CN101955979A - Application of butylated hydroxytoluene and/or alpha-linolenic acid as precursors to preparing coenzyme Q10 - Google Patents

Application of butylated hydroxytoluene and/or alpha-linolenic acid as precursors to preparing coenzyme Q10 Download PDF

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CN101955979A
CN101955979A CN 201010293424 CN201010293424A CN101955979A CN 101955979 A CN101955979 A CN 101955979A CN 201010293424 CN201010293424 CN 201010293424 CN 201010293424 A CN201010293424 A CN 201010293424A CN 101955979 A CN101955979 A CN 101955979A
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butylated hydroxytoluene
buddhist nun
eggplant buddhist
alpha
linolenic acid
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钟卫鸿
王伟建
邱乐泉
方建军
钟莉
吴石金
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Zhejiang University of Technology ZJUT
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Zhejiang University of Technology ZJUT
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Abstract

The invention provides application of butylated hydroxytoluene and/or alpha-linolenic acid as precursors to preparing coenzyme Q10 by fermenting microorganisms. The research which uses butylated hydroxytoluene and alpha-linolenic acid as precursor substances to stimulate the transformation of the microorganisms and the production of CoQ10 is used as a first report and speculates that the butylated hydroxytoluene provides a benzene structure for synthesizing CoQ10, the alpha-linolenic acid and solanesol have similar structures, provide lateral chains for synthesizing the CoQ10, and the butylated hydroxytoluene is an antioxidant and can protect the coenzyme Q10 from being oxidized. The butylated hydroxytoluene is cheaper than hydroxybenzoic acid, and the alpha-linolenic acid is cheaper than the solanesol so that the preparation cost of the coenzyme Q10 is greatly decreased.

Description

Butylated Hydroxytoluene and/or alpha-linolenic acid are as the application of precursor in the preparation Coenzyme Q10 99.0
(1) technical field
The present invention relates to be used for the new precursor that microbial fermentation prepares Coenzyme Q10 99.0---Butylated Hydroxytoluene, alpha-linolenic acid, and prepare application in the Coenzyme Q10 99.0 at microbial fermentation.
(2) background technology
Ubiquinone 10Have good medical value, be widely used in treatment of diseases such as all kinds of heart troubles, diabetes, cancer, acute, chronic hepatitis, parkinsonism, the ever-increasing demand of medicine industry, ubiquinone 10Suitability for industrialized production will become more important.
Ubiquinone 10Can pass through chemical synthesis, half chemical synthesis and microbe transformation method production [5].Restrict biological fermentation process suitability for industrialized production CoQ at present 10Principal element be that microorganism cells can not high-caliber production and accumulation CoQ 10, cause that fermentation production rate is low, production cost is higher.In recent years, many investigators transform microorganism and use gene recombination bacterium fermentative production CoQ with engineered method 10, but because CoQ 10The route of synthesis complexity makes up CoQ 10Genetically engineered high yield bacterium is not made a breakthrough both at home and abroad so far as yet.Therefore, add precursor substance or fermentation promotion thing, fermentation condition is optimized, be still and improve CoQ 10The main means of output.(" precursor substance is to the tobacco cell ubiquinone for Li quintessence, Lv Chunmao etc. 10The synthetic influence ", tobacco science and technology, 2009,6:51～55) studied precursor substance to tobacco cell CoQ 10The synthetic influence, the result shows that several precursor substances that add proper concn separately all can promote growth of NC89 tobacco cell and ubiquinone to a certain extent 10Synthetic.Liu Ping etc. (" precursor substance is to the biosynthetic influence of Coenzyme Q10 99.0 ", food and fermentation industries, 2005.31 (4): 1～5) studied with schizosaccharomyces pombe (Schizosaccharomyces promb) that the interpolation side chain is supplied with precursor eggplant Buddhist nun's alcohol and the quinone ring is supplied with the precursor P-hydroxybenzoic acid to CoQ 10The influence of output.The result shows, adds eggplant Buddhist nun alcohol separately and can reach maximum production 33.1mg/L, and comparison is according to having improved 91%; P-hydroxybenzoic acid also can improve CoQ 10Output.In addition, L-halfcystine, methionine(Met), VITMAIN B1 etc. also can improve CoQ 10Output (Jiang Shiyun, surplus Longjiang, Shen Xiaolin, Xiong Xin, letter is gorgeous, " die aromatischen Aminosaeuren is to the influence of the synthetic CoQ10 of Rhodopseudomonas palustris ", medicine biotechnology, 2008,15 (5): 380～387).
