KR100836093B1 - 2 Novel Deinococcus sp. strain A2 and method for producing astaxanthin using the same - Google Patents

Abstract

An astaxanthin bio-synthetic Deinococcus sp. strain A2 is provided to be used for producing the astaxanthin, which is used sitologically, pharmaceutically and cosmetically. A novel Deinococcus sp. strain A2 has a 16S rDNA sequence of SEQ ID : NO. 3 and is deposited as a deposition no. KCTC 11061BP. A method for producing astaxanthin comprises the steps of: (a) culturing the Deinococcus sp. strain A2; (b) recovering the cultured strain; and (c) extracting the astaxanthin from the recovered strain through HPLC(high performance liquid chromatography) or TLC(thin-layer chromatography).

Description

Novel deinococcus species A2 and methods for producing astaxanthin using the same {Novel Deinococcus sp. strain A2 and method for producing astaxanthin using the same}

1 is a photograph on a solid medium of a novel isolated strain according to the present invention (hereinafter abbreviated as A2). Orange on the medium indicates that astaxanthin is being biosynthesized.

Figure 2 is an HPLC analysis of the pigment compound extracted from the novel isolated strain A2 according to the present invention. At this time, (a) is the HPLC results of the astaxanthin product, (b) is the HPLC results of the extract of A2.

The present invention relates to a novel microorganism that produces astaxanthin, and more particularly, to Deinococcus sp. A2 producing astaxanthin and a method for producing astaxanthin using the same.

Photosynthesis is the reaction that uses light to photolyse water molecules and fix carbon dioxide to produce energy. Photosynthetic organisms have defense mechanisms, such as carotenoid and tocopherol production, to minimize damage from photoinhibition. Among them, carotenoids are C ₄₀ isoprenoid compounds with antioxidant activity, and are pigments that exist in various colors such as yellow, red, scarlet, and orange depending on their molecular structure. Carotenoids are classified into primary carotenoids and secondary carotenoids. Primary carotenoids include lutein, zeaxanthin and β-carotene, and secondary carotenoids include astaxanthin and canthaxanthin.

Astaxanthin is a kind of (3,3'-dihydroxy-β, β-carotene-4,4'-dione) carotenoids with hydroxyl and ketone groups added to both benzene rings of beta-carotene. It is a naturally occurring scarlet or light orange pigment substance. Astaxanthin is known to be present primarily in marine animal tissues such as shrimp, red seabream, salmon and lobsters. The color of astaxanthin is due to the double bond structure, and the hydroxyl and ketone groups of the benzene ring are known to control color (Andrews, et al., Phytochemistry., 15: 1003-1007, 1976). Animals rely on food intake because they do not have astaxanthin biosynthesis, which is achieved primarily by ingesting astaxanthin-containing microorganisms. However, aquaculture fish and crustaceans limit the consumption of astaxanthin-containing microorganisms in the food chain, so astaxanthin-containing microorganisms must be added to the feed to produce astaxanthin-colored products.

Astaxanthin not only inhibits free radicals from damaging DNA, proteins, lipids, etc. in the aerobic metabolic process and causes aging and carcinogenesis of cells and tissues, but also inhibits free radicals (hydroxy, peroxy radicals). It acts to suppress production. In other words, protective effect against gastric ulcer, anti-cancer effect, anti-inflammatory effect, anti-diabetic effect, corneal protection effect, muscle fatigue recovery effect, skin protection effect from UVA (ultraviolet A), antihypertensive effect, anti-arteriosclerosis effect, etc. (Hussein G et al., J. Nat. Prod., 69: 443-449, 2006). In addition, astaxanthin has the ability to enhance immune and regulate immunomodulation, and has been reported to treat autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and Crohn's disease, to prevent Helicobacter pylori infection, and to prevent Salmonella infection (Higuera). -Ciapara I et al., Crit. Rev. Food Sci. Nutr., 46: 185-196, 2006).

