JPS6053596B2 - Production method of acylase - Google Patents

Description

Detailed Description of the Invention The present invention involves culturing an acylase-producing strain belonging to the genus Aspergillus to obtain acylase, by culturing it in a medium containing at least one fatty acid or its metal salt (hereinafter referred to as fatty acid, etc.). The present invention relates to a method for producing acylase.

Acylase (E, C, 3.5.1.14) is also called aminoacylase and is used for racemic resolution of DL-amino acids because it hydrolyzes N-acyl-L-amino acids to generate L-amino acids. It is industrially important as oxygen. As a conventional method for producing acylase using filamentous fungi, Michi et al. (Special Publication No. 31-2891 and Journal of the Japanese Society of Agricultural Chemistry, Volume 2, pp. 343-345, 19M) cultured Aspergillus and Penicillium bacteria on wheat wrinkles. method, Chibata et al. (Bulletin of the Agricultural Chemical Society of Japan (Bul.
Bacterial strains of the genus Aspergillus, Penicillium, Rhizopus, Mucor, etc. (Agr, Chem, Soc, Japan) Vol. method, a method of culturing Aspergillus and Penicillium bacteria on wheat wrinkles by Rin et al. (Japanese Patent Publication No. 40-27310), and a method of culturing Aspergillus and Penicillium bacteria on wheat wrinkles containing acyl amino acids by Kokoku et al. (Japanese Patent Publication No. 40-27310). Methods such as culturing in a medium are known.

The present inventors continued intensive studies to enhance the production of acylase in strains belonging to the genus Aspergillus, and as a result, it was found that productivity was increased by adding a fatty acid having 1 to 20 carbon atoms or a metal salt thereof to the medium. I discovered that.

The present invention has been completed based on the above findings, and is extremely useful as an industrial, mass production method for acylase.
The method of the present invention will be explained in detail below. The acylase-producing strain used in the present invention may be any acylase-producing strain belonging to the genus Aspergillus, such as Aspergillus ochraceus (Aspergillus ochraceus).
illusochraceus) IF04070, Aspergillus oryzae (Asp.

"ory2ae) IAM2750, Aspergillus, sojae (Asp, sojae) IFO439l) Aspergillus oxalium (Asp, oxalium) IF
Examples include O5748. Among these strains, Aspergillus Ochraceus IFO is particularly popular due to its high productivity.
4O7O is preferred. The nutrient medium used for culturing these strains may be any conventional medium containing a carbon source, nitrogen source, inorganic salts, etc.

For example, carbon sources include wrinkles, starch, rice bran, glucose, etc. Nitrogen sources include peptone, meat factory scratches, yeast factory scratches, etc.
Examples of inorganic salts such as sodium nitrate and ammonium sulfate include magnesium sulfate and manganese sulfate. The culture form may be either solid culture or liquid culture, but solid culture based on wrinkles is preferred from the viewpoint of acylase productivity. The fatty acids having 14 to 20 carbon atoms to be added to the above nutrient medium to enhance the productivity of acylase are Cl4 myristic acid,
These include palmitic acid (Cl6), stearic acid (Cl8), oleic acid, linoleic acid, linolenic acid, and arachidic acid (C2O), regardless of whether they are saturated or unsaturated.

Metal salts of these fatty acids, such as sodium salts, potassium salts, and magnesium salts, may also be used, and mixtures thereof, such as natural fatty acids and soaps, may also be used. Among these fatty acids, saturated fatty acids myristic acid, stearic acid, palmitic acid, and metal salts thereof are particularly preferred for increasing acylase production. When fatty acids are used in the method of the present invention, they must be neutralized to bring the pH of the medium to conditions suitable for bacterial growth.

Furthermore, since fatty acids themselves are not hydrophilic, it is difficult to uniformly disperse them in the medium. Therefore, it is more convenient to use fatty acid metal salts than fatty acids. Taking fatty acid monometallic salt as an example, the concentration of these fatty acids added to the medium is 0.1% or more and 30% or less in the case of solid culture.
Preferably 1% to 15%, 0.1% for liquid culture
The content is from 3% to 3%, preferably from 0.5 to 2% (all percentages by weight, hereinafter the same).

When using fatty acids. The concentration may be equivalent to that of the fatty acid metal salt. Increased production of acylase by adding fatty acids and the like to the medium is thought to be due to the significance of these additives as nutritional sources and the maintenance of the medium pH within a range suitable for enzyme production.