(3) summary of the invention
The object of the invention provides the new precursor substance that microbial fermentation prepares Coenzyme Q10 99.0 that is used for, and prepares application in the Coenzyme Q10 99.0 at microbial fermentation.
The technical solution used in the present invention is:
Butylated Hydroxytoluene and/or alpha-linolenic acid prepare application in the Coenzyme Q10 99.0 as precursor at microbial fermentation.Butylated Hydroxytoluene, alpha-linolenic acid screen two kinds of new precursor substances that are used for the preparation of Coenzyme Q10 99.0 microbial method of discovery for the contriver, can be used as unique precursor substance and make an addition in the substratum, also can mix or be used for existing known precursor (as eggplant Buddhist nun alcohol, P-hydroxybenzoic acid etc.) combination separately the preparation of Coenzyme Q10 99.0.
Concrete, described being applied as: produce bacterium with Coenzyme Q10 99.0 and be inoculated in liquid nutrient medium, add an amount of precursor and cultivate 24～36h under 25～28 ℃, the gained fermented liquid separates gets mycelium, obtains described ubiquinone in mycelium 10Described Coenzyme Q10 99.0 produces bacterium can be known in the art the bacterial strain that is used for the Coenzyme Q10 99.0 preparation, as the schizosaccharomyces pombe mentioned in the background technology, Rhodopseudomonas palustris, Sphingol single-cell etc.
Preferably, the precursor that is added is the mixture of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol, and the Butylated Hydroxytoluene addition is that 20～40mg/L, eggplant Buddhist nun alcohol addition are 60～100mg/L.It is that 30mg/L, eggplant Buddhist nun alcohol addition are 70mg/L that choosing, Butylated Hydroxytoluene addition are more arranged.
Preferably, the precursor that is added is the mixture of Butylated Hydroxytoluene and alpha-linolenic acid, and the Butylated Hydroxytoluene addition is that 20～40mg/L, alpha-linolenic acid addition are 60～100mg/L.Choosing is more arranged, and the Butylated Hydroxytoluene addition is that 30mg/L, alpha-linolenic acid addition are 70mg/L.
Coenzyme Q10 99.0 produces bacterium and is preferably Sphingol single-cell CCTCC No:M207084 among the present invention.
As optimized technical scheme, described application method is as follows: be inoculated in liquid nutrient medium with Sphingol single-cell CCTCCNo:M 207084, add precursor and cultivate 24～36h under 25～28 ℃, get fermented liquid, fermented liquid separates gets mycelium, obtains described Coenzyme Q10 99.0 in mycelium; Described liquid nutrient medium is pressed following formulated: every 1000mL water adds glucose 10～20g, (NH 4) 2 SO 45～15g, KH 2PO 40.1～1.0g, Na 2HPO 4.05～2.0g and MgSO 40.1～1.0g, pH6.0～8.0.
Beneficial effect of the present invention is mainly reflected in: two kinds of new precursor substance and application thereof of being used for the preparation of Coenzyme Q10 99.0 microbial method are provided, Butylated Hydroxytoluene is than the low price of P-hydroxybenzoic acid, and alpha-linolenic acid also the price than eggplant Buddhist nun alcohol is low, being used to prepare Coenzyme Q10 99.0 will reduce cost greatly.
