CN112501221A - Method for improving conversion rate of threonine and saccharic acid - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a method for improving the conversion rate of threonine and sugar acid, which comprises the following steps: preparing a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 0-200mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, and V_B12mg/L，V_H50 mu g/L; inoculating the L-threonine production strain seed liquid into a fermentation tank containing a fermentation medium for fermentation for 36h, stopping fermentation, and collecting the fermentation liquid. The invention optimizes the fermentation process and improves the fermentation efficiency of threonine.

Description

Method for improving conversion rate of threonine and saccharic acid

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for improving the conversion rate of threonine and saccharic acid.

Background

Threonine, known by the scientific name 2-amino-3-hydroxybutyric acid. Threonine is an essential amino acid, and is mainly used in the fields of medicines, chemical reagents, food enhancers, feed additives and the like. Especially the amount of feed additives, which are often added to the feed of immature piglets and poultry, is the second limiting amino acid of the pig feed and the third limiting amino acid of the poultry feed, increases rapidly. The L-threonine is added into the compound feed, and has the following characteristics: the amino acid balance of the feed can be adjusted, and the growth of livestock is promoted; ② the meat quality can be improved; the nutritional value of the feed with low amino acid digestibility can be improved; fourthly, the cost of the feed raw materials can be reduced; and thus has been widely used in the feed industry in european union countries (mainly germany, belgium, denmark, etc.) and in american countries.

At present, three methods of threonine production are mainly fermentation, protein hydrolysis and chemical synthesis, and the microbial fermentation method has become the mainstream method of threonine production. The applicant is the largest amino acid biological fermentation enterprise in the world, wherein threonine is the main export amino acid. The improvement of the threonine yield and the sugar acid conversion rate can save the production cost and promote the industrial production of threonine, and is also the direction of continuous improvement and optimization of the applicant.

The optimized research of fermentation conditions in the industrial production of L-threonine, Luweining, journal of biology, 10 months 2010, takes high-yield L-threonine bacteria as an initial strain, and carries out a series of optimized researches on various fermentation conditions by combining actual industrial production conditions, and the results show that 0.2% of industrial-grade growth promoter (the growth promoter is independently researched and prepared by the company and mainly contains biotin for promoting growth and other beneficial nutritional ingredients) is added, complex sugar is used for replacing glucose as initial sugar, the concentration of the initial sugar is controlled to be 60g/L, dissolved oxygen is controlled to be 10% -20% in the fermentation process except for the growth peak period, the wet bacteria in a final fermentation tank is 45g/L, and the threonine content can reach about 110 g/L.

The inventor's prior patent technology ' CN109136299A, a method for preparing, extracting and purifying threonine ', realizes the coupling of strain culture and permeabilization treatment by adding triton X-100 in the later stage of fermentation and combining the change of temperature and pressure, can reduce the mass transfer limit of thalli cell walls and cell membranes to substrates and products under the condition of not needing subsequent permeabilization treatment on cultured cells, avoids the step of subsequent permeabilization treatment of cells and the investment of related equipment operation, and provides a simple method for improving the yield of threonine.

The patent technology 'CN 110904167A, L-threonine fermentation process optimization method', can produce certain inhibition effect on citrate dehydrogenase by feeding sodium citrate, properly weakens tricarboxylic acid cycle, can reduce the generation of byproducts and energy loss, and thus has positive significance for improving threonine yield; the glyoxylate cycle consumes a large amount of ATP and wastes carbon sources, and the isocitrate lyase is inhibited by adding the succinic acid in a flowing manner, so that the metabolic flow entering the glyoxylate cycle is reduced, and the threonine yield is increased. The pentose phosphate pathway also has a great influence on amino acid fermentation, and the prior art does not give relevant description on how to improve the synthesis efficiency of threonine fermented by the strain by enhancing the pentose phosphate pathway.

Disclosure of Invention

On the basis of the prior art, in order to improve the fermentation efficiency of threonine, the invention continuously optimizes the fermentation process and provides a method for improving the conversion rate of threonine to sugar acid.