At present, industrial production of astaxanthin is largely dependent on chemical synthesis (Ernst H., Pure Appl. Chem., 74: 1369-1382, 2002). It has a low bioabsorption rate and has a problem in safety as a food additive, so it is approved for use only in some countries in Europe. Therefore, attention is focused on the synthesis of natural astaxanthin, and there is a commercial interest in the production of astaxanthin using microorganisms producing astaxanthin. Microorganisms currently known to produce astaxanthin include microbial yeast, Phaffia rhodozyma and green algae Haematococcus pluvialis (Bhosale P and Bernstein PS, 68: 445-455, 2005). However, the cultivation of microalgae such as Haematococcus fluvialis requires a long time, serious pollution problems, and high process costs. Papia rodojima is relatively easy to ferment and has a low process cost, but has a problem in that digestion absorption is difficult due to low astaxanthin content per cell and hard cell walls.

Other recently isolated astaxanthin-producing microorganisms include Brevibacterium 103; Lizuka & Nishimura, J. Gen. Appl. Microbiol., 15: 127-134, 1969; Mycobacterium lacti Mycobacterium lacticola ; Neils HJ & DeLeenheer AP., J Appl. Bacteriol., 70: 181-191, 1991), Agrobacterium aurantiacum ; Yokoyama et al., Biosci. Biotechnol. Biochem., 58: 1842-1844, 1994), Paracoccus marcusii ; Harker et al., Int. J. Syst. Bacteriol., 48: 543-548, 1998), Paracoccus carotinifaciens ; Tsubokura et al., Int. J. Syst. Bacteriol., 49: 277-282, 1999), Paracoccus spp. MBIC 01143 ( Paracoccus sp. Strain MBIC 01143; Misawa et al., J. Bacteriol. 177: 6575-6584, 1995), Brebundimonas sp. SD212 ( Brevundimonas sp., Strain SD212; Yokoyama et al., Biosci. Biotechnol. , 58: 1842-1844, 1996), Paracoccus haeundaensis ; Lee JH et al., Int. J. Syst. Evol. Microbiol., 54: 1699-1702, 2004. They are also used as a resource for gene cloning for efficient recombinant production of astaxanthin (Sieiro C et al., Int. Micriobiol., 6: 11-16, 2003).

In order to isolate new astaxanthin-producing microorganisms, the present inventors examined the astaxanthin-producing ability of bacteria collected near Eulsukdo, Busan, and were able to isolate several astaxanthin-producing bacteria from about 20,000 colonies. as a result of identifying the astaxanthin content of the high strain A2 Day furnace Rhodococcus (Deinococcus) bar iteotneun be seen that the novel strain of the species, named it as a Day no Lactococcus species A2 strain (Deinococcus sp. strain A2) and confirm their properties The present invention has been completed.

One object of the present invention is to provide a Deinococcus sp. A2 producing astaxanthin.

Another object of the present invention is to provide a method for producing astaxanthin from Deinococcus sp. A2 containing astaxanthin.

The present invention relates to a novel microorganism that produces astaxanthin, and more particularly, Deinococcus sp. Strain A2 producing astaxanthin and astaxanthin using the same. It is about how to produce.

In one embodiment, the present invention relates to Deinococcus sp. A2, which produces astaxanthin.

Isolation and identification of Dinococcus spp. A2 according to the present invention was carried out by the following method. Strains of scarlet or bright orange color were isolated from seawater samples collected near Eulsukdo in Busan. The isolated Deinococcus sp. Strain is Gram-positive cocci, which do not form spores and do not produce CO ₂ and have no motility. And astaxanthin production. As a result of fatty acid analysis, the main fatty acid was C _{16: 1} and occupied 34.1%.