(4) description of drawings
Fig. 1 is eggplant Buddhist nun alcohol mark product, crude product HPLC collection of illustrative plates;
Fig. 2 is that different purity eggplant Buddhist nun alcohol adds fermentation product CoQ10 effect; 1: contrast; 2: eggplant Buddhist nun alcohol crude product; 3: the pure product of eggplant Buddhist nun alcohol;
Fig. 3 is the sample tomographic map that obtains after eggplant Buddhist nun alcohol crude product silicagel column separates;
Fig. 4 produces the CoQ10 effect for the different components material adds fermentation; 1: contrast; 2:12～25 pipe elutriants merge enriched material; 3:26～43 pipe elutriants merge enriched material; 4: coloring matter;
Fig. 5 is CoQ10 fermentation best composition thin-layer chromatogram;
Fig. 6 is the HPLC collection of illustrative plates of main ingredient in the CoQ10 fermentation best composition;
Fig. 7 is the mass spectrum of main ingredient in the CoQ10 fermentation best composition;
Fig. 8 is the influences of different precursors to transformation system Coenzyme Q10 99.0 output;
Fig. 9 is the influence of Butylated Hydroxytoluene concentration to Coenzyme Q10 99.0 output;
Figure 10 is the influence of Butylated Hydroxytoluene concentration to the Coenzyme Q10 99.0 transformation efficiency;
Figure 11 is the influence of eggplant Buddhist nun determining alcohol to Coenzyme Q10 99.0 output;
Figure 12 is the influence of eggplant Buddhist nun determining alcohol to the Coenzyme Q10 99.0 transformation efficiency;
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
1 materials and methods
1.1 bacterial classification
The contriver separates the product CoQ of acquisition voluntarily 10Bacterial strain Sphingol single-cell (Sphingomonas sp.) ZUTE03 is preserved in Chinese typical culture collection center, and deposit number is CCTCC No:M 207084, is being protected as novel bacterial in first to file CN200710070806.8.
1.2 instrument and reagent
SPD- 10The AVP high performance liquid chromatograph, day island proper Tianjin (SHIMADZU); Agilent5975C gas chromatograph-mass spectrometer (GC-MS), U.S. Agilent; CoQ 10Standard substance (＞99%), Japan and the pure medicine of light " WAKO " Industrial Co., Ltd; Eggplant Buddhist nun alcohol (＞99%), Weifang, Shandong group of three strongest ones; All the other reagent are all available from Hangzhou.
1.3 substratum
Solid inclined-plane/plate culture medium: yeast extract paste 10g/L, glucose 20g/L, NaCl 5g/L, peptone 10g/L, agar 20g/L, solvent are water, pH7.0.
Seed culture medium: glucose 20g/L, peptone 10g/L, yeast extract paste 10g/L, NaCl 5g/L, solvent are water, pH 7.0.
Fermention medium: glucose 10g/L, (NH 4) 2SO 410g/L, yeast extract paste 1g/L, KH 2PO 40.5g/L, Na 2HPO 41.5g/L, MgSO 47H 2O 0.5g/L, solvent are water, and pH 8.0.More than equal 115 ℃ the sterilization 30min.
1.4 method
1.4.1 the preparation of eggplant Buddhist nun alcohol crude product
Take by weighing the 120g tobacco leaf, be immersed in the distilled water, suction filtration is removed water-soluble impurity, and the tobacco leaf of handling is dried by the fire 2h in 100 ℃ of baking ovens, smashs broken 2min in the refiner to pieces at tissue.Tobacco leaf after the above-mentioned processing is dipped under 50 ℃ in the 1500ml sherwood oil, stirs extraction 10h, filter, filtrate is carried out underpressure distillation and is boiled off solvent, gets eggplant Buddhist nun alcohol crude product medicinal extract.
1.4.2 the silicagel column of eggplant Buddhist nun alcohol crude product separates and proximate analysis
Dress silicagel column (4 * 60cm) highly about 40cm, solanesol extract after the saponification is fully dissolved with a small amount of sherwood oil, be added in the silicagel column, earlier slowly wash pillar with sherwood oil, eggplant Buddhist nun alcohol is fixed on the silicagel column, coloring matter is rinsed, and treat that effusive sherwood oil uses sherwood oil after colourless again: 9: 1 (v/v) wash-outs of acetone, every pipe 40ml collects elutriant.
