JPH0157959B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a biochemical production method for guanosine diphosphate-fucose, which plays an important role as a metabolic intermediate in living organisms, and is intended to be used in pharmaceuticals and biochemical reagents. The aim is to industrially advantageously produce guanosine diphosphate-fucose, which is regarded as important as

Guanosine diphosphate-fucose is known as a substrate for fucosyltransferase, which catalyzes the synthesis of blood group-active glycolipids, and in recent years, research has begun on glycosyltransferases, which determine the type specificity of blood group substances. Conventionally, guanosine diphosphate-fucose has been difficult to prepare and not easily available, which has significantly hindered the development of medical and biochemical research.

The present inventors conducted various studies on methods for producing guanosine diphosphate-fucose at low cost and in large quantities, and found that guanosine diphosphate-fucose can be efficiently produced from guanylic acid or guanosine diphosphate-mannose using microbial enzymes. succeeded in generating it.

The presence of an enzyme that converts guanosine diphosphate-mannose to guanosine diphosphate-fucose has been found in rabbit lungs, higher plants, and microorganisms such as Klebsiella pneumoniae, Escherichia coli,
However, the present inventors conducted a wide search for microorganisms with strong convertase activity, and found that in addition to Klebsiella pneumoniae and E. He discovered microorganisms such as Bacterium and Agrobacterium rhizogenes. There is no example of producing guanosine diphosphate-fucose from guanosine diphosphate-mannose in high concentration and with good yield using such a microorganism, and the present invention is the first.

In the present invention, the microorganisms used for producing guanosine diphosphate-fucose from guanosine diphosphate-mannose include Agrobacterium,
Strains belonging to the genus Esieritia and Klebsiella pneumoniae
IFO3319, and all the usual forms of utilization of intracellular enzymes can be applied to such microorganisms. For example, a bacterial cell grind, a bacterial cell extract, a solvent-treated product, a surfactant-treated product, a freeze-dried product, etc. can be used.

The microorganisms can be cultured using commonly used culture media, such as glucose, sucrose, maltose, glycerin, molasses, starch, carbon sources such as organic acids, casamino acids,
A medium consisting of yeast extract, peptone, meat extract, soybean flour, corn steep liquor, urea, nitrogen sources such as ammonium salts, and other materials necessary for the growth of microorganisms such as potassium phosphate, magnesium phosphate, and trace metals is used.

Guanosine diphosphate-mannose is used as the reaction substrate with which the bacterial enzyme source is contacted. Furthermore, a reaction solution obtained by producing guanosine diphosphate-mannose from guanylic acid and glucose in the presence of inorganic phosphoric acid using cells of a known yeast such as baker's yeast can be used as is. .

When producing guanosine diphosphate-fucose, NADPH (reduced nicotinamide adenine dinucleotide phosphate) or NADP is added as a coenzyme, and glucose-6-phosphate is further added as necessary. The action of these compounds is to promote the conversion reaction because NADPH is regenerated by glucose-6-phosphate dehydrogenase contained in the enzyme source.

The reaction conditions of the present invention are appropriately selected within a range that allows the enzyme reaction to proceed smoothly depending on the type of strain, strength of enzyme activity, substrate concentration, and properties, but generally pH4.5 to 10,
A suitable reaction temperature is 20-40°C.

The guanosine diphosphate-fucose produced in the reaction solution can be separated using a combination of known ion exchange resin methods, activated carbon adsorption methods, cellulose adsorption methods, solvent extraction precipitation methods, and the like.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Agrobacterium radiobacter (Agrobacterium radiobacter) IAM1527 was mixed with 0.5% glucose, 1.0% casamino acid, and 0.5% yeast extract.
%, potassium phosphate 0.1%, potassium phosphate 2
The cells were cultured with shaking in a 0.3%, PH6.5 medium at 28°C, and the resulting bacterial cells were subjected to ultrasonic crushing to obtain an extract.
From this extract, the precipitate produced in the ammonium sulfate addition range of 35% to 80% was used as an enzyme source.

500mg of this enzyme source (as protein), 50μmol of guanosine diphosphate-mannose, 48μmol of NADPH
Reaction solution containing 120 μmol of glucose-6-phosphate and 1200 μmol of Tris-phosphate buffer (PH8.0)
The mixture was added to 100 ml and reacted at 30°C for 5 hours. 45.5 μmol of guanosine diphosphate-fucose was produced in the reaction solution.

The reaction solution was passed through activated carbon, washed with water, and eluted with an ammoniacal ethanol aqueous solution, and the eluate was concentrated to near neutrality. Next, the concentrated solution was adsorbed onto Dowex 1x2 resin prepared in Cl type, and then washed with water, and then eluted stepwise with a solvent system of hydrochloric acid and salt to obtain the guanosine diphosphate-fucose fraction. collected. Since this fraction still contained unreacted substrate, cellulose column chromatography was performed to purify and isolate guanosine diphosphate-fucose. The yield was approximately 31 μmol from 100 ml of the reaction solution. The measurement of guanosine diphosphate-fucose was confirmed using known paper chromatography and guanosine diphosphate-fucosyltransferase contained in blood.

