JPH07115986A - Production of levan octaose - Google Patents

Abstract

PURPOSE:To provide a method for obtained only the levan octaose to be produced substantially without producing fructooligosaccharides and fructose. CONSTITUTION:This method for producing levan octatose comprises hydrolyzing levan with levanase originated from a fungus belonging to the genus Streptomyces, hydrolyzing levan into the octaose but substantially not producing other fluctooligosaccharides and fructose, and subsequently collecting the produced levan octaose.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for microbially or enzymatically producing levan octaose from levan. Levan octaose is useful as a growth factor for enterobacteria.

[0002]

As a method for producing fructooligosaccharides such as levan octaose by hydrolyzing levan, a method of separating from hydrolyzate produced by limited decomposition with a dilute acid such as oxalic acid is known. There is. However, a microorganism producing an enzyme capable of producing levan octaose without substantially producing other fructooligosaccharides or fructose has not been reported so far.

[0003]

Levan octaose is useful as a growth factor for enterobacteria, and other fructooligosaccharides and levan octaose products free of fructose have been demanded. In order to obtain a levan octaose product without fructose or fructose, it is necessary to first produce a mixture of a plurality of fructooligosaccharides, and then isolate and purify levan octaose from the mixture, which increases the production cost, There was a problem such as a decrease in yield.

Therefore, the present invention provides a method for specifically enzymatically hydrolyzing levan to levan octaose.
The present invention provides an efficient and economical method for producing levan octaose.

[0005]

[Means for Solving the Problems] As a result of various studies to solve the above-mentioned problems, the present inventors specifically produced levan octaose from levan from actinomycetes of the genus Streptomyces. As a result, a strain that does not substantially produce other fructooligosaccharides and fructose was found, and the present invention was completed based on this finding.

The present invention therefore provides for Streptomyces ( St) which hydrolyzes levan to levan octaose, but does not substantially form other fructooligosaccharides and fructose.
and a levanase containing Streptomyces sp. actinomycete-derived revanase containing Levanase hydrolyzate to produce levan octaose and to collect the levan octaose. A method for manufacturing levan octaose is provided.

[0007]

[Detailed description] Levan has fructose β-2,6
It is a high molecular substance linked by a bond, has a branched structure, and is produced by bacteria of the genus Bacillus, the genus Leuconostoc, and further the genus Serratia. In the present invention, any levan can be used, but the longer the straight chain portion having less branches, the higher the yield.

In the present invention, any actinomycetes belonging to the genus Streptomyces, which hydrolyze levan to specifically produce levan octaose, and other fructooligosaccharides and fructose, are not substantially produced. Can be used. For example, Streptomyces an
Ulatus ) actinomycetes, and more specifically, D belonging to Streptomyces anuratus species.
-13 strains and IFO 13369 strains can be mentioned. The strain D-13 was isolated from soil according to a conventional method for separating actinomycetes, and has the following mycological properties.

[0009]

[Table 1]

Based on the above properties, this strain is classified as an actinomycete belonging to the genus Streptomyces. Further, the above D-13
From the test results of DNA-DNA hybridization between the strain and the preserved strain, the strain D-13 was Streptomyces
Anuratus ( Streptomyces anulat)
us ) IFO 13369 homology 59,77,7
It was analyzed as 3,83%. The average of the middle two exceeded 73% and 70%, so Streptomyces anulatus ( Streptomyces anulatus)
s )).

The above-mentioned actinomycetes is Streptomyces s.
p. Under the name of D-13, FERM P-13800 was established by the Institute of Biotechnology, Institute of Industrial Science and Technology as FERM P-13800.
It was deposited on the 17th of every month. In order to obtain the levan-degrading enzyme or its content by the actinomycete of the present invention, it is preferable to carry out the main culture after the pre-culture. The medium for pre-culture may be any medium in which the actinomycete of the present invention can grow. For example, as a carbon source, glycerin, glucose, sucrose,
Mannitol, inositol, starch and the like can be used alone or in combination. The amount of carbon source varies depending on its type, but is 1 to 100 g / L, preferably 5 to 20 g / L.
It is L.