Each pipe is collected liquid carry out thin-layer chromatography, silica-gel plate (GF254, Haiyang Chemical Plant, Qingdao), developping agent is a sherwood oil: ethyl acetate 4: 1 (v/v), with the colour developing of iodine fumigation.The corresponding fraction collection of identical Rf value on the silica-gel plate is also concentrated, and the material of the different components that obtains adds CoQ to 10In the fermented liquid, and investigate it to Sphingomonas sp.ZJUTE03 fermentative production CoQ 10Influence.The best component that promotes of will fermenting is carried out the separation of secondary silicagel column, and the corresponding fraction collection of identical Rf value concentrates the back and detects with HPLC, essential substance is carried out GC-MS again and analyzes.
1.4.3 the interpolation of different precursors transforms Sphingomonas sp.ZUTE03 and produces ubiquinone 10Influence
Sphingomonas sp.ZUTE03 yeast culture: CCTCC No:M 207084 is seeded to slant medium, and 28 ℃ of activation culture 24h, the bacterial classification inoculation after the activation cultivate 24h for 30 ℃ to seed culture medium, obtain seed liquor, and centrifugal, it is standby to get wet thallus.
With normal hexane configuration 100mg/L alpha-linolenic acid (LNA), 100mg/L Butylated Hydroxytoluene (BHT), 100mg/L eggplant Buddhist nun alcohol (Solanesol)+100mg/L P-hydroxybenzoic acid (PHB), 100mg/L Butylated Hydroxytoluene+100mg/L eggplant Buddhist nun alcohol, 100mg/L alpha-linolenic acid+100mg/L Butylated Hydroxytoluene, 100mg/L P-hydroxybenzoic acid+100mg/L alpha-linolenic acid and control group (not adding precursor), form Sphingomonas sp.ZUTE03 with the equal-volume water respectively and produce CoQ 10The two-phase transformation system (25ml, 0.1mol/L, pH 4.6 acetate buffer solutions are water, the 25mL normal hexane is an organic phase), add 1mL soya-bean oil again as penetrating dose of cell, insert 170mg DCW thalline, 180rpm, 37 ℃ of conversions are investigated the transformation system that adds different combination of precursors bacterial strain are produced ubiquinone 10Influence, CoQ in organic phase when HPLC measure to transform 8h 10Concentration, determine more excellent precursor or combination of precursors.
1.4.4 Butylated Hydroxytoluene concentration is to Q 10The influence of output
Mix that to set eggplant Buddhist nun determining alcohol in the precursor be 100mg/L, the concentration of Butylated Hydroxytoluene is respectively 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, and the same 1.4.3 of transformation system measures ubiquinone separately under the concentration of different Butylated Hydroxytoluenes 10Output, thus determine the more excellent concentration that Butylated Hydroxytoluene adds.
1.4.5 eggplant Buddhist nun determining alcohol is to Q 10The influence of output
Under the situation of selected Butylated Hydroxytoluene concentration, the concentration that eggplant Buddhist nun alcohol is set is respectively 60mg/L, 70mg/L, 80mg/L, 90mg/L, 100mg/L, and the same 1.4.3 of transformation system measures ubiquinone separately 10Output is determined the more excellent concentration that eggplant Buddhist nun alcohol adds.
1.4.6CoQ 10, eggplant Buddhist nun alcohol, Butylated Hydroxytoluene HPLC detect
CoQ 10The HPLC testing conditions: (4.6mm * 250mm), V (normal hexane)+V (methyl alcohol)=17+83 mixed solution is a moving phase to ZORBAX SB-C18 post, flow velocity 1.0ml/min, ultraviolet detection wavelength 275nm.
The HPLC testing conditions of eggplant Buddhist nun alcohol and leaf tobacco extract: (4.6mm * 150mm), V (normal hexane)+V (methyl alcohol)=98+2 mixed solution is a moving phase to Shim-pack HRC-SIL silicagel column, flow velocity 0.5ml/min, ultraviolet detection wavelength 215nm.