The invention is realized by the following technical scheme:

a method for increasing the conversion rate of threonine sugar acid, which comprises the following steps:

step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate and monopotassium phosphate0.5g/L, 0.5g/L dipotassium phosphate, 0.1g/L magnesium sulfate heptahydrate, 0-200 mg/L4-hydroxy-3-methoxybenzoic acid, 10mg/L ferrous sulfate heptahydrate, 10mg/L manganese sulfate monohydrate, V_B1 2mg/L，V_H 50μg/L；

Step 2) fermentation: inoculating the seed liquid of the L-threonine producing strain into a fermentation tank containing a fermentation culture medium for fermentation, controlling the temperature at 36 ℃, the stirring speed at 300rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36h, and collecting the fermentation liquid.

Preferably, the first and second electrodes are formed of a metal,

the concentration of the 4-hydroxy-3-methoxybenzoic acid is 100 mg/L.

Preferably, the first and second electrodes are formed of a metal,

the concentration of the 4-hydroxy-3-methoxybenzoic acid is 75 mg/L.

Preferably, the first and second electrodes are formed of a metal,

the concentration of the 4-hydroxy-3-methoxybenzoic acid is 50 mg/L.

Preferably, the first and second electrodes are formed of a metal,

the fermentation also comprises the step of feeding the supplement liquid.

Preferably, the first and second electrodes are formed of a metal,

the step of feeding the feed liquid in a flowing manner comprises the following steps:

1) controlling the sugar content to be 3% by feeding 50% of sucrose solution until the fermentation is finished;

2) controlling the pH value to be 7.0 by adding 20% ammonia water until the fermentation is finished;

3) after fermentation is carried out for 20 hours, adding hydrogen peroxide into each liter of fermentation liquor at a flow rate of 2ml/h in a feeding flow manner until the fermentation is finished;

4) after fermentation for 20h, adding mixed aqueous solution of succinic acid and sodium citrate into the fermentation tank in a fed-batch manner at a flow rate of 15ml/h in each liter of fermentation liquor until the fermentation is finished; in the mixed aqueous solution, the concentrations of succinic acid and sodium citrate are both 50 g/L;

5) when the fermentation time is 30h, chitosan is added into the fermentation tank at one time, and the concentration of the chitosan is controlled to be 40 mg/L.

The beneficial effects of the invention mainly comprise the following aspects:

the pentose phosphate pathway can produce glucose-6-phosphate and ribose-5-phosphate, which is beneficial to providing sufficient glyceraldehyde triphosphate to enter the threonine synthesis pathway; and a large amount of NADPH can be provided for threonine synthesis, and the threonine synthesis efficiency is improved. The research discovers that the addition of 4-hydroxy-3-methoxybenzoic acid in a fermentation medium can activate various key enzymes in a pentose phosphate pathway, including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, so that the sugar acid conversion rate and the fermentation efficiency of threonine are improved; concentration gradient tests show that the influence of the concentration of 4-hydroxy-3-methoxybenzoic acid within 50mg/L on the sugar acid conversion rate and the fermentation efficiency of threonine is small, the concentration of 50-100mg/L can obviously improve the sugar acid conversion rate and the fermentation efficiency of threonine, the concentration of 4-hydroxy-3-methoxybenzoic acid is continuously increased, the sugar acid conversion rate and the fermentation efficiency of threonine are reduced, and the 4-hydroxy-3-methoxybenzoic acid with excessive concentration possibly has an inhibiting effect on other enzymes or precursor substances for threonine synthesis.

Drawings

FIG. 1: influence of 4-hydroxy-3-methoxybenzoic acid on threonine content in the fermentation broth;

FIG. 2: influence of 4-hydroxy-3-methoxybenzoic acid on the conversion of sugar and acid.

Detailed Description

Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.

