JPS5816879B2 - Shinkinakosogaizairactum B - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention produces a novel enzyme inhibitor, lactam B, by converting nojirimycin B into lactam B using an enzyme that oxidizes nojirimycin B or a microbial cell containing this enzyme. It relates to a manufacturing method.

Nojirimycin B is a new compound with the molecular formula C6H1305N, which is a compound of nojirimycin (5-amino-5deoxy-D-glucobylanose) previously discovered by the present inventors.
It is one of the structural isomers of , and is an enzyme inhibitor that has a strong inhibitory effect on β-glucosidase.
-73748).

The present inventors have previously discovered that nojirimycin is easily oxidized and converted to D-gluconic-δ-lactam by the action of glucose oxidase or a microbial cell containing glucose oxidase. A manufacturing method was established (see specification of Japanese Patent Application No. 106928/1983).

When this method was applied to nojirimycin B, we found that nojirimycin B was also oxidized like nojirimycin, and as a result of various studies on the converted substances produced by the oxidation, we found that its properties were extremely similar to D-gluconic-δ-lactam. They discovered that this substance was similar to the previous one, but had clearly different physical and chemical properties, so they named the main substance lactam B and completed the present invention.

The nojirimycin B oxidase used in the present invention refers to an enzyme having the ability to oxidize nojirimycin B, and glucose oxidase can generally be used as the nojirimycin B oxidase.

Glucose oxidase and glucose dehydrogenase are known as glucose oxidase, and examples of glucose oxidase include crude enzyme preparation Deoxin (Nagashio Sangyo Membrane) extracted from a culture of the known bacterium Penicillium amagasakininsis, and Aspergillus. - Glucose oxidase extracted from Niger (manufactured by Miles Laboratory, USA) is known, but nojirimycin B is easily oxidized and converted to lactam B by these glucose oxidases.

On the other hand, regarding glucose dehydrogenase, although this enzyme exists widely in the animal and plant kingdoms, it is desirable to use a microbial enzyme for the purpose of the present invention.

Since microbial glucose dehydrogenase is an intracellular enzyme, it is industrially more advantageous to use microbial cells that retain and contain the enzyme, rather than separating and purifying the enzyme from microbial cells.

Gluconic acid-fermenting bacteria are known as a representative example of microorganisms that produce glucose dehydrogenase.

Gluconic acid-fermenting bacteria are widely present in molds and bacteria, and among molds, they are found in the genus Aspergillus and Penicillium.
and filamentous fungi of the genus Rhizopus, and among bacteria, Ace tobacter.
r), Gluconobacter spp.
ac ter ) and Pseudomonas spp.
onas) is known to exist.

In order to oxidize nojirimycin B by the method of the present invention using these bacteria, it is first necessary to culture them in a commonly used culture medium containing carbohydrates, proteins, amino acids, inorganic salts, and the like.

Particularly preferred media include those consisting of glucose broth, sodium glutamate, and inorganic salts.

The culture temperature is selected from the range of 20 to 37 degrees Celsius, and the culture time varies depending on the culture method such as aeration, shaking, and standing;
70 hours is appropriate.

It is preferable that the cultured bacterial cells are separated and collected from the culture process, washed with an inorganic or organic buffer solution, and then used.

On the other hand, nojirimycin B is used as a raw material in the method of the present invention, but since this substance is produced simultaneously with nojirimycin during the culture production of nojirimycin, it is purified from the culture P solution to produce only nojirimycin B. It can be isolated and used.

However, using a partially purified nojirimycin, which is a mixture with nojirimycin obtained in the process of purifying nojirimycin B, this is allowed to interact with enzymes or bacterial cells, and lactam B is separated and purified from the reaction mixture. is also an advantageous method.

In the oxidation reaction in which an oxidase or a microbial cell containing an oxidase acts on nojirimycin B, nojirimycin B or partially purified nojirimycin B (including nojirimycin) is added to an appropriate buffer solution (neutral to weakly alkaline). )
It is preferable to use it by dissolving it in a concentration of 2 to 20%.

When using microbial cells, the appropriate amount of microbial cells to be added to the nojirimycin B solution is 115 to 79174978 wet weight to allow the reaction to proceed relatively quickly.

The oxidation reaction is suitably carried out at 20 to 40°C, preferably with shaking or stirring.

The reaction can be completed in 1 to 24 hours.

The cells used in the reaction may be in a wet state, such as washed cells, or dried or freeze-dried cells.

Moreover, it is not only possible to carry out a single reaction, but also to separate and collect the bacterial cells after the completion of the reaction, and to reuse them.

