JP2017112847A - Method for producing protease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which a nitrogen source in a culture medium can be efficiently used, and protease can be produced more economically and advantageously.SOLUTION: A method for producing protease includes the following processes (A) and (B): (A) a process of culturing protease-productive microorganism in first culture medium containing extracts; and (B) a process of culturing a part or all of a bacterial cell, which is obtained from the first culture medium, again in a second culture medium after the process (A), in which nitrogen content in the second culture medium is 500 ppm or more, and a mass ratio of the concentration of extracts in the second culture medium to the concentration of extracts in the first culture medium is 0.75 or less.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a protease.

  Various detergents and bleaching agents for clothing are blended with various enzymes in order to enhance the detergency. The most industrially produced mass-produced enzymes are prostheses such as alkaline proteases, and these are usually produced by a fermentation method involving cultivation of protease-producing microorganisms.

Improvement of fermentation productivity is indispensable in order to obtain proteases in large quantities and at low prices. To date, as a technology for improving protease productivity, modification of protease-producing microorganisms by genetic techniques or continuous culture methods Culture etc. have been reported.
For example, Patent Document 1 discloses alkaline protease high productivity mutant Bacillus genus bacteria, and Patent Document 2 discloses a method for producing protease by culturing filamentous fungi having protease-producing ability in a liquid medium. A method for producing a protease is disclosed in which culture is performed while maintaining the nitrogen concentration in the culture solution at 500 mg / l or less after the end of growth.

JP 2014-161284 A Japanese Patent Laid-Open No. 62-126975

As described in Patent Documents 1 and 2, generally, a nutrient-rich liquid synthetic medium using an extract such as an amino acid or yeast extract as a part of a nitrogen source as a protease production medium. Is used.
However, adding a large amount of extracts as a part of the nitrogen source to the medium causes an increase in production cost. On the other hand, when the present inventor examined the material balance in the fermentation process, most of the nitrogen source added to the medium was consumed for cell growth, and the conversion rate from the nitrogen source to the protease was low. Efficient use for production of protease was desired.
Therefore, the present invention relates to providing a method capable of producing a protease more economically and advantageously by efficiently using a nitrogen source in a medium.

  As a result of intensive studies in order to solve the above problems, the present inventor has succeeded in cultivating protease-producing microorganisms, and then partially or entirely inoculating them, and using a medium in which the extract concentration, which is a part of the nitrogen source, is lowered. It has been found that by continuously culturing, the nitrogen source in the medium can be efficiently used for protease production while maintaining high productivity of protease.

That is, the present invention includes the following steps (A) and (B):
(A) culturing a protease-producing microorganism in a first medium containing extracts,
(B) After the step (A), including a step of culturing a part or all of the cells obtained from the first medium again in the second medium, and the amount of nitrogen in the second medium is 500 ppm or more And providing a method for producing a protease, wherein the mass ratio of the concentration of the extract in the second medium to the concentration of the extract in the first medium is 0.75 or less.

  According to the present invention, since the nitrogen source added to the medium can be efficiently used, the amount of the extract used can be reduced, and the protease can be produced in a highly economical and advantageous manner.

  The method for producing a protease of the present invention comprises (A) a step of culturing a protease-producing microorganism in a first medium containing extracts, and (B) a fungus obtained from the first medium after step (A). A step of culturing part or all of the body again in a second medium, wherein the amount of nitrogen in the second medium is 500 ppm or more, and the second medium relative to the concentration of extracts in the first medium It is a manufacturing method whose mass ratio of the density | concentration of inside extract is 0.75 or less.