As a result of analyzing the microorganism according to the present invention through the 16S rDNA sequence, it was identified as a new strain A2 of Dinococcus species by showing a similarity of about 98.83% with the known microorganisms of Daynococcus spp. On the 15th, it was deposited with KCTC (Korean Colletion for Type Cultures), Biotechnology Research Center, Biotechnology Research Institute, 52, Eun-dong, Yuseong-gu, Daejeon.

As a result of testing the optimal culture conditions of the selected strains, Dinococcus spp. A2 is a carbon source for growth, β-glucosidase, β-galactosidase, glucose, Mannose, N-acetyl-glucosamine, maltose, gluconate, malate, cytochrome oxidase, etc. can be used, and the optimum growth temperature is It was 25-35 degreeC, Preferably it was about 30 degreeC.

In another embodiment, the present invention provides a method of culturing Dinococcus sp. A2; Recovering the cultured Denococcus spp. A2; And it relates to a method for producing astaxanthin comprising the step of extracting astaxanthin from the recovered microorganisms.

Daynococcus spp. A2 having the ability of producing astaxanthin of the present invention can be cultured in various media capable of promoting the production of astaxanthin. Such culturing methods include batch culture, fed-batch culture and continuous culture. It can be illustrated. As an assimilation carbon source that may be included in the medium, glucose and polymers thereof, polyalcohols, and the like may be used. As an assimilation nitrogen source, inorganic nitrogen compounds, substances of animal, plant and microbial origin may be used, and other vitamins, Growth promoters, pigment formation promoters and the like can be included in the medium. The composition of the medium can be adjusted in various ways to increase the production capacity of astaxanthin according to the culture. As a preferred embodiment, the basic medium may be any of a variety of commercialized media known for microbial culture, for example, Luria-Bertani (LB) media.

Astaxanthin present in the cultured Deinococcus sp. A2 strain can be isolated and purified by conventional methods in the art. Astaxanthin present in the strain is an organic solvent, such as acetone, chloroform, xhloroform, methyl alcohol, hexane, dichloromethane, cyclohexane, ethanol Extraction may be performed using ethanol, benzene, carbon disulfide, diethyl ether, or a mixed solvent thereof. In addition, recovery of astaxanthin from the culture may be performed according to methods known in the art using high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Isolation and Culture of Strains

1) Isolation of Strains

Seawater was collected from Eulsuk-do coastal water in Busan and plated 100 ul each on Luria-Bertani (LB) plate and incubated at 30 ° C. for 2-4 days. After 2-4 days, orange colonies were passaged three times in the same medium to obtain a single strain, which was tentatively named A2 (see FIG. 1).

2) Culture of A2 Strain

Luria-Butani medium was used for the culture of the isolated strain A2. For liquid medium, 10 g of trypton, 5 g of yeast extract, and 5 g of sodium chloride (NaCl) were added to 1 L of distilled water and sterilized for 15 minutes at 121 ° C. 5 g of yeast extract, 5 g of sodium chloride, and 15 g of agar were placed in 1 L of distilled water and sterilized for 15 minutes at 121 ° C. The preparation of the culture was inoculated in a 250 ml baffled flask inoculated with a single colony of the strain separated in a liquid medium dispensed by 20 ml each, and the culture was shaken at 30 ℃, 180 rpm.

Example 2 Identification of Isolated Strain A2

1) Morphological characteristics of the strain

After 48 hours of incubation at 30 ° C. using Luria-Butani medium, single colonies were taken and examined for morphological characteristics under a microscope.

2) Physiological Characteristics and Carbon Source Availability of Strains

API kits were used to investigate the physiological properties and carbon source availability of isolated strain A2. The isolated strain A2 according to the present invention was inoculated in Luria-Butani medium and incubated at 30 ° C. for 3 days, and then inoculated onto the substrate of the kit, and the results are shown in Table 1.