As the nitrogen source, for example, peptone, yeast extract and the like are used. Furthermore, if necessary, inorganic salts such as phosphates and potassium salts can be included. The amount of nitrogen source varies depending on the type, but is 0.5 to 50 g / L, preferably 1 to 10 g / L. The amount of the inorganic salt also varies depending on the type, but is 0.1 to 10 g / L. As a medium for the main culture (production culture), levan is used as a sole carbon source in addition to the above carbon source or in place of the above carbon source.

The amount of the carbon source added varies depending on the kind, but is about 1 to 100 g / L, preferably 5 to 30 g / L. A particularly preferable amount of levan added is 1 to 10 g / L. Furthermore, it is preferable to use yeast extract, peptone or the like as the nitrogen source. Furthermore, it is preferable to add salts such as phosphate, potassium, magnesium, iron, and calcium as the inorganic salt. The addition amount of the nitrogen source varies depending on the type, but is 0.5 to 50 g / L, preferably 1 to 10 g / L, and the addition amount of the inorganic salt varies depending on the type, but is not more than 0.
It is 1 to 10 g / L.

The culture is carried out at 25 ° C to 30 ° C, preferably about 27 ° C.
Perform at ℃. Culture period is 1-6 for both pre-culture and main culture
It is about one day, usually about two days. Culturing is carried out under aerobic conditions, and usually aerobic conditions are secured by shaking, stirring, aeration, or the like. In the present invention, a purified levan-degrading enzyme (levanase) can be used for degrading levan to levan octaose, but it is preferable to use an enzyme-containing material from an economical point of view. Examples of the enzyme-containing material include the culture itself of the levan-degrading enzyme-producing bacterium, the culture filtrate or the centrifugally separated liquid recovered from the culture, the intermediate purified product in which the enzyme is concentrated or semi-purified to various stages, and the like.

The simplest reaction method is a method of culturing the actinomycete of the present invention in a medium containing levan as a carbon source. In this way, it produces a levan-degrading enzyme. Then, the rest of levan in the culture is decomposed by an enzyme into levan octaose, which is recovered as a target product. In this embodiment, levan octaose can be increased in yield by intermittently or continuously adding levan during the culture.

Another reaction method is a method in which cells, culture filtrate and the like are separated from the culture, and then these are used as an enzyme source to carry out the reaction as a step different from the culture. The most convenient aspect of this method is to recover the filtrate or supernatant from the culture and use this as the crude enzyme solution, eg phosphate buffer,
This is a method of degrading levan in a buffer solution such as a citrate buffer solution or a borate buffer solution.

The pH of the reaction solution in this case is 3.0 to 9.0,
Preferably, it is 5 to 7, for example pH 6.0, and the reaction temperature is 4 ° C to 70 ° C, preferably 20 ° C to 50 ° C, for example 3
It is 7 ° C. The initial concentration of substrate levan in the reaction varies depending on the enzyme concentration, but is 0.1% to 30%, preferably 1%
-10%. The reaction time varies depending on the amount of enzyme used, the amount of levan as a substrate, etc., but is 10 minutes to 1 week, preferably 1 hour to 3 days.

Levanoctaose can be recovered and purified from the reaction solution or the culture solution according to a conventional method used for the recovery and purification of saccharides. For example, it is separated by molecular sieve by various gel filtration and then dried by freeze-drying or the like. It can be a thing. According to the present invention, levan is specifically decomposed into levan octaose, and virtually no other fructooligosaccharides or fructose are produced.

Next, the present invention will be described in more detail with reference to examples. Example 1. Streptomyces anuratus D-13
(FERM P-13800) was added to a slant medium (5 g of peptone, 5 g of meat extract, 10 g of glucose, NaCl per liter).
3 g, agar 15 g; pH 7.2), and 1 platinum loop of the culture was added to 70 mL of preculture medium (3 levans per 1 L).
g, yeast extract 2g, meat extract 2g, bactopeptone 4
g, soucloth 9g, fructose 0.4g, sodium nitrate 3g, phosphoric acid-dipotassium hydrogen 1g, magnesium sulfate 0.5g, manganese chloride.4H2O 0.2g; pH
Inoculate a 500 mL Sakaguchi flask containing 7.2) 2
The cells were cultured at 7 ° C for 3 days.

