CN1912098A - Baterial for producing cozymase 10 and method for producing cozymase 10 - Google Patents
Baterial for producing cozymase 10 and method for producing cozymase 10 Download PDFInfo
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Abstract
The invention bases on new ochrobactrum sp. strain WSH-W04 separation. The strain can generate coenzyme Q10 with high base quantity. In addition the invention also offers its producing method includes culturing new bacteria in culture medium to make the coenzyme Q10 gather and extracting it.
Description
The present invention relates to a kind of new bacterial strain of producing Coenzyme Q10 99.0.In addition, the present invention relates to the suitable culture systems that the technology that comprises the fermenting broth of described microorganism and be used for Coenzyme Q10 99.0 is produced.The invention still further relates to by cultivating bacterial strain of the present invention advantageously to produce the method for Coenzyme Q10 99.0.Another aspect of the present invention also relates to the 16srRNA of described microorganism.
Ubiquinone (being also referred to as CoQ, Q or ubiquinone) is found in the film that is prevalent in microorganism, plant, fungi and zooblast.This molecule structurally is by being covalently attached to 2, the polyisoprene base side chain composition of 3-dimethoxy-5-methyl-benzoquinones.The length of side chain is different between different plant species, and its principal mode comprises 6-10 isoprene subunit.Gained ubiquinone homologue is hereinafter referred to as Q6-10.Principal mode by synthetic is a Coenzyme Q10 99.0.
Coenzyme Q10 99.0 (being also referred to as CoQ10) has certain benefit to human health, so it is widely used in makeup, medicine and healthcare products.CoQ10 is a kind of fat-soluble benzoquinones; have two kinds of main biological functions: (i) in plastosome, producing the ATP/ energy during the aerobic repiration by electron transport; (ii) protect various cellular constituents, for example lipid, protein and DNA by removing free radical.CoQ10 is used to various objectives at present:
-supply with the CoQ10 that improves some organ-/ tissue as dietary supplement (healthcare products).This is even more important to the elderly, descends along with the increase at age because scientific research has shown the CoQ10 level, finally causes many and old relevant disease.
-CoQ10 and the known medicine (Statins (statins)) that can reduce the reducing cholesterol of cell CoQ10 level are used in combination.In addition, CoQ10 is used to treat myocardosis, and has inferred that the CoQ10 defective participates in neurodegenerative disease such as Huntington ' s disease, Parkinson's disease and amyotrophic lateral sclerosis.
-CoQ10 is used as active ingredient in cosmetic formulations, so that skin avoids influencing the infringement that for example solar radiation produces free radical because of cellular metabolism or external cause.Clinical study shows that CoQ10 topical application on skin has reduced the activation that wrinkle formed and caused skin cell metabolism effectively.
Industrial, Coenzyme Q10 99.0 is semi-synthetic generation, perhaps by organism of fermentation and extract to produce from biomass subsequently.The complete chemosynthesis approach of Coenzyme Q10 99.0 is set up.Therefore yet this approach is with high costs and compare all nocompetitively with fermentation or semi-synthetic production, can't use with commercial size.
The commercial production of CoQ10 starts from Japan (DE2740614 by Kanegafuchi about 1977; DE 2747510).These patents have been described by the different yeast strain of fermentation and have been produced and reclaimed CoQ10.Yet, before that with applied for describing a large amount of patents of using different microorganisms (bacterium, yeast, filamentous fungus) to produce and extract CoQ10 from then on.The summary that the Institute of Micro-biology of the production CoQ10 that mentions in patent and/or the paper does was announced (Coenzyme Q10 by fermentation.Kanazawa, Morishige in 1981 by Kanegafuchi; Takahashi, Tamio.Pharm.Div., KanegafuchiChem.Ind.Co., Ltd., Osaka, Japan.Biomedical and Clinical Aspects of ubiquinone (1981), 3,31-42).
In the past few years, increasing technical press and patent application have been inquired into the bacteria through fermentation of the α-subgroup that belongs to protein fungus (proteobacteria) and have been produced CoQ10.Although CoQ10 is rare relatively in most of microbe as main ubiquinone, there is in described group very high percentage to produce CoQ10.In addition, belong to many bacteriums, the especially phototrophic bacteria of this group, set up the intracellular membrane structure, can store the CoQ10 of relative a large amount at this.Yet many all demonstrating hanged down the speed of growth and therefore needed the long period to gather CoQ10 in these phototrophic bacterias, causes the cost of fermenting process to increase.
