JPH066057B2 - Dextrin dextranase and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a dextrin dextranase capable of producing dextran (α1,6 glucan), which is used in the fields of medicine, biochemistry and the like, from maltooligosaccharides, starch and its degradation products. (Hereinafter referred to as DDase) and a method for producing the same.

[0002]

2. Description of the Related Art DDase was reported about 40 years ago (Hehrre, EJ & Hamilton, D. et al.
M. (1951): Journal of Biolo
musical Chemistry, 55 , 161 17
4. ) Has been conducted, but no research has been conducted since then. The purification method reported in the report is to collect the precipitate generated by adding an appropriate amount of ammonium sulfate to the cell culture medium in which the cells have not been removed, and then dissolve this in an appropriate amount of distilled water to remove insoluble cells. This is a method of obtaining the enzyme by removing it. In addition, the usual method for purifying intracellular enzymes at present is to prepare various cell-free extracts by cell disruption and then perform various chromatography using a buffer solution containing no organic solvent.

[0003]

The inventors of the present invention produced and purified DDase by experiments based on the above-mentioned report, and in the purification method, a crude product containing only a few% of the enzyme amount present in the bacterial cells was produced. Only the enzyme was obtained and could not be further purified using conventional techniques. Further, in the purification by cell disruption, DDase remained in the bacterial cells even by the usual bacterial cell disruption treatment using ultrasonic waves. Attempts were made to purify the DDase-containing solution obtained at this time by various chromatographies using a buffer solution containing no organic solvent as usual, but the purification was impossible due to almost no adsorption.

[0004]

Means for Solving the Problems The dextrin dextranase-producing bacterium belonging to the genus Gluconobacter is a Gluconobacter oxydans ATCC11894 strain (Gl.
uconobacter oxydans Ameri
can Type Culture Collectio
n strain No. 11894), Gluconobacter oxydans ATCC 11895 strain (Glucon)
objecter oxydans American
Type Culture Collection s
train No. 11895) or other DDase-producing bacterium belonging to Gluconobacter. The culture may be performed by a conventional method.

In the present invention, the organic solvent means alcohols such as n-butanol, ethylene glycol, ethanol, propanol and diethylene glycol, acetone and the like. In particular, when the enzyme is extracted from the bacterial cells, an organic solvent such as n-butanol, chloroform or the like which is hardly soluble in water or almost insoluble in water, that is, has a low polarity is preferably used. The organic solvent having a low polarity is one having a lower polarity than 5 when represented by a classification in which hexane is 0 and water is 9 (hereinafter referred to as the polarity of the organic solvent). When eluting the adsorbed enzyme in chromatography, an organic solvent having polarities of 5 or more, such as ethylene glycol, is preferably used. To collect using an organic solvent, proceed as follows. The microbial cells collected after the culturing are suspended in a buffer solution as they are or after the cells are destroyed. As the buffer solution, it is desirable to use an aqueous solution of PH such as acetate buffer solution in which DDase can stably exist. An organic solvent having a low polarity, such as n-butanol, is added to this suspension in the same volume and dispersed. Dispersion can be easily performed by gently shaking by hand. After leaving this dispersion liquid for a suitable period of time, centrifugation is performed to separate it into a layer mainly containing an organic solvent, a layer mainly containing water and a microbial cell or a disrupted cell pellet. Of these, the layer mainly composed of water was collected, dialyzed, and then subjected to hydrophobic chromatography, and the adsorbed protein was eluted with a buffer solution containing alcohols which could be easily dissolved with water such as ethylene glycol. Then, this is concentrated, and is also applied to a gel filtration column equilibrated with a buffer solution to be recovered.

The DDase of the present invention has the following features. 1) Action This enzyme acts on maltooligosaccharides having a degree of polymerization of 3 or more, including amylose, and transfers glucose residues on the non-reducing end side of those substrates to produce dextran. This action proceeds until the substance used in the reaction becomes a maltooligosaccharide having a degree of polymerization of 2, such as maltose. This action of DDase is the same even when glucose located at the reducing end of maltooligosaccharide is modified by hydrogenation or the like. 2) Optimum PH and stable PH The optimum action PH of DDase was 4.0 to 4.5, and the pH was stable at 2.5 to 6.0 when left for 30 minutes under various types of PH. 3) Optimum temperature and stable temperature The optimum working temperature of DDase was 37 to 45 ° C, and the temperature was stable at 45 ° C or lower when left for 30 minutes under various temperatures. Moreover, this enzyme showed activity up to 55 ° C. 4) Molecular weight The molecular weight of DDase by electrophoresis was about 300,000. 5) Titer measurement method Reduced and low hydrolyzed starch was used as an enzyme reaction substrate, and measurement was carried out by a conventional method. As for the titer, the amount of enzyme when 1 μmol of glucose unit was dextran per minute was 1 U.

