阅读说明：本技术 一种多级孔道吸附凝胶耦合生物催化剂生产l-哌啶甲酸的方法 (Method for producing L-piperidinecarboxylic acid by coupling multistage pore adsorption gel with biocatalyst ) 是由 陈可泉 许晟 冯娇 于 2020-07-24 设计创作，主要内容包括：本发明公开了一种多级孔道吸附凝胶耦合生物催化剂生产L-哌啶甲酸的方法,包括如下步骤：将具备细胞活性的多级孔道吸附凝胶耦合生物催化剂接种于含氯霉素和卡那霉素的发酵培养基中培养,得到第一发酵液；向第一发酵液中加入阿拉伯糖培养,得到第二发酵液；向第二发酵液中加入异丙基-β-D-硫代半乳糖苷诱导表达发酵,得到第三发酵液；将第三发酵液进一步发酵,即得含有L-哌啶甲酸的的发酵液。本发明利用金属-有机框架材料与凝胶微球的组合,兼多种孔道尺寸与结构的优点,有效提升了对于L-哌啶甲酸的吸附效果。(The invention discloses a method for producing L-piperidinecarboxylic acid by coupling a multistage pore adsorption gel with a biocatalyst, which comprises the following steps: inoculating a multistage pore adsorption gel coupling biocatalyst with cell activity into a fermentation culture medium containing chloramphenicol and kanamycin for culture to obtain a first fermentation solution; adding arabinose into the first fermentation liquid for culturing to obtain a second fermentation liquid; adding isopropyl-beta-D-thiogalactoside into the second fermentation broth to induce, express and ferment to obtain a third fermentation broth; and further fermenting the third fermentation liquid to obtain the fermentation liquid containing the L-pipecolic acid. The invention utilizes the combination of the metal-organic framework material and the gel microspheres, combines the advantages of various pore sizes and structures, and effectively improves the adsorption effect on the L-piperidinecarboxylic acid.)

1. A method for producing L-piperidinecarboxylic acid by coupling multistage pore adsorption gel with a biocatalyst is characterized by comprising the following steps: inoculating a multistage pore adsorption gel coupling biocatalyst with cell activity into a fermentation culture medium containing chloramphenicol and kanamycin for culture to obtain a first fermentation solution; adding arabinose into the first fermentation liquid for culturing to obtain a second fermentation liquid; adding isopropyl-beta-D-thiogalactoside into the second fermentation broth to induce, express and ferment to obtain a third fermentation broth; and further fermenting the third fermentation liquid to obtain the fermentation liquid containing the L-pipecolic acid.

2. The method according to claim 1, wherein the multistage pore adsorption gel-coupled biocatalyst is prepared by a method comprising:

(1) stirring and mixing a cobalt sulfate compound and an imidazole compound, centrifuging, and drying the obtained precipitate to obtain a nano metal organic framework;

(2) uniformly mixing the nano metal organic framework obtained in the step (1) with sodium alginate through water to obtain a gel-doped nano metal organic framework;

(3) mixing the gel-doped nano metal organic framework obtained in the step (2) with an escherichia coli solution, and stirring to obtain a gel-doped escherichia coli-mixed nano metal organic framework;

(4) dripping a metal ion solution into the nanometer metal organic framework doped with the gel mixed escherichia coli obtained in the step (3), and standing to obtain microspheres for adsorbing gel coupling escherichia coli through multilevel pore channels;

(5) adding the microspheres obtained in the step (4) into a culture medium for culturing, and stirring; and adding betaine into the mixture, stirring, and taking out the microspheres to obtain the multistage pore adsorption gel coupling biocatalyst.

3. The method according to claim 2, wherein in the step (1), the solvent is methanol, the concentration of the cobalt sulfate compound is 0.1-0.8mmol/mL, and the concentration of the imidazole compound is 0.5-2.5 mmol/mL; the stirring and mixing are carried out at room temperature for 6-36 h.

4. The method as claimed in claim 2, wherein in the step (2), the mass ratio of the nano metal organic framework to the sodium alginate is 1: 10-1: 30, of a nitrogen-containing gas; the mass-volume ratio of the nano metal organic framework to the water is 0.05-0.25 mg/mL.

5. The method according to claim 2, wherein in the step (3), the Escherichia coli solution OD₆₀₀Is 20; the volume ratio of the gel-doped nano metal organic framework to the escherichia coli solution is 1: 1-5: 1.

