CN106636294B - Process for producing unnatural amino acid product by immobilized double-enzyme coupling reaction - Google Patents
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Abstract
The patent discloses a process for producing an unnatural amino acid product by immobilized double-enzyme coupling reaction. Polyvinyl alcohol, sodium alginate and carrageenan are used as immobilization materials, a boric acid-borax-calcium chloride-sodium carbonate system is used as a cross-linking agent, two kinds of genetic engineering bacteria with acylation racemase and amino acylase activities are mixed according to a certain proportion and then co-immobilized, and granular immobilized cells are prepared. acetylated-DL-amino acid is used as a raw material, and the double-enzyme coupling reaction is carried out in a bioreactor to efficiently produce non-natural amino acid series products.
Description
Technical Field
The invention relates to a process for producing an unnatural amino acid product by an immobilized double-enzyme coupling reaction, belonging to the technical field of enzyme engineering in biochemistry.
Background
Unnatural amino acids are core intermediates of numerous drugs, especially numerous new drugs over nearly ten years, e.g., L-2-aminobutyric acid for levetiracetam, L-norvaline for perindopril, L-2-orthochlorophenylglycine for clopidogrel, D-tryptophan for tatanafil, D-proline for zolmitriptan, L-2-tert-leucine for atazanavir and telaprevir, D-phenylalanine for nateglinide and octreotide, etc.
The main production means of unnatural amino acids is to use biological enzyme technology, and the enzymes used include amino acylase, penicillin acylase, hydantoinase, dehydrogenase, amino acid oxidase, etc. Among them, aminoacylases have been one of the major processes and have been used for a long time for producing unnatural amino acids. Aminoacylase includes D-aminoacylase and L-aminoacylase, wherein D-aminoacylase catalyzes acetyl-D-amino acid to generate D-amino acid, and L-aminoacylase catalyzes acetyl-L-amino acid to generate L-amino acid. Industrially, the starting material for the production of unnatural amino acids by aminoacylase is acetylated-DL-amino acids, so that the conversion is only 50%.
The patent reports that DL-acetylated-DL-amino acid is converted into chiral amino acid in one step by coupling reaction after mixing acylated racemase and aminoacylase, and the conversion rate reaches 100 percent. The method has the disadvantages that the free cell method is inconvenient to reuse, the free cells are easy to break, the product cost is high, and the product quality is unstable; how to reuse the biocatalyst and reduce the cost of the catalyst is a key factor for industrial application of the technology, wherein the immobilized enzyme or immobilized cell technology is a main solution.
The immobilized cell technology is used as a new biological engineering technology and has the advantages of low cost, low pollution, high efficiency and the like. The materials used in the current immobilization technology comprise carrageenan, sodium alginate, resin, glucan, polyacrylamide and the like, and the immobilized cell material is used for industrialization and theoretically has the following characteristics: easy rapid mixing with cells or enzymes and rapid coagulation; freezing at low temperature as far as possible to avoid loss of activity of the enzyme; the granular material is granular as much as possible, is convenient to be loaded into a reactor, and obtains better reaction characteristics; has better mechanical properties such as strength and hardness; the material has porosity and good mass transfer performance; the price is low. In order to prepare immobilized cells having the above properties, researchers have introduced various immobilized cells having different properties by optimizing the immobilization material and the preparation process, selecting a high-quality cross-linking agent, and the like.
Polyvinyl alcohol as a novel biocatalyst embedding immobilization carrier has the advantages of high strength, good chemical stability, strong antimicrobial decomposition performance, low price and the like, but still has the following defects: 1. the solution for crosslinking polyvinyl alcohol has stronger acidity, which can reduce the activity of immobilized cells; 2. the high-viscosity aqueous solution has high viscosity and slow reaction with acid, so that two liquid drops with little difference in dropping time can be adhered and agglomerated, and the gel is not easy to form balls; 3. the enzyme activity retention rate after immobilization is low, and part of acid-sensitive enzymes can be gradually inactivated.
Therefore, the research of a good compound cross-linking agent system has become an important influencing factor of the enzymatic production of the unnatural amino acid by taking the polyvinyl alcohol as the immobilized material.
