JP2512769B2 - Method for producing yeast cell containing β-galactosidase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a yeast cell containing β-galactosidase.

(Prior Art) β-Galactosidase catalyzes the hydrolysis reaction and galactosyl group transfer reaction of β-galactoside bonds such as lactose and treats diarrhea due to lactose intolerance as an enzyme that decomposes lactose in dairy products. It has been used as a drug for the preparation, as a labeling enzyme for enzyme immunoassay, or as an enzyme for producing a galactooligosaccharide useful as a bifidus factor.

In addition, regarding the type, conventionally, various microorganisms,
For example, Bacillus circula
ns), Kluyveromyces la
ctis), Aspergillus oryz
ae), Escherichia coli, Cryptococcus yeast, etc., as well as those derived from plants such as Jack beans, and bovine liver and other animals. Those derived from organs are known.

As is well known, β-galactosidase catalyzes a hydrolysis reaction and, at the same time, transfers a galactosyl group,
The so-called transfer reaction is also catalyzed, but the reaction rates of the hydrolysis reaction and transfer reaction differ depending on the origin of the enzyme. For example, Aspergillus oryzae
, A β-galactosidase derived from Cryptococcus (Cryptococcus
β-galactosidase derived from yeast belonging to the genus tococcus has a relatively strong transfer activity, and a method for producing a galactooligosaccharide useful as a growth factor for Bifidobacterium has been proposed utilizing this property (Japanese Patent Publication No. 58-20266, (See JP 62-111685 A).

Other β-galactosidases that produce galacto-oligosaccharides include Bacillus circulans.
us circulans) β-galactosidase (Agric.Biol.C
hem.) 48 , 3053, (1984)], β-galactosidase of lactic acid bacteria [Journal of Daily Science (J. of
Dairy Sci.) 64, 185 ( 1981) ] it is known.

(Problems to be Solved by the Invention) As described above, various β-galactosidases that produce galactooligosaccharides are known, but there are problems in producing galactooligosaccharides using these β-galactosidases. As a result, since the hydrolysis reaction occurs at the same time, the raw material lactose is wasted, and glucose and galactose, which do not act as a growth factor of Bifidobacterium, are produced.

In this respect, β-galactosidase derived from Cryptococcus yeast is weak in hydrolysis activity and strong in translocation activity, so it is an excellent method for obtaining galacto-oligosaccharides in good yield, but it is also a raw material. A certain lactose is hydrolyzed to produce a monosaccharide (see JP-A-62-111685).

Therefore, some of the inventors of the present invention have previously reported that Lipomyces (Li
We have proposed a method for producing galacto-oligosaccharides using yeast of the genus Pomyces (see Japanese Patent Application No. 61-229223).
This strain produces β-galactosidase with strong transposition activity and produces galacto-oligosaccharides in a good yield in the cell reaction, but the β-galactosidase activity per bacterium resistance was not sufficient.

(Means for Solving Problems) As a result of intensive studies to solve such problems, the present inventors have found that by controlling the culture conditions of the yeast of the genus Lipomyces to specific conditions. The inventors have found that a β-galactosidase activity is remarkably improved and a bacterium having an improved ability to synthesize galacto-oligosaccharide is obtained, and the present invention has been completed.

That is, the present invention, when the yeast belonging to Lipomyces is cultured to produce yeast belonging to Lipomyces containing β-galactosidase as a bacterial cell, the pH during the culture is 4.5 to 8 weakly acidic or Β-galactosidase, which is characterized by carrying out a two-step culture comprising a first step of culturing while maintaining it in a weakly alkaline region and a second step of culturing while maintaining the pH during culturing in an acidic region of 3.0 or less The gist is the method for producing the yeast cells contained therein.

The yeast used in the present invention may be any strain as long as it has lactose assimilation ability and belongs to Lipomyces. Specifically, Lipomyces Starkey (Lipo
myces starkeyi) IFO 1289, Lipomyces lipofer IFO 0673, IFO 1288, etc., but especially in the present invention, Lipomyces (Lipomyces)
ces) NKD-14 (Microtechnology Research Institute of Microbiology No. 8948) is preferred.