Many patents and publication have been described by the endogenous or allos of the isoprenoid biosynthesis gene of key and have been crossed expression, and perhaps the target gene of the bypass by inactivation and CoQ10 competition isoprenoid precursor is produced CoQ10 (for example WO02/026933, WO01/027286, WO04/047763, WO05/005650).
Have at present: Yoshida, Hajime, Kotani, Yukinobu, Ochiai, Keiko, andAraki, Kazumi (1998) Production of ubiquinone-10 usingbacteria.J.Gen.Appl.Microbiol.44,19-26 about fermentation wild-type bacterium bacterial strain or by the document that the mutant strain that traditional mutagenesis produces is produced Q10.At this various bacterial strains that belong to α-protein fungus that derive from bacterial strain preservation center are screened and analyze the CoQ10 condition of production.Produce mutant strain to increase CoQ10 output.In fed-batch fermentation, the agrobacterium tumefaciens mutant reaches 180mg/l, and Rhodobacter sphaeroides bacterial strain reaches 350mg/l.
Provide about produce studying in great detail in the research by Sakato, Kuniaki, Tanaka, Hisao, Shibata, Susumu and Kuratsu of CoQ10 by the fermentation Rhodopseudomonas spheroides, Yoshiyuki in Agitation-aeration on Coenzym Q10 productionusing Rhodopseudomonas sphaeroides.Biotechnology And AppliedBiochemistry 1992,16,19-28.The attention of this piece paper concentrates on by changing different fermentation parameters, and for example oxygen supply, oxygen reduce potentiality, stirring, and method is carried out optimization.Described bacterial strain reaches 770mg CoQ10/l.The highest the tiring that this is up to now to be reported.
Summary about biosynthesizing, biotechnology production method and the application of CoQ10 is announced in " the biotechnology production and the application of Coenzyme Q10 99.0 " by Choi, Jin-Ho, Ryu, Yeon-Woo and Seo, Jin-Ho.Applied Microbiology andBiotechnology, online open: on March 3rd, 2005 (having accepted to wait to deliver).
Above-mentioned about the ameliorative measure of Coenzyme Q10 99.0 synthetic by reading, everyone has to admit to use the output of most of microbe production Coenzyme Q10 99.0 still relatively low and unsatisfactory.Attempted increasing the Coenzyme Q10 99.0 productivity, caused demonstrating the generation of the genetically modified bacterial strain that the Coenzyme Q10 99.0 productivity increases by molecular biological method.Yet, in industrial production, preferably use natural bacterial strain usually to produce desired compound.Therefore, still need to screen the more effective bacterial strain that is used for CoQ10 production in scientific circles.
Therefore the objective of the invention is further to obtain the microorganism that during fermentation has the ability of producing the high CoQ10 that tires.
This purpose reaches by the embodiment described in the claim.The invention provides a kind of new microorganism, it is named as WSH-W04.The invention still further relates to the method for the composition that comprises microorganism of the present invention or device, a kind of this new bacterial strain that is used to ferment, and the favourable application of microorganism of the present invention, in addition, the invention still further relates to a 16S rRNA gene order and comprise the microorganism of this sequence.
In the new microbe WSH-W04 that provides, the technician has the biology of quick growth, and this microorganism makes and can produce the CoQ10 that height is tired when fermentation.Especially, described new microorganism makes the amount of producing described coenzyme in rotation concussion fermentation pattern reach 64mg/l.The technician should recognize: although ferment in the rotation oscillator, bacterial strain of the prior art only shows that also the productivity of CoQ10 is lower than 100mg/l.Therefore, bacterial strain of the present invention has the high potential of producing the high CoQ10 that tires when being fermented with technical scale under optimal conditions.
