阅读说明：本技术 一种制备异烟酸的方法 (Method for preparing isonicotinic acid ) 是由 龙华 李自云 刘清华 许正宏 史劲松 龚劲松 于 2019-12-02 设计创作，主要内容包括：本发明涉及生化技术领域,尤其是一种制备异烟酸的方法。本发明使用Gibberella属、Fusarium属来源丝状真菌中的任一种作为菌种,经过发酵、水解、膜过滤、脱色、结晶、离心、干燥、包装的过程制得异烟酸。本发明的一种制备异烟酸的方法具采用微生物发酵生产异烟酸,具有反应条件温和、选择性好、流程简单、产物产率和纯度高、成本低、环境污染小等优点,而且对部分废水进行了回收利用,增加了附加值,特别适合用于大规模工业化生产。(The invention relates to the technical field of biochemistry, in particular to a method for preparing isonicotinic acid. The invention uses any one of filamentous fungi of Gibberella and Fusarium as a strain to prepare the isonicotinic acid through the processes of fermentation, hydrolysis, membrane filtration, decoloration, crystallization, centrifugation, drying and packaging. The method for preparing the isonicotinic acid has the advantages of adopting microbial fermentation to produce the isonicotinic acid, having mild reaction conditions, good selectivity, simple process, high product yield and purity, low cost, small environmental pollution and the like, recycling part of waste water, increasing the additional value and being particularly suitable for large-scale industrial production.)

1. A method for preparing isonicotinic acid, which is characterized by comprising the following steps: the method comprises the following steps:

(1) fermentation: adding a culture medium which is 40-90% of the volume of the fermentation tank into the fermentation tank, wherein the culture medium mainly comprises the following raw materials in percentage by mass: 0.1-5% of corn steep liquor, 0.1-5% of soybean cake powder, 0.1-2% of NaCl and 0.1-2% of KH₂PO₄、0.1-2%K₂HPO₄·3H₂Sterilizing O, 0.1-2% urea and 1-4% glucose, inoculating 1-10% strain, culturing for 20-80 hr while continuously introducing sterile air, controlling temperature at 20-50 deg.C during fermentation, adjusting pH to 6-9 by adding dilute hydrochloric acid or ammonia water, and adding glucose at later stage;

(2) hydrolysis: adding fermentation liquor after fermentation culture into a reaction kettle, controlling the reaction temperature to be 20-60 ℃, slowly adding 4-cyanopyridine to perform hydrolysis reaction, and tracking the residual amount of the 4-cyanopyridine by using liquid chromatography;

(3) membrane filtration: introducing the solution after the hydrolysis reaction into microfiltration equipment for filtering to obtain filtrate and filter residue, wherein the filtrate is an isonicotinic acid ammonium solution, and the filter residue is waste cells and trace 4-cyanopyridine after the reaction;

(4) and (3) decoloring: adding activated carbon into the filtrate obtained in the step (3), stirring for 1 hour, and filtering by using an activated carbon filter;

(5) and (3) crystallization: adding the filtrate obtained in the step (4) into a crystallization kettle with a stirring mechanism, stirring, slowly adding 31% hydrochloric acid until the pH value is 2-5, controlling the temperature of the crystallization kettle at 20-70 ℃ during the period, and reacting to generate white precipitate, namely isonicotinic acid crystals;

(6) centrifuging, drying and packaging: and (4) putting the mixed liquid produced in the step (5) into a centrifugal machine for centrifugation and washing to obtain mother liquid and isonicotinic acid crystals, drying the isonicotinic acid crystals in a fluidized bed until the moisture content is lower than 0.5%, and packaging.

2. The method of claim 1, wherein the ratio of: the strain used in the fermentation in step (1) may be selected from any one of filamentous fungi of Gibberella and Fusarium.

3. The method of claim 2, wherein: the fermentation pretreatment process of the enzyme producing strain Gibberella comprises the following steps: activating the slant strain Gibberellaintermedia after long-term domestication and breeding, inoculating 500 mu L of bacterial liquid into a 500mL conical flask containing 50mL of seed culture medium, performing shake culture on a reciprocating shaking table at 30 ℃ and 180r/min for 36h, and diluting the bacterial liquid by 10^-4、10^-5、10^-6Then coating the strain on a solid screening culture medium, culturing in a constant temperature incubator at 30 ℃, selecting a single bacterial colony in a screening plate, transferring the single bacterial colony into a fermentation culture medium, culturing for 60 hours, measuring the enzyme activity, and storing the strain with high activity in a glycerin pipe or a slant culture medium.

