JPS63304994A - Production of leucrose by dextran sucrase produced by streptococcus bovis - Google Patents

Abstract

PURPOSE:To obtain leucrose, an ligosaccharide useful as a sweetener for preventing fatness, by treating sucrose with dextran sucrase produced by Streptococcus bovis. CONSTITUTION:Streptococcus bovis is anaerobically cultivated in a medium containing a carbon source (sucrose or glucose) to introduce dextran sucrase to produce dextran sucrase. The dextran sucrase as an acceptor is treated with sucrose in the presence or absence of fructose. 148 strain (FERM P-9390) belonging to the Streptococcus bovis collected from a bovine rumen is preferably used as the Streptococcus bovis.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applicable to Streptococcus and Streptococcus bovis.
ococcus bovis s) produced by dextransucrase (dextr
a, n5ucrase) by leucrose (1euc
The present invention relates to a method of manufacturing rose.

Oligosaccharides such as leuclose are expected to have a wide range of uses in the pharmaceutical and food industries, including as sweeteners to prevent obesity, growth factors for bifidobacteria, and sweeteners to prevent dental caries.

[Conventional technology]

Roy Claus is HΦ H Stodola (H,Hy
Stodola et al.
It has been reported that a small amount of leuclose is produced when dextran is synthesized using L.
Journal of the American Chemical Society (J, As, Ches.

Soc, Vol. 78, p. 2514 (19513)).

It is also known that leuclose is generated by the translocation action of dextransucrase (E. J. Bourne et al., Biochemical
Journal (Bloches, J., vol. 79, p. 549 (1981)).

Conventional methods for producing leuclose involve separating leuucrose produced as a by-product during dextran synthesis, and are basically divided into fermentation methods and enzymatic methods, both of which involve the use of L. mesenteroides. It is being done.

In the fermentation method, L. mesenteroides is cultured in a medium containing sucrose, and a small amount of leucrose, which is bundled as a by-product in the medium, is separated and purified through various steps.

In the enzymatic method, L. mesenteroides is cultured in a medium containing sucrose, and dextransucrase produced in the medium is used as an enzyme source to perform an enzymatic reaction in a sucrose-fructose mixed system to produce leuucrose.

[Problem to be solved by the invention]

However, the L. mesenteroides used in this production method has a high nutritional requirement, so its cultivation requires relatively expensive nutritional components such as various vitamins and amino acids.Moreover, its growth rate is slow, so L. It takes several days for production.

In addition, in the enzymatic method, textransucrase, which is a complete substrate-inducing enzyme, is produced by L. mesenteroides only in sucrose medium, so a large amount of dextran accumulates in the fermentation solution, making it difficult to recover the produced enzyme. At the same time, it is easily inactivated by complex formation with dextran, and the optimum temperature for enzyme action is relatively low at 30°C, so the reaction rate is low.

Further, the yield in the fermentation method is extremely low, and the production efficiency in the enzyme method is low at 2 to 6% based on consumed sugar under enzyme reaction conditions of 2% sucrose.

Therefore, it is desired to develop a new process that can rapidly produce leuclose in high yield.

The present inventors have conducted intensive research in view of the above circumstances, and have found that a method for producing leucrose using dextransucrase produced by Nis bovis isolated from the rumen of cows (hereinafter referred to as cow rumen) has been developed in culture.・Bovis has low auxotrophy and requires only biotin as a vitamin and grows on only inorganic nitrogen as nitrogen nutrition, and its growth rate is in the log phase 2~
It is extremely fast with a generation change time of about 10 minutes and produces dextransucrase efficiently in a glucose medium, and since no dextran is produced at this time, the sterilization effect can be easily performed. However, because it does not form a complex with dextran, there is almost no decrease in enzyme activity during the reaction, and the optimum temperature for the enzyme action of the produced enzyme is relatively high at 40°C. It has the advantage that the yield of leucrose under enzymatic reaction conditions is as high as 30% based on consumed sugar, and is much superior to the conventional production method using dextransucrase produced by L. mesenteroides. This discovery led to the completion of the present invention.

[Means for solving problems]

That is, the present invention cultivates Nis bovis anaerobically in a medium containing a carbon source that induces dextransucrase,
The present invention relates to a method for producing leuucrose using dextransucrase produced by Nis-Bovis, which is characterized by reacting the produced dextransucrase with sucrose or with sucrose in the presence of a receptor.

[Function and Examples]

A representative example of the dextransucrase-producing bacteria used in the method of the present invention is strain 148 isolated from bovine rumen.

This strain is non-motile, Durham staining is positive, and the bacteriological form is streptococci. This strain also showed a negative result in the catalase test, showing so-called homo-lactic acid fermentation in which glucose is metabolized to dextrorotatory lactic acid through the EMP pathway.