Disclosure of Invention
In order to overcome the defects, the invention provides a process for producing an unnatural amino acid product by an immobilized double-enzyme coupling one-step reaction.
The method is characterized in that: the method comprises the steps of mixing genetically engineered bacteria with amino acylase and acylation racemase and co-immobilizing the genetically engineered bacteria by taking a mixture of polyvinyl alcohol, sodium alginate and carrageenan as an immobilization material and a boric acid-borax-calcium chloride-sodium carbonate system as a cross-linking agent to prepare the granular immobilized cells. acetylated-DL-amino acid is used as a raw material, and a double-enzyme coupling reaction is utilized in a bioreactor to produce unnatural amino acid series products in one step. The specific operation process is as follows:
the preparation process of the cross-linking agent solution comprises the following steps:
adding water into a stirring container, adding a certain proportion of boric acid, borax, calcium chloride and sodium carbonate, and adjusting the pH value to obtain a cross-linking agent solution. The proportion of the raw materials can be increased or decreased according to the requirements.
Cell co-immobilization process:
adding a certain amount of polyvinyl alcohol into water to swell for 24 hours, then adding sodium alginate and carrageenan, heating a sterilization pot to fully dissolve the polyvinyl alcohol, stirring the polyvinyl alcohol while the polyvinyl alcohol is hot, cooling the polyvinyl alcohol to 40-50 ℃, then adding a mixed bacterial suspension of L-type aminoacylase and acylation racemase which is preserved at 50 ℃ in advance into the liquid, stirring the mixture evenly, sucking the mixture by using an injector, then dropwise adding the mixture into a cross-linking agent solution of the boric acid-borax-calcium chloride-sodium carbonate system to form small balls with a certain diameter, and soaking the small balls in the cross-linking agent for 3-12 hours. Washing with physiological saline to obtain L-type amino acid immobilized cells for transformation. Replacing the L-aminoacylase with D-aminoacylase to prepare the D-type amino acid immobilized cell.
The biological catalysis process comprises the following steps:
adding acetylated-DL-amino acid into distilled water to obtain 0.1-0.2M solution, adjusting pH =7.5 with caustic soda flakes, adding the L-amino acid immobilized cell, and converting at 37 deg.C for 5-10 hr to obtain acetylated-DL-amino acid with a conversion rate of less than 0.001% (more than 99.9% conversion, the same below). Is L-alanine, and is subjected to concentration and purification treatment to obtain the L-amino acid.
Adding acetylated-DL-amino acid into distilled water, preparing into 0.1-0.2M solution, adjusting pH =7.5 with caustic soda flakes, adding the D-type amino acid immobilized cell for conversion, measuring at 37 deg.C for 5-10 hr to obtain acetylated-DL-amino acid with content of less than 0.001%, converting into D-amino acid, concentrating, and purifying to obtain L-amino acid.
The immobilization material is a mixture of polyvinyl alcohol, sodium alginate and carrageenan, and the concentration of the polyvinyl alcohol material is 5-15%, preferably 8-12%.
The weight ratio of the main material to the auxiliary material is as follows: sodium alginate: carrageenan =100:0.1-0.5: 0.2-1.0.
The cross-linking agent is a boric acid-borax-calcium chloride-sodium carbonate system, and low-temperature cross-linking is adopted for cross-linking, wherein the cross-linking temperature is-10-20 ℃.
The immobilized cells prepared according to the above method are in the form of granules. The thallus with the activity of amino acylase and acylation racemase is mixed according to the enzyme activity proportion and then immobilized to prepare granular immobilized cells, and the two-enzyme coupling reaction is utilized to produce the non-natural amino acid series products in one step.
Advantageous effects of the invention
The invention adopts the novel polyvinyl alcohol immobilized compound material, better solves the problems of granularity, stability and rapid solidification speed of the immobilized cells;
the invention adopts the novel cross-linking agent to cross-link under the condition of near-neutral environment, thereby avoiding the damage to enzyme. The process is successfully used for immobilizing mixed thalli of aminoacylase and acylation racemase, and produces the unnatural amino acid in one step by a coupling reaction technology.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The analytical method used in this example was as follows:
immobilized cell strength: the immobilized beads were placed in a 100mL syringe, and the breakage of the beads was observed by applying a certain pressure.