6. The method according to claim 2, wherein in the step (4), the metal ion solution is an aqueous solution containing ferrous ions and calcium ions, the concentration of the ferrous ions is 20-120 mmol/L, and the concentration of the calcium ions is 20-120 mmol/L; the volume ratio of the metal ion solution to the nano metal organic framework of the doped gel mixed escherichia coli is 100: 1-100: 25; the dropping speed is 1-5 drops/s; the standing time is 15-60 min.

7. The method according to claim 2, wherein in the step (5), the microspheres obtained in the step (4) are added into an LB culture medium according to the ratio of 1g/100 mL-10 g/100mL of the culture medium, and are stirred at 15-35 ℃ for 5-10 min; and adding 100-600 mg of betaine into the mixture, stirring the mixture for 15-60 min at 15-35 ℃, and taking out microspheres to obtain the multistage pore adsorption gel coupling biocatalyst.

8. The method according to claim 1, wherein the multistage pore adsorption gel coupled biocatalyst with cell activity is prepared by placing the multistage pore adsorption gel coupled biocatalyst in LB medium, culturing at 37 ℃ and 100-300 rpm for 3 h.

9. The method according to claim 1, wherein the content of chloramphenicol and kanamycin in the fermentation medium containing chloramphenicol and kanamycin is 10-40 mg/L and 20-60 mg/L, respectively; the final concentration of the arabinose is 0.5-2.5 g/L; the final concentration of the isopropyl-beta-D-thiogalactoside is 50-300 mg/L.

10. The method according to claim 1, wherein the multistage pore adsorption gel coupling biocatalyst is taken out from the fermentation broth containing the L-pipecolic acid, added into an LB culture medium according to the ratio of 1g/100 mL-10 g/100mL culture medium, and stirred at 15-35 ℃ for 5-10 min; and adding 100-600 mg of betaine into the mixture, stirring the mixture for 15-60 min at 15-35 ℃, taking out microspheres, and obtaining the multistage pore adsorption gel coupling biocatalyst with cell activity, wherein the multistage pore adsorption gel coupling biocatalyst is reused.

Technical Field

The invention belongs to the fields of whole-cell catalysis, L-piperidinecarboxylic acid production and material adsorption separation, and relates to a method for producing L-piperidinecarboxylic acid by using a multistage pore adsorption gel coupling biocatalyst.

Background

L-pipecolic acid is a key precursor of medicaments such as various immunosuppressants and the like as a nitrogen-containing heterocyclic compound. Most of the drugs are chiral heterocyclic polyketone molecules, and L-pipecolic acid is one of the core components of the chiral heterocyclic polyketone molecules and is an irreplaceable part of natural metabolic synthesis and artificial production synthesis processes of the compounds. For example, L-pipecolic acid is involved in synthesizing antitumor drugs VX710, swainsonine and the like, and antibiotics such as jojobin and vancomycin and the like, so that the L-pipecolic acid has very high application value in the fields of medicine, physiology and the like. The research on the synthesis of the piperidinecarboxylic acid can increase the yield of the piperidinecarboxylic acid and enhance the production of the drugs, and can also promote the research on the action mechanism of a large class of drugs taking the piperidinecarboxylic acid as a core structure and even promote the development of new drugs of compounds with similar molecular structures.

With the continuous development of the pharmaceutical synthesis industry, the demand of people for such key small molecules is also increasing. Before 2001, L-piperidinecarboxylic acid was produced mainly by chemical synthesis. However, the synthesis of optically pure chiral organic compounds using chemical methods is generally not an easy problem to solve. The chemical synthesis of L-pipecolic acid can only be achieved by methods of directly taking chiral substances such as L-lysine and the like as reactants, introducing chiral groups and the like. The chemical synthesis method has the problems of complicated steps and environmental pollution in the process. A safer, green and economical method for biosynthesis of L-pipecolic acid is receiving attention from the industry. The biological method for preparing the L-piperidinecarboxylic acid has the advantages of less reaction steps, green and environment-friendly process and the like, but the biological method still has the defects of relatively low yield, complex operation steps and the like, so that the biological method is not suitable for large-scale industrial production of the L-piperidinecarboxylic acid. Therefore, it is necessary to develop a new method to solve the problems of the prior art.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problems of low yield, low thallus tolerance, difficult product separation and complicated continuous production steps in the process of producing L-piperidinecarboxylic acid by biological catalysis in the prior art, and provides a method for producing L-piperidinecarboxylic acid by a multistage pore adsorption gel coupling biocatalyst.