Further, no growth was observed at 10°C, growth was observed at 45°C, and no growth was observed at a NaCl concentration of 6.5% or at a pH of 9.8 or higher. In addition, it strongly decomposes starch, 40%
Bile blood agar sedl
It has the property of growing in um). From the stated mycological properties,
It is clear that strain 148 belongs to Nis bovis, which is described in Bersey's Manual of Determinative Bacteriology, 8th edition. Therefore, this bacterial strain was designated as the 14g strain belonging to Nis bovis. This strain has been assigned the accession number FERN P-939 to the Institute of Microbiology, Agency of Industrial Science and Technology.
0).

It should be noted that mycological properties are extremely susceptible to mutation, and it is a well-known fact that mutations can occur due to natural or artificial mutations (for example, ultraviolet irradiation, use of mutagens such as nitrosoguanidine, etc.). All strains belonging to Nis bovis, including naturally occurring mutant strains, can be used in the present invention as dextransucrase-producing bacteria.

To produce leuclose, first, dextransucrase-producing bacteria belonging to Nis bovis are inoculated into a nutrient medium containing a carbon source that induces dextransucrase, and cultured anaerobically to obtain dextransucrase.

Nutrient media used for culturing dextransucrase-producing bacteria include carbon sources such as sugars that can induce dextransucrase such as sucrose and glucose, inorganic nitrogen sources such as ammonium sulfate and ammonium nitrate, organic nitrogen sources such as yeast extract and meat extract, and sodium chloride. ,
A medium to which inorganic salts such as phosphates and calcium chloride, and trace amounts of vitamins such as biotin are added as necessary, is usually used, and liquid culture is preferred as the culture method.

In particular, it is preferable to use glucose as a carbon source to avoid producing dextran, which can interfere with the purification of dextransucrase and cause a decrease in enzyme activity. Sucrose with high efficiency may also be used.

Dextransucrase can be produced by inoculating Nis bovis 148iii strain into a nutrient medium, for example, while passing carbon dioxide gas through the medium, at a temperature of 25 to 50°C, preferably around 40°C, and at a pH of 5.0°C, for example, while adding Na2CO3. 7, preferably 6 to 6.5, and is obtained in a culture solution by terminating the culture at the end of the logarithmic growth phase.

The obtained culture solution can be used as is for the enzyme reaction, but usually after the culture is complete, the bacterial cells are removed by centrifugation or other operations and the supernatant is used.

Furthermore, the supernatant may be purified and used according to a conventional purification method, or may be used after being immobilized.

As such a purification method, the enzyme in the culture solution is directly purified by Sephadex gel (5ephadex" G-50 ~
200 (manufactured by Pharmacia Fine Φ Chemicals), and the enzyme is eluted after the impurities are removed by washing with cold water. By this method, the enzyme is electrophoretically purified to a nearly single state, and this purified enzyme is highly specific in that no sugars are detected. Sephadex gel is G-50~200
(product number) has a high enzyme adsorption rate, with a maximum of approximately 10
Shows adsorption rate close to 0%. Although a batch method is also possible for the adsorption method, preferably an adsorption method using a column shows good results. When the maximum amount of enzyme adsorbed to the gel was analyzed, 180,000 units of enzyme (conditions for units will be described later) were adsorbed per 1 g (dry weight equivalent) of Sephadex gel (G-200), which is equivalent to 2 g of culture medium. corresponds to the amount of enzyme produced in In addition, cold water used for washing (5 to 2
(0°C), adding a suitable protective agent such as a buffer is effective in stabilizing the enzyme. For elution of adsorbed enzyme,
It is effective to add various saccharides to the eluate and elute at 45°C, but elution of about 7Q% is also possible with warm water elution at 45°C using an eluate without the addition of saccharides.

This phenomenon of efficient adsorption to Sephadex gel and the fact that sugars are not detected in the resulting enzyme are specific to the enzyme produced by this strain, and are not observed in L. mesenteroides. It is a phenomenon.

Next, a method for producing leucrose using the dextransucrase thus obtained will be explained.

In the present invention, sucrose is used as a substrate for Leucrose production. Furthermore, when sucrose is used in the presence of fructose as an acceptor, leuucrose is efficiently produced as a single oligosaccharide. The concentration of added sugars is
It may be determined by considering the enzyme unit.

However, when the molar concentration ratio of fructose to sucrose is low, the polymer dextran is produced as the main product, and as the molar ratio of fructose is increased, dextran synthesis is suppressed and the production rate of leuucrose tends to increase. Furthermore, if the molar ratio of fructose to sucrose is greater than 1:4, not only will dextran synthesis be inhibited, but also leuclose synthesis itself will be inhibited. is 1:1~1:
4 is preferred.

