JP2687239B2 - Method for producing β-galactosidase bacterial enzyme - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a β-galactosidase microbial enzyme.

[Conventional technology]

β-Galactosidase has an action of hydrolyzing lactose into glucose and galactose and an action of causing β-galactosyl transfer reaction of lactose, and is used for producing lactose-decomposed milk and galacto-olicosaccharide.

β-galactosidase is Aspergillus oryzae,
It is produced in the cells of various bacteria such as Streptococcus thermophilus, yeast, etc., and conventionally, it is taken out of the cells and used for the enzymatic reaction. If the activity of β-galactosidase can be expressed in the cells and used as it is for enzyme reaction in the form of so-called cell enzyme, separation / purification of the enzyme becomes unnecessary and the utilization rate is improved. In the case of enzymes, it is hardly done. The main reason seems to be that the use of this enzyme is mostly limited to the food manufacturing field. That is, bacterial enzymes are generally produced by a method of treating bacterial cells with an organic solvent such as toluene, acetone, methanol, ethanol or a surfactant such as SDS (sodium dodecylbenzene sulfonate). Many activators are also toxic. In particular, the conventionally used surfactants such as SDS are highly toxic and it is difficult to completely remove them after the treatment, so that it was difficult to use them for the production of bacterial enzyme for food production. Furthermore, the organic solvent is flammable and requires careful handling and recovery. Since it is also a protein denaturing agent, the enzyme may be inactivated unless the treatment conditions are strictly controlled.

[Problems to be solved by the invention]

Therefore, an object of the present invention is to provide a method for inexpensively and easily producing a β-galactosidase microbial enzyme that can be safely used in food production.

[Means for solving the problem]

Generally, it is explained that the enzyme in a microbial cell exerts its activity by treatment with an organic solvent or a detergent because the selective permeability of the cell wall or cell membrane is changed. That is, in living cells, selective permeation of substances by cell walls and cell membranes impedes the permeation of substrates into cells, but when cells are treated with an organic solvent or a surfactant, they are present in the surface layer or gaps of cell walls or cell membranes. The lipid is extracted and the selective permeability is changed, and the permeability of the substrate and the reaction product is increased to allow the reaction between the intracellular enzyme and the extracellular substrate.

However, whether or not it is possible to produce a practical bacterial enzyme by expressing the enzyme activity by the mechanism as described above,
It depends on the type of enzyme and substrate, and not all organic solvents and surfactants are effective. In the case of β-galactosidase-producing bacteria, the surfactants that give β-galactosidase microbial enzyme having selective permeability capable of taking up lactose as a substrate and releasing transfer oligosaccharide are extremely limited, and its HLB value is particularly important. It was confirmed that in most cases, it should be 16 or more.

The present invention has been completed based on the above findings,
It is characterized by culturing a microorganism that produces β-galactosidase in the cell, and treating the obtained bacterial cells with a surfactant having an HLB value of 16 or more consisting of sucrose fatty acid ester or lysophospholipid or chirasaraponin. .

Among the surfactants used in the β-galactosidase microbial enzyme production method of the present invention, as the sucrose fatty acid ester-based surfactant, those having an HLB value of 16 or more may be selected from commercial products and used. Since quillaja saponin is a natural product and does not have various HLB values, commercial products can be used as they are. In addition, HLB value of 16 or more lysophospholipid, soybean lecithin, egg yolk lecithin natural phospholipids and polyhydric alcohol produced by phosphatidyl group transfer reaction lecithin such as phosphatidylglycerol lipase or phospholipase.
It is a substance represented by the following general formula, which can be produced by treatment with A ₂ and partial hydrolysis.

(In the formula, R ¹ and R ² represent a hydrogen atom or an acyl residue of a fatty acid. However, ^one of R ¹ and R ² is a hydrogen atom and the other is an acyl group. X is a polyhydric alcohol. From the above, an organic group remaining after removing one hydrogen atom of the arbitrary hydroxyl group is shown.) Particularly preferred in this lysophospholipid is a polyhydric alcohol corresponding to the organic group X in the above general formula, ethylene glycol, glycerol, sorbitol, Mannitol, glucose, galactose, fructose, sucrose, or lactose.

