution.

[0039] Material properties: Na content 20%; pH 7.7; temperature 25-30℃; conductivity 33-38 mS / cm.

[0040] Processing technology: molecular weight cutoff 500D, temperature 25-30℃, pressure 20 bar, compression 20 times, content 170-190g / L; after membrane filtration, the material transmittance is 70%, the clear liquid is collected, and the turbid liquid is collected and returned to ultrafiltration.

[0041] Furthermore, the nanofiltration decolorization process includes:

[0042] The molecular weight cutoff is 500D, the temperature is 25-30℃, the pressure is 20 bar and the compression is 20 times, the content is 170-190g / L, the material after membrane filtration has 70% light transmittance, the clear liquid is collected to obtain nanofiltration clear liquid, and the turbid liquid is collected and returned to ultrafiltration.

[0043] Acidification crystallization conditions:

[0044] Feed solution: nanofiltration solution.

[0045] Material properties: Na content 17-20%; pH 7.7; temperature 25-30℃; conductivity 30 mS / cm.

[0046] Processing procedure: Heat the material to 85 degrees Celsius, slowly add 50% sulfuric acid dropwise, adjusting the pH to approximately 3.5-3.7. The acid addition time is about 1 hour. Maintain this temperature for 30 minutes until the pH stabilizes, then begin cooling. Monitor the pH throughout the process. During cooling, the pH may rise; if necessary, add more sulfuric acid to control the pH and stabilize it at 3.6-3.7. Once the temperature drops to 15-20 degrees Celsius, maintain this temperature for 1 hour before discharging and centrifuging.

[0047] Furthermore, the acidification crystallization process includes:

[0048] Heat the nanofiltration solution to 85 degrees Celsius, slowly add 50% sulfuric acid to adjust the pH to 3.5-3.7, add acid over 1 hour, maintain for 30 minutes until the pH stabilizes, then begin cooling while monitoring the pH. If the pH rises during cooling, add more sulfuric acid to control the pH and stabilize it at 3.6-3.7. After the temperature drops to 15-20 degrees Celsius, maintain for 1 hour, then discharge and centrifuge.

[0049] Furthermore, the gentle heating is achieved through a 60°C water bath.

[0050] Compared with the prior art, the beneficial effects of the present invention are:

[0051] 1. This invention uses calcium alginate to immobilize nitrile hydrolase. The nitrile hydrolase immobilized by this method has very little loss of enzyme activity, protects the enzyme from shear force, has a high substrate diffusion rate, and can be reused up to 14 times. The encapsulated enzyme can be reused multiple times, which greatly reduces production costs.

[0052] 2. The conversion reaction process of this invention is simple and convenient. It has a high conversion rate at a reaction temperature of about 30°C and in a slightly alkaline reaction environment. The encapsulated and immobilized enzyme reaction system has fewer impurities and pigments, reducing pollution and by-product generation. It is also easier to handle subsequent decolorization and deodorization, saving on the separation and purification of subsequent products, and has certain potential for industrial application.

[0053] 3. The immobilized enzyme selected in this invention has small and uniform encapsulated particle diameter, high mechanical strength, low enzyme activity loss rate and easy preservation. The separation and purification method adopted in this invention produces products with few impurities and a high yield, which can reach more than 90%. Detailed Implementation

[0054] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Example 1: Weigh 3g of alginic acid powder and add it to sterile deionized water to prepare a 3% calcium alginate solution. Stir and dissolve in a 60℃ water bath until a homogeneous, transparent, particle-free viscous solution is formed. Cool to room temperature. Weigh 12g of anhydrous calcium chloride (CaCl2) and dissolve it in sterile deionized water to prepare a 600mL, 2% calcium chloride solution. Place the solution in a beaker and thoroughly and gently mix the cooled calcium alginate solution with the prepared enzyme solution under sterile conditions. Then, using a 5mL syringe, uniformly add the mixture dropwise to the 2% calcium chloride aqueous solution, controlling the encapsulated particle size to within the 2mM range. After the addition is complete, continue stirring for 1 hour. Wash with pure water 3-4 times before the reaction.