Another aspect of the present invention relates to the fermenting broth that comprises microorganism of the present invention.Favourable fermenting broth is that the technician is known, typically contains a kind of carbon source, a kind of nitrogenous source and a kind of phosphate source, and salt, VITAMIN and trace element, forms to optimize cell growth and product.One preferred embodiment in, the invention provides a kind of fermenting broth that also comprises additive, this additive fermentation during microorganism of the present invention forward influence CoQ10 production.This additive is selected from the group of being made up of following: CoQ10 precursor p-Hydroxybenzoate (PHB), prenol, Geraniol, tyrosine, shikimate and chorismic acid, and the composite vegetable extract of soya-bean oil, tomato extract and Radix Dauci Sativae extract.Can know that by following paragraph preferred especially use is vegetable extract in the present invention.
Another aspect of the present invention also relates to a kind of culture systems, and it comprises:
I) a kind of container that can ferment;
Ii) a kind of fermenting broth that comprises microorganism of the present invention;
The instrument of the described microorganism of iii) a kind of fragmentation;
Iv) a kind of device that from fermenting broth, separates coenzyme CoQ10.
This culture systems can be assembled according to technician's knowledge.Wherein the container that can ferment can be any container well known by persons skilled in the art.Preferred container is to be present in container of the prior art (for example at Stanbury et al, 1998, Principles ofFermentation T echnology, the container among the B utterworth-Heinemann).This container comprises, for example, and the Erlenmeyer culturing bottle or the fermentor tank of custom size and shape.Fermenting broth such as above-mentioned generation preferably comprise described additive.After fermentation is finished, the CoQ10 that produces must be separated from reaction mixture.Because CoQ10 is stored in the microorganism, so cell was broken before separating.Therefore, culture systems of the present invention also has a kind of instrument of disruption of microorganisms.This equipment can be any equipment well known by persons skilled in the art, Hopkins for example, 1991 mentioned equipment.
The method that reclaims CoQ10 is known, comprises by extracting CoQ10 with organic solvent from cytolemma, chooses wantonly and carries out further purifying subsequently, for example carries out purifying by precipitation, crystallization, column chromatography, thin-layer chromatography etc.Discriminating and quantized analytical procedure comprise HPLC, mass spectrometry, mass spectrum of nuclear magnetic resonance method etc.Yet the technician can oneself select correct method.
The cultivation of bacterial strain can be carried out at Erlenmeyer culturing bottle middle and small scale, perhaps fed batch cultivation or lasting cultivation the in fermentor tank.Cultivation in fermentor tank obtains higher cell yield usually; And need the crucial fermentation parameter of stricter control, for example pH, dissolved oxygen concentration, redox potential (ORP), nutrient consumption/loss.The mass production that these parameters had before demonstrated CoQ10 is crucial (Kuratsu et al., 1984; Wu et al., 2003; Urakami and Hori-Okubo, 1988; Sakato et al., 1992).
The pH scope of substratum is suitable for fermentation between 6 and 8.Preferred pH scope is fermented between 6.5 and 7.5, most preferably pH7.2.Consider the oxygen supply problem, fermentation should be stirred and be carried out.For the Erlenmeyer culturing bottle, suitable stirring velocity is 100-300rpm.Preferred stirring velocity 150 and 250rpm between, most preferred stirring velocity is 200rpm.The experiment that multiple CoQ10 production bacterial strain is carried out has before proved that the CoQ10 amount increases along with fermentation time in the born of the same parents.Use microorganism of the present invention, suitable fermentation time is at least 58 hours.Preferred fermentation time is 72 hours, and preferred fermentation time is 120 hours.Certainly, this can change according to the application of best fermentation condition.Fermentation should a suitable temperature between 20 and 40 ℃ be carried out.Preferred temperature is 25-35 ℃, and preferred especially temperature is 30 ℃.
A kind of cultivation method of microorganism of the present invention that relates in one aspect to again of the present invention, this method is to carry out in the mode that the additive that during fermentation will have the potentiality that stimulate CoQ10 production contacts with described microorganism, described additive is selected from the group of being made up of following: CoQ10 precursor p-Hydroxybenzoate (PHB), prenol, Geraniol, tyrosine, shikimate and chorismic acid, and the composite vegetable extract of soya-bean oil, tomato extract and Radix Dauci Sativae extract.Confirmed to use such additive, the output of CoQ10 almost can double (seeing Table 4).In the present invention, preferably use PHB, soya-bean oil, tomato extract or Radix Dauci Sativae extract.What more preferably use is soya-bean oil, tomato extract or Radix Dauci Sativae extract.These additives are commercially available.
