CN104293749B - Method for preparing high-yield leucine aminopeptidase through fermentation of recombinant bacillus subtilis - Google Patents

Abstract

The invention discloses a method for preparing high-yield leucine aminopeptidase by recombinant Bacillus subtilis fermentation, which belongs to the technical field of enzyme preparation and fermentation. The present invention optimizes the combination of medium optimization and fermentation process control to obtain a suitable method for culturing recombinant Bacillus subtilis; after the recombinant Bacillus subtilis is inserted into the seed medium, the shaker is cultivated to the logarithmic growth phase, and a certain amount of seed liquid is inoculated Into the adjusted fermentation medium, use a fermenter to carry out fermentation culture, and take samples every 3 hours to measure parameters such as enzyme activity. The present invention adopts the strategy of combinatorial optimization to optimize the conditions for the fermentation of recombinant Bacillus subtilis to produce leucine aminopeptidase, and optimize the combination of fermentation medium and fermentation culture conditions, so that the recombinant Bacillus subtilis can ferment and produce leucine aminopeptidase The ability of the enzyme has been greatly improved, and the enzyme activity of leucine aminopeptidase reaches 605 U/mL.

Description

A method for preparing high-yield leucine aminopeptidase by fermentation of recombinant Bacillus subtilis

technical field

The invention relates to a method for preparing high-yield leucine aminopeptidase by fermentation of recombinant Bacillus subtilis, belonging to the technical fields of enzyme preparation and fermentation.

Background technique

Aminopeptidases are a class of exopeptidases that selectively cleave amino acid residues from the N-terminus of proteins or peptide chains, and have a wide range of actions. According to the cleavage characteristics of aminopeptidase, the enzymatic hydrolysis products of aminopeptidase are small peptides and free amino acids. Therefore, aminopeptidase has very important industrial application value. It can effectively and environmentally friendly hydrolyze proteins. Aminopeptidase has a wide range of applications and can help cut off hydrophobic amino acids at the end of the peptide chain and remove protein hydrolysates. bitter taste; compound with different endonucleases, participate in deep hydrolysis of proteins; prepare multifunctional active peptides; can also be used as N-terminal sequencing tool enzymes and enzymes for medical diagnosis. Tools for protein sequence determination can use leucyl aminopeptidase as a fixative for fusion products or recombinant proteins. Aminopeptidase is found in medical research, it is helpful to the treatment of many diseases, and can be used to study many physiological phenomena from the molecular level. Prolypeptidase can be used to remove neurotoxic chemical poisons and organophosphorus pesticides, on the one hand as an antidote, and on the other hand as a disinfectant for the environment.

Microbial fermentation culture optimization strategies often start from the optimization of the medium and the control of the fermentation process. At present, it is not possible to infer and calculate the medium formulation and fermentation process control strategy suitable for a certain microorganism from the basic principles of biochemical reactions. Different microorganisms are suitable for different culture media and fermentation process control strategies. There is no single culture method suitable for the fermentation and cultivation of a variety of microorganisms. Therefore, small fermentation equipment such as shake flasks and glass tanks are required for a specific microorganism. According to a certain experimental design and test method, the most suitable culture method can be selected by combining and optimizing the fermentation strategies such as medium composition and fermentation process control.

Aminopeptidase has important research value and application value in food, industry, medicine, etc., but the current fermentation production of aminopeptidase has the problem of low yield, and how to increase the yield has become an urgent problem to be solved.

Contents of the invention

The purpose of the present invention is to provide a combined optimization strategy for the preparation of high-yield leucine aminopeptidase by recombinant Bacillus subtilis fermentation, so as to solve the problems of low yield and difficult industrial application in the existing fermentation process.

The technical solution of the present invention is a method for preparing a high-yield leucine aminopeptidase by recombinant Bacillus subtilis fermentation, the steps of which are: using recombinant Bacillus subtilis WB600 as the starting strain, optimizing the combination of medium optimization and fermentation process control, Obtain a method suitable for the cultivation of recombinant Bacillus subtilis; after the recombinant Bacillus subtilis is inserted into the seed medium, the shaker is cultivated to the logarithmic growth phase, and a certain amount of seed liquid is inoculated into the adjusted fermentation medium, and the fermenter is used to carry out For fermentation culture, samples were taken every 3 hours to determine parameters such as enzyme activity.