[0056] Example 2: The encapsulated particles obtained above were dissolved in 700 mL of water, the pH was adjusted to 7.5, and the temperature was controlled at 30°C. 70% 3-cyanopyridine was added at a flow rate of 27 mL / h, and the stirring speed was controlled at 250 rpm. Samples were taken for testing after 8 hours or 15 hours. 0.05% calcium chloride was added before each batch of reaction to harden the encapsulated particles. The resulting conversion reaction solution was microfiltered at 50-60°C, 2-3 bar pressure, a compression ratio of 7.5 times, and 3 times the volume of dialysis water. Activated carbon was then added at 0.5% by volume, the temperature was raised to 60-80°C, and the mixture was stirred for 1 hour to obtain a decolorized solution with a transmittance of 30-50%. The decolorized solution was ultrafiltered, with a molecular weight cutoff of 1 KD, at a temperature of 25-30°C, a pressure of 20 bar, and a compression ratio of 20 times. The clear liquid was collected, with a concentration of 200 g / L; the turbid liquid was collected and returned to activated carbon for decolorization. The material was subjected to nanofiltration at a molecular weight cutoff of 500D, a temperature of 25-30℃, a pressure of 20 bar, a compression ratio of 20 times, and a content of 170-190 g / L. After filtration, the material was subjected to nanofiltration with 70% light transmittance. The clear liquid was collected, and the turbid liquid was collected and returned to ultrafiltration. The obtained material was heated to 85℃, and 50% sulfuric acid was slowly added dropwise to adjust the pH to about 3.5-3.7. The acid addition time was about 1 hour, and the pH was maintained for 30 minutes until it stabilized. Then, the temperature was lowered, and the pH was monitored throughout the process. During the cooling process, the pH may rise. Sulfuric acid was added to control the pH and stabilize it at 3.6-3.7. After the temperature dropped to 15-20℃, it was maintained for 1 hour before centrifugation. The purification yield of the immobilized enzyme conversion solution using this method is over 90%, and the obtained product is free of impurities, discoloration, and off-odors.

[0057] Comparative Example 1: 5g of bacterial cells were dissolved in 700mL of water, the pH was adjusted to 7.5, and the temperature was controlled at 30℃. 70% 3-cyanopyridine was added at a flow rate of 27mL / h, with stirring at 250rpm. Samples were taken after 8 hours for analysis. The resulting conversion reaction solution was then microfiltered at 50-60℃, 2-3 bar, and a compression ratio of 7.5 times, followed by 3 times dialysis water. Activated carbon was then added at 0.5% by volume, the temperature was raised to 60-80℃, and the mixture was stirred for 1 hour to obtain a decolorized solution with a transmittance of 30-50%. The decolorized solution was ultrafiltered, with a molecular weight cutoff of 1KD, at 25-30℃ and a pressure of 20 bar, compressed 20 times, and the clear liquid was collected, with a concentration of 200g / L. The turbid liquid was collected and returned to activated carbon for further decolorization. The material was subjected to nanofiltration at a molecular weight cutoff of 500D, a temperature of 25-30℃, a pressure of 20 bar, a compression ratio of 20 times, and a content of 170-190 g / L. After filtration, the material was subjected to nanofiltration with 70% light transmittance. The clear liquid was collected, and the turbid liquid was collected and returned to ultrafiltration. The obtained material was heated to 85℃, and 50% sulfuric acid was slowly added dropwise to adjust the pH to about 3.5-3.7. The acid addition time was about 1 hour, and the pH was maintained for 30 minutes until it stabilized. Then, the temperature was lowered, and the pH was monitored throughout the process. During the cooling process, the pH may rise. Sulfuric acid was added to control the pH and stabilize it at 3.6-3.7. After the temperature dropped to 15-20℃, it was maintained for 1 hour before centrifugation. The material was then separated and purified using whole-cell conversion fluid. The final yield was about 85%. The obtained nicotinic acid product had some pigment residue and a stronger odor than the immobilized enzyme conversion fluid.

[0058] To quickly verify the reusability of the batches, 70% of 3-cyanopyridine was catalyzed for conversion to nicotinic acid under conditions of pH 7.5 and 30°C. The immobilized enzyme used in the first catalytic reaction was recovered and reused for the second batch reaction, and so on. The number of recovered batches of immobilized enzyme was calculated using the residual amount of 3-cyanopyridine as a reference index. The results for each batch are shown in the table below:

[0059]

[0060]

[0061] As shown in the table above, the enzyme activity decreases slowly with the increase of the number of reactions. By extending the reaction time, the immobilized nitrile hydrolase can continuously carry out 16 batches of catalytic reactions, and the substrate residue can reach 0, with a conversion rate of more than 99%. If the reaction time is further extended or the substrate 3-cyanopyridine is fed in, the number of times it can be reused will be even higher.

[0062] By comparing Examples 1 and 2 of the present invention, it is clear that the embodiments of the present invention employ encapsulated enzyme treatment, resulting in a purification yield of over 90%, and the obtained product is free of impurities, discoloration, and off-odors. In contrast, Comparative Example 1, which did not employ encapsulated enzyme treatment, yielded nicotinic acid with some pigment residue, and the odor was stronger than that of the immobilized enzyme conversion solution. This is because the membrane separation method of the present invention can highly selectively separate enzymes and products, operates at low temperatures, efficiently desaltes and removes impurities, and maintains product stability. Furthermore, the encapsulated immobilized enzyme reaction system of the present invention contains fewer impurities and pigments, reducing pollution and by-product generation. It also allows for better handling of subsequent decolorization and deodorization, saving on subsequent product separation and purification.

