JPS5857155B2 - Production method of coenzyme Q↓1↓0 by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides coenzyme Q1o (hereinafter referred to as C0
Q1o).

More specifically, a microorganism belonging to the genus Agrobacterium that does not have bark hypertrophy-inducing activity on plants and has the ability to produce C0QIO is cultured in a nutrient medium to produce C0QIJ) in the culture solution and the bacterial cells. The present invention relates to a method for producing COQIO by a fermentation method, which is characterized in that COQIO is collected.

The purpose is to provide a new method for producing C0Q1o, which has a wide range of uses as a pharmaceutical.

C0Q1o is a compound represented by the following formula, and is known to be contained in the cells of relatively many types of microorganisms.

Conventionally, methods for producing C0QIO by fermentation using microorganisms include culturing various yeasts (Japanese Patent Publication No. 48-
8836, 48-25517゜51-19034. JP 52-105288 et al.) or Rhodopseudomonas (JP 47-7954, JP 48-21519)
, Alcaligenes (Special Publication No. 51-19034), Pseudomonas (Special Publication No. 36-14293. Japanese Patent Publication No. 52-44)
290.52-47990), Proteus (Tokuko Sho 3)
Methods using bacteria of various genera such as 6-14293) are known.

However, in all of these methods, the amount of COQIO produced is low and still unsatisfactory.

The present inventors have discovered that C0Q1o is produced by an even better fermentation method.
As a result of various studies on manufacturing methods, we found that COQ□ belongs to the genus Agrobacterium and does not have bark thickening-inducing activity on plants.

It has been found that COQIO can be industrially advantageously produced by using a producing bacterium.

Conventionally, bacteria of the genus Agrobacterium are COQ□.

Containing Agrobacterium tumefaciens N-C-T-C397 or Agrobacterium tumefaciens B6, which is a type of plant cancer, has a strong activity of inducing thickening of plant bark. COQ□ of about 600 μg per 1 g of dry weight.

is known to be included together with C0Q9 and C0Qs (Biochem-J, vol. 111, 46
1 page, 1969: Arch-Biochjm-Bi
ophys,.

(Vol. 113, p. 548, 1966) called this C0QI.
No attempt has been made to utilize O for industrial production.

The main reasons for this are the low production of C0Q1o, the fact that other COQ congeners (COQ9 and C0Qs) are produced by-products and are difficult to separate from C0Q1o, and this type of plant pathological fungus. The reason for this is that if used in industrial production where bacterial cells are handled on a large scale, it may have an adverse effect on agricultural crops, and that processing the bacterial cells is time-consuming.

In order to overcome these difficulties, the present inventors selected a strain that produces C0Q1o in high yield from among microorganisms that do not have the activity of inducing plant bark thickening.
Bacteria belonging to Agrobacterium radiobacter, which is a non-plant pathogen, and other Agrobacterium bacteria that do not have bark thickening-inducing activity on plants produce a significant amount of COQIO, and COQ other than C0QIO We found a strain that does not produce congeners.

Until now, it has never been known that microorganisms belonging to Agrobacterium radiobacter produce C0QIO, and there have been no examples of using other non-phytopathogenic bacteria of the genus Agrobacterium for the fermentation production of C0QIO. This is not known and the present invention is based on such novel findings.

The microorganism used in the present invention belongs to the genus Agrobacterium and does not have bark thickening-inducing activity on plants.
As for 0Q1o production bacteria, both wild strains and mutant strains can be used; representative examples include Agrobacterium radiobacter ATCC4718° ATCC6466, Agrobacterium 5p-0363, Agrobacterium tumefaciens IPO417, ICPB TR6, and NCPP.
B1641 can be mentioned.

Furthermore, non-phytopathogenic mutant strains derived from Agrobacterium tumefaciens that have bark hypertrophy-inducing activity on plants can also be used in the present invention.

In addition, various drugs (antibiotics, etc.) derived from the above-mentioned Agrobacterium genus C0QIO-producing bacteria that do not have bark hypertrophy-inducing activity on plants are used as parent strains.

Amino acid analogs, nucleic acid analogs, vitamin analogs,
Mutant strains with resistance or sensitivity to sulfa drugs, respiratory inhibitors, sterol synthesis inhibitors, etc.), various auxotrophic mutant strains (amino acid auxotrophy, nucleic acid auxotrophy, vitamin auxotrophy, etc.), morphological mutant strains, Mutant strains lacking the ability to synthesize polymeric substances, mutant strains resistant to catabolite repression, and temperature-sensitive strains can also be used in the present invention.

The mycological properties of the above-mentioned Agrobacterium bacteria are described in the Verge Eighth Manual of Determinative Bacteriology, 8th edition (1974) and are well known, and the present invention was carried out in accordance with this description. It is something that

Ta! However, according to the classification method in this manual, the aforementioned Agrobacterium tumefaciens lPO417,
Since ICPBTR6 and NCPPB 1641 are non-plant pathogenic bacteria, it is not appropriate to classify them as ``Agrobacterium tumefaciens'' and they should be classified as other species of the genus Agrobacterium, but these strains The description of the present invention is based on the nomenclature of the isolate.

Furthermore, it is reported in the literature (e.g., J, Gen, ]!14rob
icl.

78, p. 227, 1973) and is publicly known.

Furthermore, mutant strains that do not have bark hypertrophy-inducing activity on plants can be easily derived from strains that do not have bark hypertrophy-inducing activity by known methods such as acridine dye treatment and heat treatment.

