JPS6019993B2 - Method for producing β-galactosidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved method for producing 8-galactosidase.

8-Galactosidase has been increasingly used in the food and pharmaceutical fields in recent years as an enzyme that decomposes lactose present in dairy products, but its range of use has been limited due to economic reasons, and cheaper enzymes have been Involvement in manufacturing methods is requested.

In general, it is difficult to automate the production of inexpensive enzymes.
In addition, liquid culture methods are considered to be more advantageous than solid culture methods, which tend to cause bacterial contamination.

Until now, acid B-galactosidase produced by filamentous fungi has often been produced using bacteria belonging to the genus Asbergillus. Compared to the 8-galactosidase produced by Asbergillus-producing bacteria, the 3-galactosidase produced by Venicillium-producing bacteria has superior yeast characteristics, but this Benicillium strain has significantly inferior enzyme productivity in liquid culture. However, it was not available for industrial use. Furthermore, although 8-galactosidase produced by yeast and bacteria can be induced and produced by lactose, these are both intracellular enzymes and are not secreted into the culture solution. Although 3-galactosidase produced by Benicillium-producing bacteria is also secretory, no secretion production occurs at all depending on lactose. Furthermore, other high-molecular plant polysaccharides do not show significant enzyme productivity for the blind strain of the genus Benicillium, even if the culture conditions such as feeding and feeding are changed. As a result of studies aimed at producing inexpensive enzymes, the present inventors discovered that the presence of two types of substances in the medium during culture dramatically increases enzyme productivity. The present invention has been made based on this. That is, the production method of the present invention consists of allowing a vegetable crop having galactose as a part of its constituent components and a fine water-insoluble monster substance to coexist in a liquid column.

Generally, it is preferable to dissolve 1 to 10% (w/v) of the vegetable polysaccharide and suspend about 0.5 to 5% (w/v) of the fine water-insoluble substances. In addition, as a nutrient source for the growth of microorganisms, it is preferable to add organic and inorganic nitrogen sources, various inorganic salts, vitamins, and if necessary, some pulp as a carbon source. After sterilizing the medium containing these essential substances and nutrient sources, Kugalactosidase-producing bacteria of the genus Benicillium are inoculated, and liquid culture is performed under aerobic conditions. 8-galactosidase is secreted and accumulated in the medium at a culture temperature of 25 to 35 p.m. and a culture period of 3 to 7 days. For culture, usual liquid culture methods such as rotary shaking culture and aerated culture are used.
The 8-galactosidase produced in the medium can be recovered from the culture solution section from which the bacterial cells and solid matter have been separated after the completion of the culture using general enzyme recovery and purification techniques. That is, if necessary, the concentrated culture solution is subjected to salting with an inorganic salt such as ammonium sulfate or precipitation separation using an organic solvent such as alcohol or acetone. These precipitates can be dissolved in a small amount of water and freeze-dried to obtain enzyme powder. Further purification of the enzyme can be achieved by using enzyme purification methods such as ion exchange column chromatography, gel filtration, affinity chromatography, and open air permanence. The plant polysaccharides used in the present invention always contain galactose as a constituent sugar, and many of them are generally classified as polysaccharides called plant gums.

For example, pectin, locust bean gum, guar gum, gum arabic, tamarind gum, chikaraya gum, gatsutei gum, tragacanth gum, larch gum,
These include grain rubber or galactomanna gum. Some of these produce high viscosity gels when dissolved in water, but generally they can be used as nutritional sources for induction of less than a few percent, and if necessary, they can be treated to reduce viscosity. You can also go there. Generally, the amount of these plant-based polygonums added to the culture medium is often in the range of 1 to 10%, but larch gum and the like can also be used in higher concentrations. Next, water-insoluble substances include organic substances such as grain powder, legume powder, cereal cortex powder, legume cortex powder, bran powder, bran, dried yeast powder, dried algae powder, and cellulose powder, as well as talc, clay, alumina, Examples include powdered inorganic substances such as diatomite, calcium carbonate, and silica.