[0063] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for nicotinic acid immobilized enzyme conversion, characterized in that, include Wet bacterial cells containing nitrile hydrolase were taken, and the flocculated solution was passed through an ultrafiltration membrane for immobilization to obtain embedded particles no larger than 2 mM. After the encapsulated particles harden, the resulting conversion solution is separated and purified using membrane separation. The process involves microfiltration, activated carbon decolorization, ultrafiltration, nanofiltration decolorization, acidification crystallization, and centrifugation to finally obtain the nicotinic acid product.

2. The nicotinic acid immobilized enzyme conversion method as described in claim 1, characterized in that, The preparation process of the flocculant solution through an ultrafiltration membrane specifically includes: Take wet bacterial cells containing nitrile hydrolase and prepare a 200 g / L bacterial suspension with 50 mM pH 8.0 phosphate buffer. Use a high-pressure homogenizer to break the cells three times under a pressure of 80 MPa to obtain the cell lysis solution. Add 5 ml / L of 40% polyquaternium-10 solution to the cell lysis solution to flocculate the cell fragments. After stirring for 1 hour, centrifuge and collect the supernatant to obtain the flocculated clear liquid. Pass the obtained flocculated clear liquid through an ultrafiltration membrane to remove the salt in the supernatant.

3. The nicotinic acid immobilized enzyme conversion method as described in claim 1, characterized in that, The embedding and immobilization process specifically includes: Preparation of sodium alginate solution: Weigh an appropriate amount of calcium alginate powder, add sterile deionized water, and prepare a 3% calcium alginate solution. Dissolve the solution under gentle heating and continuous stirring until a uniform, transparent, particle-free viscous solution is formed, and then cool to room temperature. Preparation of calcium chloride solution: Weigh an appropriate amount of anhydrous calcium chloride, dissolve it in sterile deionized water to prepare a 2% calcium chloride solution, and place it in a beaker for easy collection of subsequent dripping calcium alginate droplets. Preparation of embedding mixture: The cooled calcium alginate solution and the enzyme solution to be immobilized were thoroughly and gently mixed under sterile conditions, and then added dropwise to a 2% calcium chloride aqueous solution. After the addition was completed, stirring was continued for 1 hour. The solution was washed 3-4 times with pure water before the reaction.

4. The nicotinic acid immobilized enzyme conversion method as described in claim 1, characterized in that, The process of hardening the embedded particles includes: The encapsulated particles were dissolved in 700 mL of water, the pH was adjusted to 7.5, and the temperature was controlled at 30℃. 70% 3-cyanopyridine was added at a flow rate of 27 mL / h and the stirring speed was controlled at 250 rpm. Samples were taken for testing after 8 h or 15 h. 0.05% calcium chloride was added before each batch of reaction to harden the encapsulated particles.

5. The nicotinic acid immobilized enzyme conversion method as described in claim 1, characterized in that, The microfiltration process includes: The resulting conversion reaction solution was microfiltered at 50-60℃, 2-3 bar pressure, 7.5 times compression ratio, and 3 times dialysis water.

6. The method for nicotinic acid immobilized enzyme conversion as described in claim 1, characterized in that, The activated carbon decolorization process includes: Add 0.5% activated carbon by volume, heat to 60-80 degrees Celsius, stir for 1 hour, and obtain a decolorizing solution with a light transmittance of 30-50% for activated carbon decolorization.

7. The nicotinic acid immobilized enzyme conversion method as described in claim 1, characterized in that, The ultrafiltration process includes: The molecular weight cutoff is 1 kDa. The temperature is 25-30℃ and the pressure is 20 bar, which is compressed 20 times. The clear liquid is collected with a content of 200 g / L. The turbid liquid is collected and then decolorized with activated carbon.

8. The method for nicotinic acid immobilized enzyme conversion as described in claim 1, characterized in that, The nanofiltration decolorization process includes: The molecular weight cutoff is 500D, the temperature is 25-30℃, the pressure is 20 bar and the compression is 20 times, the content is 170-190g / L, the material after membrane filtration has 70% light transmittance, the clear liquid is collected to obtain nanofiltration clear liquid, and the turbid liquid is collected and returned to ultrafiltration.

9. The nicotinic acid immobilized enzyme conversion method as described in claim 8, characterized in that, The acidification crystallization process includes: Heat the nanofiltration solution to 85 degrees Celsius, slowly add 50% sulfuric acid to adjust the pH to 3.5-3.7, add acid over 1 hour, maintain for 30 minutes until the pH stabilizes, then begin cooling while monitoring the pH. If the pH rises during cooling, add more sulfuric acid to control the pH and stabilize it at 3.6-3.

7. After the temperature drops to 15-20 degrees Celsius, maintain for 1 hour, then discharge and centrifuge.

10. The nicotinic acid immobilized enzyme conversion method according to claim 3, characterized in that, Mild heating is achieved through a 60°C water bath.

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