JP2006160699A - New isoprenoid quinone - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new indicator compounds for the chemical classification of Nocardia microorganisms, to provide a method for producing the compounds using such microorganisms, and to provide a chemical classification method of the Nocardia microorganisms. <P>SOLUTION: The compounds are represented by formula(I) and formula(II) respectively. The method for producing the compounds using the microorganisms is provided. The method for classifying the microorganisms with these compounds as indicators is also provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel compound useful as an indicator for chemical classification, a method for producing the same using microorganisms, and a method for classifying microorganisms using these compounds as indicators.

  Conventionally, intracellular fatty acid composition and isoprenoid quinone (hereinafter simply referred to as “quinone”) composition, which are generally contained in most bacteria, have been mainly used as important indicators of chemical classification of bacteria (non-patented). Reference 1). Quinones are essential components of respiratory chains and photosynthetic electron transport chains contained in bacterial cell membranes. Structurally, it is roughly divided into naphthoquinone type and benzoquinone type, and menaquinone (2-Methyl-3-polyprenyl-1,4-naphthoquinone) and ubiquinone (2-methyl-1,3-methoxy-3-polyprenyl), respectively. -1,4-benzquinone). There are several biosynthetic derivatives in both quinone groups. In addition to structural differences of the quinone skeleton, the isoprenoid side chain length and hydrogen saturation, the presence / absence of a modifying group, and the like can be useful indicators for chemical classification. The structural differences of quinones peculiar to such taxonomic groups are genetically stable, and there is almost no change in dominant quinone species due to the influence of culture environment conditions.

  Microorganisms belonging to the Nocardia family of Gram-positive bacteria that have hydrocarbon degradability play an important role in activated sludge (Patent Documents 1 and 2), and may cause fish infections Often, taxonomic identification of fungi is necessary. Research so far has revealed that the dominant quinone species of Nocardia microorganisms are unusual in which the end of the isoprenoid side chain of menaquinone is a cyclohexene ring (Non-patent Documents 2 and 3). . However, since the microorganisms examined are only a limited genus in the Nocardiaceae family, it is desired to elucidate new dominant quinone species of other genera and provide the new quinone compound.

Goodfellow, M. and O’Donnell, A. G. (eds.) (1994) Chemical methods in prokaryotic systematics. John Wiley & Son, Chichester. Howarth, O. W., Grund, E., and Kroppenstedt, R. M. Biochem. Res. Commun., 140, 916-923 (1986). Collins, M. D., Howarth, O. W., Grund, E., and Kroppenstedt, R. M., FEBS Microbial. Lett., 41, 35-39 (1987). JP 2002-101872 A JP 2004-222526 A

  In view of the above circumstances, the present invention provides a novel quinone compound, a production method thereof, and a chemical classification method using the novel quinone compound as an index, by searching for a new dominant quinone species from a novel microorganism of the Nocardia family. With the goal.

  As a result of intensive studies in order to solve the above problems, the present inventors have found that the dominant quinone species produced by a novel microorganism of the Nocardiaceae newly isolated from the natural world is a novel quinone compound. The present invention has been completed.

  That is, the present invention provides the following (1) to (6).

(1) A compound represented by the following formula (I).

(2) A compound represented by the following formula (II).

(3) A microorganism belonging to the family Nocardia and producing the compound described in (1) and the compound described in (2).

(4) The microorganism according to (3) having a 16S rRNA gene represented by a base sequence having 97% or more homology with the base sequence described in SEQ ID NO: 1.

(5) The compound of (1) or the Nocardiaceae microorganism having the ability to produce the compound of (2) is cultured in a medium, and the compound of (1) or the compound of (2) is contained in the culture. A method for producing the compound according to (1) or the compound according to (2), wherein the compound according to (1) or the compound according to (2) is collected from the culture and collected.

(6) A method for classifying a microorganism belonging to the Nocardia family using the compound described in (1) or the compound described in (2) as an index.

  The present invention provides a novel chemical classification indicator substance for microorganisms and a method for producing the substance using microorganisms. The compounds of the present invention are useful as an indicator of the classification of Nocardiaceae microorganisms.

  The present invention is described in detail below.

  The compound of the present invention is a compound that was first isolated and purified from Hou_blue strain, a novel (new genus and new species) microorganism of the Nocardia family. The properties of the compound of the present invention and the production method thereof will be described in detail below.

1. Properties of the Compound of the Present Invention The compound of the present invention is a compound represented by the following formulas (I) and (II).