[Process (A)]
This step is a step of culturing a protease-producing microorganism in a first medium containing extracts.
Examples of the protease-producing microorganism include microorganisms belonging to the genus Aspergillus, the genus Rhizopus, the genus Streptomyces, the genus Penicillium, and the genus Bacillus. Among them, from the viewpoint of protease productivity, bacteria belonging to the genus Bacillus are preferable, and Bacillus subtilis is more preferable.
Examples of Bacillus bacteria include Bacillus sp. KSM-64 strain (FERM P-10482), Bacillus clausii KSM K-16 strain (FERM BP-), which are alkaline protease-producing microorganisms. 3376), Bacillus sp. KSM-KP43 strain (FERM BP-6532), Bacillus sp. KSM-KP9860 strain (FERM BP-6534), Bacillus no. D-6 (FERM P-1592) (protease E-1), Bacillus sp Y (FERM BP-1029), Bacillus SD521 (FERM P-11162), Bacillus sp (Bacillus sp.) KSM-9865 strain (FERM P- 18566), NCIB12289 strain, NCIB12513 strain and the like.
The microorganism may be a wild type strain or a mutant strain in which mutations such as insertion, substitution, deletion, etc. of the base sequence have been caused by various genetic manipulations, and production of a desired protease by applying known artificial modifications. The ability may be added.

The first medium used in the present invention contains extracts. Here, the medium may be a synthetic medium, a natural medium, or a semi-synthetic medium obtained by adding a natural component to a synthetic medium.
The extract is not particularly limited, and examples thereof include yeast extract, meat extract, fish meat extract, etc., yeast extract and fish meat extract are preferable, and yeast extract is more preferable. Yeast extract is an extract obtained by subjecting yeast cells to treatments such as autolysis, enzyme treatment, and hot water treatment. Usually, as a raw material yeast, Saccharomyces cerevisiae or Candida Examples include Candida utilis.

Moreover, the 1st culture medium used by this invention may contain the nitrogen source which does not originate in extracts other than extracts as a nitrogen source.
Examples of nitrogen sources not derived from extracts include nitrogen-containing compounds such as ammonia, urea, inorganic / organic ammonium salts, potassium nitrate, sodium nitrate, corn gluten meal, soybean flour, polypeptone, tryptone, peptone, various amino acids, soy bean meal Etc.
A commercial item may be used for these nitrogen sources, and what was manufactured and acquired suitably may be used for them.

The amount of nitrogen in the first medium is preferably 2000 to 15000 ppm (parts per million by mass), more preferably 3000 to 10000 ppm, and still more preferably 4000 to 8000 ppm from the viewpoint of cell growth and productivity. The amount of nitrogen in the medium can be determined by total nitrogen analysis described later.
In the first medium, the concentration of the extract is preferably 0.1 to 2% (w / v), more preferably 1 to 2% (w / v) as a dry solid content from the same point. is there.

The first medium can contain a carbon source, inorganic salts, other necessary nutrient sources, and the like.
Examples of the carbon source include saccharides. Examples of the saccharide include monosaccharides such as glucose, fructose and xylose, and disaccharides such as sucrose, lactose and maltose. The saccharide may be an anhydride or a hydrate. In addition, a sugar solution containing saccharides, for example, a sugar solution obtained from starch, molasses (waste molasses), a sugar solution obtained from cellulosic biomass, or the like can also be used. Of these, glucose and maltose are preferable from the viewpoint of the growth of microorganisms.
The concentration of the carbon source in the first medium is preferably 5 to 25% (w / v).

Examples of inorganic salts include sulfates, magnesium salts, zinc salts, and phosphates.
The concentration of inorganic salts in the first medium is preferably 0.5 to 1% (w / v).
Moreover, you may add an antibiotic and a trace component suitably in a culture medium as needed.

The pH of the first medium (25 ° C., hereinafter the same) is preferably 4-9, more preferably 5-8. The pH of the medium can be appropriately adjusted using a buffer.
In this specification, the amount of nitrogen in the medium, the concentration of extracts, the concentration of carbon source, the concentration of inorganic salts, pH, etc. are the values at the beginning (at the time of medium preparation or at the start of culture) unless otherwise specified. .

The culture of the protease-producing microorganism may be performed according to normal culture conditions.
For example, the culture temperature is not particularly limited as long as it does not adversely affect the growth of microorganisms. Usually, it is preferably 20 to 48 ° C, more preferably 25 to 45 ° C, still more preferably 28 to 39 ° C. is there.

  For example, in the case of the Bacillus subtilis, the inoculation amount of the microorganism in the first medium is preferably 0.1 to 5% (v / v).

  The culture period is preferably 10 hours to 7 days, further 12 hours to 4 days, more preferably 24 hours to 3 days, depending on the growth of microorganisms.