The culture medium for microorganisms used in the present invention includes a carbon source,
Both synthetic and natural media can be used as long as they contain adequate amounts of nitrogen sources, inorganic substances, and other nutrients.

Any type of carbon source may be used in the culture medium as long as it can be used by the bacteria used.

i.e. glucose, fructose. Carbohydrates such as sucrose, blackstrap molasses, starch, and starch hydrolysates, sugar alcohols such as glycerin and sorbitol, amino acids such as aspartic acid, glutamic acid, lysine, alanine, and glycine, lactic acid, pyruvic acid, acetic acid, malic acid, and formic acid. Acids such as , succinic acid, fumaric acid, citric acid', and fatty acids, hydrocarbons such as n-paraffin, and alcohols such as methanol, ethanol, propatool, and butanol can be used alone or in combination. If the desired substance is shown, the required substance is added to the culture medium.

In addition, various substances such as nucleic acid-related substances, amino acids, vitamins, organic acids, fatty acids, alcohols, sterols, and other precursors for C0Q1o biosynthesis and related compounds are added to the medium or culture solution. This may increase the amount of C0Q1o produced.

Cultivation is carried out under aerobic conditions such as shaking culture and aerated agitation culture.

During cultivation, the pH of the culture solution is preferably about 5 to 9.

As a neutralizing agent, ammonia water, sodium hydroxide, calcium hydroxide, calcium carbonate, magnesium phosphate,
Potassium hydroxide, urea, etc. are used.

The culture period is usually 3 to 7 days, and C0Q1° accumulates both in the culture solution and in the bacterial cells, but most of it accumulates in the bacterial cells.

C0QIO can be isolated from the culture by conventional methods such as solvent extraction and other operations.

Examples are shown below.

Example l Glucose 2 g/cil t Peptone 1 g/d
l, yeast extract 1 g/d, l>salt 0-5 g
300TLl of seed medium with a pH of 7.2 and a composition of /dl
Pour into a 21-volume Erlenmeyer flask and sterilize.

In addition, Agrobacterium radiobacter ATCC
4718 and cultured with shaking at 30°C for 24 hours.

250 ml of the seed culture solution was inoculated into a 51-volume jar fermenter containing fermentation medium 31 with the following composition, and the mixture was heated at 600 rpm.
When cultured with aeration and agitation for 30 hours under culture conditions with a rotational speed of 31 per minute and a temperature of 30°C, the culture solution was 11 per 26
■COQ□.

was produced, and the by-product amount of C0Qs and C0Q9 was less than 0.2 m9.

Composition of fermentation medium Nigelcose 5 g/dl, ammonium sulfate 1. g/dl.

Potassium phosphate 0.05 g/dl, dipotassium phosphate O-0' 5 g/dl, magnesium sulfate heptahydrate 0.025 g/dl, corn stew liquor 2 g/dl, calcium carbonate 2 g/dl dl(p
Adjust H to 7.2 with ammonia water before sterilization).

Culture solution 21 was centrifuged to obtain wet bacterial cells with a dry weight of 41 g.

After suspending this in 150ml of water, 400ml of methanol
．． Add 80 g of sodium hydroxide and 15 g of pyrogallol, reduce and saponify at 85°C for 40 minutes, let stand to cool, and add n-
Perform extraction twice by adding hexane.

The n-hexane layer was collected, anhydrous sodium sulfate was added thereto, and after dehydration, it was concentrated under reduced pressure. The residue was dissolved in 401 rL of acetone, and after removing insoluble matter, it was concentrated again, and the residue was reduced to 10 m
After dissolving 0COQIO in acetone, it was poured into a silica gel column and eluted with benzene. Fractions containing 0COQIO were collected and concentrated, and the residue was dissolved in 5 Tnl of ethanol and cooled to obtain 13 squares of yellow crude crystals of COQIO.

The crystals obtained from this product by further recrystallization with ethanol have the Rf value of reversed phase thin layer chromatography, high performance liquid chromatography glue tension time, melting point, nuclear magnetic resonance spectrum, elemental analysis value, infrared absorption spectrum, ultraviolet absorption spectrum. The curve matched that of C0Q1o.

Example 2 300ml containing 20TIll of seed medium with the same composition as Example 1
A conical Erlenmeyer flask is inoculated with the Agrobacterium radiobacter strains shown in Table 1, and cultured with shaking on a rotary shaker for 24 hours at a rotation speed of 210 rpm and a culture temperature of 30°C.

2 ml of the obtained seed culture solution was inoculated into a 3001711 volume Erlenmeyer flask containing 20 ml of fermentation medium having the same composition as that used in Example 1, and cultured with shaking in the same manner as the seed culture for 30 hours.

The amount of C0Q1o produced by each strain is shown in Table 1.

Example 3 The same procedure as in Example 2 was used except that the strain of Agrobacterium tumefaciens shown in Table 2 that does not have bark thickening inducing activity on plants was used as the bacteria used. The results were COQ 10 as shown in Table 2. was generated.

Example 4 The same procedure as in Example 2 was carried out except that the non-plant pathogenic bacterium NP-1 derived from Agrobacterium tumefaciens ATCC4452 (a strain with bark hypertrophy-inducing activity on plants) was used as the bacterium used. The results were 26 ml. ;//l
of C0Q1o was generated.

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Agrobacterium that does not have bark hypertrophy-inducing activity on plants and has the ability to produce Coenchyme "Q□." A method for producing coenzyme Q1o, which comprises culturing in a medium containing a moderate amount of coenzyme Q1o to accumulate coenzyme Q1o in the culture, and collecting coenzyme Q1o from the culture.