The method for producing lactam B from nojirimycin B using bacterial cells described above can be applied almost directly to the conversion reaction of nojirimycin B to lactam B using glucose oxidase or crude enzyme preparations containing these enzymes. I can do it.

That is, a glucose oxidase preparation is added to an aqueous solution in which nojirimycin B or nojirimycin 8-part purified product is dissolved at an appropriate concentration sufficient for the enzyme reaction to proceed, and the pH is adjusted to 5 to 85.
Aerate and stir between

The reaction temperature is permissible from room temperature to about 60°C, but from 30 to 60°C.
40°C is most preferred.

The reaction time depends on the reaction temperature and enzyme concentration, but is usually 2 to 7 days.
Completed in 0 hours.

In separating lactam B from the reaction-completed solution, if necessary, microbial cells and the like are separated from the reaction solution by filtration or centrifugation to obtain solution P.

This F solution is passed through a tower made of a strongly acidic ion exchange resin, such as Amberlite t-IR-120 (H type), and unreacted nojirimycin B and nojirimycin are removed, and the passed liquid is passed through a column made of a strong acidic ion exchange resin, such as Amberlite t-IR-120 (H type). Amber Light ■R-45 (
Neutralize with OH type) and concentrate.

At this time, if the oxidation reaction is carried out with high yield, lactam B crystals or lactam B and D-gluconic-δ
-Lactam mixed crystals precipitate directly.

If lactam B contains impurities or D-gluconic-δ
- If it is precipitated as a mixed crystal with a lactam, it is possible to separate and purify lactam B by carbon column chromatography or other means and then concentrate it to obtain the desired product as a crystal.

If necessary, it can also be purified by a recrystallization method from water-alcohol.

Although this is an assay method for lactam B, this substance has no antibacterial activity.
Although there is no suitable assay method, it has a strong β-glucosidase inhibitory effect.

For example, according to the measurement method described in Agricultural and Biological Chemistry, Vol. 34, p. 966, 1970, 50% of lactam B to apricot emulsin
The % inhibitory concentration is 1. I×10”-6M.

Therefore 50% of lactam B for β-glucosidase
The inhibitory concentration can be determined and the content of lactam B can be determined, but only if enzyme inhibitors other than lactam B are not included.

Therefore, it is convenient to use silica gel thin layer chromatography, which will be described later, to determine the state of conversion of nojirimycin B to lactam B by enzymes or microbial cells.

The physicochemical properties of the novel enzyme inhibitor lactam B produced by the method of the present invention are as shown below.

This substance is a white needle-shaped crystal with a melting point of 140-141°C.

It is well soluble in water, but slightly soluble or insoluble in common organic solvents.

The ultraviolet absorption of this substance shows no specific absorption in the range 210-360 mμ, and the optical external absorption curve is shown in FIG.

The specific rotation measured in an aqueous solution at a concentration of 1% is [a] 20
It was +1.6°.

The elemental analysis values of this substance are C40,53, H6,28, N
It showed a value of 7.54%.

Lactam B was acetylated with acetic anhydride-pyridine to give a tetraacetylated product, and when this product was subjected to mass spectrometry, it showed an M+1 peak at m/e 346.

Therefore, the molecular weight of lactam B is 177, and the most suitable molecular formula is 06H1105N.

Since this molecular formula is the same as D-gluconic-δ-lactam, which is an oxide of nojirimycin, lactam B
is strongly suggested to be a structural variant of D-gluconic-δ-lactam.

In silica gel thin layer chromatography using a system of n-butanol-acetic acid-water (3:1:1), the Rf value of D-gluconic-δ-lactam is 0.29, and the Rf value of lactam B is Since it is 0.33, it is clear that the two are different substances.

Example 1 Take Nojirimycin B200■ in a test tube and add 10% of distilled water.
m1 was added and dissolved, and deoxin (iooo unit/
7IIg, manufactured by Nagashio Sangyo) was added and the pH was adjusted to 6.0.
After adjusting the temperature, a shaking reaction was performed at 28° C. for 6 hours using a tube shaker.

The reaction completed liquid was passed through a small column (3 ml) of Amberlite IR-120 (H type), further 5 Tll of distilled water was passed through the column, the passed liquid and the washing liquid were combined, and the mixture was transferred to Amberlite IR-45 (OH type). After neutralization, a small amount of charcoal powder (100
7III is added to decolorize.

When the coal powder is filtered and the slurry is concentrated under reduced pressure, lactam B crystals are precipitated.

The crystals were left to stand for a while at 5°C to allow photons to precipitate, and then the crystals were separated and dried under vacuum to produce 135 mI of white needle-like crystals of lactam B! I got it.