This effect of keeping the pH within a suitable range can also be obtained to some extent by adding weakly alkaline salts. These salts are preferably salts consisting of weak acids or organic acids whose constituent acids have relatively low dissociation constants, such as sodium phosphate, potassium phosphate, sodium carbonate, sodium bicarbonate, sodium fumarate, sodium lactate, etc. is exemplified. That is, acylase can also be inhibited by adding appropriate amounts of these inorganic or organic salts to the culture medium to maintain the pH during oxygen production within the range of approximately 7.5 to 8.5, preferably 7.8 to 8.2. It is possible to increase production to some extent. In this case, the effect will be further enhanced if fatty acids used in the present invention are added in combination. In other words, by adding these salts and fatty acids in combination, the amount of fatty acids added can be made smaller than when they are used alone, and the same effect can be obtained. That is, by using this method, the amount of expensive fatty acids and the like used can be reduced. Another advantage of the method of the invention is that acylase productivity is not affected by medium sterilization conditions.

In general, when culturing microorganisms, the milder the sterilization conditions, the more favorable results are often obtained. Medium sterilization in the method of the present invention is performed at a vapor pressure of approximately 1.2 kgIcF11130.
It is carried out within the range of 1.3k91cIt17 minutes,
In the past, acylase productivity decreased as the conditions became more severe, but this effect was completely eliminated by adding fatty acids and the like. The method for culturing the microorganism of the present invention may be carried out in accordance with a conventional method for culturing filamentous fungi.

For example, the culture temperature is 20-357C, preferably 22-357C.
The culture period is 0°C and 1 to 7 days. Acylase can be obtained from a culture according to the method of the present invention by appropriately combining known methods.

For example, solid cultured koji is extracted with an aqueous medium to obtain an extract containing acylase. The extract is concentrated by ultrafiltration, vacuum concentration, or the like, and then purified by salting out with ammonium sulfate or the like or organic solvent precipitation with ethanol to obtain a crude enzyme powder. Furthermore, a purified product can be obtained by ion exchange, gel filtration, dialysis, etc., if necessary. In the case of liquid culture, acylase accumulates within the bacterial cells, so acylase was extracted in advance, for example, according to the extraction method using enzyme-A produced by Achromobacter lunatus (Japanese Patent Publication No. 13271/1983). Afterwards, it is best to separate the bacterial cells. The subsequent purification can be performed in the same manner as in the case of the solid culture extract. The present invention will be explained in detail below using test examples and examples.

The activity of acylase was determined by carrying out an oxygen reaction at 37°C using N-acetyl-DL-methionine (pH 8.0, containing 5 x 10-4 MC0C12) as a substrate, and quantifying the L-methionine produced by ninhydrin colorimetry. The enzyme activity that produced 1 μMOL(7)L-methionine in 3 minutes was defined as 1 unit. Test Example 1 5ml of water and various concentrations of fatty acid metal salts shown in Table 1 were added to 5y of wheat planks, mixed well, and then transferred to a 100ml Erlenmeyer flask and sterilized with 1.2k91c1113 powder steam.

Next, a stock strain of Aspergillus ochraceus IFO4O7O was inoculated and cultured at 30°C for 5 days. After culturing, 100 ml of water was added to the koji, and extraction was performed for about an additional time, and the acylase activity of the extract was measured. 10 without fatty acid metal salt addition
Table 1 shows the relative activity when it is 0%. Nonsal SN-1 is a stearic acid soap manufactured by NOF Corporation, and SS-40N is a stearic acid-based soap manufactured by Kao Soap, with a composition of 65% stearic acid, 65% palmitic acid, and 65% myristic acid. , 30%, and 5%. NS Sorb is a soap made by Kao Soap whose main ingredient is oleic acid, and its composition is 40%, 35%, and 25% of oleic acid, palmitic acid, and stearic acid, respectively. According to the results in Table 1, it is clear that the acylase production ability of this strain is increased by adding fatty acid salts to the medium.

Particularly effective are saturated fatty acid salts such as sodium myristate, sodium palmitate, and sodium stearate, and soap products containing these. Salts of unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid inhibit the growth of bacterial strains as the number of double bonds increases.
The effect will be lower. Test Example 2 Wheat wrinkle 5y, water 5ml, sodium stearate 0.5
After mixing well, the mixture was transferred to a 100 ml Erlenmeyer flask and 0.7 kg Icr117α steam sterilized.