4. The method of claim 3, wherein: the formula of the shake flask stage fermentation medium comprises: 0.75% of yeast powder, 1% of glucose, 0.116% of NaCl0 and KH₂PO₄0.272%, urea 0.3%, pH7.0.

5. The method of claim 2, wherein: the fermentation pretreatment process of the zymogenic strain Fusarium comprises the following steps: activating the slant strain Fusarium oxysporum bred by long-term domestication, inoculating 500 μ L of the bacterial liquid into a 500mL conical flask containing 50mL of seed culture medium, performing shake culture on a reciprocating shaking table at 30 ℃ and 120r/min for 36h, and diluting the bacterial liquid by 10^-4、10^-5、10^-6Then coating the strain on a solid screening culture medium, culturing in a constant temperature incubator at 30 ℃, selecting a single bacterial colony in a screening plate, transferring the single bacterial colony into a fermentation culture medium, culturing for 60 hours, measuring the enzyme activity, and storing the strain with high activity in a glycerin pipe or a slant culture medium.

6. The method of claim 5, wherein: the formula of the shake flask stage fermentation medium comprises: 0.5% of yeast powder, 1.5% of glucose, 0.1% of NaCl0 and KH₂PO₄0.272%, urea 0.3%, pH7.0.

7. The method of claim 1, wherein the ratio of: and (3) recovering the mother liquor generated in the step (6) by evaporation crystallization, wherein the main component of the mother liquor is ammonium chloride.

Technical Field

The invention relates to the technical field of biochemistry, in particular to a method for preparing isonicotinic acid.

Background

Isonicotinic acid, also known as 4-picolinic acid, participates in the oxidation-reduction process in the organism, is an important intermediate for synthesizing the anti-tuberculosis drug ramification (isoniazid), can also be used as an anti-corrosion agent, an electroplating additive, a photosensitive resin stabilizer, a polyvinyl chloride heat stabilizer, a nonferrous metal flotation reagent and the like, and is used as a precursor for synthesizing the plant growth regulator trinexamine in agriculture. Has wide market prospect.

There are many methods for synthesizing isonicotinic acid, which are mainly classified into oxidation, hydrolysis, biocatalysis, and the like. The oxidation method is classified into a potassium permanganate oxidation method, a nitric acid oxidation method, a catalytic air oxidation method, an ozone oxidation method and other traditional chemical oxidation methods and electrochemical oxidation methods.

The isonicotinic acid synthesized by the traditional chemical oxidation methods such as a potassium permanganate oxidation method, a nitric acid oxidation method, a catalytic air oxidation method, an ozone oxidation method and the like generally has the problems of large environmental pollution, complex process, easy deep oxidation and the like.

The isonicotinic acid synthesized by the electrochemical oxidation method can be synthesized under the condition of being treated at normal temperature and normal pressure without a catalyst, has the advantages of good reaction selectivity, low investment, low cost, good product quality and the like, but has low yield due to the problems of the electrode and the diaphragm.

The hydrolysis method for synthesizing isonicotinic acid is a method generally applied in industry. Hydrolyzing 4-cyanopyridine under high temperature and high pressure in alkaline condition to obtain 4-picolinate, and adding acid for reaction to obtain isonicotinic acid. The process needs high temperature and high pressure, and has the disadvantages of low product yield, environmental pollution and the like.

Compared with a Chemical method, the microbial catalysis method for producing the isonicotinic acid has the advantages of high selectivity, high efficiency, low cost, mild reaction conditions, small Environmental pollution and the like, the existing biocatalysis method for producing the isonicotinic acid is still less reported, the strains are Nocardia globerula and Pseudomonas putida (Applied and Environmental Microbiology,54: 1030-. The biological method has good economic and environmental protection values and lays a foundation for the large-scale production of the isonicotinic acid.