The HPLC testing conditions of Butylated Hydroxytoluene (BHT): ZORBAX SB-C18 post (4.6 * 150mm, 5 μ m), 100% chromatographic grade ethanol is moving phase, flow velocity 1.0ml/min, ultraviolet detection wavelength 215nm.
2 results and analysis
2.1 the eggplant Buddhist nun alcohol content analysis in the eggplant Buddhist nun alcohol crude product
Method 1.4.1 is obtained eggplant Buddhist nun alcohol crude product carry out the HPLC detection, result such as Fig. 1, eggplant Buddhist nun alcohol content is 14.43% in HPLC detection eggplant Buddhist nun alcohol crude product.
2.2 add eggplant Buddhist nun alcohol crude product to Sphingomonas sp.ZUTE03 fermentation product CoQ 10Influence
A certain amount of eggplant Buddhist nun alcohol crude product and standard substance are added fermention medium, and making eggplant Buddhist nun alcohol final concentration is 100mg/L, and inoculation Sphingomonas sp.ZUTE03 cultivates 36h, measures CoQ 10Output and cellular biomass.The result adds the fermentation of eggplant Buddhist nun alcohol crude product and produces CoQ as shown in Figure 2 10Effect best, can reach 3.12mg/L, and add the CoQ of the pure product of eggplant Buddhist nun alcohol 10Output has only 2.09mg/L, illustrate in the eggplant Buddhist nun alcohol crude product and contain the better material of facilitation effect, but eggplant Buddhist nun alcohol crude product remains compounding substances, separates so the application continues that eggplant Buddhist nun alcohol crude product is carried out silicagel column, investigates the concrete component that in the crude product bio-transformation is worked.
2.3 the silicagel column of eggplant Buddhist nun alcohol crude product separates
Eggplant Buddhist nun alcohol crude product is carried out silica gel column chromatography, with the colour developing of iodine fumigation, the result as shown in Figure 3,1～No. 7 pipe does not almost have material, mainly is owing to behind adding sherwood oil and acetone mixing elutriant, do not have material to wash out substantially, so colour developing is not arranged; 8～No. 11 pipe begins to occur the substance that show color of obvious Rf value greater than eggplant Buddhist nun alcohol Rf value, also has a small amount of eggplant Buddhist nun alcohol to be washed out simultaneously; It is the darkest that 12～No. 20 pipes equal the spot that occurs on the position of eggplant Buddhist nun alcohol Rf value in the Rf value, and essential substance is an eggplant Buddhist nun alcohol; Manage the locational spot colors of corresponding eggplant Buddhist nun alcohol for 21～No. 25 and shoal gradually, eggplant Buddhist nun alcohol reduces gradually; On 26～No. 43 pipe eggplant Buddhist nun alcohol positions almost immaculate occur, be Rf value all less than the substance that show color of the pure Rf value of eggplant Buddhist nun.Merge 12～20 pipe elutriants, concentrate the higher eggplant Buddhist nun alcohol of purity, merge 26～43 pipe elutriants, concentrate and obtain the following material of eggplant Buddhist nun alcohol spot.
2.4 the additive effect of different silicagel column separated portions research
The material of the different components that will be obtained by method 1.4.2 is configured to the concentration of 0.75g/L respectively, adds in the fermented liquid behind the fermentation 12h, and total fermentation time 36h measures cell yield and CoQ 10Content.The result adds CoQ behind the enriched material of 26～43 pipe elutriants as shown in Figure 4 10Ferment effect best, CoQ 10Output reaches 11.73mg/L, is 653% of control group, is that 12～25 pipe elutriant enriched materials add back CoQ 10572% of output is that coloring matter (column chromatography for separation by sherwood oil the material that goes out of wash-out) at first adds back CoQ 10156% of output, advantage are clearly.So determining the enriched material of 26-43 pipe elutriant is that the following material of eggplant Buddhist nun alcohol spot is a best composition on the thin plate.
