JPS5852639B2 - Method for producing phosphorous oxide - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing phosphorous oxide using microorganisms.

More specifically, the present invention provides the desired phosphoric oxide,
Using a microorganism that has the ability to produce a phosphoric acid precursor in the presence of a high-energy phosphoric acid compound or a processed product thereof, the precursor and the high-energy phosphoric acid compound are reacted in the presence of an energy source. , a method for producing a target phosphoric acid, characterized in that a microorganism having the ability to regenerate a low-energy phosphoric acid compound generated by the reaction into the high-energy phosphoric acid compound or a processed product thereof is used in combination. Concerning a method for producing oxides.

Conventionally, phosphorylates such as cytidine diphosphate choline (CDP-choline), nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), and coenzyme A (CoA) have been combined with their respective precursors and high-energy phosphoric acid compounds. (For example, adenosine diphosphoric acid, ATP) is produced using microorganisms (Japanese Patent Publication No. 4840757, 1741).
-15516 Publication, Publication No. 45-22518, Publication No. 4
9-13994).

However, in these conventional methods, when producing a phosphoric acid such as CDP-choline, cytidine-57-phosphate and choline or phosphorylcholine, N
When producing AD, use nicotinic acid; when producing FAD, use flavin mononucleotide (FMN), CoA
When producing phosphoric acid, it is essential to use high-energy phosphoric acid compounds such as ATP as raw materials in addition to precursors corresponding to the respective phosphoric oxides, such as pantothenic acid and cysteine, which improves reaction efficiency and production. Further improvements in terms of cost are desired.

The present inventors conducted various studies to improve the above problems.

As a result, energy sources (e.g. sugars such as glucose) and phosphate donors (KH2PO4, 2HPO4,
3PO4, NaH2PO4゜Na2HPO4, Na3
It has the ability to generate high-energy phosphate compounds (e.g., ATP) from phosphates such as PO4, and converts low-energy phosphate compounds (e.g., ADP, adenosine diphosphate) into the high-energy phosphate compounds (ATP). )
We have discovered the fact that phosphorylation can be efficiently and inexpensively produced by carrying out the phosphorylation reaction in combination with microorganisms that have the ability to regenerate.

That is, in the case of the above method, it is possible to directly involve the generated high-energy phosphoric acid compound such as ATP in the reaction without separating it from the reaction system,
By supplying an energy source and a phosphate donor to the reaction system, high-energy phosphoric acid compounds such as ATP are regenerated and can be used for the reaction, making it possible to obtain phosphoric oxides extremely efficiently and at low cost.

The present invention will be explained in detail below.

In the present invention, as microorganisms, a microorganism (hereinafter referred to as a first microorganism) capable of producing a target phosphoric oxide from a precursor of the phosphoric oxide and a high-energy phosphoric acid compound, an energy source and a phosphoric acid compound are used. Using both a microorganism (hereinafter referred to as a second microorganism) that has the ability to generate a high-energy phosphoric acid compound from an acid donor and has the ability to regenerate a low-energy phosphoric acid compound into the high-energy phosphoric acid compound, By allowing these two types of microbial cells or their processed products to coexist in an aqueous medium containing a precursor of the desired phosphoric oxide, an energy source, and a phosphoric acid donor, and causing a reaction, the desired A phosphoric oxide can be produced.

Examples of the phosphorylated compound in the present invention include CDP-
Examples include choline, NADP, cotinamide adenine dinucleotide phosphate), CoA, and FAD.

As the first microorganism used in the present invention, any microorganism can be used as long as it has the ability to produce the target phosphoric oxide from a precursor of the phosphoric oxide and a high-energy phosphoric acid compound. Examples include Brevibacterium ammoniagenes ATCC6872.

In addition, the second microorganism is a microorganism that has the ability to generate a high-energy phosphoric acid compound from an energy source and a phosphoric acid donor, and has the ability to regenerate a low-energy phosphoric acid compound into the high-energy phosphoric acid compound. Any of them can be used, and specific examples include Satucharomyces cerevisiae ATCC 15248 and Corynebacterium glutamicum ATCC 13761.