At last, the present invention relates to the application of microorganism of the present invention in producing CoQ10.The CoQ10 method of producing can be carried out as the method for institute's illustration in this application.Yet the embodiment that is provided is not considered to the restriction to each invention.Known alternative and the method for carrying out the inventive method of those skilled in the art produced CoQ10.Preferred especially this method is carried out according to the production method of the microorganism that proposes previously self.In addition, microorganism of the present invention also can be used to produce the mutant strain and the variant of this bacterial strain.Same those skilled in the art possess this mutant strain that how to obtain bacterial strain of the present invention and the knowledge of variant.Such method is known in the art, comprises cellular exposure in UV-light, X ray or radioactive substance.In addition, cell can be handled with mutagenic compound, and described mutagenic compound for example are N-methyl-N '-nitro-N-nitrosoguanidine (NG), ethylmethane sulfonate (EMS), methyl-sulfate (DMS) and all similar mutagenic compound.In such technology some are at for example Ausubel etal., and 1997, Current protocols in molecular biology is summarized among the John Wiley and Sons.
For obtaining microorganism of the present invention, pedotheque is selected from the different zones of China, comprises herbary, garden, brewery and anaerobic grain sludge.Select the microorganism that comprises in these samples and test the ability that it produces ubiquinone.
The most of microbe bacterial strain of being tested does not up to now all produce Coenzyme Q10 99.0, but produces the derivative (being Q6-Q9) with shorter isoprenoid side chain.The present application people has carried out HPLC and has analyzed those bacterial strains that have formation Coenzyme Q10 99.0 ability with initial discriminating.Generally speaking, therefore separate 982 pure growths, tested the ability that it forms Coenzyme Q10 99.0 subsequently.
Totally 10 bacterial strains are proved and produce Coenzyme Q10 99.0 as main ubiquinone.In these bacterial strains, the bacterial strain of called after WSH-WO4 demonstrates the highest Coenzyme Q10 99.0 productivity (16mg/l), and therefore is selected to develop the method for fermentation preparation of cozymase Q 10.The isolating subculture of the bacterial strain of called after WSH-W04 is sent to Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH, and (DSMZ, Braunschweig Germany) carry out taxonomy, morphology and biochemical identification.DSMZ identifies as follows to the culture that bacterial strain WSH-W04 is done:
Morphology:
Cell is a Gram-negative, and is directly bar-shaped, and width is 0.6-0.7 μ m, and length is 1.5-2.5 μ m.Cell be active movement and have＞1 polar flagellum.
Biochemistry and physiologic character:
Table 1-3 has shown main biochemistry and the physiology characteristic of bacterial strain WSH-W04.Conjugated fatty acid distribution plan (not shown), WSH-W04 is accredited as α-subgroup (member of α-subgroup) of protein fungus (Proteobacteria).
Table 1: biochemical and physiology test
| Gram-reaction is dissolved aminopeptidase (Cerny) oxidizing ferment catalase ADH urase indoles from NO by 3%KOH3NO 2The hydrolysis of the amylatic hydrolysis DNA of the hydrolysis Tween 80 of the hydrolysis Vitamin C2 of gelatin | - + - + + - + - + - - - - - |
Table 2: utilization of carbon source
| Dextrose fructose citric acid arabinose mannose sweet mellow wine maltose malic acid N-acetyl-glucosamine | - + - - - - - + + |
| Phenethyl ester D-wood sugar trehalose sorbierite inositol methyl alcohol methylamine acetic acid betaine ethanol | - + + + + - - - + + |
Table 3: the generation of oxidizing acid
| From the acid of D-glucose from the acid of D-wood sugar from the acid of L-arabinose acid from D-fructose | + + + + |
Determine that by 16S rRNA sequence carrying out kind is to grow to analyze:
After amplification from genomic dna, complete 16S rRNA encoding gene is checked order.Corresponding DNA sequence is shown in SEQ ID No:1.Therefore, another aspect of the present invention is a kind of isolated nucleic acid sequences, it has sequence shown in the SEQ ID No:1 or corresponding rRNA sequence or have on nucleic acid level with the sequence of these sequence hybridizations or with SEQ ID No:1 or corresponding rRNA sequence under stringent condition＞and the sequence of 97.1% homology.