The physicochemical properties of the compound represented by the above formula (I) are as follows:
(1) Material color: Yellow (2) Molecular weight: 720
(3) Molecular formula: C ₅₁ H ₇₆ O ₂
(4) Mass spectrometry: High resolution FABMS (fast neutral particle impact ionization mass spectrometry)
Actual value 720.5858 [M] ⁺
Calculated value 720.5842 (C ₅₁ H ₇₆ O ₂ )
(5) UV absorption spectrum (in methanol) λmax (logε) 248nm (4.07), 262nm (3.99), 328nm (3.29)
(6) ¹ H NMR (measured in deuterated chloroform, 750 MHz)
δppm 0.81 (3H, d, J = 6.6Hz), 0.82 (3H, s), 0.83 (3H, d, J = 6.6Hz), 0.90 (3H, s), 0.96-1.16 (5H, m), 1.17- 1.40 (13H, m), 1.44 (1H, m), 1.52 (3H, m), 1.57 (3H, s), 1.58 (6H, s), 1.65 (2H, m), 1.71 (1H, m), 1.76 (3H, s), 1.92 (4H, m), 1.96 (6H, m), 2.05 (5H, m), 2.18 (3H, s), 3.36 (2H, d, 6.9Hz), 4.52 (1H, s) , 4.74 (1H, s), 5.00 (1H, t, 6.8Hz), 5.10 (3H, m), 7.68 (1H, m), 8.07 (1H, m)
(7) ¹³ C NMR (measured in deuterated chloroform, 125 MHz)
δppm 12.77, 16.05, 16.11, 16.19, 16.40, 16.73, 19.78, 23.84, 24.50, 24.90, 25.40, 25.57, 26.09, 23.36, 26.76, 26.80, 28.53, 32.53, 32.63, 32.72, 34.95, 36.46, 36.74, 37.19, 37.36 , 37.46, 38.29, 39.83, 39.85, 40.09, 53.65, 108.74, 108.77, 123.95, 124.24, 124.89, 126.12, 126.24, 132.11, 132.16, 133.17, 133.22, 134.49, 134.81, 135.40, 137.88, 143.25, 146.15, 143.149 , 185.32
(8) Solubility: Soluble in chloroform, acetone and methanol.

The physicochemical properties of the compound represented by the above formula (II) are as follows:
(1) Material color: Yellow (2) Molecular weight: 734
(3) Molecular formula: C ₅₂ H ₇₈ O ₂
(4) Mass spectrometry: High resolution FABMS
Actual value 734.5914 [M] ⁺
Calculated value 734.6002 (C ₅₂ H ₇₈ O ₂ )
(5) UV absorption spectrum (in methanol) λmax (logε) 248nm (4.08), 262nm (4.00), 329nm (3.29)
(6) ¹ H NMR (measured in deuterated chloroform, 750 MHz)
δppm 0.80 (3H, d, J = 6.6Hz), 0.81 (3H, s), 0.82 (3H, s), 0.83 (3H, d, J = 6.6Hz), 0.90 (3H, s), 1.00 (3H, s), 0.96-1.16 (6H, m), 1.17-1.40 (14H, m), 1.40-1.52 (5H, m), 1.56 (6H, s), 1.57 (1H, m), 1.65 (2H, m) , 1.76 (3H, s), 1.88-1.94 (6H, m), 1.96-2.06 (4H, m), 2.10-2.22 (2H, m), 2.18 (3H, s), 3.36 (2H, d, 6.9Hz ), 4.55 (1H, s), 4.75 (1H, s), 5.00 (1H, t, 6.8Hz), 5.06 (1H, t, 6.8Hz), 7.68 (1H, m), 8.07 (1H, m)
(7) ¹³ C NMR (measured in deuterated chloroform, 125 MHz)
δppm 12.77, 16.34, 16.40, 18.49, 19.74, 19.78, 23.82, 24.50, 24.59, 25.41, 25.41, 25.59, 26.09, 26.48, 27.57, 28.52, 29.74, 30.96, 32.58, 32.58, 32.73, 34.78, 34.97, 36.35, 36.74 , 37.01, 37.23, 37.37, 37.47, 39.01, 40.09, 41.40, 51.90, 54.67, 108.72, 118.77, 124.08, 126.12, 126.24, 127.24, 132.11, 132.16, 133.18, 133.23, 135.65, 137.89, 140.04, 143.25, 146. , 184.38, 185.32
(8) Solubility: Soluble in chloroform, acetone and methanol.

2. Production of compounds of the invention 2.1. Production of Compound of Formula (I) or (II) The compound of formula (I) or (II) of the present invention is obtained by culturing a microorganism in a medium, producing and accumulating the compound in a culture, and It can be produced by collecting the compound.