Optimization of fermentation medium:

(1) Preparation of seed liquid: Recombinant Bacillus subtilis strain Bacillus subtilis preserved in a glycerol tube at -40°C Insert WB600 into the seed culture medium, use a rotary constant temperature shaker for cultivation, control the rotation speed of the shaker at 220rpm, cultivate at a temperature of 37°C, and cultivate for 10 hours to obtain a seed liquid;

(2) Shaking table fermentation culture: Add the enzyme production accelerator to the fermentation medium, and then inoculate the seed liquid into the above fermentation medium at an amount of 5%, and use a rotary constant temperature shaker for cultivation, and control the speed of the shaker 220rpm, culture temperature is 37°C, culture 36h;

The fermentation medium is: 6 g/L of soluble starch, 20 g/L of soybean meal, 18 g/L of yeast extract g/L, glycerin 2 mL/L, dipotassium hydrogen phosphate 6 g/L, cobalt chloride 0.6 mmol/L, prepared with deionized water;

The enzyme-producing accelerator includes osmotic pressure regulators, organic acid salts, and surfactants;

(3) After centrifuging the obtained fermentation broth, take the supernatant, measure the enzyme activity, and determine the type and dosage of the enzyme production accelerator;

The enzyme production accelerator includes osmotic pressure regulators (such as potassium chloride), surfactants (such as Tween 80), organic acid salts (such as sodium glutamate, potassium tartrate) and the like.

The specific steps of the fermenter fermentation process are:

(1) Preparation of seed liquid: Recombinant Bacillus subtilis strain Bacillus subtilis preserved in a glycerol tube at -40°C Insert WB600 into the seed culture medium, use a rotary constant temperature shaker for cultivation, control the rotation speed of the shaker at 220rpm, cultivate at a temperature of 37°C, and cultivate for 10 hours to obtain a seed solution;

Described seed medium is sodium chloride 10g/L, tryptone 10g/L, yeast extract 5g/L;

(2) Fermentation culture: Inoculate 5% of the cultivated seed liquid into a 7L fermenter, the fermenter has a liquid volume of 4L, inject sterile air, the stirring speed is 250-450rpm, and the ventilation ratio is 1.2: 1. Couple the dissolved oxygen with the stirring speed, keep the minimum dissolved oxygen at 30%, and ferment for 30 hours;

The fermentation culture temperature in the first 12 hours of the fermentation culture process was 37°C, and the fermentation culture temperature was adjusted to 30°C after 12 hours; when the fermentation culture reached 15 hours, 500g/L of glucose feed solution was added at one time to make the concentration of reducing sugar in the fermentation liquid 5g/L;

The fermentation medium is 20 g/L of soluble starch, 20 g/L of soybean meal, and 18 g/L of yeast extract. g/L, glycerin 2 mL/L, dipotassium hydrogen phosphate 6 g/L, cobalt chloride 0.6 mmol/L, potassium chloride 0.1 mol/L, sodium glutamate 0.6%, potassium tartrate 0.7%, Tween- 80 2.4 mL/L, prepared with deionized water;

(3) Parameter measurement: During the fermentation process, samples were taken every 3 hours to measure the activity and biomass of leucine aminopeptidase.

Leucine aminopeptidase enzyme activity assay method is the use of ultraviolet spectrophotometry (LNA method):

To 2mL 50mmol/L Tris-HCl buffer solution with pH 8.5, add 1mL of enzyme solution diluted by a certain factor, then add 1mL of L-leucine-p-nitroaniline, mix well, and react in a water bath at 50°C for 10min. Absorbance was measured at 405 nm. The calculation formula is:

In the formula:

X—Enzyme activity of the sample in U/mL;

N—absorbance value measured at a wavelength of 405 nm;

Y—the dilution factor of the crude enzyme solution.

Biological material sample: Bacteria: Recombinant Bacillus subtilis (contains PMA5 plasmid connected with the complete sequence of aminopeptidase gene from Bacillus subtilis Zj016, host bacteria Bacillus subtilis WB600), see Chinese patent CN102492645A for details.