Sequence of the present invention can be used as probe and get other and the relevant bacterial strain of kind system growth with high CoQ10 production potential to detect to angle.
The following embodiment of the present invention relates to the microorganism that comprises above-mentioned one or more sequences.
Term " under stringent condition " is at implication such as Sambrook et al. (Sambrook, the J. of this paper; Fritsch, E.F.and Maniatis, T. (1989), Molecular cloning:a laboratory manual, 2
NdEd., Cold Spring Harbor Laboratory Press, NewYork) described.Preferably, if with 1 * SSC (pH 7.0 for 150mM sodium-chlor, 15mM Trisodium Citrate) and 0.1%SDS (sodium lauryl sulphate), 80 ℃, preferred 85 ℃, more preferably 88 ℃, most preferably after 90 ℃ of washings 1 hour; More preferably with 0.2 * SSC and 0.1%SDS 80 ℃, preferred 85 ℃, more preferably 88 ℃, most preferably after 90 ℃ of washings 1 hour, still observe the male hybridization signal, then explanation exists according to strict hybridization according to the present invention.
This dna sequence dna is compared with the available 16S rRNA gene order from reference α-protein fungus, with Ochrobactrum sp. 97.1% maximum comparability (Relman1999) is arranged, different subspecies with Bacterium melitense (Brucella melitensis) have 96.5% similarity subsequently, have 96.2% similarity with Ochrobactrum gallinifaecis DSM 15295 and Ochrobactrum intermedium LMG3301.Consider rRNA similarity, fatty acid distribution figure, biochemistry and physiological data, WSH-W04 can be appointed as the novel species that Ochrobactrum belongs to.
From as can be seen above-mentioned, the invention provides a kind of candidate bacterium new and high potentiality of fermentation preparation of cozymase Q 10.
The pure growth of bacterial strain WSH-W04 on June 5th, 2005 regulation according to budapest treaty be deposited in Chinese typical culture collection center (CCTCC), preserving number is CCTCC M 205054.
Term microorganism of the present invention is meant the intact cell that comprised complete organism form and the cell of broken form.
Nucleotide sequence required for protection comprises also that according to the present invention homology with SEQ ID No:1 or its corresponding rRNA sequence is greater than 97.1%; be preferably greater than 97.2%, 97.3% or 97.4%; more preferably greater than 97.5% or 97.6%, be preferably greater than 98%, 98.5% or 99% sequence especially.The available formula H of used term " homology " (or homogeny) (%) in the literary composition=[1-V/X] * 100 definition, wherein H is meant homology, X is a contrast sequence center base sum, the number of different nuclear bases in the sequence that V is with the contrast sequence compares.
Embodiment:
The separation of the bacterial strain of new mass production Coenzyme Q10 99.0
Pedotheque is selected from the different zones of China, comprises herbary, garden, brewery and anaerobic grain sludge.With sample separately in 30 ℃, under anaerobism and the illumination condition, at 100ml enrichment medium (1% peptone, 1%NH
4Cl, 2%NaCl, 1%NaHCO
3, 0.5%Na
2HPO
4, 0.2%MgCl
2, pH 7.0) in cultivated 168 hours.The sample of enrichment culture is coated on the flat board that contains solid complex medium (1% peptone, 0.5% yeast extract, 0.5%NaCl, 2% agar, pH 7.0).Dull and stereotyped 30 ℃ of aerobic insulations 72 hours.One colony is transferred on the agar slant of same medium to obtain pure growth.In order to determine Coenzyme Q10 99.0, each pure growth is inoculated into the Q10 that contains 25ml produces in the 250ml culturing bottle of substratum, described Q10 produces substratum by 4% sucrose, 5% molasses, 1% (NH
4)
2SO
4, 0.05%KH
2PO
4, 0.05%K
2HPO
4, 0.025%MgSO
47H
2O, 4% corn immersion liquid and 1ml/l trace element solution are formed.This trace element solution contains 88mg Na in 1 liter of deionized water
2B
4O
710H
2O, 37mg (NH
4)
6Mo
7O
274H
2O, 8.8mg ZnSO
47H
2O, 270mg CuSO
45H
2O, 7.2mg MnCl
24H
2O and 970mg FeCl
36H
2O.Before autoclaving, the pH that Q10 produces substratum is adjusted to 7.2 with 28% ammoniacal liquor.At 30 ℃, on rotary shaker, cultivated 72 hours with 200rpm, afterwards harvested cell and analyze CoQ10 and form situation (seeing below).