2.5CoQ 10The best separation and purification that promotes component of fermentation
With CoQ 10The best component that promotes of fermenting is that 26～43 enriched materials of managing elutriants carry out the separation of secondary silicagel column, and the silica-gel plate thin-layer chromatography, the colour developing of iodine fumigation, and the result is as shown in Figure 5.12～17 pipes that Rf value among Fig. 5 is identical merge the back and concentrate, and carry out HPLC and detect, and result such as Fig. 6, HPLC collection of illustrative plates show that 12～17 pipe condensed matters have two class main ingredients, and retention time is respectively 6.488min and 9.715min.This two classes main ingredient is carried out GC-MS analyze, detect through GC-MS, active principle is the mixture of multiple material, more structure is arranged, but mainly be two big classes, a class is a linear olefin, the structure of this and eggplant Buddhist nun alcohol has certain similarity, and another kind of is the material that contains benzene ring structure, and this is CoQ 10The synthetic advantageous conditions of having created.Fig. 7 is two kinds of representative materials, and the former is a Butylated Hydroxytoluene, and the latter is an alpha-linolenic acid.The present invention is a precursor with these two kinds of materials, studies it Sphingomonas sp.ZUTE03 is transformed product CoQ 10Promoter action.
2.6 add different precursors Sphingomonas sp.ZUTE03 is transformed production CoQ 10Influence
According to method 1.4.3, the precursor that adds 7 kinds of various combinations transforms produces CoQ 10, result such as Fig. 8.Experiment shows, adds two kinds of precursors all than the ubiquinone that adds a kind of precursor 10The output height is wherein to add the combination ubiquinone of Butylated Hydroxytoluene+eggplant Buddhist nun alcohol 10Output the highest, reach 72.05mg/L.What added alpha-linolenic acid or Butylated Hydroxytoluene during secondly, each makes up obviously produces ubiquinone than eggplant Buddhist nun alcohol+P-hydroxybenzoic acid again 10Effective, and latter's ubiquinone 10Output with add separately alpha-linolenic acid or Butylated Hydroxytoluene and remain basically stable, illustrate that new precursor alpha-linolenic acid, Butylated Hydroxytoluene are better than the changing effect of eggplant Buddhist nun alcohol, P-hydroxybenzoic acid.Therefore, the present invention selects the combination of precursors of Butylated Hydroxytoluene+eggplant Buddhist nun alcohol.
2.7 Butylated Hydroxytoluene concentration is to Q 10The influence of output
As shown in Figure 9, when eggplant Buddhist nun determining alcohol is set at 100mg/L, ubiquinone when Butylated Hydroxytoluene concentration is 30mg/L 10Output the highest, reach 74.52mg/L, when Butylated Hydroxytoluene concentration is 40mg/L, 50mg/L, 60mg/L, ubiquinone 10Output all improve, illustrate that precursor had been excessive when Butylated Hydroxytoluene concentration was 30mg/L, improve Butylated Hydroxytoluene concentration again and can not improve ubiquinone 10Output.
Transformation efficiency such as Figure 10 of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol in the experiment, result show that the transformation efficiency of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol all reached maximum when Butylated Hydroxytoluene concentration was 30mg/L, and wherein the transformation efficiency of Butylated Hydroxytoluene is 92.15%, and the transformation efficiency of eggplant Buddhist nun alcohol is 90.31%.So, be under the 100mg/L at eggplant Buddhist nun determining alcohol, the more excellent concentration that Butylated Hydroxytoluene adds should be 30mg/L, at this moment ubiquinone 10Output reached the highest, and the transformation efficiency of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol all reaches maximum.
2.8 eggplant Buddhist nun determining alcohol is to Q 10The influence of output
As shown in Figure 11, under the situation of Butylated Hydroxytoluene concentration certain (being 30mg/L), ubiquinone when eggplant Buddhist nun determining alcohol is 70mg/L 10Output the highest, reach 75.81mg/L; And Butylated Hydroxytoluene concentration is when being 80mg/L, 90mg/L, 100mg/L, ubiquinone 10Output ubiquinone when all being lower than eggplant Buddhist nun determining alcohol and being 70mg/L 10Output, illustrate that precursor had been excessive when eggplant Buddhist nun determining alcohol was 70mg/L, improve eggplant Buddhist nun determining alcohol again and can not improve ubiquinone 10Output.