Example 1

A method for increasing the conversion rate of threonine sugar acid, which comprises the following steps:

step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 100mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, V_B1 2mg/L，V_H 50μg/L。

Step 2) fermentation: inoculating seed solution of L-threonine producing strain (such as Escherichia coli engineering bacteria TRFC) into fermentation tank containing fermentation medium at inoculation amount of 1.5%, fermenting, and inoculating with inoculation density OD₆₀₀At the temperature of 0.4 ℃, the stirring speed of 300rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36 hours, and collecting fermentation liquor;

in the fermentation process, a feed liquid needs to be fed in a flowing mode, and the method specifically comprises the following steps:

1) controlling the sugar content to be 3% by adding 50% of sucrose solution (50 g of sucrose is dissolved in water until 100ml is a 50% solution) until the fermentation is finished;

2) controlling the pH value to be 7.0 by adding 20% ammonia water until the fermentation is finished;

3) after fermentation is carried out for about 20 hours, adding hydrogen peroxide into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor until the fermentation is finished;

4) after fermentation is carried out for about 20 hours, adding mixed aqueous solution of succinic acid and sodium citrate into the fermentation tank in a feeding flow manner at a flow rate of 15ml/h in each liter of fermentation liquor until the fermentation is finished; in the mixed aqueous solution, the concentrations of succinic acid and sodium citrate are both 50 g/L;

5) after fermenting for about 30h, chitosan is added into the fermentation tank at one time, and the concentration of the chitosan is controlled to be 40 mg/L.

Example 2

A method for increasing the conversion rate of threonine sugar acid, which comprises the following steps:

step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 75mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate,manganese sulfate monohydrate 10mg/L, V_B1 2mg/L，V_H 50μg/L。

Step 2) fermentation: inoculating seed solution of L-threonine producing strain (such as Escherichia coli engineering bacteria TRFC) into fermentation tank containing fermentation medium at inoculation amount of 1.5%, fermenting, and inoculating with inoculation density OD₆₀₀At the temperature of 0.4 ℃, the stirring speed of 300rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36 hours, and collecting fermentation liquor;

in the fermentation process, a feed liquid needs to be fed in a flowing mode, and the method specifically comprises the following steps:

1) controlling the sugar content to be 3% by adding 50% of sucrose solution (50 g of sucrose is dissolved in water until 100ml is a 50% solution) until the fermentation is finished;

2) controlling the pH value to be 7.0 by adding 20% ammonia water until the fermentation is finished;

3) after fermentation is carried out for about 20 hours, adding hydrogen peroxide into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor until the fermentation is finished;

4) after fermentation is carried out for about 20 hours, adding mixed aqueous solution of succinic acid and sodium citrate into the fermentation tank in a feeding flow manner at a flow rate of 15ml/h in each liter of fermentation liquor until the fermentation is finished; in the mixed aqueous solution, the concentrations of succinic acid and sodium citrate are both 50 g/L;

5) after fermenting for about 30h, chitosan is added into the fermentation tank at one time, and the concentration of the chitosan is controlled to be 40 mg/L.

COMPARATIVE EXAMPLE 1 (CN 110904167A EXAMPLE 1)

A method for increasing the conversion rate of threonine sugar acid, which comprises the following steps:

step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, V_B1 2mg/L，V_H 50μg/L。

Step 2) fermentation: inoculating seed solution of L-threonine producing strain (e.g. Escherichia coli-engineered bacterium TRFC) into fermentation medium containing 1.5% of the strainFermenting in a fermenter, inoculating with OD₆₀₀At the temperature of 36 ℃, the stirring speed of 300-500rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36 hours, and collecting fermentation liquor;

in the fermentation process, a feed liquid needs to be fed in a flowing mode, and the method specifically comprises the following steps:

1) controlling the sugar content to be 3% by adding 50% of sucrose solution (50 g of sucrose is dissolved in water until 100ml is a 50% solution) until the fermentation is finished;

2) controlling the pH value to be 7.0 by adding 20% ammonia water until the fermentation is finished;

3) after fermentation is carried out for about 20 hours, adding hydrogen peroxide into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor until the fermentation is finished;

4) after fermentation is carried out for about 20 hours, adding mixed aqueous solution of succinic acid and sodium citrate into the fermentation tank in a feeding flow manner at a flow rate of 15ml/h in each liter of fermentation liquor until the fermentation is finished; in the mixed aqueous solution, the concentrations of succinic acid and sodium citrate are both 50 g/L;

5) after fermenting for about 30h, chitosan is added into the fermentation tank at one time, and the concentration of the chitosan is controlled to be 40 mg/L.

