KR930008974B1 - Method for preparation of fructosyl transferase & beta-galactosidase - Google Patents

Abstract

A mutant producing the fructosyl transferase and beta- galactosidase is screened by UV treatment of the parent strain Aureobasidium pullulans (NRRL Y-2567). For production of the enzymes, the mutant was cultured at 25-35 deg.C in a nutrient medium containing sucrose or lactose as a carbon source at neutral pH range, under aerobic condition. IPTG (2.0-10mM) was added into the medium to induce production of the enzymes.

Description

Method for preparing fructosyltransferase and β-galactosidase

The present invention is characterized by aerobic culture of microorganism strain Aureobasidium pullulans belonging to the genus Aureobasidium (Aureobasidium pullulans) aerobic culture in a nutrient medium containing a specific carbon source fructosyl transferase or β-galacto Preparation of fructosyltransferase or β-galactosidase, characterized by the accumulation of β-galactosidase in cells and in the culture medium, from which fructosyltransferase or β-galactosidase is recovered. It is about a method.

Fructosyltransferase is an enzyme that transfers and binds one molecule of fructose to the β (1,2) position to another fructose. The genus Aureobasidium, Aspergillus, and Fusarium It can be obtained from microorganisms such as the genus Fusarium and plants such as asparagus and swine potatoes.

In general, by using the fructose metastasis of this enzyme, it has a structure in which 1 to 4 fructose is combined with sugar, has a bifidus proliferative activity that is indigestible and effective in preventing tooth decay, and is a low-calorie sweetener, health food, animal feed, and pharmaceuticals. The fructooligosaccharide which is widely used by the above is manufactured.

On the other hand, β-galactosidase is an enzyme having a function of degrading lactose to glucose and galactose, and also has a transacting activity of galactose depending on its origin.

β-galactosidase is a fungus such as the genus Aspergillus, genus Penicillum, E. coli, Streptococcus, Lactobacillus, Bifidobacterium, etc. It can be obtained from bacteria and yeasts such as the genus Kluyveromyces and Candida.

The inventors have found that the two enzymes have the following common characteristics.

1) Both enzymes break down glycoside bonds of the substrate.

2) Both enzymes transfer β-bonds by transferring sugars (fractose grooves, galactose) produced by the breakdown of glycoside bonds to another substrate.

Thus, it is expected that the mechanism of action of the two enzymes will be similar.

Under these circumstances, the present inventors have conducted a long research to produce the above two enzymes in one strain, and as a result, have discovered that the above object can be achieved by using an oreovacsidium pullulan mutant strain. Completed.

That is, the present invention is cultured orocobadium pullulans mutants capable of selectively producing fructosyltransferase or β-galactosidase according to the type of carbon source in the medium, fructosyltransferase or β-galacto Provided is a method for preparing a sidase.

Hereinafter, the present invention will be described in more detail.

Oreobashdium flulans variance according to the present invention can be prepared as follows.

Parent Oreobacidium flulans (NRRL Y-2567) was prepared in medium 1 (pH) containing 6% sugar, 0.3% yeast extract, 0.5% dibasic potassium phosphate, 0.1% sodium chloride, 0.06% ammonium nitrate and 0.02% magnesium sulfate. 6.8, 120 ℃ × 20 minutes sterilization) for 36 to 48 hours, then centrifuged to separate the supernatant and bacteria, discard the supernatant and suspended in sterilized 0.05M phosphate buffer (pH 6.8) and then 15W ultraviolet light 30 cm exposure there for 30 minutes to cause mutations. Survival after UV mutation was 0.1-0.5%. Then, plate in medium 2 (120 ° C. × 20 min sterilization) containing 20% potato leach, 2% glucose and 1.5% agar, incubate for 60 to 84 hours at 30 ° C., and then culture the separated colonies in a flask. By multiplying to obtain a primary mutant strain.

The primary mutant was resuspended in medium 2, and the suspension of the clonie was well suspended in sterile 0.05M phosphate buffer solution (pH 6.8), followed by N-methyl-N'-nitro-N-nitorose- at a concentration of 50 g / ml. After adding 2 ml of guanidine solution and shaking for 30 minutes at room temperature, the cells and the mutant were separated by centrifugation and washing, and then plated in medium 2, and then cultured at 30 ° C. for 60 to 84 hours, and then the chlorine was excellent. Got a tea variation.