(1) Microorganism The microorganism that can be used in the production method of the present invention is a microorganism that belongs to the Nocardia family and can produce the compound represented by the above formula (I) or (II). There is no particular limitation. Examples of such microorganisms include Nocardiaaceae Hou_blue strain. The Nocardiaceae Hou_blue strain is a newly isolated strain from nature, and its bacteriological properties are as follows.

a. Morphological properties
1) Cell shape and size: Cocci, 0.86 x 0.86μm
2) Presence or absence of mobility: None
b. Culture properties
1) ISP medium No.2 Plate culture: Grayish green colony formation
2) ISP medium No.4 Plate culture: No growth
3) ISP medium No.5 Plate culture: No growth
4) ISP medium No.6 Plate culture: Grayish green colony formation
5) ISP medium No.7 Plate culture: No growth
6) 1/10 Tryptic Soy Agar plate culture: Gray green colony formation
c. Physiological properties
1) Growth temperature range: 4-37 degrees
2) Formation of melanin-like pigment: Not generated
3) Alkaline phosphatase activity: Negative
4) Esterase (C4) activity: Positive
5) Esterase lipase (C8) activity: positive
6) Esterase (C4) activity: Positive
7) Leucine allylamidase activity: positive
8) Valine allylamidase activity: negative
9) Cystine allylamidase activity: negative
10) Trypsin activity: negative
11) Chymotrypsin activity: Negative
12) Acid phosphatase activity: Negative
13) Naphthol-AS-BI-phosphohydrolase activity: positive
14) α-Galactosidase activity: Negative
15) β-galactosidase activity: negative
16) β-glucuronidase activity: negative
17) α-Glucosidase activity: Negative
18) β-glucosidase activity: negative
19) α-Mannosidase activity: Negative
20) α-fucosidase activity: negative
21) Use of carbon sources
L-arabinose ： −
D-fructose: +
D-glucose: +
Inositol:-
D-mannitol:-
L-rhamnose:-
Raffinose:-
Sucrose:-
D-xylose:-
Butyric acid: +
Hexadecane: +
W medium (composition below) was used as the basal medium.

G + C content of genomic DNA 63.7 mol% (measured by HPLC method)
The base sequence of 16S rRNA gene of Nocardiaaceae Hou_blue strain is as shown in SEQ ID NO: 1. Using the GenBank database and BLAST program, we searched for the closest microbial species based on the homology of the base sequence of the 16S rRNA gene, and found the closest known strain of Rhodococcus sp. 16S rRNA gene, strain GW- There was only 94% homology with 86a, and the microorganism of the present invention was found to be a microorganism of a new genus and new species of the Nocardia family.

  In addition, Nocardia Department Hou_blue shares were registered with the National Institute of Technology and Evaluation Biotechnology Headquarters (2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture) on June 8, 2004 with the deposit number NITE P- Deposited as 3.

  Instead of the Nocardia family Hou_blue strain, a mutant strain derived from the strain or a similar strain of the strain can be mentioned. Examples of the “mutant strain” and “similar strain” herein include a strain having a 16S rRNA gene having high homology with the 16S rRNA gene (SEQ ID NO: 1) of the Nocardia department Hou_blue strain. “High homology” usually means 97% or more homology, preferably 98% or more homology, and most preferably 100% homology.

(2) Microbial culture In the present invention, a normal microorganism culture method is used for the culture of the microorganism. As the medium, any of a synthetic medium or a natural medium can be used as long as it contains an assimilated carbon source, nitrogen source, inorganic substance, and necessary growth / production promoting substances as appropriate. As the carbon source, glucose, starch, dextrin, mannose, fructose, molasses and the like are used alone or in combination. Furthermore, hydrocarbons, alcohols, organic acids, amino acids (such as tryptophan) and the like are used as necessary. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean flour, cottonseed meal, casamino acid, etc. are used alone or in combination. . In addition, inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, and zinc sulfate are added as necessary. Furthermore, trace components that promote the growth of the microorganisms to be used and the production of the compounds of the present invention can be appropriately added, and such components can be appropriately selected by those skilled in the art.

  As a culture method, liquid culture is suitable, but is not limited thereto. The culture temperature is suitably 20-30 ° C., the pH of the medium during culture is desirably maintained at 7-9, and it is desirable to carry out rotation or reciprocal shaking culture at a shaking speed of 30-120 rpm. When culturing is usually carried out for 10 to 14 days in liquid culture, the target compound is produced and accumulated in the cells. The culture is stopped when the production amount in the culture reaches the maximum.