Mass transfer degree of immobilized cells: taking several immobilized pellets, putting the immobilized pellets into a 250mL conical flask containing 200mL of water, adding a drop of methylene blue, and observing the condition that the color permeates into the immobilized pellets at regular time
Degree of swelling (%) of immobilized cells: 100mL of the immobilized cells were taken and stored in 0.05M phosphate buffer at 25 ℃ for 24 hours, increasing the volume.
The preservation rate of the enzyme activity of the immobilized cells is as follows: after the immobilized cells react for 5 hours in 0.05M phosphate buffer solution and 0.1M acetyl-DL-phenylalanine concentration, the specific activity of the immobilized cells is measured, and compared with the initial specific activity, the preservation rate of the enzyme activity is
Immobilized cell particle perfection ratio: the percentage of the immobilized cells remaining after eliminating the particles damaged by more than one quarter of the volume after 5 hours of reaction in 0.05M phosphate buffer at 0.1M acetyl-DL-phenylalanine concentration.
Balling time: the immobilized cells were dropped from a syringe and completely hardened in the crosslinking agent solution.
Adhesion: visually, the immobilized cells were dropped from the syringe and were adhered to each other in the crosslinking agent solution.
Example 1
(A) Preparation of crosslinker solution
Adding 800mL of water into a flask, adding 15g of boric acid and 15g of borax, mixing, then adding 10g of calcium chloride and 50g of sodium carbonate, measuring the pH value, adjusting the pH value to 7.0 by using sodium carbonate or boric acid, metering the volume to 1000mL, and uniformly mixing for later use. The proportion of the raw materials can be increased or decreased according to the requirements.
(B) Cell immobilization method
Adding 10g of polyvinyl alcohol into 90mL of water to swell for 24h, adding 0.2 g of sodium alginate and 0.2 g of carrageenan, heating a sterilization pot to fully dissolve the polyvinyl alcohol, stirring while the polyvinyl alcohol is hot, cooling to 40-50 ℃, then adding 100mL of mixed bacterial suspension of aminoacylase and acylation racemase (the specific activity of L-aminoacylase is 40umol/g.min, and the specific activity of acylation racemase is 80 umol/g.min) which is preserved at 50 ℃ in advance into the liquid, and uniformly stirring. Sucking the mixture by using a 50mL syringe, then dropwise adding the mixture into the cross-linking agent solution of the boric acid-borax-calcium chloride-sodium carbonate system to form small balls with a certain diameter, and soaking the small balls in the cross-linking agent for 3-12 hours. Washing with normal saline to obtain L-type amino acid immobilized cell with specific activity of 15.5umol/g.min, and storing in refrigerator for conversion and performance measurement.
The above preparation process and immobilized cells were further tested, and the data obtained were as follows: the immobilized particles are about 3mm, the strength is high, the balling time is 12 hours, the mass transfer effect is good, the swelling degree of the immobilized cells is 12 percent, the cohesiveness is avoided, and the particle perfectness rate is 95 percent. The preservation rate of the enzyme activity of the immobilized cells is 91 percent.
(C) Transformation test method:
adding 27 g of acetylated-DL-alanine into 1L of distilled water to prepare a 0.2M solution, adjusting the pH with caustic soda flakes =7.5, adding 50g of the L-type amino acid immobilized cells to convert, measuring at 37 ℃ for 10 hours, wherein the acetylated-DL-alanine is less than 0.001 percent and is almost completely converted into the L-alanine, and concentrating and purifying to obtain 18 g of the L-alanine.
DL-acetyl-leucine, DL-acetyl-valine, DL-acetylphenylalanine, DL-acetylnorleucine are used as raw materials, 0.2M solution is prepared, pH =7.5 is adjusted by caustic soda flakes, 50g of immobilized cells are added for conversion, acetylation-DL-amino acid is less than 0.001% and is completely converted into L-amino acid after 10 hours at 37 ℃. The raw materials respectively obtain L-leucine, L-valine, L-phenylalanine and L-norleucine.