The concentration (OD600 value) of the bacterial cells after completion of the culture is preferably 40 to 100. This OD600 value can be measured by the method described in Examples below.
In addition, the method for obtaining the bacterial cells from the first medium after completion of the culture is not particularly limited, and examples thereof include appropriate separation means such as a gradient method, centrifugation, and filtration.

[Process (B)]
This step is a step of culturing part or all of the cells obtained from the first medium after the step (A) in the second medium. This step can be carried out, for example, by inoculating a part or all of the cells into the second medium. Moreover, it can also carry out by leaving a part or all of the said microbial cell in a culture tank, and adding a 2nd culture medium to this. From the viewpoint of production efficiency, the method is preferably a method of planting cells separated by centrifugation or the like.
The amount of cells to be planted from the first medium to the second medium is 6 as a percentage of the cell concentration (OD600 value) determined by the following calculation formula from the viewpoint of nitrogen conversion rate and protease productivity. % Or more, preferably 10% or more, and more preferably 20% or more. Further, from the same point, it is preferably 100% or less, more preferably 50% or less, and still more preferably 30% or less.
Amount of bacterial cells to be transferred from the first medium to the second medium (%) =
[OD600 value of second medium after cell transfer to second medium] / [OD600 value of first medium at end of culture in first medium] × 100

  The extract concentration (dry solid content) in the second medium is 0.75 or less in mass ratio with respect to the extract concentration (dry solid content) in the first medium. The mass ratio of the concentration of the extract in the second medium to the concentration of the extract in the first medium is preferably 0.5 or less, more preferably from the viewpoint of efficient use of the nitrogen source. Is 0.3 or less. The mass ratio of the concentration of the extract in the second medium to the concentration of the extract in the first medium may be 0, but is preferably 0.15 or more from the viewpoint of protease productivity. is there.

  The amount of nitrogen in the second medium is 500 ppm or more, but is preferably 1000 ppm or more, more preferably 2000 ppm or more, and further preferably 3500 ppm or more from the viewpoint of protease productivity. The amount of nitrogen in the second medium is preferably 15000 ppm or less, more preferably 10,000 ppm or less, and even more preferably 7500 ppm or less, from the viewpoint of efficient use of the nitrogen source.

The amount of nitrogen in the second medium is a mass ratio to the amount of nitrogen in the first medium, and is preferably 0.1 or more, more preferably 0.5 or more, and still more preferably 0, from the viewpoint of protease productivity. .7 or more, more preferably 0.8 or more. Moreover, from the point of aiming at efficient utilization of a nitrogen source, Preferably it is 0.95 or less, More preferably, it is 0.9 or less, More preferably, it is 0.85 or less.
Nitrogen in the medium while maintaining protease productivity by continuously culturing cells inherited from the first medium in a medium with reduced extract concentration and nitrogen content as described above. The source can be efficiently utilized for protease production.

  Similar to the first medium, the second medium can contain a nitrogen source, a carbon source, inorganic salts, other necessary nutrients, and the like. Specifically, it is as described above.

The culture of the microorganism in the step (B) may be the same culture condition as that in the process (A) or may be different culture conditions.
In this step, the culture is preferably performed at a temperature of 28 to 39 ° C. for 48 hours to 4 days.

By such culturing, the nitrogen source in the medium is efficiently utilized for protease production, and protease is produced at high yield. In the present invention, the protease is preferably an alkaline protease.
In the present invention, the nitrogen conversion rate (%) from the nitrogen source to the protease in the medium is preferably 28% or more, more preferably 30% or more, and further preferably 32% or more.
Here, the nitrogen conversion rate is a value obtained by dividing the nitrogen concentration of the protease by the nitrogen concentration in the medium. Details of the method for calculating the nitrogen conversion rate are described in the Examples.

The enzyme solution obtained according to the present invention can be used as it is, but if necessary, it can be purified, crystallized or granulated by a known method.
On the other hand, the protease-producing microorganism separated from the culture solution can be reused again for protease production. That is, after the step (B), a part or all of the protease-producing microorganism may be used to repeat the culture again in the second medium once or more, further twice or more, and further three or more times. Good.

