JP2731100B2 - Dextran sucrase-producing novel microorganism and method for producing dextran sucrase using the novel microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel dextran sucrase-producing microorganism which uses a saccharide other than sucrose as a carbon source and produces dextran sucrase without by-producing dextran, and a dextran sucrase using the novel microorganism. It relates to a method of producing.

[0002]

2. Description of the Related Art Dextran, which is a polysaccharide, is composed of α-1,
It is a polymer of glucose mainly composed of 6-glucosidic bonds, and is conventionally useful in the field of medicine or biochemistry, and its safety has been widely recognized. In the natural world, such dextran is mainly produced by dextransucrase (hereinafter referred to as DSase), which is present in lactic acid bacteria such as Leuconostoc sp., Using sucrose or the like as a substrate. DSase is a type of α-glucosyltransferase and acts on sucrose to produce dextran.

[0003] As a method for industrially producing dextran, a fermentation method of culturing Leuconostoc spp. Using sucrose as a raw material and synthesizing dextran into cells, and a DSas production method using sucrose as a raw material.
There is an enzymatic synthesis method of enzymatically synthesizing e itself.

[0004] In this case, the enzymatic synthesis method is industrially more advantageous than the fermentation method in the following points. The concentration of sucrose, which is a fermentation raw material, can be set high. Adjustment of the molecular weight of dextran to be synthesized is easier than in the fermentation method, and dextran of uniform quality can be obtained for each production batch. Continuous synthesis is possible by immobilizing the enzyme. Since the cells are not included, dextran can be easily purified, and the cost of purification to meet strict specifications is low.

[0005] DSase required for such enzymatic synthesis method
Is an enzyme whose synthesis is induced only by sucrose in a medium which is a substrate of a reaction catalyzed by DSase, and is usually produced only in a medium containing sucrose. Accordingly, DSase is produced by culturing Leuconostoc spp. Using sucrose as a carbon source by using the fermentation method described above.

[0006]

However, newly induced and produced DSase uses sucrose in the medium as a substrate to produce dextran which is only a by-product for this purpose. Since dextran is a very viscous polysaccharide, if it is accumulated in the culture solution, the step of separating and purifying the enzyme from the culture solution becomes extremely complicated, and the purification efficiency decreases. Since mass production of DSase is difficult in this way, it is difficult to provide inexpensive DSase,
Dextran synthesis by enzymatic synthesis was hindered. Accordingly, an object of the present invention is to provide a novel microorganism derived from the genus Leuconostoc that produces DSase without producing dextran.
An object of the present invention is to provide a method capable of obtaining ase.

[0007]

In order to solve the above-mentioned problems, the present inventors have developed DSase without sucrose.
A novel microorganism which is a constitutive mutant of DSase capable of producing E. coli was constructed and obtained. In addition, as a carbon source
By culturing this novel microorganism strain in the absence of sucrose, it was found that the enzyme can be produced in a culture solution without producing dextran, and at the same time, the enzyme can be produced in high units, thus completing the present invention.

The microorganism according to the present invention includes DSas
belonging to Leuconostoc mesenteroides producing e
And a strain having the property of producing DSase even without sucrose as a carbon source in the medium . In general, Leuconostoc Mese to produce DSase
From a mutant strain obtained by subjecting a bacterium belonging to the interroides to a mutagenesis treatment as a parent strain, even without sucrose,
A substance that constantly produces DSase is selected and used.

As a method for mutagenesis, known mutagenesis means which is generally used can be used. That is, general mutation means such as nitrosoguanidine (hereinafter referred to as NTG) treatment, UV treatment, and ethyl methanesulfonate treatment can be used. In addition, stable mutants can be obtained by natural mutation. To select a strain that produces DSase from saccharides other than sucrose from among the mutated mutants, the mutated strain is cultured in a medium containing saccharides other than sucrose as a carbon source, and then a new protein synthesis is performed. A strain which can confirm the synthesis of dextran by giving sucrose as a carbon source in a state in which sucrose is suppressed may be selected. As a saccharide used as a carbon source for selection, glucose, fructose, and maltose can be used alone or in combination of two or more.