The carbon source used in the culture in the present invention is most preferably a β-galactosidase-derived substrate such as lactose, and the nitrogen source is, for example, an organic nitrogen source such as peptone, casein, corn steep liquor, meat extract, yeast extract, or the like. An inorganic nitrogen source such as ammonium sulfate or ammonium chloride can be used. Especially when ammonium sulfate or ammonium chloride is used, the pH of the culture medium can be lowered by stopping the addition of alkali, since silver sulfate or silver chloride liberated with the consumption of nitrogen lowers the pH of the culture solution. Therefore, it is preferable. The concentration of the carbon source used is preferably 0.2 wt% to 30 wt%, preferably 10 wt% to 25 wt%, particularly preferably 15 wt% to 22 wt%. The concentration of nitrogen source is preferably 0.1 wt% to 10 wt%, and 1 wt% to 6 wt%.
% Is preferable, and 3 wt% to 5 wt% is particularly preferable. further,
If necessary, various vitamins, minerals, inorganic salts and the like may be added. The culture temperature is, for example, 20 ° C to 40 ° C.
℃, preferably 25 ℃ ~ 35 ℃, more preferably 28 ℃ ~ 32
° C.

In the present invention, the pH at the start of culturing is close to the growth extreme pH of the strain to be used, and the culturing is performed while adjusting the pH in the weakly acidic or weakly alkaline region of pH 4,5 to 8, and then at pH 3 in the logarithmic growth phase. Decrease the pH to a weak zone below 0.0 and continue the culture. The β-galactosidase activity gradually increases after the pH is lowered, and reaches its maximum after 12 to 48 hours. The culture can be terminated when the β-galactosidase activity is maximized, and the cells can be collected by a commonly used method such as centrifugation, filtration or decantation.

To produce galacto-oligosaccharides with the obtained bacterial cells,
For example, suspend the cells in 20% (w / v) lactose solution,
The reaction may be carried out at pH 6.5 at pH 6.5 for 24 hours. After the reaction was completed, the amount of galacto-oligosaccharide produced was measured by high performance liquid chromatography (column: Waters, Micro Bonder Pack / N).
H _2, mobile phase: quantified with acetonitrile / water = 7/3), β-
Galactosidase activity can be measured.

(Example) Next, the present invention will be specifically described with reference to Examples.

In the examples,% means% by weight unless otherwise specified.
Represents

Example 1 Lactose 20%, ammonium sulfate 4%, KH ₂ PO ₄ 0.3%, MgSO ₄ .7H ₂ O
Medium 20 consisting of 0.15%, biotin 30 μg /, vitamin B ₁₃ 3 mg / was charged into a 30-volume jar fermenter, and after sterilization, the pH was adjusted to 6.0.

A jar fermenter was inoculated with Lipomyces NKD-14 (Microtech Lab. No. 8948) 1 pre-incubated at 30 ℃ for 24 hours in a medium of the same composition, at 30 ℃, aeration rate 20 / min, impeller Rotating speed 600rpm, adjusting pH to 6 with 4N NaOH
It was cultured for 34 hours. The bacterial concentration at this time is 2.0% (dry weight)
Met.

Next, at this point, the dropping of 4N NOH was stopped and the p
While lowering H to 2.0 and maintaining pH at 2.0,
The culture was continued for another 14 hours. The bacterial concentration after the culture is 3.
It was 8% (dry weight).

Table 1 shows the relationship between the culture time in the main culture, the pH of the medium, the β-galactosidase activity, and the bacterial concentration.

For the measurement of β-galactosidase activity, 20% lactose was used as a substrate, 2% dry cells were added, and pH was adjusted to 6.
After reacting at 5,30 ℃ for 24 hours, the supernatant was analyzed with a Waters high performance liquid chromatography column, Microbonder Pack / NH ₂ (mobile phase: acetonitrile / water = 7/3), and the galacto-oligosaccharide after reaction was analyzed. It was displayed as the production concentration (%, w / v).

From the results in Table 1, the β-galactosidase activity increased 5.0 times after the medium pH was shifted to 2.0, and the effect of the present invention is clear. At the end of the culture, the yield of lactose from the cells was 19%.

Comparative Example 1 Culture was carried out in the same manner as in Example 1 except that the pH was adjusted to 6.0 until the 48th hour of culture without lowering the pH to 2.0.

 The results are summarized in Table-2.

From the results shown in Table-2, β-
It is clear that little increase in galactosidase activity is seen. In addition, the yield of sugar to the bacterium at the end of culture is
It was 19.5%.

(Effect of the Invention) According to the present invention, a yeast cell containing a highly active β-galactosidase can be obtained in a high yield only by a simple operation of changing pH during culture.

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Claims (1)

(57) [Claims] 1. β- by culturing yeast belonging to Lipomyces
When producing yeast cells belonging to lipomyces containing galactosidase, the first step of culturing while maintaining the pH during culturing in the weakly acidic or weakly alkaline region of 4.5 to 8, and the acidic region of 3.0 or less in the culturing pH A method for producing a yeast cell containing β-galactosidase, which comprises culturing in two steps, that is, a second step of culturing while maintaining the above.