The invention idea is as follows: as shown in figure 1, a sodium alginate gel doped nano metal-organic framework material and escherichia coli are mixed together to prepare a multi-stage pore adsorption gel coupling biocatalyst, namely, a gel microsphere with multi-stage pores is constructed to wrap and fix the escherichia coli, a surfactant or an organic reagent is used to perform permeability modification on escherichia coli cell membranes, the catalytic activity of the escherichia coli for producing L-piperidinecarboxylic acid is improved, then the gel microsphere doped with the nano metal-organic framework material can be used for selectively enriching the L-piperidinecarboxylic acid by using a multi-stage pore structure, and further the product recovery process is simplified. According to the invention, through material design and escherichia coli biocatalysis process research, a multistage pore adsorption gel coupling biocatalyst is established, and production and separation of L-piperidinecarboxylic acid are considered at the same time. Firstly, sodium alginate gel is doped with a nano metal-organic framework material, so that the selective enrichment of L-pipecolic acid in the catalytic reaction process is realized; secondly, the porous adsorption gel is combined with escherichia coli, so that the yield of the L-piperidinecarboxylic acid produced by catalysis of the escherichia coli is increased; and thirdly, the continuous production of the L-piperidinecarboxylic acid by a biological method is realized, the operation steps are reduced, and the method has practical application value for improving the yield of the L-piperidinecarboxylic acid.

In order to solve the technical problem, the invention discloses a method for producing L-pipecolic acid by coupling a multistage pore adsorption gel with a biocatalyst, which comprises the following steps: (i) inoculating a multistage pore adsorption gel coupling biocatalyst with cell activity into a fermentation culture medium containing chloramphenicol and kanamycin according to the dosage of 0.08-0.16 g/mL for culture to obtain a first fermentation solution; (ii) adding arabinose into the first fermentation liquid for culturing to obtain a second fermentation liquid; (iii) adding isopropyl-beta-D-thiogalactoside into the second fermentation broth to induce, express and ferment to obtain a third fermentation broth; (iv) and further fermenting the third fermentation liquid to obtain the fermentation liquid containing the L-pipecolic acid.

In the step (i), the preparation method of the multistage pore adsorption gel coupling biocatalyst comprises the following steps:

(1) stirring and mixing a cobalt sulfate compound and an imidazole compound, centrifuging, and drying the obtained precipitate to obtain a nano metal organic framework;

(2) uniformly mixing the nano metal organic framework obtained in the step (1) with sodium alginate through water to obtain a gel-doped nano metal organic framework;

(3) mixing the gel-doped nano metal organic framework obtained in the step (2) with an escherichia coli solution, and stirring to obtain a gel-doped escherichia coli-mixed nano metal organic framework;

(4) dripping a metal ion solution into the gel-mixed escherichia coli-doped nano metal organic framework obtained in the step (3), and standing to obtain a microsphere with the diameter of 1-3 mm and capable of adsorbing gel-coupled escherichia coli through a multistage pore channel;

(5) adding the microspheres obtained in the step (4) into a culture medium for culturing, and stirring; and adding betaine into the mixture, stirring, and taking out the microspheres to obtain the multistage pore adsorption gel coupling biocatalyst.

In the step (1), the cobalt sulfate compound is preferably CoSO₄·6H₂O; the imidazole is 2-methylimidazole, imidazole or other imidazole derivatives; in the solution, the solvent is methanol, the concentration of the cobalt sulfate compound is 0.1-0.8mmol/mL, preferably 0.4mmol/mL, and the concentration of the imidazole compound is 0.5-2.5mmol/mL, preferably 1.6 mmol/mL; the stirring and mixing are carried out at room temperature at 500rpm for 6-36h, preferably 12 h; the centrifugation is 6000rpm for 15 min.

In the step (1), the nano metal organic framework is one or more of ZIF-67, ZIF-8 or derivatives thereof.

In the step (2), the mass ratio of the nano metal organic framework to the sodium alginate is 1: 10-1: 30, of a nitrogen-containing gas; the mass-volume ratio of the nano metal organic framework to the water is 0.05-0.25 mg/mL; preferably, the mass ratio of the nano metal organic framework to the sodium alginate is 1: 20; the mass-volume ratio of the nano metal organic framework to the water is 0.1 mg/mL.

Wherein the molecular weight of the sodium alginate is that of Shanghai national medicine Shanghai test.

In the step (2), the uniform mixing is microwave heating for 5-10 s until the sodium alginate powder is completely dissolved in water, and stirring at 500rpm for 15min until the nano metal organic framework powder is uniformly mixed in the sodium alginate gel.