The secondary mutants thus obtained were treated with medium 3 (pH 6.8, 120 ° C. × 20 min sterilization) and lactose containing 20% sugar, 5% corn steep solution, 0.5% dibasic potassium phosphate, 1% sodium nitrate and 0.05% magnesium sulfate. 10%, peptone 1.5%, yeast extract 0.5%, monobasic phosphate 0.5%, sodium nitrate 0.2%, magnesium sulfate 0.05% and calcium chloride 0.05%, respectively (pH 6.0, 120 ℃ x 20 minutes sterilization) Variants with high activity of both fructosyltransferase and β-galactosidase were selected and named MW-4218.

Variation strain Oreobashdium flulans MW-4218 according to the present invention was deposited on December 20, 1991 with the deposit number of (KFCC) -10754 to the Korean spawn association (KFCC).

Mutant strain of the present invention was observed to have the following strain characteristics.

1) It is an incomplete fungus in which the form of mycelium changes from spores to mycelium form → yeast form → mycelium form → spores depending on the growth environment. That is, in the early stage, germination of spores grows in the form of mycelium, and as time passes, the surrounding growth environment improves rapidly and grows in the yeast form.

Depletion of nutrients or accumulation of metabolites inside and outside the cells converts the growth back to mycelium form, eventually forming spores.

2) In the growth phase of logarithmic growth, that is, the yeast form, the cell type is circular or oval, and the production and secretion of enzymes is active, and in the second half of growth under environmental stress, polysaccharides are produced and separated to increase the viscosity of the culture solution rapidly.

3) In the early stage of growth and logarithmic growth, yellow or flesh-colored color is formed, and then brown and black pigment is formed in the stage of growth. At this time, the pigment to be produced varies greatly depending on the pH conditions of the culture medium, the pH is near the midline to show flesh or yellow, and the weakly acidic to produce a brown pigment. In addition, it varies depending on the growth temperature, but when the temperature is around 25 ℃, the production of pigments is vigorous.

4) The enzymes produced vary depending on the type of carbon source of the culture. In other words, if the carbon source uses a carbon source composed mainly of sucrose, such as sugar, raw sugar, and molasses, the enzyme produced is mainly fructosyltransferase. When lactose is used as a main carbon source, such as lactose and whey powder, β- Mainly produces galactosidase.

5) Compared with parent strain, the activity of fructosyltransferase did not change significantly, whereas β-galactosidase showed a significant increase in hydrolytic and metastatic activity.

As mentioned above, the carbon source that can be used for culturing the mutant strain of the present invention may select a sucrose-containing or lactose-containing carbon source according to the purpose of culture, that is, the type of enzyme to be obtained. Nitrogen sources such as yeast extract, corn steep liquor, peptone, sodium nitrate, ammonium nitrate, polypeptone and amino acids can be used.

Moreover, magnesium sulfate, sodium chloride, a first potassium phosphate, a second potassium phosphate, etc. can be used as mineral and inorganic salts.

The culture temperature of the mutant strain is suitable in the range of 25 to 35 ° C, preferably in the range of 28 to 32 ° C, and the optimum pH for culturing is near neutral.

When cultured under the above conditions, fructosyltransferase or β-galactosidase accumulates in the cells and in the culture medium depending on the type of carbon source.

In the enzyme activity measurement, the activity of fructosyltransferase was determined by measuring the amount of free and produced glucose by the enzyme oxidation reaction, and the glucose concentration was determined by the glucose oxidation method. The substrate concentration was 60% (w / v), the temperature was 55 ° C, Under the condition of pH 5.5, the amount of enzyme that produces 1 μmole of glucose was defined as 1 unit.

The activity of β-galactosidase was calculated by using O-nitrilephenyl-β-galactoside (ONPG) as a substrate, 0.05% calcium chloride, pH 6.0, 120 ° C x 20 minutes sterilization, substrate concentration 5mM ONPG, temperature 55 ° C, The amount of enzyme that produces 1 μmole of O-nitrophenol per minute under pH 6.5 was defined as 1 unit.

The activity of β-galactosidase can also be measured using lactose as a substrate. At this time, the substrate concentration was 10% (w / v) and reacted under the same conditions as in the case of ONPG. The amount of glucose produced was measured by glucose oxidation, and the amount of enzyme that produced 1 μmole of glucose per minute was defined as 1 unit. In addition, the concentration of galactose was analyzed and quantified by HPLC.