In the present invention, these two types of microorganisms can be allowed to coexist in the reaction system as they are as bacterial cells or their processed products, but it is also possible to immobilize the two types of microorganisms simultaneously in a polymer carrier and utilize this. It is also possible to carry out the reaction by

Cultivation of microorganisms to obtain microbial cells can be carried out according to conventional microbial culture methods.

Any known method can be used to immobilize microorganisms or their processed products (for example, "Immobilized Enzymes" edited by Chibata, Kodansha, 1975), but Japanese Patent Application No. 51-138414 and Showa 52-4851
The method described in the specification of No. 9 is effectively utilized.

The specification of Japanese Patent Application No. 51-138414 states that ``Acrylic acid amide, acrylic acid, a metal salt of acrylic acid, and a crosslinking agent are made to contain enzymes or microorganisms, and a polymerization initiator is added to cause a polymerization reaction. The specification of Japanese Patent Application No. 52-48519 describes a method for immobilizing enzymes or microbial cells by producing a gel containing microbial cells, and the specification of Japanese Patent Application No. 52-48519 describes a method for immobilizing enzymes or microbial cells by producing a gel containing microbial cells. ``immobilized enzyme or immobilized microbial cell composition obtained by contacting in a liquid to form a complex, and then encapsulating the resulting complex with a water-insoluble polymeric substance.'' These methods can be used in the present invention when microbial cells or processed products thereof are immobilized.

When immobilizing two types of microorganisms (a first microorganism and a second microorganism) at the same time, these two types of microorganism cells or treated products are mixed in any ratio and then mixed according to the above immobilization method. By this operation, two types of microbial cells can be immobilized on the polymer carrier at the same time (in the case of immobilizing microbial cells, it is desirable to dry or freeze-dry the cells in advance).

Used in the present invention.

Precursors of the target phosphoric oxide include NAD (target product: NADP), pantothenic acid (target product: CoA), F
Examples include MN (target product: FAD), CMP (cytidine-57-phosphate) (target product: CDP-choline), and the like.

As the energy source, for example, sugars such as glucose, sugar alcohols, alcohols, organic acids, etc. are used, and the use of alcohols and organic acids reduces bacterial contamination and is advantageous for long-term operation of the reaction.

Phosphate donors include KH2PO4, 2HPO4゜K3PO4, NaH2PO4, Na2HPO4, Na5
A phosphate salt such as PO, etc. is used, and preferably a phosphate buffer is used.

Furthermore, in the present invention, the reaction is carried out in the presence of a metal salt such as magnesium in the aqueous medium, if necessary.

In the reaction of the present invention, the cells of two types of microorganisms or the treated product are allowed to coexist in an aqueous medium containing a precursor of the desired phosphoric oxide, an energy source, a phosphoric acid donor, and a metal salt such as magnesium. to perform the reaction.

The reaction can be carried out under either aerobic or anaerobic conditions, with a pH of 5.0 to 8.5, preferably 6.0 to 8.
, 01 reaction rate at 20 to 45°C, preferably around 30°C, and usually for 5 to 40 hours.

Purification of the phosphorus oxide from the reaction solution after the completion of the reaction can be carried out by an ion exchange treatment method or the like.

Examples will be described below.

Example 1 Glucose 0.4 g/d11 yeast extract 0.4 g/
d11 malt extract 1.0 g/dl medium 300ml!
(pH 6,0) using 31 flasks containing 1.30
'C, 200r, p, fTL with shaking culture.

Saccharomyces cerevisiae
Brevibacter cultured according to ATCC 15248 and Example 1 of Japanese Patent Publication No. 41-15516
ium ammoniagenes ATCC6872
After collecting, washing, and freeze-drying each, the former bacterial cells 1
2 g of the latter bacterial cells mixed at a ratio of 2 parts to 5 parts of physiological saline. ! i 2.1% chitosan suspended in 5 g of water was mixed with 29% benzene under strong stirring. Ethyl cellulose (1% solution with 100 CPS) in 11 g of p, n-hexane
Dissolve 3g of W
10 emulsions were produced.