Neither sucrose fatty acid ester nor lysophospholipid having an HLB value of less than 16 can be used because they do not provide the cell membrane with the selective permeability necessary for achieving the object of the present invention.
Soybean lecithin, lyso-type lecithin, and transfer lecithin (eg, phosphatidylglycerol) are commonly used as phospholipid-based surfactants, but these have HLB values of 12 to 1
Probably because there are only about 5 of them, the enzyme activity cannot be expressed in any of them.

When the β-galactosidase bacterial enzyme is produced by the production method of the present invention, a microorganism that produces β-galactosidase in the bacterial cell and is suitable for use as the bacterial enzyme, for example, Streptococcus thermophilus. , Lactobacillus bulgaricus, Streptococcus lactis, Lactobacillus salivarius, Lactobacillus reichmannii, Lactobacillus
Helvetics, Bacillus brevis, Bacillus stearothermophilus, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium addressacetis, Kluyveromyces fragilis, Kluyveromyces lactis, Candida pseudotropicalis, Brera singularis and the like are cultured by a conventional method.
The obtained culture broth may be subjected to a detergent treatment as it is, but it is desirable to carry out an enzyme treatment after making a concentrated cell suspension from which medium components have been removed by operations such as centrifugation and washing.

The treatment temperature may be 20 ° C. or higher as long as the β-galactosidase is not inactivated, and the treatment effect is higher at a temperature as high as possible, but usually 30 to 55 ° C. is suitable. In the treatment liquid, substances such as glycerin and sugar liquid that improve the thermal stability of the enzyme can coexist.
A temperature rising process up to ℃ is possible. Since the enzyme is unstable and the storage stability of the product is poor if the microorganisms are alive even after the treatment with the surfactant, it is advantageous to raise the treatment temperature to improve the stability by sterilization.

Surfactant has a concentration of about 0.
Add 3 to 3%. The required treatment time depends on the treatment temperature, but when treating at around 50 ° C, it takes about 15
Minutes to 2 hours.

Since all the surfactants used in the present invention are highly safe, it is sufficient to centrifuge the cells after the treatment to simply wash them, and the surfactants may be left as they are.

The β-galactosidase microbial enzyme of the present invention is a reaction for hydrolyzing lactose in milk as a measure for lactose intolerance, a transfer reaction for producing galactooligosaccharide as a Bifidobacterium growth promoter from lactose, β- As an enzyme for the reaction using galactosidase, it can be used in the same manner as the extracellular enzyme.

〔Example〕

Hereinafter, the present invention will be described with reference to Examples and Comparative Examples. The bacterial cell suspension used in each example was cultured in a medium containing 0.5% yeast extract in skim milk powder to give β-into the bacterial cells.
The culture solution of Streptococcus thermophilus accumulating galactosidase was centrifuged to collect the bacterial cells, which was dispersed in water and then centrifuged again in the same manner,
It was prepared by adding 0.01 M potassium phosphate buffer to the obtained cells and stirring. The β-galactosidase activity of the bacterial cells after the treatment with the surfactant was measured as follows.

β-galactosidase activity measurement method: The bacterial cells after the treatment with the surfactant were diluted with 0.01 M potassium phosphate buffer, and 0.1 ml thereof was diluted.
0.01M potassium phosphate buffer containing 2% lactose (pH
7.0, containing Mg ²⁺ 10ppm) 0.9 ml, and react exactly at 40 ° C for 30 minutes. Then, the enzyme is inactivated by heating at 100 ° C. for 5 minutes, and glucose produced by the enzymatic reaction is colorimetrically determined.

Examples 1 to 6 and Comparative Examples 1 to 7 To 2 ml of the above-mentioned cell suspension of Streptococcus thermophilus, 0.2 ml of a 10% aqueous solution of a surfactant was added, and the mixture was stirred at 50 ° C. for 30 minutes.
A heating process was performed for a minute.