These water-insoluble substances need to be powdered and dispersed in the culture medium, and for this purpose they must be powdered. The effective particle size standard is JIS 42 mesh particle size, 350 microns or less, and it can be assumed that the smaller the particle size, the more likely the effect will be produced, and preferably fine powder of 100 microns to several microns is suitable for use. Among these, organic powder such as soybean powder has been conventionally used as a medium component for microorganisms, and is added as a nutrient source such as a carbon source, nitrogen source, and vitamin source. However, unlike these nutritional sources (which are generally water-solubilized), the present invention uses so-called "lees" or "husks" that remain in an immovable state to produce physical effects in the soil. This is what we seek. The 8-galactosidase production ability can be similarly improved by using products treated with hot water, alcohol, or caustic soda. As an organic nitrogen source,
Beptone, malt extract, meat extract, enzyme extract, cornstap IJ car, etc. are common, and inorganic nitrogen sources include ammonium sulfate, ammonium nitrate, sodium nitrate, etc.

As various inorganic salts, it is effective to add salts having a phosphate group such as monopotassium phosphate, dipotassium phosphate, disodium phosphate, etc., and salts such as magnesium chloride, sodium chloride, and dibasic sulfate. Examples of vitamins include enzyme extracts, malt extracts, chlorella extracts, and the like as various vitamin-containing substances. As the bran, it is also possible to add general sugars such as glucose, sucrose, maltose, dextrin, and starch. As the 3-galactosidase-producing bacteria used in the present invention, any 8-galactosidase-producing bacteria belonging to the genus Benicillium can be used.
Multicolor KU-0-132 heat is effective.

This bacterium is the same strain used for the production of B-galactosidase in JP-A-54-10529, and its mycological properties are as follows.
Observation records on various differentiation media are as follows.

[Toji] [Growth condition] Growth is strong on wort agar medium.

On the 5th day of culture, the mycelia became yellow to clear in color, with some parts tinged with red.
The central part is raised and there is a Z. Some greenish-gray conidia are observed. The underside of the hyphae is clear brown. Elution of a yellow diffusible dye is observed. On the 7th day of culture, the central part of the bacterial flora turns red, and green conidia are attached to the surrounding area.
J Potato Glucose Agar Medium Growth is moderate.

On the 5th day of culture, the outside of the bacterial flora turns yellow, the inside turns light brown, and green conidia grow in the center.

The back side of the bacterial flora is yellowish brown, and no pigment elution is observed. Culture 7
As the day progresses, the clear-colored area bacterial flora 2 becomes covered with conidia. Growth on Abec agar medium is vigorous.

The bacterial flora swells and becomes velvety, yellowish in color, with a pleated surface, and eventually the entire surface is covered with dark green to greenish-gray conidia. The back side of the 2 bacterial flora exhibits a clear brown color, and a diffusible pigment of the same color is eluted.

oatmeal agar medium Growth is moderate.

After 5 days of culture, the outside of the bacterial flora turned white and the inside turned yellow, and three pale greenish-gray conidia were observed on top. Short, spider-like aerial mycelium can be seen in the center. The underside of the bacterial flora is yellow, with no diffusible pigment. On the 7th day of culture, the entire surface is covered with linear gray conidia. YpSs agar medium
3 Growth is vigorous.

After 5 days of culture, the bacterial flora leaves 2 to 3 white hyphae on the outside and a large number of velvety conidia settle on the entire surface. The underside of the bacterial flora is yellowish brown and no diffusible pigments are observed. Saburo agar medium
4 Growth is poor.

The mycelium is thin and white. After 5 days of culture, some green conidia were observed on the cuff-shaped bacterial mass in the center. The back side of the mycelium is white. There are no diffusible pigments. Cornmeal agar medium: Growth is extremely poor.

The vegetative hyphae are thin and white. A yellow sticky substance appears in the center. Adhesion of conidia is not observed until the 7th day of culture. The back side is almost the same as the front side. There are no diffusible pigments. Growth on Enzyme YM agar soil is good.

After 5 days of culture, the bacterial flora had a pleated appearance, the outer edge was white with a width of 1 to 3 skins, the inner part was pale and clear, and greenish-gray conidia were attached to the center.