Physiological Characteristics and Carbon Sources of Strains  Analysis item  Growth  Reduction of nitrates to nitrites -  Indole production -  Glucose acidification -  Arginine dihydrolase -  Urease -  Esculin hydrolysis (β-glucosidase) +  Gelatine hydrolysis (protease) -  β-galactosidase +  Glucose assimilation +  Arabinose assimilation -  Mannose assimilation +  Mannitol assimilation -  N-acetyl-glucosamine assimilation +  Maltose assimilation +  Gluconate assimilation +  Caprate assimilation -  Adipate assimilation -  Malate assimilation +  Citrate assimilation -  Phenyl-acetate assimilation -  Cytochrome oxidase +

3) Identification via 16S rDNA base sequence of isolated strain A2

The isolate strain A2 was inoculated in Luria-Butani and incubated at 30 ° C. for 24 hours, and the chromosome extracted therefrom was used as a template for PCR reaction. The primers of SEQ ID NO: 1 and SEQ ID NO: 2 were used as Jung-Hoon Yoon et al., Int. J. Syst. Bacteriol., 48: 187-194, 1998).

16S-F: 5'-GAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 1)

16S-R: 5'-AGAAAGGAGGTGATCCAGCC-3 '(SEQ ID NO: 2)

The polymerase chain reaction was performed using a thermal cycler (manufacturer: Takara, Japan), and then reacted at 95 ° C. for 5 minutes, 30 seconds at 95 ° C., 30 seconds at 55 ° C., 1 minute 30 seconds at 72 ° C. After performing a round, it was carried out under the conditions of reacting for 7 minutes at 72 ℃ to obtain a fragment of 1.5kb. The obtained fragment was extracted from 0.9% agarose gel and then cloned into pGEM-T easy (Promega, USA) and analyzed for sequencing (SEQ ID NO: 3). As a result of confirming the flexible relationship between different species through the analyzed BLAST-N of NCBI, Deinococcus strain ( Deinococcus strain) aquaticus strain) 98.83% similarity (similarity) with PB314 was identified as a new strain belonging to Deinococcus species (Table 2). The isolated strain was named Deinococcus sp. Strain A2, and was deposited with KCTC accession number KCTC 11061BP as of January 15, 2007.

16S rDNA Similarity with Other Species of Deinococcus Species A2   Strain  % Similarity in:  One  2  3  4  5  6  7  1.Strain A2 2. Deinococcus aquaticus strain PB314  98.83 3. Deinococcus indicus Wt / 1aT  97.10  96.44 4. Deinococcus grandis  96.39  96.39  95.01 5. Deinococcus hohokamensis strain KR-245  95.89  94.48  93.80  95.13 6. Deinococcus deserti strain VCD117T  96.20  95.61  93.64  95.54  96.23 7. Deinococcus radiodurans  94.08  93.47  96.12  93.66  93.00  92.68 8. Deinococcus radiopugnans  94.34  92.46  91.72  93.12  93.87  94.48  92.48

4) Isolated strain Deinococcus Identification through fatty acid analysis of species A2

In order to identify the isolated strain Deinococcus spp. A2 according to the present invention, the fatty acid composition in the cells was analyzed. To this end, isolated strain Deinococcus spp. A2 was incubated for 48 hours at 30 ° C. in Luria-Butani plates. The analyzer used a Microbial Identifcation System MIDI (Microbial ID. Inc., De, USA) and the analyzed data was retrieved from TSBA40 (Sherlock Version 4.9; manufactured by MIDI, USA). 40 mg of the culture was taken in a cap tube with a loop, and then 1 ml of a reagent containing 15% sodium hydroxide was added to a methanol: distilled water (methanol: DW) 1: 1 solution, followed by reaction at 100 ° C. for 25 minutes to extract intracellular fatty acids. For methylation of the extracted fatty acid, 2 ml of 6N HCl was added and reacted at 80 ° C. for 10 minutes, and then rapidly cooled. In order to convert an aqueous fatty acid into an organic phase, hexane: methyl-tert-butyl ether (Hexane: Methyl- tert Butyl Ether) After adding 1.25 ml of a 1: 1 mixed solution, the mixture was rotated for 10 minutes, and only the supernatant was taken. Finally, the sample was washed with 3 ml of saturated sodium hydroxide solution and analyzed through an analyzer. After a review of distribution to perform fatty acid analysis The main fatty acids C _{16: 1} were compared to the total occupying ratio was reached 34.1% (Table 3).