[Total nitrogen analysis]
It measured using total nitrogen analyzer TN-100 (made by Mitsubishi Chemical Corporation).

[Method for measuring protease production]
After the culture, the alkaline protease activity of the culture supernatant excluding the bacterial cells was measured by the following procedure, and the amount of alkaline protease secreted and produced outside the bacterial cells was determined.
Take 0.9 mL of 1/15 M phosphate buffer (pH 7.4) and 0.05 mL of 40 mM Glt-Ala-Ala-Pro-Leu-p-nitroanilide / dimethylsulfoxide solution in a test tube and incubate at 30 ° C. for 5 minutes. did. To this was added 0.05 mL of enzyme solution (culture supernatant) and reacted at 30 ° C. for 10 minutes, then 2.0 mL of 5% (w / v) aqueous citric acid solution was added to stop the reaction, and the spectrophotometer Was used to measure the absorbance at 420 nm. Based on the change in absorbance, the amount of alkaline protease in the sample was quantified. The enzyme activity for producing 1 μmol of p-nitroaniline per minute was defined as 1 U.

[Measurement method of bacterial cell concentration]
A portion of the culture broth was diluted and mixed 100 times with a 5% (w / v) aqueous sodium chloride solution and then used at a wavelength of 600 nm using a U-2000 type Hitachi spectrophotometer (Hitachi). Turbidity was measured and OD600 value was calculated from the dilution rate.

[Calculation of nitrogen conversion rate]
The nitrogen conversion rate was calculated by the following formula.
Nitrogen conversion rate (%) = [nitrogen concentration of protease (ppm)] / [initially charged nitrogen concentration (ppm)] × 100
(Wherein protease nitrogen concentration (ppm) = protease activity (U) × 0.428 (ppm / U))

Example 1
(1) As shown in JP2014-161284, a recombinant plasmid pHY-SP64-E-1 containing an alkaline protease gene was obtained.
Subsequently, the recombinant plasmid pHY-SP64-E-1 was transformed into a protoplast transformation method (Mol. Gen.) on a recA gene deletion strain (kao119 strain) which is a Bacillus subtilis mutant constructed as shown in JP-A-2014-158430. Genet., 1979, vol.168, p.111) to obtain a transformant.

  The transformant obtained above was inoculated into 30 mL of LB medium (1% by mass tryptone peptone, 0.5% by mass yeast extract, 0.5% by mass NaCl, 15 ppm tetracycline hydrochloride) and shaken at 39 ° C. for 20 hours. It was cultured and used as an inoculum.

Subsequently, the inoculum was added to the first medium (1% by mass fish meat extract, 0.5% by mass yeast extract, 3.6% by mass amino acid mixture, 0.07% by mass metal salt, 0.4% by mass ammonium salt, 0.0% by mass, 2 mass% dipotassium phosphate, 16 mass% maltose, 0.002 mass% antifoam, 15 ppm tetracycline hydrochloride, nitrogen content: 5756 ppm, pH: 6.1) 1600 mL inoculated with 2% (v / v), The culture was aerated and stirred at 36 ° C., 0.5 vvm, 800 rpm for 3 days.
After completion of the culture, the cells were collected by aseptic centrifugation (5 ° C., 8000 r / min, 15 minutes). The bacterial cell concentration (OD600 value) after completion of the culture was 89, and the alkaline protease production amount of the culture solution excluding the bacterial cells was 2185 U / L.

(2) With respect to 20 mL of the second medium (nitrogen concentration: 4310 ppm) having the same composition, except that the cells recovered in (1) above were obtained by removing the extracts (fish meat extract and yeast extract) from the first medium, Inoculate the second medium after microbial cell transplantation with an OD600 value of 89 (100% with respect to the OD600 value after completion of the culture of the first medium), and fluted flask at 36 ° C. and 210 rpm for 4 days And continued culture.
After completion of the culture, aseptic centrifugation (5 ° C., 8000 r / min, 15 minutes) was performed to remove the bacterial cells and obtain an enzyme solution. The production amount of alkaline protease in the enzyme solution was 3154 U / L.