Comparative example 1:
adding 10g of polyvinyl alcohol into 90mL of water to swell for 24h, heating a sterilization pot to fully dissolve the polyvinyl alcohol, stirring the polyvinyl alcohol while the polyvinyl alcohol is hot, cooling the polyvinyl alcohol to 40-50 ℃, adding 100mL of mixed bacterial suspension of aminoacylase and acylation racemase (the specific activity of L-aminoacylase is 40umol/g.min, and the specific activity of acylation racemase is 80 umol/g.min) which is preserved at 50 ℃ in advance, and stirring the mixture uniformly. Sucking the mixture by using a 50mL syringe, then dropwise adding the mixture into a cross-linking agent solution (15 g/L boric acid, 10g/L calcium chloride and 50g/L sodium carbonate) of a boric acid-calcium chloride-sodium carbonate system to form small balls with a certain diameter, and soaking the small balls in the cross-linking agent for 3-12 hours. Washing with normal saline to obtain L-type amino acid immobilized cells, and measuring performance.
The above tests were repeated with the respective concentrations of 2%, 5%, 8%, 10%, 15%, and 20% polyvinyl alcohol, and the other conditions were unchanged, with the following results:
the experimental results show that the swelling rate of the single polyvinyl alcohol immobilized cells is high, the balling speed is low, the balls are mutually adhered, and meanwhile, the viscosity is increased along with the increase of the concentration of the polyvinyl alcohol, and even the balls cannot be balling. The polyvinyl alcohol concentration is suitably 10%.
Comparative example 2
10g of polyvinyl alcohol is taken and added into 90mL of water to swell for 24h, sodium alginate with different proportions is added to repeat the test, other conditions are the same as those of comparative example 1, and the results are as follows:
the experimental results show that: after the sodium alginate is added, the strength of the small ball is increased, the swelling rate is reduced, the small ball is easy to form the ball and is not adhered, and the optimum content is 0.2%.
Comparative example 3
10g of polyvinyl alcohol is added into 90mL of water to be swelled for 24h, carrageenan with different proportions is added to repeat the test, other conditions are the same as those of comparative example 1, and the results are as follows:
the experimental results show that: after the carrageenan is added, the strength of the pellets is increased, the swelling ratio is reduced, the pellets are still difficult to form, but the recovery rate of the immobilized enzyme is improved and is most suitable to be 0.5 percent.
Comparative example 4
10g of polyvinyl alcohol is added into 90mL of water to swell for 24h, sodium alginate is 0.2%, carrageenan with different proportions is added to repeat the test, and other conditions are the same as those of comparative example 1, and the results are as follows:
through experimental comparison, the optimal proportion of 10 percent of polyvinyl alcohol, 0.2 percent of sodium alginate and 0.5 percent of carrageenan is optimized.
Comparative example 5
The test conditions are the same as those of comparative example 4, the results are as follows:
by comparison, boric acid 15g/L, borax 15g/L, calcium chloride 10g/L, sodium carbonate 50g/L are preferred as the optimal crosslinking system.
Example 2
(A) Preparation of crosslinker solution
Same as example 1
(B) Cell immobilization method
Adding 10g of polyvinyl alcohol into 90mL of water to swell for 24h, adding 0.2 g of sodium alginate and 0.2 g of carrageenan, heating a sterilization pot to fully dissolve the polyvinyl alcohol, stirring the polyvinyl alcohol while the polyvinyl alcohol is hot, cooling the polyvinyl alcohol to 40-50 ℃, then adding 100mL of mixed bacterial suspension of D-aminoacylase and racemase (the specific activity of D-aminoacylase is 30umol/g.min, and the specific activity of racemase is 98 umol/g.min) which is preserved at 50 ℃ in advance, and stirring the mixture uniformly. Sucking the mixture by using a 50mL syringe, then dropwise adding the mixture into the cross-linking agent solution of the boric acid-borax-calcium chloride-sodium carbonate system to form small balls with a certain diameter, and soaking the small balls in the cross-linking agent for 3-12 hours. Washing with normal saline to obtain D-type amino acid immobilized cell, measuring to obtain immobilized cell with specific activity of 12.2umol/g.min, and storing in refrigerator for conversion.