Formation detects to Coenzyme Q10 99.0 by strain separated
With each culture of 1 milliliter as above-mentioned cultivation by in the Glass tubing of 10ml with 10, centrifugal 10 minutes results of 000rpm.Supernatant discarded is also freezing to promote cytoclasis at-70 ℃ with cell.Subsequently, cell material is passed through supersound process in refrigerative water-bath ultra-sonic generator, use twice of 3ml acetone extraction.The combination acetone fractions is also analyzed by reverse hplc in Agilent 1100 systems that are equipped with the Zorbax-SB-C18 post.50: 50 (v/v) carries out elution with methyl alcohol-ethanol.Coenzyme Q10 99.0 is by comparing and differentiated with the reliable sample that derives from Sigma, and estimates its amount from working curve.
By determining and optimization that the Coenzyme Q10 99.0 of WSH-W04 is produced
Produce the high potential of Coenzyme Q10 99.0 based on it, bacterial strain WSH-W04 is selected to develop a kind of industrial method that Coenzyme Q10 99.0 is produced that is suitable for.At first with WSH-W04 agar slant (2% sucrose, 1% peptone, 1% yeast extract, 0.5%NaCl, pH7.2) on, cultivated 24 hours in 30 ℃.This inclined-plane is washed (each inclined-plane 3ml) with sterilized water, and suspension is combined.Subsequently, with the ratio (glycerine: cell suspending liquid, v/v) adding of aseptic glycerine with 1: 4.Cell suspending liquid is divided into the equal portions of 1ml, and freezing in polypropylene tube in-70 ℃.For cultivating, 1 tubule original seed culture is inoculated in contains the shaking in the bottle of 25ml planted medium, and on rotary shaker, be incubated with 200rpm at 30 ℃, described planted medium is made up of 2% sucrose, 1% peptone, 1% yeast extract and 0.5%NaCl (pH7.2).The 2.5ml kind is planted culture be transferred in the 250ml culturing bottle that contains the 25ml minimum medium, described minimum medium is by 4% sucrose, 0.7% (NH
4)
2SO
4, 0.05%KH
2PO
4, 0.05%K
2HPO
4, 0.025%MgSO
4.7H
2O, 8% corn immersion liquid, 8mg/l thiamines, 8mg/l nicotinic acid and 1ml/l trace element solution are formed.Described trace element solution contains 88mgNa in 1 liter of deionized water
2B
4O
710H
2O, 37mg (NH
4)
6Mo
7O
274H
2O, 8.8mg ZnSO
47H
2O, 270mg CuSO
45H
2O, 7.2mg MnCl
24H
2O and 970mg FeCl
36H
2O.
Fermention medium was adjusted to pH 7.2 with 28% ammoniacal liquor before autoclaving, and cultivated on rotary shaker 120 hours with 200rpm at 30 ℃.When needs, gather in the crops, extract and analyze Coenzyme Q10 99.0 with 1 milliliter/part by HPLC as above-mentioned.
With bacterial cultures with behind 100 times of the distilled water dilutings, measure its in the optical density(OD) at 660nm place to monitor its growth, from working curve, estimate dry cell weight (cdw).
The raising of Q10 output
For increasing the Coenzyme Q10 99.0 production among the bacterial strain WSH-W04, in the training period different composite nutrient additives is added in the minimum medium, described additive is p-Hydroxybenzoate (PHB), soya-bean oil, tomato extract and Radix Dauci Sativae extract (table 4).Basic Coenzyme Q10 99.0 tire (do not have any specific additives and obtain) be 37mg/l.Add different additives and produce maximum 64mg/l Coenzyme Q10 99.0s, equal to increase by 73%.These results show that clearly bacterial strain WSH-W04 is particularly suitable for the production microorganism as fermentation preparation of cozymase Q 10.