Beneficial effects of the present invention: the present invention adopts the strategy of combinatorial optimization to optimize the conditions for the fermentation of recombinant Bacillus subtilis to produce aminopeptidase, and optimize the combination of fermentation medium and fermentation culture conditions, so that the recombinant Bacillus subtilis can ferment and produce leucine The ability of leucine aminopeptidase has been greatly improved, and the enzyme activity of leucine aminopeptidase has reached 605 U/mL.

Description of drawings

Fig. 1 Potassium chloride is on the influence of producing enzyme and biomass;

The influence of Fig. 2 Tween-80 on producing enzyme and biomass;

The influence of Fig. 3 potassium tartrate on producing enzyme and biomass;

The influence of Fig. 4 L-sodium glutamate on producing enzyme and biomass;

Fig. 5 initial sugar concentration is 20 g/L fermentation;

Fig. 6 variable temperature fermentation;

Figure 7 Fed-fed fermentation.

detailed description

Example 1

Bacterial species: Recombinant Bacillus subtilis (containing the PMA5 plasmid connected with the complete sequence of the aminopeptidase gene from Bacillus subtilis Zj016, and the host strain Bacillus subtilis WB600).

Seed medium: sodium chloride 10 g/L, tryptone 10 g/L, yeast extract 5 g/L.

Seed culture: Inoculate the recombinant Bacillus subtilis strains stored in glycerol tubes at -40°C into the seed medium, and cultivate them on a rotary constant temperature shaker with the speed of the shaker controlled at 220 rpm and the culture temperature at 37°C for 10 hours.

Add enzyme production promoters (osmotic pressure regulators, surfactants, organic acid salts) in a certain amount to the unadjusted fermentation medium (soluble starch 6 g/L, soybean meal 20 g/L, yeast extract 18 g/L, glycerin 2mL/L, dipotassium hydrogen phosphate 6 g/L, cobalt chloride 0.6 mmol/L, potassium chloride 0.1 mmol/L), after inoculating 5% seed solution, use a rotary Cultivation was carried out on a constant temperature shaking table, the rotation speed of the shaking table was controlled at 220 rpm, the cultivation temperature was 37° C., and the cultivation was carried out for 36 hours. After the cultivation, the fermentation broth was centrifuged to obtain the supernatant to measure the activity of leucine aminopeptidase.

The results show that: Potassium chloride in the osmotic pressure regulator has a greater promotion effect on the production of enzymes, and the enzyme activity reaches 110U/mL, which is an increase of 40U per milliliter compared with the control; Tween 80 in the surfactant has a greater effect on the production of enzymes. The enzyme activity reached 152U/mL, an increase of 82U per milliliter compared with the control; sodium glutamate and potassium tartrate in organic acid salts had a greater promotion effect on enzyme production, and the enzyme activity reached 122U/mL and 157U respectively /mL, relative to the control per milliliter increased by 52U, 87U.

The effect of potassium chloride on enzyme production and biomass is shown in Figure 1, the effect of Tween-80 on enzyme production and biomass is shown in Figure 2, and the effect of potassium tartrate on enzyme production and biomass is shown in Figure 3 , the effect of L-sodium glutamate on enzyme production and biomass is shown in Figure 4.

Four factors and three levels of orthogonal experiments were carried out on the four kinds of enzyme production accelerators that can promote enzyme production, and the results are shown in Table 1.

Determine the dosage and combination of 4 kinds of enzyme-producing accelerators as potassium chloride 0.1 mol/L, sodium glutamate 0.6%, potassium tartrate 0.7%, Tween-80 2.4 mL/L. Under this combination, the enzyme activity of the fermentation broth reached 207.6 U/mL.

Table 1 Orthogonal experiment design and results

Example 2

Bacterial classification, seed culture condition and seed culture medium are with embodiment 1.

Fermentation medium: soluble starch 20 g/L, soybean meal 20 g/L, yeast extract 18 g/L, glycerin 2mL/L, dipotassium hydrogen phosphate 6 g/L, cobalt chloride 0.6 mmol/L, potassium chloride 0.1 mol/L, sodium glutamate 0.6%, potassium tartrate 0.7%, Tween-80 2.4 mL/L.