Anchovies can be seen on the back side of the bacterial flora. The underside of the bacterial flora is light brown, with pigment diffusion of the same color. In addition, as for other physiological and ecological properties, coral growth range: 3.5-8.5
(Optimal 4.5-6.0) Growth temperature range: 20-3
Growth on nitrite medium (optimum 25-30℃):
Impossible.

Benicillus: Unicycle. Conidiophore: Stands upright from the base and has an enormous tip. Munnko:
8 to 1 are also densely clustered. Chain of conidia:
Cylindrical. Conidia: spherical, 2-3 mounds. Ruined face. and the above strain is Venicillium
Belongs to multicolor. Benicillium multicolor (
Penicilli Mountain (mmulticolor) is the
Japanese Federation Collection of Microorganisms (TheJa
paneseFederation of Cul
tme Collection of Microoo
rganIsms), 1957 edition, page 81, and the above-mentioned KU-○-132 compound has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as accession number 4375.

The enzymatic properties of the enzyme obtained by the present invention were
The following is a representative example of the Umu multicolor KU-○-132 strain.

'1) Substrate specificity: It shows strong degrading power for substrates such as lactose, 0-nitrophenyl-31D-galactopyranoside, and P-nitrophenyl-81D-galactopyranoside; It has no effect on nitrophenyl-Q1D-galactopyranoside or phenyl-B-DO-glucopyranoside.

'21 Very thin shaft. Action pH: The optimum pH is 4.5, and enzyme action is exhibited within the pH range of 2.5 to 7.0.

'31 Optimum temperature, action temperature: It has an optimal temperature at 55°C, but has enzymatic action if it is below 75:00. [41
pH stabilization method: has a stable range between pH 2.5 and 8.0).

[5} Effect of metal ions: Iron ions (10-3 mol) make it slightly unstable.

■ Isoelectric point: PI from the aerophoresis method of ampholine
The value was 5.37.

'7} Molecular weight: The molecular weight estimated from the gel furnace superdissolved mother position is thought to be about 120,000.

According to the method of the present invention, 8-galactosidase can be produced in high units.

In other words, in order for Benicillium strains to exhibit high enzyme productivity, it is necessary for hyphae to grow in a fine state throughout the medium, and this role is played by water-insoluble substances of 350 microns or less. It can be said that the desired high-potency 3-galactosidase was produced for the first time by coexisting with a vegetable polysaccharide containing galactose as one of its constituent components. On the other hand, when this water-insoluble substance is present in the beach and lactose is used as a nutrient source for transfer instead of vegetable polysaccharide, a similar effect is seen on the growth of mycelia, but 8 -No secretion of galactosidase is observed. Next, the effects will be explained using test examples. The activity of 8-galactosidase was measured using 0-nit. Using phenyl-81D-galactopyranoside as a substrate, 1 unit (IU) is defined as the production of 1 France mol of 0 nitropherne per minute under the conditions of 370 and pH 4.5. Put 1g of monopotassium phosphate, 1g of ammonium sulfate, 1g of cornstarch liquor, and 0.1g of enzyme extract into an Erlenmeyer flask, add the following substances to the basal medium (pH 5.0) dissolved in 100ml of water, and sterilize by autoclaving. Thereafter, Benicillium multicolor KU-○-132 vermilion was inoculated, cultured with shaking at 30°C for 5 days, and the 8-galactosidase activity of the culture solution was measured.

The results are shown in Table 1. In Table 1, 1 to 13 are comparative areas, and 14 to 20 are inventive areas.

Test example Put gum arabic, 1 g of monopotassium phosphate, 1 g of ammonium sulfate, 1 g of cornstarch liquor, and 0.1 g of enzyme extract in a 2500M Erlenmeyer flask.
Defatted soybeans, yeast powder, and rice bran residue (extracted lees) that had been treated as described below were added to a medium (pH 5.0) dissolved in water.

Processing 1: Add 100 ml of water to each tank and overflow to 100 ml to elute the water and hot K dissolved matter, then centrifuge to remove the water section and use the water.

Process 2: Add 100% Z of alcohol to each juice, extract vigorously, and centrifuge to remove the alcohol category and keep the residue.