(C) Transformation test method:
adding 27 g of acetylated-DL-alanine into 1L of distilled water to prepare a 0.2M solution, adjusting the pH with caustic soda flakes =7.5, adding 50g of the D-type amino acid immobilized cells, converting, measuring at 37 ℃ for 10 hours, converting all acetylated-DL-alanine into D-alanine, concentrating and purifying to obtain 18 g of D-alanine.
DL-acetyl-leucine, DL-acetyl-valine, DL-acetylphenylalanine, DL-acetylnorleucine are used as raw materials, 0.2M solution is prepared, pH =7.5 is adjusted by caustic soda flakes, 50g of immobilized cells are added for conversion, acetylation-DL-amino acid is less than 0.001 percent and is completely converted into D-amino acid after 10 hours at 37 ℃. The raw materials are respectively used for obtaining D-leucine, D-valine, D-phenylalanine and D-norleucine.
Example 3
(A) Preparation of crosslinker solution
Same as example 1
(B) Cell immobilization method
Adding 10g of polyvinyl alcohol into 90mL of water to swell for 24h, adding 0.2 g of sodium alginate and 0.2 g of carrageenan, heating a sterilization pot to fully dissolve the polyvinyl alcohol, stirring while the polyvinyl alcohol is hot, cooling to 40-50 ℃, adding 100mL of aminoacylase bacterial suspension (the specific activity of L-aminoacylase is 80 umol/g.min) which is preserved at 50 ℃ in advance, and stirring uniformly. Sucking the mixture by using a 50mL syringe, then dropwise adding the mixture into the cross-linking agent solution of the boric acid-borax-calcium chloride-sodium carbonate system to form small balls with a certain diameter, and soaking the small balls in the cross-linking agent for 3-12 hours. Washing with normal saline to obtain L-type amino acid immobilized cell, measuring to obtain immobilized cell with specific activity of 36.5umol/g.min, and storing in refrigerator for conversion.
(C) Transformation test method:
adding 27 g of acetylated-DL-alanine into 1L of distilled water to prepare a 0.2M solubility solution, adjusting the pH value to be 7.5 by using caustic soda flakes, adding 50g of the L-type amino acid immobilized cells for conversion, measuring the temperature at 37 ℃ after 10 hours, determining the conversion rate of the L-alanine to be 50 percent, and concentrating and purifying to obtain 6.8 g of L-alanine.
Taking DL-acetyl-leucine, DL-acetyl-valine, DL-acetylphenylalanine and DL-acetylnorleucine as raw materials to respectively obtain L-leucine, L-valine, L-phenylalanine and L-norleucine. Raw material test: all the above were prepared into 0.2M solution, pH =7.5 was adjusted with caustic soda flakes, 50g of the immobilized cells were added for transformation, and acetylated-DL-amino acid was converted into L-amino acid at 50% conversion rate after 10 hours at 37 ℃.
Example 4
(A) Preparation of crosslinker solution
Same as example 1
(B) Cell immobilization method
Adding 10g of polyvinyl alcohol into 90mL of water, swelling for 24h, adding 0.2 g of sodium alginate and 0.2 g of carrageenan, heating a sterilization pot to fully dissolve the polyvinyl alcohol, stirring while the polyvinyl alcohol is hot, cooling to 40-50 ℃, adding 100mL of D-aminoacylase bacterial suspension (the specific activity of D-aminoacylase is 65 umol/g.min) which is preserved at 50 ℃ in advance, and stirring uniformly. Sucking the mixture by using a 50mL syringe, then dropwise adding the mixture into the cross-linking agent solution of the boric acid-borax-calcium chloride-sodium carbonate system to form small balls with a certain diameter, and soaking the small balls in the cross-linking agent for 3-12 hours. Washing with normal saline to obtain D-type amino acid immobilized cell, determining that the specific activity of the immobilized cell is 28.5umol/g.min, and storing in refrigerator for conversion.