The reaction may normally be carried out at 20 to 50°C, but the reaction rate,
From the viewpoint of enzyme stability, it is preferable to carry out the reaction at around 40°C. The pH of the reaction solution is preferably 5 to 7, preferably 5.5 to 6.5. Although the reaction time varies depending on the enzyme unit and substrate concentration, the reaction is terminated when sucrose is almost completely consumed. In addition, trace amounts of metal salts such as calcium chloride may be added to promote the reaction.

The obtained leucrose can be separated and purified using an activated carbon column. For example, an enzyme reaction solution is passed through a column packed with activated carbon, and leuclose and dextran are adsorbed onto the activated carbon. Next, the activated carbon is sufficiently washed with distilled water, and then a 2 to 4% ethyl alcohol aqueous solution is passed therethrough to separate and purify the target leucrose.

Next, the present invention will be explained in more detail using Examples, but the present invention is not limited to these Examples.

The reaction solution obtained in the example was subjected to the following analytical test.

(Quantitative analysis by high performance liquid chromatography (hereinafter referred to as HPLC)) 0.4 ml of the enzyme reaction solution deionized with an ion exchange resin (IR (II") 120) etc. was mixed with 1 ml of acetonitrile.
．． 5 ml of methanol and 0.2 ml of methanol while stirring, and then centrifuged bone R (15,000 rpm x
After 501 in), add internal standard solution (0,2 in) to 1 ml of supernatant.
% lactose aqueous solution), add 1 ml, 0.45, ia
Samples for HPLC were prepared by treatment with a non-aqueous filter. 10 μg of this was subjected to IPI, C,
Fructose, glucose, sucrose, palatinose, and leuucrose in the reaction solution were identified and quantified.

The measurement was carried out using a Trirotor ■ (trade name, manufactured by JASCO ■) using the following analysis conditions, and a high-sensitivity differential refractometer (manufactured by Senshu Kagaku ■) was used as a detector. Note that Leucrose was eluted after about 1002 minutes.

Analysis conditions; Column: Senshu φ pack NII = -1251
-N (product name, manufactured by Senshu Kagaku ■), 4. Bφ x 250 flow rate: 1ml/■in Column temperature = 30℃ Eluate (volume ratio) Niacetonitrile/water The enzymatic reaction was carried out for 1 hour in pH 5.5), and the liberated fructose was removed using the Somogi method (Tucha et al., Journal of Bacteriology
, Bact, ).

64, p. 521 (1982)). The enzyme activity that produced 0.52 ml/ml of fructose under these reaction conditions was defined as one unit.

The amount of dextran produced in the reaction solution was determined by the following method. That is, 1 ml of distilled water for 1 ml of reaction solution.
After stirring thoroughly, add 1 ml of absolute ethanol to 1 ml of this solution while stirring, and centrifuge (15,000
0 rpm x 15 ml), wash the precipitate twice with the same ethanol, dissolve the obtained precipitate in distilled water, and
After adjusting the volume to l, collect 0.5 ml from this and add 10 m
1, and the amount of dextran was estimated from the amount of sugar determined by the phenol-sulfuric acid method.

Example 1 In a 2R flask, 50 g of glucose, 10 g of Horibebuton, 5 g of yeast extract, and 2 g of ammonium sulfate were added.

Baking soda 3g, cysteine hydrochloride o, sg, potassium phosphate 2.25g, dipotassium phosphate 2.25g5NaC&
0.9g %Mg5Oa 0.09g and Ca
09 g of CIzo was added and dissolved in 1 g of tap water. The flask was then plugged with cotton and sterilized at 120°C for 15 minutes to prepare a medium (hereinafter referred to as (A) medium).

After cooling, carbon dioxide gas was aseptically introduced into the medium to bring the culture system under anaerobic conditions.
RN P-9390) 20ml was inoculated into (A) medium, and the pH was adjusted by adding Na2COs over time at a temperature of 40°C.
The cells were cultured while adjusting the ratio to 6 to 6.5.

The culture was terminated at the end of the logarithmic growth phase, and a supernatant was obtained by centrifugation (lo, 000 rpm x 20 m1n).

The supernatant was dialyzed in running water day and night, then at a temperature of 5-10
Sephadex gel (G-200) column (30φ
x 30 ha) at a flow rate of 2 ml/win,
After adsorbing the enzyme, 0.05M phosphate buffer (pH
5, 5) was washed at the same flow rate.

Further remove the gel from the column with 0.05M phosphate buffer (pH
After washing the gel into a beaker with 5, 5) and warming it to 45°C, perform suction filtration to remove the gel on the filter paper at 45°C.
The solution was washed with the same buffer solution at °C, and the strained filtrate (approximately 50 ml) was used as an enzyme solution in the enzyme reaction.

