JPS6334B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a method for isolating β-galactosidase,
Specifically, the present invention relates to an improved method for efficiently separating β-galactosidase into two β-galactosidases that differ in substrate specificity, particularly in oligosaccharide production ability, using simpler operations.

Conventional technology β-galactosidase
is an enzyme that catalyzes the hydrolysis of β-galactoside bonds contained in lactose, etc. and galactoside rearrangement reaction, and is classified as enzyme number EC3.2.1.23 by the International Union of Biochemistry Committee, and its systematic name is β-D. −
Galactoside galactohydrolase (β-D
It is an enzyme called -galactoside (galactohydrolase). This enzyme has been used in various microorganisms such as Bacillus circulans.
circulans), Escherichia coli, Kluyveromyces lactis, Aspergillus
In addition to those originating from Aspergillus oryzae, Aspergillus niger, etc., those originating from plants and animals are known.

The present inventors previously obtained a β-galactosidase specimen (purified by ultrafiltration) originating from Bacillus circulans and further performed gel filtration using Sephadex G-150, ion exchange chromatography, and chromatography using a polybuffer exchanger. It has been found that when purified by focusing and electrophoresis using polyacrylamide gel, the above enzyme is separated and purified into two types of β-galactosidase () and () with different enzyme forms [Agricultural and Biological Chemistry (Agric.Biol.Chem.)
48(12), 3053-3061 (1984)].

Problems to be Solved by the Invention The two types of β-galactosidase () and () (hereinafter sometimes simply referred to as "enzyme" and "enzyme") have remarkable differences, especially in their oligosaccharide-producing ability. By separating these enzymes, enzymes with very weak oligosaccharide-producing ability can be used, for example, to degrade lactose in milk, and enzymes with strong oligosaccharide-producing ability can be used to produce galactooligosaccharides.
It was thought that each could be used advantageously. However, the above separation method requires very complicated steps and operations, and is completely difficult to implement on an industrial scale.Instead of the above method, there is a method that efficiently separates the enzyme with simpler operations. development was desired.

Means for Solving the Problems In view of the above-mentioned current situation, the present inventors have conducted extensive research with the aim of eliminating the shortcomings seen in the previously developed separation and purification method, and have developed a column chromatography method using hydroxyapatite gel. According to Graphi, it was discovered that the above object was achieved, and the present invention was completed.

That is, the present invention uses β-galactosidase derived from Bacillus circulans to differentiate β-galactosidase and β-galactosidase having different oligosaccharide-producing abilities by chromatography using hydroxyapatite gel.
The present invention relates to a method for separating β-galactosidase and galactosidase.

According to the method of the present invention, the above-mentioned enzymes can be efficiently separated with a very easy operation,
Each of the enzymes thus isolated can be used as a purified product in an extremely effective manner for unique purposes that take advantage of their properties.

In the method of the present invention, Bacillus is used as a raw material.
β-galactosidase derived from P. circulans is used. The enzyme, as also described in the above-mentioned literature,
Bacillus circulans (ATCC No. 31382), which is available in powder form or as a partially purified solution by ultrafiltration,
It can also be obtained by culturing known enzyme-producing bacteria belonging to the genus Circulans. This culture method and the physicochemical properties of the resulting enzyme are described, for example, in US Pat. No. 4,237,230.

In the present invention, it is essential that the β-galactosidase be subjected to chromatography using hydroxyapatite gel. As the hydroxyapatite used here, various known ones such as "Hydroxyapatite 25%
In addition to ``solids No. HO252'' (manufactured by Sigma), examples include hydroxyapatite gel manufactured by Taihei Kagaku Sangyo, sold by Hani Kagaku, manufactured by Nippon Chemical, and sold by Seikagaku Kogyo. Chromatography using this gel can be carried out in the same way as normal chromatography of this type, and generally involves filling a suitable column with the gel and passing the enzyme solution through it. The enzyme may be adsorbed onto the gel using a gel, and then the adsorbed enzyme may be eluted. A phosphate buffer can be used as an eluent for eluating the enzyme.

In the method of the present invention, it is particularly appropriate to elute the enzyme using the above-mentioned phosphate buffer according to the concentration gradient method. That is, the separation of the target enzyme requires a concentration of approximately 20 to
This can be carried out by sequentially passing the above-mentioned phosphoric acid buffer with increasing concentration in the range of 300 mM through the column. Through this procedure, contaminant proteins are first eluted with approximately 20mM phosphate buffer, and the target β-galactosidase is eluted with 110mM phosphate buffer.
The β-galactosidase of interest is eluted with 250-300 mM phosphate buffer.