Add this to a 1% polyethylene glycol solution (pH 8.5)
1201111 to form a W10/W' emulsion.

10-15 TL of n-hexane cooled on ice at 5-10℃
/rrm for 1 hour to completely remove benzene.

The thus obtained microencapsulated microbial mixture was taken from house to house, washed twice with n-hexane, and then the n-hexane was completely removed under vacuum.

10ml of this microcapsule, 66g of NADO,
Cystine hydrochloride 0.011 Magnesium sulfate heptahydrate 0
．． 1g, glucose 7.2g, phosphate buffer (pH
7,9) was added to 21 Erlenmeyer flasks containing 1001 rL of a reaction solution containing 3.8 g as PO4.
After shaking at ℃ for 5 hours, the reaction supernatant contained NADP.
was produced at a ratio of 0.1:l.

The molar yield of the product was 18%.

Example 2 Brevibacterium ammonia
genesATCC6872 was cultured.

In addition, Corynebacterium glutamicum ATCC13761 was cultured with shaking using 30011 L of glucose broth medium supplemented with 5 g/11 yeast extract.
Seed culture of glucose 30g/L KH2PO40
,5g/l, 2HPO41,5g/L NH4(12
g/11. , MgSO4・7H200, 59/ 111
Mn 804 ・4H2020”9/ lj 1FeS
04+ 7 H2O20”9/ 11ZnS04+ 7
H2O5Tr19/11 Biotin 3.0μg/13
1 cone steeple force 2g/111 Thiamine-HCJ
'1mg/A (separate steaming), urea 3 g/l (separate steaming)
Transplanted into a 101 jar fermenter containing medium 31 (pH 7,2) and incubated at 30°C, 500r, p, m, I
Culture was carried out for 36 hours in II air/l/yniyr.

After collecting, washing, and freeze-drying each, 2 g of a mixture of 2 bacterial cells of the former and 1 bacterial cell of the latter was microencapsulated in the same manner as in Example-1.

This microencapsulated microorganism mixture 1011Ll,
Sodium pantothenate 0.2g, cystine hydrochloride 0
．． 1.9゜AMP (5'-adenylic acid) 0. ! 11 Magnesium sulfate heptahydrate 0.1 g, phosphate buffer (
p) (3.8g with 7,0 as PO4, glucose 10
The reaction mixture was added to 21 Erlenmeyer flasks containing 10 g of QmA' and subjected to a shaking reaction at 30° C. for 10 hours. As a result, 0.1 g of coenzyme-A was produced in the reaction solution supernatant.

The molar yield from pantothenic acid was 14%.

Example 3 Sacc-h cultured in the same manner as in Example-1
aromyces cerevisiae ATCC1
Example-1 of 5248 and Japanese Patent Publication No. 45-22518
Brevibacterium a cultured according to
mmoniagenes ATCC6872, respectively.
After collecting, washing, and freeze-drying, 2 g of a mixture of 1 bacterial cell of the former and 2 bacterial cells of the latter was added to FMN100.
■, Mg5O, 7H200,i, phosphate buffer (
pH 7.0) as PO4, 3.8g, glucose 7.
The mixture was added to 21 Erlenmeyer flasks containing 100M of a reaction solution consisting of 2g, and shaken at 37°C for 20 hours, resulting in 78m9 of FAD produced in the reaction solution.

Claims (1)

[Claims] 1 Using a microorganism that has the ability to produce a target phosphoric oxide from a precursor of the phosphoric oxide in the presence of a high-energy phosphoric acid compound, or a processed product thereof, the precursor and the high-energy phosphoric acid compound are combined. In a method for producing a target phosphoric acid by reacting in the presence of an energy source, the method uses a phosphoric acid compound of the genus Satucharomyces or Corynebacterium, which has the ability to regenerate a low-energy phosphoric acid compound produced by the reaction into the high-energy phosphoric acid compound A method characterized by using in combination a microorganism belonging to the genus Umbrella or a processed product thereof.