The present invention will be described in more detail with reference to the following Examples, but the present invention is not limited to these Examples.

Example 1

〈G germination Culture〉

50 ml of medium 1 was placed in a 250 ml shaking Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes, inoculated with platinum of Oreobashdium pullulans MW-4218 (KFCC 10754), and incubated for 36 hours with rotation shaking at 30 ° C. It was.

〈All Cultures〉

The culture solution obtained in the germination culture was inoculated at 5% (v / v) in a 500 ml shaking Erlenmeyer flask containing 100 ml of medium 1, and cultured for 24 hours under the same conditions as the germination culture, and then used as a spawn seed.

<Main culture>

The previously obtained seed was aseptically inoculated into a 1 L shake flask containing medium 3 at a concentration of 5% (v / v). Then incubated for 25 hours under the same conditions as the germination culture. After completion of the culture, the activity of the produced fructosyltransferase in and outside the cells was 400 to 450 units per ml of culture medium.

Example 2

〈G germination Culture〉

Oreobasidium pullulan MW-428 (KFCC 10754) colony grown in Medium 2 was a medium containing 5% lactose, 1% glucose, 0.5% peptone, 0.5% potassium monophosphate and 0.02% magnesium sulfate. After aseptically inoculated into a shaking Erlenmeyer flask containing 50 ml (pH 6.0, 120 ℃ × 20 minutes sterilization) and incubated at 30 ℃ 48 hours.

〈All Cultures〉

The culture solution obtained in the germination culture was inoculated at 5% (v / v) in a 500 ml shaking Erlenmeyer flask containing 100 ml of medium 5, and cultured for 24 hours in the same manner as the germination culture, and used as a spawn seed.

<Main culture>

The seed spawn obtained above was inoculated in a 1 L shake flask containing 200 ml of medium 4 at a concentration of 5% (v / v), and then cultured for 48 to 60 hours in the same manner as in germination culture. The activity of β-galactosidase produced after the culture was 10-20 units per ml.

Example 3

In the same manner as in Example 2, the concentration of isopropyl-β-thiogalactofuranoside (IPTG), known as a derivative for inducing the production of β-galactosidase during the logarithmic growth phase of the present culture, was 0.5-10 mM. Cultured by addition of

The enzyme activity of the culture medium according to the concentration of IPTG was measured and the results are shown in Table 1.

Table 1 Activity of β-galactosidase by IPTG Concentration

Example 4

The enzymes were obtained by culturing in the same manner as in Example 1 and Example 2 using NRRL Y-2567, which is a parent strain instead of the variant strain Oreobashicium pullulan MW-4218 of the present invention, and the results are shown in Table 2. It was.

TABLE 2

As can be seen from the results of Table 2, the present invention shows a β-galactosidase generating ability about 7 to 8 times higher than the parent strain.

Example 5

The hydrolytic and transition activities of β-galactosidase obtained in Example 2 were measured using lactose as a substrate. Meanwhile, NRRL Y-2567, a parent strain, was cultured in the same manner to obtain β-galactosidase, and their activities were measured and compared. Hydrolysis activity and transition activity are expressed by the hydrolysis rate and the transfer rate, respectively, and the results are shown in Table 3.

TABLE 3

As can be seen from the results of Table 3, the β-galactosidase produced by the mutant strain of the present invention is at least 2 times higher hydrolytic activity and 7 times higher transfer activity than the β-galactosidase produced by the parent strain. Indicates.

Claims (3)

Aureobasidium pullulans MW-4218 (KFCC 10754), which can produce fructosyltransferase or β-galactosidase, depending on the type of carbon source, is used as a carbon source in a nutrient medium containing sugar or lactose, respectively. Fructosyltransferase, characterized by accumulating fructosyltransferase or β-galactosidase in cells or in culture by aerobic incubation at 25 to 35 ° C. and neutral pH, and recovering the enzyme therefrom. Method for producing β-galactosidase. The method according to claim 1, wherein when producing β-galactosidase, isopropyl-β-thiogalactofuranoside (IPTG), which is a β-galactosidase production inducer, is added at a concentration of 2.0 to 10 mM. A method for producing fructosyltransferase and β-galactosidase. The method for producing fructosyltransferase and β-galactosidase according to claim 1, wherein the produced β-galactosidase has a transition activity of 50% or more of the hydrolytic activity.