Separation of both enzymes can thus be carried out. The isolated β-galactosidase and its interaction, substrate specificity, optimal pH, optimal temperature, molecular weight, temperature stability, metal ions, and other chemicals as described in the literature previously reported by the present inventors. They are the same in terms of physicochemical properties such as their effects on enzyme activity.

Examples Examples will be given below to explain the present invention in more detail.

Note that β-galactosidase activity in Examples was measured by the following method.

<β-galactosidase activity> Add 1 ml of this enzyme to 4 ml of 0.1M acetate buffer (PH6.0) containing 0.25% 2-nitrophenyl-β-D-galactopyranoside (ONPG), and incubate at 40°C for 15 minutes.
After reacting for a minute, 1 ml of the reaction solution was poured into 1 ml of 10% sodium carbonate to stop the reaction, and the o
- Determine the amount of ONPG decomposed by measuring the amount of nitrophenol. The amount of enzyme that hydrolyzes 1 μM ONPG per minute is defined as 1-ONPG unit.

Example 1 Add 0.3 g of lactose, 3 g of polypeptone, 1.5 g of meat extract, and 0.2 g of NaCl to water and mix the whole amount.
The volume was adjusted to 100 ml, and after adjusting the pH to 7.0, it was placed in a Sakaguchi flask and sterilized. One platinum loop of Bacillus circulans (ATCC31382) was inoculated into this,
The cells were cultured with shaking at 38°C for 1 day.

Ingredients listed below: Lactose 48g Fish powder 36g Pharmamedea (Traders Oil Mill) 18g Corn Day Tailor's Soluble 10g Yeast extract 1.95g CaCO ₃ 4g (NH ₄ ) ₂ SO ₄ 1.5g MnCl ₂ 0.02g Na ₂ CO ₃ 2.1 g of soybean oil (5 ml) was placed in a mini-jar fermentor, water was added to bring the volume to 1, and the mixture was sterilized. This was inoculated with 10 ml of the culture solution obtained above, and cultured with aeration at 38°C for 2 days.

A portion of the culture solution obtained above was centrifuged to remove bacterial cells to obtain an enzyme solution. This enzyme solution was subjected to salting out with 55% saturated ammonium sulfate, and centrifuged to obtain an enzyme precipitate. This was added to 20mM phosphate buffer (PH
6.0) to obtain a dialyzed enzyme solution.

The total activity of this enzyme solution was 4800 ONPG units.

Hydroxyapatite gel (Hani Kagakusha) 60
ml was packed into a column, and 250 ml of 20 mM phosphate buffer (PH6.0) was passed through the column at room temperature, and then the enzyme solution obtained above was passed through the column to adsorb the enzyme.

Next, add 20mM phosphate buffer (PH) to the above column.
6.0) 250ml, 50mM phosphate buffer (PH6.0) 450
ml, 110mM phosphate buffer (PH6.0) 450ml, 150
350 ml of mM phosphate buffer (PH6.0), 500 ml of 200 mM phosphate buffer (PH6.0), and 250 ml of 250 mM phosphate buffer (PH6.0) were sequentially passed through the tube to elute the enzyme.

The elution curve of the enzyme obtained above is shown in FIG. In Figure 1, the vertical axis represents the absorbance of the protein at 280 mμ (UV280, shown by the solid line) and the ONPG activity of β-galactosidase (shown by the broken line).
The horizontal axis shows the elution volume (ml). The figure also shows the concentration of phosphate buffer.

As is clear from FIG. 1 above, β-galactosidase is eluted with 250 mM phosphate buffer, and β-galactosidase is eluted with 110 mM phosphate buffer.

The β-galactosidase thus isolated had properties consistent with the physicochemical properties described in the above-mentioned literature.

[Brief explanation of the drawing]

FIG. 1 shows an elution curve of β-galactosidase according to an example of the present invention.

Claims (1)

[Claims] 1. A method for separating β-galactosidase, which comprises separating β-galactosidase derived from Bacillus circulans into β-galactosidase and β-galactosidase having different oligosaccharide-producing abilities by chromatography using hydroxyapatite gel.