Therefore, even without sucrose, DSa
is capable of producing DSase without induction of sucrose, a substrate of dextran synthesis by DSase.
Means always producing . A strain having such properties due to the mutation treatment is referred to as a DSase constitutive mutant.

Hereinafter, Leuconostoc mesenteroides NRRL B-512F (ATCC 10830) is an example of a strain that is specifically suitable for use in the present invention.
a) (Leuconostoc mesenteroi)
des NorthernUtilization R
search and DevelopmentDi
vision, U.S.A. S. Department of
Agriculture B-512F (Americic
an Type Culture Collection
n 10830a)) as a parent strain, which is a DSase constitutive mutant of Leuconostoc spp.
An example of obtaining the strain M898 (hereinafter simply referred to as strain M898) is shown. In addition, this bacterium
FERM No.P for Biotechnology Industrial Technology Research Institute
No. -13385 has been deposited on January 25, 1993.

First, the parent strain was transformed into an MRS liquid medium (US DI)
Lactobacilli MRS Br manufactured by FCO
oth, the carbon source is glucose and does not contain sucrose), and cultured at 30 ° C. for 24 hours.
It was treated with TG (0.5 mg / ml, 60 minutes). Further, after washing the cells, the cells were appropriately diluted, applied to an MRS agar plate medium, and cultured at 30 ° C. for 2 days. After this, 400
10% sucrose agar containing 0 ppm of tetracycline was overlaid and left at 25 ° C. for 2 to 6 hours. Here, tetracycline in the overlay agar inhibits novel protein synthesis of the mutant strain in the MRS agar medium. For this reason, only DSase that has been produced by culturing in an MRS agar medium before overlaying agar can synthesize dextran using sucrose in the overlayed agar as a substrate.

By observing the plate, strains that produced viscous dextran around the colonies were isolated as DSase constitutive mutants. For these mutants, strain M898 was selected by examining bacterial stability and DSase productivity in a liquid medium containing various carbon sources. Table 1 shows the composition of this liquid medium.

[0014]

[0015] The medium for producing DSase by the obtained microorganisms may be a medium containing carbon sources, nitrogen sources, organic natural nutrients, phosphates, metal ions, vitamins, etc. in good balance. However, it is not limited to a synthetic medium. As a carbon source, glucose, fructose, maltose, liquid sugar (a mixture of glucose and fructose) and the like can be used as carbon sources superior to sucrose. In other words, if saccharides other than sucrose are used, dextran as a by-product does not accumulate in the medium, so that culturing is started at a high sugar concentration or the concentration is maintained by intermittently adding sugar. Or continuous culture. For example, the initial sugar concentration, which was conventionally 2%, can be 10%. These saccharides can be used alone or in combination of two or more.

As the culturing conditions, those generally used for the enzymatic synthesis of dextran can be used. That is, the cells are cultured by static culture or slow stirring culture for 12 to 48 hours. The culture temperature is 15
３５35 ° C., preferably 25-30 ° C. The pH at the time of culturing is 4.0 to 7.5, preferably 5.0 to 7.0.
It is.

The culture solution obtained by culturing under predetermined conditions does not contain dextran, but contains a high concentration of DSase produced and secreted by the DSase constitutive mutant. Therefore, this culture solution can be used as it is for enzyme reactions such as dextran synthesis. Further, DSase purified to a desired purity by a known separation and purification method generally used from a supernatant or the like obtained by removing the cells from the culture solution by centrifugation or filtration, or a DSase enzymatically synthesized from dextran It can also be used in synthetic methods.
Further, the extracted and purified enzyme can be immobilized on a suitable carrier and used continuously. As the separation and purification method, a method used as a normal enzyme purification method, for example, a salting out method using ammonium sulfate, a precipitation method using an organic solvent, dialysis, an ultrafiltration method, a gel filtration method using Sephadex, a diethylaminoethylcellulose method, and the like Ion exchange column chromatography and the like can be used in an appropriate combination depending on the intended use of the purified enzyme and the desired purity.