Table 4: nutritional additive is to bacterial strain WSH-W04
aProduce the effect of Coenzyme Q10 99.0
| Additive | dcw(g/l) | CoQ10(mg/l) | mg CoQ10/g cdw |
| PHB (10mg/l) PHB (20mg/l) PHB (30mg/l) PHB (40mg/l) soya-bean oil (0.4%) soya-bean oil (1.6%) soya-bean oil (4.0%) tomato extract (20%) carrot extract (10%) | 16.33 16.94 17.75 16.90 16.15 19.23 26.62 32.39 17.23 16.42 | 36.75 41.13 52.32 54.44 52.91 48.82 58.23 63.94 56.79 55.75 | 1.95 2.13 2.60 2.84 2.86 2.21 1.98 1.83 2.87 3.07 |
aValue is three independent mean values of cultivating.
Reference:
Sakato，Kuniaki；Tanaka，Hisao；Shibata，Susumu；Kuratsu，Yoshiyuki.Agitation-aeration studies on Coenzyme Q10 production usingRhodopseudomonas spheroides.Biotechnology and AppliedBiochemistry(1992)，16(1)，19-28.
Aeration-agitation effect on Coenzyme Q10 production byAgrobacterium species.Kuratsu，Yoshiyuki；Sakurai，Minoru；Hagino，Hiroshi；Inuzuka，Keiichi.Tech.Res.Lab.，Kyowa Hakko Kogyo Co.Ltd.，Hofu，Japan.Journal of Fermentation Technology(1984)，62(3)，305-8.
Urakami，Teizi；Hori-Okubo，Makiko.Production of isoprenoidcompounds in the facultative methylotroph Protomonas extorquens.Journal of Fermentation Technology(1988)，66(3)，323-32.
Wu，Zufang；Du，Guocheng；Chen，Jian.Effects of dissolved oxygenconcentration and DO-stat feeding strategy on CoQ10 production withRhizobium radiobacter.World Journal of Microbiology & Biotechnology(2003)，19(9)，925-928.
Yoshida，Hajime；Kotani，Yukinobu；Ochiai，Keiko；Araki，Kazumi.Production of ubiquinone-10 using bacteria.Journal of General andApplied Microbiology(1998)，44(1)，19-26.
Yoshida，Hajime；Araki，Kazumi；Nakayama，Kiyoshi.Fermentativeproduction of L-arginine.Part II.Mechanism of L-arginine productionby L-arginine-producing mutants of Corynebacterium glutamicum.Agricultural and Biological Chemistry(1979)，43(1)，105-11.
Hopkins，T.R.Physical and chemical cell disruption for the recovery ofintracellular proteins.Bioprocess Technology (1991)，12(Purif.Anal.Recomb.Proteins)，57-83.
Kuratsu，Yoshiyuki；Sakurai，Minoru；Hagino，Hiroshi；Inuzuka，Keiichi.Aeration-agitation effect on Coenzyme Q10 production byAgrobacterium species.Journal of Fermentation Technology(1984)，62(3)，305-8.
Kanazawa，Morishige；Takahashi，Tamio.Coenzyme Q10 byfermentation.Biomedical and Clinical Aspects of Coenzyme Q(1981)，3 31-42.