(1) Effect of initial sugar concentration on fermentation culture

Cultivation method: Inoculate 5% of the cultivated seed culture solution into a 7L fermenter for fermentation (the liquid volume is 4L), the fermentation temperature is 37°C, and sterile air is introduced during the fermentation process, and the ventilation rate is 1.2:1, the minimum dissolved oxygen is maintained at 30%, the dissolved oxygen is coupled with the rotation speed, the stirring speed is controlled at 250-450rpm, and the fermentation and cultivation time is 30h. During the fermentation process, samples were taken every 3 hours, and parameters such as leucine aminopeptidase activity and biomass were measured.

The results showed that adjusting the concentration of soluble starch in the fermentation medium from 6 g/L to 20 After 1 g/L, the activity of leucine aminopeptidase reached 205 U/mL.

(2) Effect of temperature control in stages on fermentation culture

Culture method: Except for temperature control, other fermentation culture conditions are the same as in Example 2 (1), only the fermentation temperature is changed, and the fermentation temperature is maintained at 37°C during 0-12 hours of the fermentation process. When the fermentation progresses to 12 hours, gradually reduce The temperature of the fermentation culture was adjusted to 30° C. and maintained until the end of the fermentation. During the fermentation process, samples were taken every 3 hours to measure the activity of leucine aminopeptidase, biomass and other parameters.

The results show that the temperature control of the fermentation culture in stages can improve the enzyme production capacity of the recombinant Bacillus subtilis, and the enzyme activity of the fermentation can reach 567 U/mL.

(3) Effect of feed on fermentation culture

Cultivation method: The cultivation method is the same as that in Example 2 (2). When the fermentation progresses to 15 hours, 500 g/L of glucose feed solution is added to the fermenter at one time, so that the concentration of reducing sugar in the fermentation liquid is 5 g/L. During the fermentation process, samples were taken every 3 hours, and parameters such as leucine aminopeptidase activity and biomass were measured.

The results showed that feeding during the fermentation process made the enzyme activity of the leucine aminopeptidase reach 605 U/mL, and the fermentation period was shortened by 6 hours.

Claims (1)

1. A method for preparing high-yield leucine aminopeptidase by recombinant bacillus subtilis fermentation, characterized in that the specific steps of the fermenter fermentation process are: (1) Preparation of seed liquid: insert recombinant Bacillus subtilis WB600 stored in a glycerol tube at -40°C into the seed medium, and use a rotary constant temperature shaker for cultivation. The rotation speed of the shaker is controlled at 220rpm. The temperature is 37°C, cultured for 10 hours, and the seed liquid is obtained; Described seed medium is sodium chloride 10g/L, tryptone 10g/L, yeast extract 5g/L; Recombinant Bacillus subtilis, containing the PMA5 plasmid connected with the complete sequence of the aminopeptidase gene from Bacillus subtilis Zj016, host bacteria Bacillus subtilis WB600; (2) Fermentation culture: inoculate 5% of the cultivated seed liquid into a 7L fermenter, the liquid volume of the fermenter is 4L, feed sterile air, the stirring speed is 250-450rpm, and the ventilation ratio is 1.2: 1. Couple the dissolved oxygen with the stirring speed, keep the minimum dissolved oxygen at 30%, and ferment for 30 hours; The fermentation culture temperature in the first 12 hours of the fermentation culture process was 37°C, and the fermentation culture temperature was adjusted to 30°C after 12 hours; when the fermentation culture reached 15 hours, 500g/L of glucose feed solution was added at one time to make the concentration of reducing sugar in the fermentation liquid 5g/L; The fermentation medium is 20 g/L of soluble starch, 20 g/L of soybean meal, 18 g/L of yeast extract, 2 mL/L of glycerol, 6 g/L of dipotassium hydrogen phosphate, 0.6 mmol/L of cobalt chloride, and Prepared with deionized water; enzyme production accelerators: potassium chloride 0.1 mol/L, sodium glutamate 0.6%, potassium tartrate 0.7%, Tween-80 2.4 mL/L; (3) Parameter measurement: During the fermentation process, samples were taken every 3 hours to measure the activity and biomass of leucine aminopeptidase.