(Yield 68%) Example 2 2% sucrose, 0°5% peptone, 0.2% yeast extract
, Sodium glutamate 0.2%, K2HPO4°0.2
%, MgC720,01%, FeSO40,001% and MnSO40,001% (pH 6,
After dispensing 11 IC of 8) into test tubes and sterilizing them, Aspergillus niger IAM 2094, IJ Zopus deremer IAM 6015, Gluconobacter suboxidans IAM 1829 and Pseudomonas oparis IFO1 were added.
2051 was inoculated with one platinum mushroom, and cultured for 42 hours in a tube shaker at 28°C.

Centrifuge each cultured bacterial cell and adjust the pH to 7,5,0,0.
Wash with 5M phosphate buffer to obtain washed bacterial cells.

2% Nojirimycin B solution (dissolved in pH 7, 5, 0,05M phosphate buffer) ' Transfer 2 ml to a test tube, add 40 μ of each washed bacterial cell (wet), and shake in a tube shaker at 28°C for 16 hours. After the reaction, each reaction slurry is obtained by centrifugation.

Pour the mud into a small column (2ml) of Amberlite 1R120.
The column was washed with 5 ml of distilled water, the passed through liquid and the washing liquid were combined, and the dilution factor required to show 50% inhibition of apricot emulsin was determined, and the content of lactam B was calculated.

The results are shown in the table below.

Table 1 Production of lactam B by microbial oxidation of nojirimycin B Microorganism used Lactam B yield Aspergillus niger 23.1% AM20
9 Rhizopus telemer 33.2%IAM
6015 Microorganisms used Lactam B yield Pluconobacter suboxidans IAM1829 98.0% Pseudomonas obalis IFO1205178.5% Example 3 500 ml of the same liquid medium as in Example 2, 15 bottles of 100 ml per Yosaka Lough flask After dispensing and sterilizing with cotton plugs (120°C, 15 minutes), one platinum loop of Gluconobacter suboxidans IAM1829 was inoculated.
Incubate in a reciprocating shaker at ℃ for 44 hours.

Centrifuge the culture process to collect bacteria. The obtained bacterial cells were adjusted to pH 7,
Washed with 5,0,05M phosphate buffer, washed bacterial cells 11
．． Obtained 5S'.

30 g of partially purified nojirimycin B (lyophilized powder, containing about 90% nojirimycin) obtained in the nojirimycin purification process was dissolved in a 3% concentration of pH 7,5,0,05M phosphate buffer. Add 10 g (wet) of the previously obtained washed Gluconobacter suboxycens cells and heat at 30°C.
The reaction was stirred for 7 hours.

The reaction mixture was centrifuged to remove bacteria, and the supernatant liquid was passed through a column of Amberlite IR-120 (H type) 4007121, and then 600'/Ill of distilled water was passed through the column to wash the column with water. The liquids were combined to obtain 1.5 tons of liquid.

When this is neutralized using Amberly 1-IR-45 (OH type) and the neutralized solution is concentrated under reduced pressure, D-gluconic-δ-lactam crystals (18,7P) are precipitated.

Further concentration under reduced pressure yields lactam B and D-gluconic.
A mixed crystal with δ-lactam was precipitated, which was separated and dried under vacuum to obtain a mixed crystal of lactam B 3.1f (lactam B content: about 50%).

Example 4 20m of 2.9v of the lactam B mixed crystal obtained in Example 3
1 in distilled water, applied to a 900 ml carbon column (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd., for chromatography), and developed with distilled water.

When the mixture is fractionated into 20 ml fractions in each test tube, D-gluconic-δ-lactam is eluted first, and then lactam B is eluted.

Each fraction was examined by thin layer chromatography, and both fractions containing only lactam B (fractions! 103 to 162) were collected (approximately 1.27) and concentrated under reduced pressure to a volume of approximately 30 ml. To this, an equal amount of methanol was added and further concentrated. Crystals of B precipitate.

This was vacuum-dried separately, and white needle-shaped crystals of lactam B 88
01 ng was obtained.

An equal amount of methanol was added to the crystal mother liquor and concentrated under reduced pressure again to precipitate crystals, which were separated and dried under vacuum to obtain a second crystal of lactam B 590~.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption curve diagram of lactam B produced by the method of the present invention.

Claims (1)

[Claims] 1 Nojirimycin B or a partially purified product of Nojirimycin B is reacted with microbial cells that produce and contain Nojirimycin B oxidase or this fermentation enzyme to produce Nojirimycin B.
A method for producing lactam B, which comprises converting lactam B into lactam B, and collecting lactam B from the reaction mixture.