Fatty Acid Distribution of Deinococcus Species A2 and Other Similar Species  fatty acid  % in: Deinococcus A2 Deinococcus indicus Wt / 1a ^{T (1)} Deinococcus grandis DSM 3963 ^{T (2)} Deinococcus radiodurans DSM 20539 ^{T (2)} Deinococcus deserti VCD117 ^{T (3)} Deinococcus radiopugnans ATCC 19172 ^{T (2)}  Straight-chain: C _{14 : 0}  1.3  1.9  -  -  0.7  - C _{15 : 0}  5.2  -  18.1  3.4  6.7  12.0 C _{16 : 0}  16.2  12.2  5.0  14.1  12.1  5.2 C _{17 : 0}  3.8  -  8.7  5.4  2.5  5.2 C _{18 : 0}  0.4  0.1  -  -  -  -  Branched-chain: iso-C _{15 : 0}  8.9  0.1  5.6  -  0.7  18.3 anteiso - C _{15 : 0}  0.2  4.8  -  -  -  - iso-C _{16 : 0}  0.5  -  2.0  -  6.2  - iso-C _{17 : 0}  6.3  -  2.8  -  1.7  8.1 anteiso-C _{17 : 0}  0.1  9.8  -  -  -  -  Unsaturated: C _{15 : 1}  9.9  12.5  30.8  3.1  4.9  9.1 C _{16 : 1}  34.1  33.7  11.7  42.8  43.1  5.1 C _{17 : 1}  6.2  10.7  8.6  21.6  17.1  27.0 C _{18 : 1}  0.3  0.3  -  1.3  2.4  -

Data from (1) K. Suresh et al., Int. J. Syst. Evol. Microbiol. 54: 457-461, 2004 (2) Ferreira et al ,, Int. J. Syst. Bacteriol. 47: 939-947, 1997 (3) Groot et al., Int. J. Syst. Evol. Microbiol., 55: 2241-2246, 2005

Example  3. HPLC  ( high performance liquid chromatography Through) Astaxanthin of isolated strain A2 Productive  black

HPLC (high performance liquid chromatography) confirmed that isolate strain A2 produced astaxanthin. The pigment compound contained in the isolated strain A2 was extracted through acetone and concentrated under reduced pressure. The pigment extract concentrated under reduced pressure was dissolved in a solvent mixed with acetonitrile: methanol: isopropanol 90: 6: 4. The column used for high performance liquid chromatography (HPLC) was a C18 reversed phase column (Ultrasphere ODS 4.6 mm × 25 cm, manufactured by Beckman Coulter, CA, USA), and the same solvent used as the solvent in which the pigment extract was dissolved. . The flow rate was 1 ml / min, and the absorbance was detected at 475 nm.

The results are shown in FIG. HPLC analysis of astaxanthin samples showed peaks at 6.5 components (see (a)), and isolated strain A2 also showed the same peak at 6.4 components (see (b)). You can see that it generates.

Astaxanthin biosynthetic bacteria Dinococcus sp. A2 provided by the present invention can stably supply astaxanthin, which is used for food, pharmaceutical and cosmetic applications.

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Claims (4)

A new Deinococcus sp. Microorganism having the astaxanthin of accession number KCTC 11061BP having the 16S rDNA sequence of SEQ ID NO: 3. delete (a) culturing the microorganism of claim 1; (b) recovering the cultured microorganisms; And (c) extracting astaxanthin from the recovered microorganisms. The method of claim 3, wherein the astaxanthin extraction of step (c) is characterized in that it uses high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

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