Various strains of the genus Aspergillus shown in Table 2 were inoculated into this, and the same procedure as in Test Example 1 was carried out. As a control, a sample without sodium stearate was also operated in the same manner. The acylase activity of the extract is shown in Table 2. As is clear from Table 2, Aspergillus ochraceus IF
An increase in acylase production by addition of fatty acid metal salts was also observed in other Aspergillus genus '04070.

Test Example 3 Wheat Wrinkle 5f, Water 5WLt and Nonsal SN-
1. Various inorganic and organic salts were added in the proportions shown in Table 3, mixed well, and the following operations were carried out in the same manner as in Test Example 1.

The relative activities of the extracts are shown in Table 3. As is clear from Table 3, by adding the fatty acid metal salt and the salts exemplified in the same table in combination, the same effect can be obtained with the addition of less fatty acid metal salt.

Test Example 4 3.5 ml of water, 0.5 y of stearic acid or oleic acid, and 1.5 ml of caustic soda aqueous solutions of various concentrations shown in Table 4 were added to 5 g of wrinkled wheat, mixed well, and operated in the same manner as in Test Example 1.

Table 4 shows the relative activity when no fatty acid was added as 100%. The results shown in Table 4 show that when fatty acids are used, the production of acylase can be increased by adding an alkali together.

Test Example 5 Wheat wrinkle 5y, water 5mt and sodium stearate 0
．． After adding 5y or 0.5y of sodium oleate and mixing well, the mixture was sterilized under various conditions shown in Table 5, and then operated in the same manner as in Test Example 1.

The relative activities of the extracts are shown in Table 5. When fatty acid metal salts are not added, acylase productivity decreases as the sterilization conditions become stricter, but when they are added, no effect is observed at all.

Test Example 6 Add 1% each of sodium stearate, sodium oleate, or Nonsal to a medium with the composition shown below.
1.2kg IC in a 00m Yosaka Lof flask! Il
After sterilization, a seed culture of Aspergillus ochraceus IF'04070 prepared by culturing in a medium of the same composition was inoculated.

Glucose 3g/100ml Corn Steep Liquor 2 Sodium Nitrate
0.2 Magnesium sulfate 0.05
After culturing at pH 6.5 at 30° C. for 5 days, 2 ml of a 1.5% aqueous solution of enzyme-A produced by Achromobacter lunatus was added to the culture solution to extract acylase.

The activity of the extract is 154% and 132% when sodium stearate, sodium oleate, and Nonsal are added, respectively, when the control (no fatty acid salt added) is 100%.
%, 155%. Example 1 20 g of Nonsal 50f1 disodium phosphate (anhydrous) and 0.5 f of water were added to Wheat Wrinkle 1k9, mixed well, placed on a 50 x 70 cm aluminum koji lid, and sterilized with 1.2k91d17 powder.

0.3e of the seed culture of Aspergillus ochraceus IFO4O7O, which had been previously cultured at 30°C for 2 days, was inoculated into a 10% wheat wrinkle suspension medium, mixed, and cultured at 30°C for 5 days. The extracted koji was crushed, water 2' was added thereto, extracted for 1 hour, and then filtered to obtain an enzyme solution. Add 2 more water to the residue
' was added and the same operation was performed to obtain a total of 3e enzyme solution.
The acylase activity of the solution was 153 uImt. The activity of a control enzyme solution, which was operated in the same manner as above except that Nonsal was not added, was 96 uImt. The enzyme solution 3e was concentrated to 500 ml using an ultrafiltration membrane (manufactured by Asahi Kasei, Model N4010), then 300 mt of ethanol was added to precipitate impurities, and the precipitate was removed by centrifugation to obtain a clear liquid.

Claims (1)

[Claims] 1. Cultivating an acylase-producing strain belonging to the genus Aspergillus in a medium containing at least one linear fatty acid having 14 to 20 carbon atoms or a metal salt thereof, and obtaining acylase from the culture. A method for producing acylase, characterized by: 2 Claim 1 in which the culture medium contains salts in addition to linear fatty acids having 14 to 20 carbon atoms or metal salts thereof
Method for producing acylase as described in Section 1. 3. The method for producing acylase according to claim 2, wherein the medium contains a salt whose constituent acid is a weak acid or an organic acid.