The enzyme thus obtained was electrophoretically single.

Using this purified enzyme, the effects of temperature, pi, and metal salts on leuclose production were investigated.

For the enzyme reaction, 2.0 ml of 5% sucrose aqueous solution, 0.0
2.0 ml of 5M phosphate buffer (pH 5.0-7.0)
, 0.5 ml of enzyme solution (100 units), and 0.5 ml of distilled water for 24 hours.

When examining the influence of temperature, the enzymatic reaction was carried out in the range of 10 to 45° C. using a PNS, S phosphate buffer.

When examining the influence of pH, a phosphate buffer solution with a pH of 5.0 to 7.0 was used and the reaction was carried out at 40°C.

Also, when looking at the influence of metal salts, instead of 0.5 ml of distilled water, use a 0.005 M aqueous solution of various metal salts, CCaCl.
2, HgCf12, HnCI2, Pe5Oa,
Using 0.5 ml of CoCl2, Cu5O4), the reaction was further carried out at 40°C in a phosphate buffer solution of pH 5.5.

The oligosaccharide obtained by the enzymatic reaction was separated and purified using an activated carbon column, and the hydrolyzate was analyzed by HPLC, which revealed that it was a trisaccharide consisting of glucose and fructose. Furthermore, this oligosaccharide was chromatographically clearly different from sucrose and palatinose, which are also trisaccharides composed of glucose and fructose, and was identified as leuucrose by HPLC.

In addition, the sucrose remaining in the obtained reaction solution and the produced leucrose were analyzed by HPLC, and the produced dextran was analyzed by the above-mentioned phenol-sulfuric acid method, and the results were calculated based on the amount of sucrose consumed and the initial addition amount of the produced leucrose. It is shown in Table 1 along with the yield relative to sugar (yield relative to sugar).

[Margin below]

Example 2 As in Example 1, using a purified enzyme, the effects of the substrate (sucrose) concentration and the amount of receptor (fructose) added on leucrose production were examined.

For the enzyme reaction, 2.0 ml of sucrose aqueous solution or sucrose/fructose mixed aqueous solution, 2.0 ml of 0.125H phosphate buffer (pH 5.5), enzyme solution (100 units)
With a composition of 0.5 ml, and 0.5 ml of distilled water, 4
The test was carried out at 0°C for 24 hours.

The effect of substrate concentration was investigated using a 1625-25% sucrose aqueous solution at a final concentration shown in Table 2.

In addition, the effect of the amount of receptor added is that the final concentration of sucrose is 5
% and 10% respectively, the molar concentration ratio of fructose to sucrose is 1:0.25~
The investigation was conducted using an aqueous solution containing a mixture of 1:4.

In both reaction systems, a single oligosaccharide was produced, but the oligosaccharide production rate was higher with the amount of receptor added than with the amount of receptor added.

The oligosaccharide obtained by the enzymatic reaction was separated and purified using an activated carbon column, and the hydrolyzate was analyzed by HPLC and was found to be a trisaccharide consisting of glucose and fructose. Furthermore, this oligosaccharide was chromatographically clearly different from sucrose and palatinose, which are also trisaccharides composed of glucose and fructose, and was identified as leuucrose by HPLC.

In addition, the sucrose remaining in the obtained reaction solution and the produced leucrose were analyzed by HPLC, and the produced dextran was analyzed by the above-mentioned phenol-sulfuric acid method, and the results were calculated based on the amount of sucrose consumed and the initial addition amount of the produced leucrose. It is shown in Table 2 along with the yield relative to sugar (yield relative to sugar).

[Margin below]

[Effects of the Invention] The method of the present invention has the advantage that leuclose, which is expected to have a wide range of applications in the pharmaceutical and food industries, can be rapidly produced in high yield.

Furthermore, the method of the present invention is economically advantageous because it uses Streptococcus bovis, which does not require expensive nutrients for culturing, as the dextransucrase-producing bacterium.

Claims (1)

[Claims] 1. Cultivating Streptococcus bovis anaerobically in a medium containing a carbon source that induces dextransucrase,
A method for producing leuucrose using dextransucrase produced by Streptococcus bovis, which comprises reacting the produced dextransucrase with sucrose or with sucrose in the presence of a receptor. 2 148 strains belonging to Streptococcus bovis as the Streptococcus bovis (FERM
The manufacturing method according to claim 1 using P-9390). 3. The production method according to claim 1 or 2, wherein the carbon source is glucose or sucrose. 4. The manufacturing method according to claim 1 or 2, wherein the receptor is fructose. 5 The molar concentration ratio of sucrose and fructose is 1
:1 to 1:4.