The enzymatic activity of DSase in the present invention is determined by the method of Kobayashi et al. (Biochemical et al., Biophysica Actor).
ysica Acta), vol. 614, pp. 46-62, 1
980). That is, this is a method in which, when the enzyme is allowed to act at 30 ° C. in a sucrose solution dissolved in an acetate buffer having a pH of 5.2, the amount of the enzyme that releases 1 μmol of fructose per minute is defined as one unit. The amount of released fructose can be measured by high performance liquid chromatography. Also add ethanol
Utilizes the property that dextran precipitates
Then, add 2 volumes of ethanol to the reaction solution after the enzyme reaction.
Dextran formation can be confirmed by adding
You.

[0019]

The present invention uses a constitutive mutant of DSase which does not undergo induction of sucrose, a substrate for dextran production by DSase , using sucrose as a carbon source.
Without culturing, DSase can be produced without producing viscous dextran in the medium. Therefore, a complicated process for removing dextran in the step of extracting and purifying the enzyme can be eliminated. Also,
By using a constitutive mutant strain, high units of DSase can be produced.

[0020]

EXAMPLES As an embodiment of the present invention, a method for producing DSase using the aforementioned strain M898 will be described. The M898 strain cultured on the MRS agar medium for 24 hours was inoculated into a test tube containing 10 ml of MRS liquid medium, and cultured for 24 hours as a seed, and glucose, fructose, maltose, sucrose, glucose and fructose were used as carbon sources. Mixture of (1: 1)
Were respectively inoculated to 1% in the medium shown in Table 1 (total 5 types) prepared so as to contain 2%.
The culture was allowed to stand at 5 ° C.

At 24 hours and 48 hours after the start of the culture, a part of the culture was collected, the DSase activity was measured, and the presence or absence of dextran was tested. DSas
Table 2 shows the measurement results of the e-activity. In addition, D of M898 strain
In order to compare the Sase productivity with that of a conventional Leuconostoc genus, the same test as described above was performed on the parent strain Leuconostoc mesenteroides NRRL B-512F. Table 2 also shows the measurement results of the DSase activity.

[0022]

[Table 2]

As is clear from Table 2, the strain M898 is:
DSase was produced using sugars other than sucrose as a carbon source. Its enzymatic activity was significantly higher than the activity obtained when the parent strain B-512F produced DSase using sucrose as a carbon source. In addition, even when sucrose was used as a carbon source, the activity was higher than that of the B-512F strain. From this, it is clear that this mutant is a DSase constitutive mutant capable of mass-producing DSase.

Dextran was produced in a culture solution using sucrose as a carbon source, but no dextran was produced in a culture solution using a sugar other than sucrose as a carbon source. As a result, scrolling
Culture without the use of DSase, purification of DSase,
It was confirmed that it could be easily performed without going through complicated procedures as in the past. As described above, the microorganism of the present invention has a
Since it is an adult mutant, the method of the present invention
DSase can be obtained each time, and the production efficiency of DSase
Rate. Further, the microorganism of the present invention and
According to the method, DSase can be provided in large quantities and at low cost.
Dextran production by enzymatic synthesis
Dextran
Wide use is possible. Also, for immobilization of DSase
Of DSase itself, such as application to continuous synthesis methods
Can be planned.

[0025]

As described above in detail, it DSase constitutive mutants was constructed acquired by the present invention, novel capable of DSase to production of DSase without induction of sucrose is the substrate in generating dextran It is a microorganism. Therefore, this by the novel microorganism can be the production of DSase without made sub viscous dextran to the culture solution, DSase separation and purification process from the culture broth Ru significantly simplified.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 9/10 C12R 1:01)

Claims (3)

(57) [Claims] 1. A genus belonging to Leuconostoc mesenteroides
A bacterium which produces dextransucrase even without sucrose . 2. The microorganism is Leuconostoc mesenteroides M898 (Accession No.
FERM No. P-13385). 3. The use of sucrose as a carbon source.
A method for producing dextransucrase, which comprises culturing the microorganism according to claim 1 or 2 .