Example 3

Influence of 4-hydroxy-3-methoxybenzoic acid on threonine content and sugar acid conversion rate in fermentation broth.

On the basis of comparative example 1, the influence of 4-hydroxy-3-methoxybenzoic acid on threonine fermentation was verified, and the amount of 4-hydroxy-3-methoxybenzoic acid added to the fermentation medium was set as follows: 0, 5, 25, 50, 75,100, 125,150,200 in mg/L, as shown in FIG. 1, when the concentration is low, 0-50mg/L, the yield of threonine is slightly improved along with the addition of 4-hydroxy-3-methoxybenzoic acid, when the concentration is increased to 75mg/L, the content of 4-hydroxy-3-methoxybenzoic acid is obviously increased, 4-hydroxy-3-methoxybenzoic acid is continuously increased to 100mg/L, 4-hydroxy-3-methoxybenzoic acid is still slightly improved, when the addition of 4-hydroxy-3-methoxybenzoic acid is 125mg/L, the addition is reduced by about 3% rather than 100mg/L, when the addition of 4-hydroxy-3-methoxybenzoic acid is 150 mg/L, the apparent decrease in threonine production may be due to the inhibitory effect of excessive concentrations of 4-hydroxy-3-methoxybenzoic acid on other enzymes or precursors of threonine synthesis. As shown in FIG. 2, the trends of the sugar acid conversion rate and the threonine production curve are almost consistent, indicating that 4-hydroxy-3-methoxybenzoic acid increases the fermentation yield of threonine mainly by increasing the sugar acid conversion rate.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (6)

1. A method for increasing the conversion rate of threonine sugar acid, which comprises the following steps:

step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 0-200mg/L of 4-hydroxy-3-methoxybenzoic acid, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, and V_B1 2mg/L，V_H 50μg/L；

Step 2) fermentation: inoculating the seed liquid of the L-threonine producing strain into a fermentation tank containing a fermentation culture medium for fermentation, controlling the temperature at 36 ℃, the stirring speed at 300rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36h, and collecting the fermentation liquid.

2. The method of claim 1, wherein the concentration of 4-hydroxy-3-methoxybenzoic acid is 100 mg/L.

3. The method of claim 1, wherein the concentration of 4-hydroxy-3-methoxybenzoic acid is 75 mg/L.

4. The method of claim 1, wherein the concentration of 4-hydroxy-3-methoxybenzoic acid is 50 mg/L.

5. The method of claim 1, wherein the fermentation further comprises the step of feeding a feed solution.

6. The method of claim 5, wherein the step of feeding a make-up liquid comprises:

1) controlling the sugar content to be 3% by feeding 50% of sucrose solution until the fermentation is finished;

2) controlling the pH value to be 7.0 by adding 20% ammonia water until the fermentation is finished;

3) after fermentation is carried out for 20 hours, adding hydrogen peroxide into each liter of fermentation liquor at a flow rate of 2ml/h in a feeding flow manner until the fermentation is finished;

4) after fermentation for 20h, adding mixed aqueous solution of succinic acid and sodium citrate into the fermentation tank in a fed-batch manner at a flow rate of 15ml/h in each liter of fermentation liquor until the fermentation is finished; in the mixed aqueous solution, the concentrations of succinic acid and sodium citrate are both 50 g/L;

5) when the fermentation time is 30h, chitosan is added into the fermentation tank at one time, and the concentration of the chitosan is controlled to be 40 mg/L.

Images