In the step (3), the Escherichia coli solution OD₆₀₀Is 20; the volume ratio of the gel-doped nano metal organic framework to the escherichia coli solution is 1: 1-5: 1, preferably 5: 1; the stirring was 300rpm and was at 4 ℃ for 30 min.

In the step (4), the metal ion solution is an aqueous solution containing ferrous ions and calcium ions, the concentration of the ferrous ions is 20-120 mmol/L, preferably 60mmol/L, and the concentration of the calcium ions is 20-120 mmol/L, preferably 60 mmol/L; the volume ratio of the metal ion solution to the nano metal organic framework of the doped gel mixed escherichia coli is 100: 1-100: 25, and preferably 100: 6; the dropping speed is 1-5 drops/s; the standing time is 15-60 min, preferably 30 min.

In the step (5), the microspheres obtained in the step (4) are added into an LB culture medium according to the ratio of 1g/100 mL-10 g/100mL, preferably 8g/100mL, and stirred for 5-10 min at 15-35 ℃, preferably 5min at 20 ℃ to activate escherichia coli; and then adding 100-600 mg of betaine/8 g of microspheres, preferably 500mg of betaine/8 g of microspheres, stirring at 15-35 ℃ for 15-60 min, preferably at 20 ℃ for 30min, and taking out the microspheres to obtain the multistage pore adsorption gel coupling biocatalyst.

In the step (i), the multistage pore adsorption gel coupling biocatalyst with cell activity is prepared by placing the multistage pore adsorption gel coupling biocatalyst in an LB culture medium, culturing at 37 ℃ and 100-300 rpm for 3h to allow cells to propagate or grow in a small amount, and ensuring that the cells are not seriously damaged by a surfactant.

In the step (i), the contents of chloramphenicol and kanamycin in the fermentation medium containing chloramphenicol and kanamycin are 10-40 mg/L and 20-60 mg/L respectively, preferably 34mg/L and 50mg/L respectively; the concrete components are as follows: 30g/L glucose, 10g/L ammonium sulfate, 0.3g/L ferrous sulfate, 2g/L yeast powder, 5g/L peptone, 0.5g/L potassium chloride, 1.6g/L magnesium sulfate heptahydrate and 160 mg/L vitamin B; further comprising: 34mg/L chloramphenicol and 50mg/L kanamycin. The fermentation medium was adjusted to pH 7 using MOPOS buffer (100 mM).

In step (i), the culture is carried out at 37 ℃ and 200rpm for 2 h.

In the step (ii), the final concentration of the arabinose is 0.5-2.5 g/L, preferably 1 g/L; the culture is carried out at 37 ℃ and 200rpm for 1 h.

In the step (iii), the final concentration of the isopropyl-beta-D-thiogalactoside is 50-300 mg/L, preferably 100mg/L, and the induction expression is carried out for 12h at 25 ℃ and 200 rpm.

In step (iv), the further fermentation is to ferment the third fermentation liquid at 37 ℃ and 200rpm for 48 h.

In the step (iv), the multilevel pore canal adsorption gel coupling biocatalyst is taken out from the fermentation liquor containing the L-pipecolic acid, added into an LB culture medium according to the ratio of 1g/100 mL-10 g/100mL culture medium, and stirred for 5-10 min at 15-35 ℃; then adding 100-600 mg of betaine into the mixture, stirring the mixture for 15-60 min at 15-35 ℃, taking out microspheres to obtain the multistage pore adsorption gel coupling biocatalyst with cell activity, and reusing the catalyst; preferably, it is added to LB medium at a ratio of 8g/100mL medium and stirred at 20 ℃ for 5 min; then, 500mg of betaine was added thereto, and stirred at 20 ℃ for 30 min.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the system for producing the L-piperidine formic acid by using the immobilized whole cell catalysis is obtained by a synthetic method with lower material cost and simple and convenient operation and a strategy of simplifying production operation steps.

2. The method for constructing the immobilized whole-cell gel microspheres improves the catalytic activity of the cells in the catalytic process.

3. The combination of the metal-organic framework material and the gel microspheres is utilized, and the advantages of various pore sizes and structures are combined, so that the adsorption effect on the L-piperidinecarboxylic acid is effectively improved.

4. The combination has strong designability, can be used for carrying out the combination of pore sizes and the specific adsorption modification according to requirements, has good biocompatibility, and maintains better microbial tolerance and reutilization property.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a schematic diagram of a multistage pore adsorption gel coupled biocatalyst.

Detailed Description