Example 2
The amount of cells transferred to the second medium in Example 1 was changed to the values shown in Table 2, the extract concentration in the second medium was changed to 0% by mass, and the nitrogen amount (4310 ppm) shown in Table 2 Except for the above, the same culture as in Example 1 was carried out to obtain an enzyme solution.

Examples 3-6
The amount of cells transferred to the second medium in Example 1 was changed to the values shown in Table 2, and the extract concentrations in the second medium were 0.15% by mass (Example 3) and 0.3, respectively. The amount of nitrogen (4455-5394 ppm) shown in Table 2 was changed to mass% (Example 4), 0.75 mass% (Example 5), and 1.125 mass% (Example 6), and cultured for 3 days. Were cultured in the same manner as in Example 1 to obtain an enzyme solution.

Examples 7-10
The amount of cells transferred to the second medium in Example 1 was changed to the values shown in Table 1, and the extract concentrations in the second medium were 0.15% by mass (Example 7) and 0.3, respectively. The amount of nitrogen (4455-5394 ppm) shown in Table 1 was changed to mass% (Example 8), 0.75 mass% (Example 9), and 1.125 mass% (Example 10), and the cells were cultured for 3 days. The same operation as in Example 1 was performed.

Comparative Example 1
The same operation as in Example 1 was performed except that the second medium was changed to a medium having the same composition as the first medium (nitrogen amount: 5756 ppm).

Comparative Example 2
Except for changing the amount of cells transferred to the second medium in Example 1 to the values shown in Table 2, and changing the second medium to a medium having the same composition as the first medium (nitrogen amount: 5756 ppm). The same operation as in Example 1 was performed.

Comparative Example 3
The amount of cells transferred to the second medium in Example 1 was changed to the values shown in Table 1, and the second medium was changed to a medium having the same composition as the first medium (nitrogen amount: 5756 ppm) for 3 days. The same operation as in Example 1 was performed except that the cells were cultured.

Comparative Example 4
The amount of cells transferred to the second medium in Example 1 was changed to the values shown in Table 2, and the second medium was changed to a medium (0.08 mass% fish meat extract, 0.04 mass% yeast extract, 0 .3% by weight amino acid mixture, 0.07% by weight metal salt, 0.03% by weight ammonium salt, 0.2% by weight dipotassium phosphate, 16% maltose, 0.002% antifoaming agent, 15 ppm tetracycline hydrochloride, The amount of nitrogen was 468 ppm), and the same operation as in Example 1 was performed except that the cells were cultured for 3 days.

  Tables 1 and 2 show the culture conditions, nitrogen conversion rate, and alkaline protease production amount in Examples 1 to 10 and Comparative Examples 1 to 4, respectively.

  As described above, according to the method of the present invention, it was confirmed that the nitrogen conversion rate (%) from the nitrogen source in the culture medium to the protease was high. It was also confirmed that protease productivity was maintained even when the amount of nitrogen in the medium, particularly extracts, was reduced.

Claims (5)

Next steps (A) and (B):
(A) culturing a protease-producing microorganism in a first medium containing extracts,
(B) After the step (A), including a step of culturing a part or all of the cells obtained from the first medium again in the second medium, and the amount of nitrogen in the second medium is 500 ppm or more A method for producing a protease, wherein the mass ratio of the concentration of the extract in the second medium to the concentration of the extract in the first medium is 0.75 or less.   The method for producing a protease according to claim 1, wherein the concentration of the extract in the first medium is 0.1 to 2% (w / v) as a dry solid content. The protease according to claim 1 or 2, wherein the amount of bacterial cells to be planted from the first culture medium to the second culture medium is 6% or more as a percentage of the bacterial cell concentration (OD600 value) determined by the following calculation formula: Manufacturing method.
(Amount of bacterial cells to be transferred from the first medium to the second medium (%) =
[OD600 value of the second medium after cell transfer to the second medium] / [OD600 value of the first medium at the end of the culture in the first medium] × 100)   The method for producing a protease according to any one of claims 1 to 3, wherein the protease-producing microorganism is Bacillus subtilis.   The method for producing a protease according to any one of claims 1 to 4, wherein the protease is an alkaline protease.