Choi，Jin-Ho，Ryu，Yeon-Woo，and Seo，Jin-Ho.Biotechnologicalproduction and applications of Coenzyme Q10.Applied Microbiologyand Biotechnology，Published online before printing：3 March 2005。
Sequence table
＜110〉Degussa
＜120〉produce the bacteria culture of Coenzyme Q10 99.0 and the method for producing Coenzyme Q10 99.0
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tcagggaaac ttgtgctaat accctatacg tccttcggga gaaagattta tcggagatgg 180
atgagcccgc gttggattag ctagttggtg gggtaaaggc ctaccaaggc gacgatccat 240
agctggtctg agaggatgat cagccacact gggactgaga cacggcccag actcctacgg 300
gaggcagcag tggggaatat tggacaatgg gcgcaagcct gatccagcta tgccgcgtga 360
gtgatgaagg ccctagggtt gtaaagctct ttcaccggtg aagataatga cggtaaccgg 420
agaagaagcc ccggctaact tcgtgccagc agccgcggta atacgaaggg ggctagcgtt 480
gttcggattt actgggcgta aagcgcacgt aggcggattg gtcagttagg ggtgaaatcc 540
cagggctcaa ccctggaact gcctttaata ctgccagtct tgagtatgga agaggtgagt 600
ggaattccga gtgtagaggt gaaattcgta gatattcgga ggaacaccag tggcgaaggc 660
ggctcactgg tccattactg acgctgaggt gcgaaagcgt ggggagcaaa caggattaga 720
taccctggta gtccacgccg taaacgatga atgttagccg ttggggagtt tactcttcgg 780
tggcgcagct aacgcattaa acattccgcc tggggagtac ggtcgcaaga ttaaaactca 840
aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 900
cagaacctta ccagcccttg acatcccgat cgcggttagt ggagacacta tccttcagtt 960
tggctggatc ggagacaggt gctgcatggc tgttgtcagc tcgtgtcgtg agatgttggg 1020
ttaagtcccg caacgagcgc aaccctcgcc cttagttgcc agcattgagt tgggcactct 1080
aaggggactg ccggtgataa gccgagagga aggtggggat gacgtcaagt cctcatggcc 1140
cttacgggct gggctacaca cgtgctacaa tggtggtgac agtgggcagc gagcacgcga 1200
gtgtgagcta atctccaaaa gccatctcag ttcggattgc actctgcaac tcgagtgcat 1260
gaagttggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1320
tgtacacacc gcccgtcaca ccatgggagt tggttttacc cgaaggcgct gtgctaaccg 1380
caaggaggca ggcgaccacg gtagggtcag cgactggggt gaagtcgtaa caaggtagcc 1440
gtaggggaac ctgcggctgg atcacc 1466
Claims (8)
1. microorganism WSH-W04, preserving number: CCTCC M 205054.
2. fermenting broth that comprises the microorganism of claim 1.
3. culture systems, it comprises:
I) a kind of container that wherein can ferment;
Ii) a kind of fermenting broth that comprises the microorganism of claim 1;
The instrument of the described microorganism of iii) a kind of fragmentation;
Iv) a kind of device that from fermenting broth, separates Coenzyme Q10 99.0.
4. the method for microorganism of production claim 1, wherein during fermentation, having stimulates the additive of Coenzyme Q10 99.0 production potential to contact with described microorganism, and described additive is selected from the group of being made up of following: the composite vegetable extract of Coenzyme Q10 99.0 precursor p-Hydroxybenzoate and soya-bean oil, tomato extract and Radix Dauci Sativae extract.
5. the application of the microorganism of claim 1 in producing Coenzyme Q10 99.0.
6. the microorganism of claim 1 is in the mutant strain of producing this bacterial strain and the application in the variant.
7. isolating nucleic acid, it has sequence shown in the SEQ ID NO:1 or corresponding rRNA sequence or have in nucleic acid level with the sequence of these sequence hybridize under stringent condition or with SEQ ID NO:1 or corresponding rRNA sequence＞sequence of 97.1% homology.
8. the microorganism that comprises one or more sequence of claim 7.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200510091417 CN1912098A (en) | 2005-08-10 | 2005-08-10 | Baterial for producing cozymase 10 and method for producing cozymase 10 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510091417 CN1912098A (en) | 2005-08-10 | 2005-08-10 | Baterial for producing cozymase 10 and method for producing cozymase 10 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101314782B (en) * | 2007-05-30 | 2012-05-30 | 宁波天安生物材料有限公司 | Method for fermentation preparation of cozymase Q10 |
| CN108048496A (en) * | 2017-12-25 | 2018-05-18 | 浙江新和成股份有限公司 | The fermentation method for producing of CoQ10 and the high-content CoQ10 being prepared by it |
-
2005
- 2005-08-10 CN CN 200510091417 patent/CN1912098A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101314782B (en) * | 2007-05-30 | 2012-05-30 | 宁波天安生物材料有限公司 | Method for fermentation preparation of cozymase Q10 |
| CN108048496A (en) * | 2017-12-25 | 2018-05-18 | 浙江新和成股份有限公司 | The fermentation method for producing of CoQ10 and the high-content CoQ10 being prepared by it |
| CN108048496B (en) * | 2017-12-25 | 2020-11-10 | 浙江新和成股份有限公司 | Method for producing oxidized coenzyme Q10 by fermentation and high-content oxidized coenzyme Q10 prepared by same |
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