 The surfactants used for the treatment are as follows.

Example 1: Sucrose fatty acid ester / DK-SS (Daiichi Kogyo Seiyaku Co., Ltd.) Example 2: Sucrose fatty acid ester / DK-SL (Daiichi Kogyo Seiyaku Co., Ltd.) Example 3: Sucrose fatty acid ester / DK -SL18A (Daiichi Kogyo Seiyaku Co., Ltd.) Example 4: A product obtained by treating transferred lecithin containing 84% by weight of phosphatidylglycerol with pancreatin (Sigma) and hydrolyzing the β-position. Contains 89% by weight of lysophosphatidylglycerol.

Example 5: A product containing 60% by weight of lysophosphatidylglycerol, which was obtained in the same manner as in Example 4 from the transferred lecithin containing 30% by weight of phosphatidylglycerol.

Example 6: Kirasayaponin (Maruzen Kasei Co., Ltd.) Comparative Example 1: Sorbitan fatty acid ester span 20 (Tokyo Kasei Co., Ltd.) Comparative Example 2: Glycerin fatty acid ester Suntone (Derky Co.) Comparative Example 3: Enzyme-treated lecithin Hermizer A (Kyowa Hakko Co., Ltd.) Comparative Examples 4 to 7: Sucrose fatty acid ester Reference Example 1 20 ml of ethanol was added to 20 ml of the bacterial cell suspension, and the mixture was stirred at 20 ° C. for 30 minutes.
Minutes. Then 0.01M potassium phosphate buffer 10ml
Was added to dilute, and the mixture was centrifuged to remove ethanol, and the total volume was adjusted to 2 ml with the same buffer.

Reference Example 2 Untreated bacterial cell suspension Reference Example 3 The bacterial cell suspension was sonicated for 15 minutes to crush the bacterial cells to obtain an enzyme solution.

Reference Example 4 The same treatment as in Example was carried out using sodium dodecylbenzenesulfonate as a surfactant.

Table 1 shows the results of measuring the β-galactosidase activity of the bacterial cells or enzyme solutions of the above examples. The enzyme activity shown in the table is a relative value based on the enzyme activity of Reference Example 1 as 100. Since the untreated cells of Reference Example 2 also exhibited 15 activities, it is considered that those having an activity of 20 or less had virtually no treatment effect.

Table 1 HLB value Enzyme activity Example 1 19 99 Example 2 20 108 Example 3 16 108 Example 4 22 116 Example 5 25 98 Example 6-98 Comparative Example 1 8 18 Comparative Example 2 2 17 Comparative Example 3 11 18 Comparative Example 4 2 18 Comparative Example 5 6 18 Comparative Example 6 11 16 Comparative Example 7 15 20 Reference Example 1 100 Reference Example 2 15 Reference Example 3 104 Reference Example 4 40 118 [Effect of the Invention] As described above, the present invention According to the method, a β-galactosidase microbial enzyme for food production, which has not been put into practical use because there is no suitable activation method, can be produced safely and easily. The activity of the bacterial enzyme obtained by the production method of the present invention is comparable to that obtained by the conventional typical activation method using ethanol or sodium dodecylbenzesulfonate.

Since the β-galactosidase microbial enzyme is a kind of immobilized enzyme in which the enzyme is immobilized on the microbial cells, it can be repeatedly used after being used for the enzymatic reaction and then easily recovered by centrifugation or filtration. It can also be used as an immobilized enzyme which is further immobilized on a carrier. Therefore, the enzymatic reaction can be performed at a much lower cost than using β-galactosidase taken out by destroying cells,
Further, there is an advantage that the reaction product can be easily purified.

Claims (1)

(57) [Claims] 1. A method of culturing a β-galactosidase-producing microorganism in a cell, and treating the resulting cell body with a surfactant having an HLB value of 16 or more composed of sucrose fatty acid ester or lysophospholipid or Kirasa yaponin. The method for producing a β-galactosidase microbial cell enzyme.