Disclosure of Invention

The invention aims to provide a method for preparing isonicotinic acid, overcomes the defects of the prior art, and has the advantages of mild reaction conditions, good selectivity, simple process, high product yield and purity, low cost, small environmental pollution and the like.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for preparing isonicotinic acid, comprising the steps of:

(1) fermentation: adding a culture medium which is 40-90% of the volume of the fermentation tank into the fermentation tank, wherein the culture medium mainly comprises the following raw materials in percentage by mass: 0.1-5% of corn steep liquor, 0.1-5% of soybean cake powder, 0.1-2% of NaCl and 0.1-2% of KH₂PO₄、0.1-2%K₂HPO₄·3H₂Sterilizing O, 0.1-2% urea and 1-4% glucose, inoculating 1-10% strain, culturing for 20-80 hr while continuously introducing sterile air, controlling temperature at 20-50 deg.C during fermentation, adjusting pH to 6-9 by adding dilute hydrochloric acid or ammonia water, and adding glucose at later stage;

(2) hydrolysis: adding fermentation liquor after fermentation culture into a reaction kettle, controlling the reaction temperature to be 20-60 ℃, slowly adding 4-cyanopyridine to perform hydrolysis reaction, and tracking the residual amount of the 4-cyanopyridine by using liquid chromatography;

(3) membrane filtration: introducing the solution after the hydrolysis reaction into microfiltration equipment for filtering to obtain filtrate and filter residue, wherein the filtrate is an isonicotinic acid ammonium solution, and the filter residue is waste cells and trace 4-cyanopyridine after the reaction;

(4) and (3) decoloring: adding activated carbon into the filtrate obtained in the step (3), stirring for 1 hour, and filtering by using an activated carbon filter;

(5) and (3) crystallization: adding the filtrate obtained in the step (4) into a crystallization kettle with a stirring mechanism, stirring, slowly adding 31% hydrochloric acid until the pH value is 2-5, controlling the temperature of the crystallization kettle at 20-70 ℃ during the period, and reacting to generate white precipitate, namely isonicotinic acid crystals;

(6) centrifuging, drying and packaging: and (4) putting the mixed liquid produced in the step (5) into a centrifugal machine for centrifugation and washing to obtain mother liquid and isonicotinic acid crystals, drying the isonicotinic acid crystals in a fluidized bed until the moisture content is lower than 0.5%, and packaging.

Preferably, the strain used in the fermentation in step (1) may be selected from any one of filamentous fungi of genus Gibberella and genus Fusarium.

Preferably, the fermentation pretreatment process of the enzyme producing strain Gibberella comprises the following steps: activating the slant strain Gibberella aintermedia bred by long-term domestication, inoculating 500 mu L of bacterial liquid into a 500mL conical flask containing 50mL of seed culture medium, performing shake culture on a reciprocating shaking table at 30 ℃ and 180r/min for 36h, diluting the bacterial liquid by 10-4, 10-5 and 10-6, coating the bacterial liquid on a solid screening culture medium, culturing in a 30 ℃ constant temperature incubator, selecting a single bacterial colony in a screening plate, transferring the bacterial colony into a fermentation culture medium, culturing for 60h, measuring the enzymatic activity, and storing the bacterial strain with high activity in a glycerinum tube or a slant culture medium.

Preferably, the shake flask stage fermentation medium formula consists of: 0.75% of yeast powder, 1% of glucose, 0.116% of NaCl0, KH2PO40.272% of urea and pH 7.0.

Preferably, the pretreatment process for fermentation of the enzyme-producing strain Fusarium comprises the following steps: activating a slant strain Fusarium oxysporum bred by long-term domestication, inoculating 500 mu L of bacterial liquid into a 500mL conical flask containing 50mL of seed culture medium, performing shake culture on a reciprocating shaking table at 30 ℃ and 120r/min for 36h, diluting the bacterial liquid by 10-4, 10-5 and 10-6, coating the diluted bacterial liquid on a solid screening culture medium, culturing in a 30 ℃ constant temperature incubator, selecting a single bacterial colony in a screening plate, transferring the single bacterial colony into a fermentation culture medium, culturing for 60h, measuring the enzymatic activity, and storing the strain with high activity in a glycerinum tube or a slant culture medium.

Preferably, the shake flask stage fermentation medium formula consists of: 0.5% of yeast powder, 1.5% of glucose, 0.1% of NaCl0, KH2PO40.272%, 0.3% of urea and pH 7.0.

Preferably, the mother liquor produced in the step (6) mainly contains ammonium chloride, and is recovered by evaporation crystallization.

The invention has the beneficial effects that: compared with the prior art, the method for preparing the isonicotinic acid has the following advantages: the invention adopts the microbial fermentation to produce the isonicotinic acid, has the advantages of mild reaction conditions, good selectivity, simple process, high product yield and purity, low cost, small environmental pollution and the like, recycles part of wastewater, increases the additional value, and is particularly suitable for large-scale industrial production.

Detailed Description