(3) Isolation / Purification of Compound Isolation / purification of the compound of the present invention from cultured cells is carried out according to a method commonly used for isolating / purifying a microbial metabolic product from the culture. For example, the culture is separated into culture broth and cells by filtration or centrifugation, and the cells are freeze-dried and extracted with a chloroform / methanol (2: 1) mixed solvent or the like. Next, the bacterial cell extract is concentrated and purified by column chromatography, HPLC or the like to obtain the compound of the present invention. The obtained compound is determined to be the compound of the present invention by examining whether or not it exhibits the properties described in “1. Properties of the compound of the present invention” by an ordinary chemical method such as NMR analysis. Can be confirmed.

  The trend of the compound of the present invention during the culturing and purification operations can be traced by HPLC with a photodiode array detector (PDA) and HLPC / APCI-MS using ultraviolet absorption and mass spectrometry values as indicators.

3. Use of the Compound of the Present Invention The compound of the present invention can be used as an index for classification of Nocardiaceae microorganisms as follows.

(1) Compositional analysis of isoprenoid quinone in cells After lyophilized cells (10-100 mg) are dissolved in about 5 mL of a chloroform / methanol (2: 1) mixed solvent, the supernatant is separated by centrifugation. It is concentrated to dryness, and the residue is dissolved in 5 to 10 μL of acetone and used as a sample for HPLC / PDA / APCI-MS / MS analysis. The quinone molecular species is identified by comparing the retention time in HPLC, ultraviolet absorption spectrum by PDA, and MS / MS pattern with the retention times and patterns of known quinones and the compounds of the present invention for the quinones in the samples provided. . For the identified quinones, the composition ratio is calculated from the ratio of peak areas in HPLC and used as data for chemical classification.

(2) Bacterial community analysis in activated sludge by quinone profile A chloroform / methanol (2: 1) mixed solvent is added to the sludge suspension and homogenized or sonicated. After centrifugation, the upper aqueous layer is discarded, and the intermediate layer and the lower layer (chloroform layer) are filtered with a filter paper. It is concentrated to dryness and further dissolved with hexane-water (1: 1), and then the hexane layer is separated. The hexane layer is concentrated to dryness, dissolved in a small amount of acetone, and used as a sample for HPLC / PDA / APCI-MS / MS analysis. The composition analysis is performed in the same manner as the composition analysis of the isoprenoid quinone in the above (1) bacterial cells to create a quinone profile of activated sludge. In general, one major quinone species exists in one bacterial species / genus. Therefore, the abundance ratio of quinone species in the activated sludge is considered to reflect the distribution ratio of each quinone taxon as it is. The abundance ratio of bacteria of each genus of Nocardia family that plays an important role such as hydrocarbon decomposition in activated sludge can be estimated using known quinone species and compounds of the present invention as indicators.

  Hereinafter, the present invention will be described specifically by way of examples. However, the technical scope of the present invention is not limited by the examples.

[Example 1] Production of compounds of formulas (I) and (II) Hou_blue strain was used as a bacterium producing the compounds of formulas (I) and (II). The strain was cultured for 10 days at 30 ° C. in a conical flask with a 1 L baffle containing a liquid medium obtained by adding 1 g of hexadecane to 300 mL of W medium. During the culture, the pH of the medium was not particularly controlled.

  10 L of the culture solution thus obtained was centrifuged (6000 × g, 20 minutes), and only the cells were collected, followed by extraction with chloroform / methanol (2: 1). Next, the obtained bacterial cell extract was purified by silica gel column chromatography. Using Wako Gel C200 (Wako Pure Chemical Industries) as a carrier, and elution with hexane-ethyl acetate (8: 2) (7: 3) (1: 1) as a moving layer, a fraction containing the compound was obtained.

  Next, these fractions were purified by HPLC (column: Nomura Chemical Develosil ODS-HG-5 (diameter 4.6 mm, length 250 mm), mobile phase methanol-isopropanol (7: 3, v / v), and the present invention. The compounds of formulas (I) and (II) were obtained in an amount of 3 mg and 1.5 mg, respectively, and the obtained compounds of (I) and (II) exhibited the above-mentioned physicochemical properties.

Claims (6)

A compound represented by the following formula (I).

A compound represented by the following formula (II).

  A microorganism belonging to the family Nocardia and producing the compound according to claim 1 and the compound according to claim 2.   The microorganism according to claim 3, which has a 16S rRNA gene represented by a base sequence having 97% or more homology with the base sequence described in SEQ ID NO: 1.   A Nocardiaceae microorganism having the ability to produce the compound according to claim 1 or the compound according to claim 2 is cultured in a medium, and the compound according to claim 1 or the compound according to claim 2 is produced and accumulated in the culture. The method according to claim 1 or the compound according to claim 2, wherein the compound according to claim 1 or the compound according to claim 2 is collected from the culture.   The classification method of the microorganisms which belong to the Nocardia family which uses the compound of Claim 1 or the compound of Claim 2 as a parameter | index.