(C) Transformation test method:
adding 27 g of acetylated-DL-alanine into 1L of distilled water to prepare a 0.2M solubility solution, adjusting the pH with caustic soda flakes to =7.5, adding 50g of the D-type amino acid immobilized cells, converting, measuring at 37 ℃ after 10 hours, determining that the conversion rate of the D-alanine is 50%, and concentrating and purifying to obtain 7.0 g of D-alanine.
Respectively obtaining D-leucine, D-valine, D-phenylalanine and D-norleucine by using DL-acetyl-leucine, DL-acetyl-valine, DL-acetylphenylalanine and DL-acetylnorleucine as raw materials. Raw material test: all of the above were prepared into 0.2M solution, pH =7.5 was adjusted with caustic soda flakes, 50g of the immobilized cells were added for transformation, and acetylated-DL-amino acid was converted into D-amino acid at 50% conversion rate, measured 10 hours after 37 ℃.
Claims (3)
1. A process for producing unnatural amino acid products by immobilized double-enzyme coupling reaction is characterized in that: mixing two genetic engineering bacteria with aminoacylase and acylation racemase in the presence of an immobilization material and a cross-linking agent, co-immobilizing to obtain an immobilized cell, and performing biocatalytic reaction by using acetylated-DL-amino acid as a raw material to obtain an unnatural amino acid product; the immobilized material is a mixture of polyvinyl alcohol, sodium alginate and carrageenan, wherein the concentration of the polyvinyl alcohol is 10%; the weight ratio is, polyvinyl alcohol: sodium alginate: carrageenan =100: 0.2: 0.5; the cross-linking agent is a boric acid-borax-calcium chloride-sodium carbonate system, and the concentration ratio is 15g/L of boric acid, 15g/L of borax, 10g/L of calcium chloride and 50g/L of sodium carbonate; the co-immobilization operation comprises the steps of adding polyvinyl alcohol into water to swell for 24 hours, then adding sodium alginate and carrageenan, heating to fully dissolve the polyvinyl alcohol, stirring while the polyvinyl alcohol is hot, cooling to 40-50 ℃, then adding amino acylase and acylation racemase mixed bacterial suspension which is preserved at 50 ℃ in advance into the liquid, stirring uniformly, then dripping into a cross-linking agent solution in a dripping mode to form small balls, and soaking in the cross-linking agent for 3-12 hours; washing with physiological saline to obtain amino acid immobilized cells.
2. The process for producing unnatural amino acid according to claim 1, wherein the step of performing the immobilized double-enzyme coupling reaction comprises: the immobilized cells are granular.
3. The process for producing unnatural amino acid according to claim 1, wherein the step of performing the immobilized double-enzyme coupling reaction comprises: adding acetylated-DL-amino acid into water to prepare 0.1-0.5M solution, adjusting pH =7.5 with caustic soda flakes, adding the D-type or L-type amino acid immobilized cells for conversion, measuring at 37 ℃ for 5-10 hours, converting the acetylated-DL-amino acid to D-type or L-alanine with the concentration and purification treatment, and obtaining the D-type or L-amino acid.
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| CN101603063A (en) * | 2009-07-23 | 2009-12-16 | 南京大学 | The fixation of microbial cell enzyme process prepares the method for the amino lipid acid of L-2- |
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| CN105368913A (en) * | 2015-12-22 | 2016-03-02 | 滨海瀚鸿生化有限公司 | Bi-enzyme preparation method for industrial production of chiral unnatural amino acid |
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| CN102174632A (en) * | 2009-05-19 | 2011-09-07 | 重庆邮电大学 | Bio-catalytic deracemization preparation method of non-natural L-amino acid |
| CN101603063A (en) * | 2009-07-23 | 2009-12-16 | 南京大学 | The fixation of microbial cell enzyme process prepares the method for the amino lipid acid of L-2- |
| CN105368913A (en) * | 2015-12-22 | 2016-03-02 | 滨海瀚鸿生化有限公司 | Bi-enzyme preparation method for industrial production of chiral unnatural amino acid |
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