Example 2 Klebsiella pneumoniae
pneumoniae) IFO3319 was cultured in the same manner as in Example 1 to prepare an enzyme source. This enzyme source 500mg
(as protein) Guanosine diphosphate - Mannose 50 μmol, NADPH 50 μmol, Glucose - 6
- 120 μmol of phosphoric acid, Tris-HCl buffer (PH8.0)
Add 100 ml of reaction solution containing 1200 μmol,
The reaction was carried out at 30°C for 6 hours. 38.5 μmol of guanosine diphosphate-fucose was produced in the reaction solution.

Example 3 Echerichia coli
Using IFO3806, live bacterial cells obtained by culturing in the same manner as in Example 1 were freeze-dried. 2.5g of this enzyme source,
Guanosine diphosphate-mannose 20μmol,
20μmol of NADPH, 60μmol of glucose-6-phosphate, 1.5ml of toluene, Tris-HCl buffer (PH
8.0) Add to 50 ml of reaction solution containing 600 μmol,
The reaction was carried out at 30°C for 5 hours. 12.5 μmol of guanosine diphosphate-fucose was produced in the reaction solution.

Example 4 Using Agrobacterium rhizogenes IFO13257,
The cells obtained by culturing in the same manner as in Example 1 were subjected to ultrasonic treatment and extracted. The obtained enzyme solution (protein
50ml of guanosine diphosphate (containing 420mg), 50μmol of guanosine diphosphate-mannose, 50μmol of NADPH, glucose-
6-Phosphoric acid 120μmol, Tris-HCl buffer (PH8.0)
The mixture was mixed with 50 ml containing 1200 μmol and reacted at 30° C. for 8 hours. 32 μmol of guanosine diphosphate-fucose was produced in the reaction solution.

Example 5 Echerichia intermedia
The cells were cultured using IFO13544 (intermedia) in the same manner as in Example 1, and the resulting bacterial cells were subjected to ultrasonication to extract the enzyme. The precipitate produced when less than 80% ammonium sulfate was added to this extract was used as the crude enzyme. This crude enzyme
500mg (as protein) of guanosine diphosphate
The mixture was added to 100 ml of a reaction solution containing 40 μmol of mannose, 40 μmol of NADPH, 120 μmol of glucose-6-phosphate, and 1200 μmol of Tris-HCl buffer (PH8.0), and reacted at 30° C. for 8 hours. 28 μmol of guanosine diphosphate-fucose was produced in the reaction solution.

Example 6 5 g of dried baker's yeast was mixed with 1 mmol of sodium guanylate, 40 mmol of glucose, and 1 m of magnesium sulfate.
When the mixture was added to a composition of 18 mmol of phosphate buffer (PH7.0) and subjected to a shaking reaction at 28° C. in a total volume of 100 ml, 0.42 mmol of guanosine diphosphate-mannose was produced in 9 hours of reaction.

To this reaction solution, 2 g of the enzyme preparation of Agrobacterium radiobacter IAM1527 prepared in the same manner as in Example 1, 0.4 mmol of NADP, and glucose-6-
1 mmol of phosphoric acid was added to each, and a standing reaction was carried out at 30°C. As a result, 0.22 mmol of guanosine diphosphate-fucose was produced in 7 hours of reaction.

Example 7 10 g of dried baker's yeast was mixed with 2 mmol of sodium guanylate, 80 mmol of glucose, and 2 m of magnesium sulfate.
When the mixture was added to a composition of 36 mmol of phosphate buffer (PH 7.0) and a total volume of 200 ml was subjected to a shaking reaction at 30°C for 7 hours, 1.2 mmol of guanosine diphosphate-mannose was produced.

Aerobacter aerogenes prepared in the same manner as in Example 2 was added to this reaction solution.
aerogenes) IFO3319 enzyme preparation 4g and NADP0.6
mmol and 2 mmol of glucose-6-phosphoric acid were added, and the mixture was left to react at 30°C. During the 7 hours of reaction, 0.46 mmol of guanosine diphosphate-fucose was produced.

Example 8 5 g of dried baker's yeast was mixed with 1 mmol of sodium guanylate, 40 mmol of glucose, and 1 m of magnesium sulfate.
When the mixture was added to a composition of 18 mmol of phosphate buffer (PH7.0) to a total volume of 100 ml and subjected to a shaking reaction at 28°C, 0.32 mmol of guanosine diphosphate-mannose was produced in 7 hours of reaction.

Echerichia intermedia prepared in the same manner as in Example 5 was added to this reaction solution.
intermedia) 2g of enzyme preparation of IFO13544 and
0.4 mmol of NADP and 1 mmol of glucose-6-phosphoric acid were added, and a standing reaction was carried out at 30°C.
As a result, 0.18 mmol of guanosine diphosphate-fucose was produced in 9 hours of reaction.

Claims (1)

[Scope of Claims] 1. A microorganism characterized by efficiently producing guanosine diphosphate-fucose from guanosine diphosphate-mannose using an enzyme of a microorganism selected from the genus Agrobacterium, the genus Escherichia, and Klebsiella pneumoniae IFO3319. A method for producing guanosine diphosphate-fucose. 2. A method for producing guanosine diphosphate-fucose using a microorganism according to claim 1, wherein NADPH (reduced nicotinamide adenine dinucleotide phosphate) or NADP and glucose-6-phosphate are added to the enzyme reaction solution. 3. The method for producing guanosine diphosphate-fucose according to claim 1, wherein guanosine diphosphate-mannose is produced from glucose, inorganic phosphoric acid, and guanylic acid using yeast.