Next, 1.4 mL of this preculture was placed in 500 mL of 70 mL of the main culture medium (the composition is the same as that of the preculture medium).
The cells were inoculated in a Sakaguchi flask of mL and cultured with shaking at 27 ° C. for 48 hours. The culture solution was filtered, 100 mL of the supernatant was concentrated to 10 mL with an evaporator, and then column chromatography was performed using a Biogel P-2 column (4.8 x 60 cm). The result is shown in FIG. Fractions No. 90 to No. 100 were collected, concentrated by a rotary evaporator, and subjected to thin layer chromatography (thin layer plate: silica gel, developing solvent ethyl acetate:
Isopropanol: water / 18: 13: 9). From this result, generation of levan octaose was confirmed.

Example 2. 500 mL of the culture filtrate cultured by the method of Example 1 was dialyzed against a 20 mM acetate buffer (pH 6.0). This is a DEAE-cellulose column (3.6
(× 23 cm) to adsorb the enzyme. 0 → 1M Na
The active fraction was collected by elution with a Cl gradient. 30% enzyme
Make a solution of ammonium sulphate and add this to Butyrut Yopal 6
It was adsorbed on a 50M column (2.6 × 28 cm).

The active fraction was collected by elution with a gradient of 30 → 0% ammonium sulfate. The enzyme was dissolved in 20 mM acetate buffer (pH 6.0) containing 0.2 M NaCl, and this was adsorbed on a Biogel A-0.5 m column (2.6 × 126 cm). The active fraction was collected by elution with the same buffer and used as the enzyme evaluation product. The enzyme activity 1 u was defined as an enzyme activity that produces 1 μmol of fructose-equivalent reducing sugar in 1 minute.

Example 3. 20 mM acetate buffer (pH 6.0)
1.5 u of the enzyme prepared in Example 2 and 200 mg of levan prepared in Example 2 were added to 20 mL and reacted at 30 ° C for 3 days. The reaction solution was loaded on a Biogel P-6 column (4.5 × 31.5 cm) and chromatographed. The results are shown in Fig. 2. When the obtained fraction of levan octaose was decomposed with invertase (from Candida utilis), the reaction was terminated when the reducing sugar amount became 8 times. It was confirmed by thin layer chromatography shown in Example 1 that the decomposed product was fructose.

Example 4. In Example 1, Streptomyces sp. D-13 (FERM P-13800)
Instead of Streptomyces anuratus IFO
The culture was performed in exactly the same manner except that 13369 was used.
Then, the culture solution was filtered in the same manner as in Example 1 to obtain 100 mL of the supernatant.
Was concentrated to 10 mL with an evaporator, and then subjected to column chromatography on a Biogel P-2 column, No90-10
The 0 fractions were collected, concentrated, and confirmed by thin layer chromatography that levanoctaose was produced.

[Brief description of drawings]

FIG. 1 is a medium containing levan containing Streptomyces
sp. It is a result of biogel P-2 column chromatography showing the production of levan octaose when D-13 was cultured.

FIG. 2 shows a reaction solution containing levan, Streptomyces sp. It is a result of the biogel P-6 column chromatography showing the production of levan octaose when the D-13-derived levan degrading enzyme was reacted.

Claims (4)

[Claims] 1. A Streptomyces hydrolyze the levan oct ose levan is not substantially generate other fructooligosaccharides and fructose (Streptomyc
es ) characterized in that levan octaose is produced by hydrolyzing levan using a repanase derived from genus actinomycete or a revanase-containing substance derived from streptomyces genus actinomycete, and the levan octaose is collected. Levan octaose manufacturing method. 2. The Streptomyces actinomycete is Streptomyces anuratus ( Streptomyc).
es anulatus ). 3. The Streptomyces anuratus actinomycete is Streptomyces anuratus D-13.
FERM P-13800 or Streptomyces
The method of claim 2, which is Anuratus IFO 13369. 4. The revanase-containing material is a culture of a revanase-producing bacterium, a microbial cell of a revanase-producing bacterium, a culture supernatant of a revanase-producing bacterium, or a partially purified levanase preparation. the method of.