[0007]

[Action] DDase has a strong affinity for organic solvents and a specificity that it exists near the cell membrane of bacteria.
Further, the organic solvent can dissolve the lipid, which is a component of the cell membrane. Therefore, DDase can be extracted by directly acting with an organic solvent without destroying cells. Generally, the enzyme rapidly loses its activity in the presence of an organic catalyst, but DDase does not inactivate it in an organic solvent.

[0008]

[Example] (Example 1) 0.5% yeast extract, 0.3% polypeptone, 0.5%
After sterilizing 4 liters of a medium containing glucose and 1% ethanol, Gluconobacter oxydans ATCC1189
Four strains were inoculated and cultured with shaking at 30 ° C. for 16 hours. The obtained cells were suspended in 10 mM acetate buffer, and an equal volume of n-
After adding butanol and mixing well, centrifugation was performed to obtain DDase activity in the aqueous fraction. The enzyme activity at this time was 0.54 U / ml. Next, the DDase fraction obtained at this time was dialyzed to remove n-butanol and then subjected to phenyl Toyopearl and Toyopearl HW-65S chromatography using 40% ethylene glycol to purify DDase. The purified enzyme thus obtained showed a single band by gel electrophoresis, and the enzyme recovery rate at this time was 49.7%.

(Example 2) 0.5% yeast extract, 0.3% polypeptone, 0.5%
After sterilizing 1 liter of medium containing glucose and 2% glycerin, Gluconobacter oxydans ATCC 1189
Four strains were inoculated and cultured with shaking at 30 ° C. for 16 hours. After culturing, 2.0 g of the bacterial cells obtained by centrifugation was suspended in 25 ml of 10 mM acetate buffer, an equal volume of n-butanol was added and mixed well, and then centrifuged to DDase the aqueous layer fraction. The activity was obtained. The enzyme activity at this time was 0.39 U / ml. Next, the DDase fraction obtained at this time was dialyzed to remove n-butanol, and then subjected to phenyl Toyopearl and Toyopearl HW-65S chromatography using 40% ethylene glycol to purify DDase. The resulting purified enzyme showed a single band by gel electrophoresis. The enzyme recovery calculated from the enzyme activity was 21.8%.

(Example 3) 0.5% yeast extract, 0.3% polypeptone, 0.2%
After sterilizing 2 liters of a medium containing glucose and 1% glycerin, Gluconobacter oxydans ATCC1189
Five strains were inoculated and cultured with shaking at 30 ° C. for 40 hours. After culturing, 3.9 g of the bacterial cells obtained by centrifugation was suspended in 45 ml of 10 mM acetate buffer, an equal volume of n-butanol was added and mixed well, and then centrifuged to separate the aqueous layer fraction into DDase. The activity was obtained. The enzyme activity at this time was 0.24 U / ml. Next, the DDase fraction obtained at this time was dialyzed to remove n-butanol, and then subjected to phenyl Toyopearl and Toyopearl HW-65S chromatography using 40% n-propanol to purify DDase. The resulting purified enzyme showed a single band by gel electrophoresis. The enzyme recovery rate calculated from the enzyme activity was 23.4%.

[0011]

[Effect] DDase could be collected by the present invention. In addition, it is an easy method that does not cause cell destruction, and has a high recovery rate.
se could be purified. By purifying DDase, for example, when dextran is produced from maltooligosaccharide or the like, it is not affected by enzymes other than DDase or other components contained in the crude enzyme obtained by the conventional method. In addition, impurities are hardly mixed in the target dextran, and its purification becomes extremely easy. In particular, dextran, which is a product, is a high-molecular substance, so it is difficult to remove foreign substances mixed in, so
It is better to use purified enzyme.

Claims (2)

[Claims] 1. A dextrin dextranase characterized by being obtained by culturing a dextrin dextranase-producing bacterium belonging to the genus Gluconobacter and harvesting it from an organic solvent. 2. A method for producing dextrin dextranase, which comprises culturing a dextrin dextranase-producing bacterium belonging to the genus Gluconobacter and collecting the bacterium from the bacterium by using an organic solvent.