Treatment 3: 0 for each crab. After adding NaOH solution LOOO' and extracting proteins etc., centrifugation is performed. After removing the sieve, the remaining liquid is suspended and neutralized with water, and then centrifuged again to collect the remaining liquid.

Next, autoclave sterilization was performed, Benicillium multicolor KU-○-132 strain was inoculated, and after shaking culture was performed at 30q0 for 5 days, the 3-galactosidase activity of the culture solution was measured.

The results are shown in Table 2. Table 2 Test Example 3 In a 500' Erlenmeyer flask, larch gum sato, phosphoric acid 1
To a culture medium (pH 5.0) containing 1 g of potassium, 1 g of ammonium sulfate, 1 g of cornstarch liquor, and 0.1 g of enzyme extract dissolved in 100 g of water, pulverized defatted soybean powder was added, and after sterilization in an autoclave, Benicillium. Multicolor KU-○-132 strain was inoculated and cultured with shaking at 30qC for 5 days, and then the 8-galactosidase activity of the culture solution was measured.

The results are shown in Table 3. In Table 3, pigs 1 to 2 are comparative areas, and 3 to 7 are inventive areas. Examples of the present invention are listed next, but the present invention is not limited to these examples. .

Example 1 3% gum arabic, 1% beptone, 1% malt extract, 2% cellulose powder (42-100 mesh), 0.5% ammonium sulfate, 0.5% monopotassium phosphate, 0.05% magnesium sulfate. Transfer 100 ml of culture medium (bait 5.2) into 500 Erlenmeyer flasks, sterilize by autoclave, and add Benicillium multicolor KU-○-132.
The beads were inoculated and cultured with rotary shaking at 30°C for 5 days.
8-galactosidase was produced.

The culture fluid had an 8-galactosidase activity of 130 U/. Example 2 The same procedure as in Example 1 was carried out except that gum arabic was replaced with larch gum, and cellulose powder was replaced with diatom powder.

The 8-galactosidase activity of the culture solution was 125 U/. Example 3 A medium containing 4% larch gum, 3% defatted soybean powder (100-200 mesh), 1.0% potassium phosphate, 1.0% ammonium sulfate, and 0.1% enzyme extract was mixed for 20 to 30 days.
〆Put in the customer's jar fermentor and heat at 120qC for 20 minutes.
After steam sterilization, Benicillium multicolor KU-○-132 was cultured separately in the same medium 400' in advance.
The Mori seed fungus was inoculated, and Aedato Azusa culture was carried out for 6 days at 300°C.

After culturing, solid substances such as bacterial cells were removed by centrifugation to obtain a clear culture broth. This culture had 150 m/i 3-galactosidase. Next, 15% of the culture broth was concentrated to 1/10 using an ultrafilter membrane with a molecular weight cutoff of 13,000.

Claims (1)

[Scope of Claims] 1. When a β-galactosidase-producing strain belonging to the genus Penicillium is cultured in liquid under aerobic conditions and β-galactosidase is collected from the culture, a plant having galactose as a component in the medium is used. Polysaccharides with an average particle size of 42
A method for producing β-galactosidase, which comprises culturing in the presence of a water-insoluble substance with a size of 350 microns or less. 2 Plant polysaccharides include vectin, locust bean gum,
The β-galactosidase according to claim 1, which is a polysaccharide containing galactose as a part of its constituents, such as guar gum, gum arabic, tamarind gum, gum gatu, gum karaya, gum tragacanth, gum larch, gum gum, or gum galactomanna. manufacturing method. 2 Water-insoluble substances with an average particle size of 42 mesh (350 microns) or less are organic substances such as grain powder, legume powder, cereal cortex powder, legume coat powder, germ powder, bran, dried yeast powder, dried algae powder, cellulose powder, etc. A method for producing β-galactosidase according to claim 1. 4 Water-insoluble substances with an average particle size of 42 mesh (350 microns) or less include talc, clay, alumina, diatomaceous earth,
The method for producing β-galactosidase according to claim 1, which is an inorganic substance such as calcium carbonate or silica.