Transformation efficiency such as Figure 12 of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol in the experiment, result show that when eggplant Buddhist nun determining alcohol was 70mg/L, it is maximum that the transformation efficiency of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol all reaches, and wherein the transformation efficiency of Butylated Hydroxytoluene is 93.19%, and the transformation efficiency of eggplant Buddhist nun alcohol is 92.28%.So, be under the 30mg/L in Butylated Hydroxytoluene concentration, the more excellent concentration that eggplant Buddhist nun alcohol adds should be 70mg/L, at this moment ubiquinone 10Output reached the highest, and the transformation efficiency of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol all reaches maximum.
To sum up, the more excellent concentration combination of precursor Butylated Hydroxytoluene and eggplant Buddhist nun alcohol should be 30mg/L Butylated Hydroxytoluene, 70mg/L eggplant Buddhist nun alcohol, and this moment, the transformation efficiency of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol all reached more than 90%.
4 conclusions
Studies show that, if two or more can improve the condition combined action of secondary metabolite productive rate, will be to the raising performance synergy of secondary metabolite output.Found can be used for ubiquinone at present 10The synthetic precursor substance has eggplant Buddhist nun alcohol, P-hydroxybenzoic acid, L-phenylalanine, and L-halfcystine etc. are that precursor research is maximum with eggplant Buddhist nun alcohol, P-hydroxybenzoic acid wherein, eggplant Buddhist nun alcohol and CoQ 10Side chain---the isoprene tetra-sodium structurally has certain similarity, P-hydroxybenzoic acid is then as CoQ 10The precursor substance of quinone ring in synthetic.The application carries out the silicagel column separation to the eggplant Buddhist nun alcohol crude product in tobacco leaf source, through promoting fermentation contrast experiment and GC-MS to analyze, therefrom obtain two kinds of new precursor substances---Butylated Hydroxytoluene and alpha-linolenic acid, research is also found to transform production CoQ at Sphingomonas sp.ZUTE03 10The two-phase transformation system in, adding with 30mg/L Butylated Hydroxytoluene+70mg/L eggplant Buddhist nun alcohol is that the output of combination of precursors is the highest, can reach 75.81mg/L, the transformation efficiency of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol reaches 93.19% and 92.28% respectively.With Butylated Hydroxytoluene and alpha-linolenic acid is that precursor substance promotes microbial transformation to produce CoQ 10Research be reported first, the supposition Butylated Hydroxytoluene is CoQ 10The synthetic benzene ring structure that provides, and the structure of alpha-linolenic acid and eggplant Buddhist nun alcohol has similarity, is CoQ 10Synthetic provide side chain, and Butylated Hydroxytoluene is a kind of antioxidant, can protects ubiquinone 10Not oxidized.In addition, Butylated Hydroxytoluene is than the low price of P-hydroxybenzoic acid, and alpha-linolenic acid also the price than eggplant Buddhist nun alcohol is low, will help reducing production costs to the further research of novel precursor.

Claims (8)

1. Butylated Hydroxytoluene and/or alpha-linolenic acid prepare application in the Coenzyme Q10 99.0 as precursor at microbial fermentation.
2. application as claimed in claim 2, it is characterized in that described being applied as: produce bacterium with Coenzyme Q10 99.0 and be inoculated in liquid nutrient medium, add an amount of precursor and cultivate 24～36h under 25～28 ℃, the gained fermented liquid separates gets mycelium, obtains described ubiquinone in mycelium 10
3. application as claimed in claim 2 is characterized in that the precursor that is added is the mixture of Butylated Hydroxytoluene and eggplant Buddhist nun alcohol, and the Butylated Hydroxytoluene addition is that 20～40mg/L, eggplant Buddhist nun alcohol addition are 60～100mg/L.
4. application as claimed in claim 3 is characterized in that: the Butylated Hydroxytoluene addition is that 30mg/L, eggplant Buddhist nun alcohol addition are 70mg/L.
5. application as claimed in claim 2 is characterized in that the precursor that is added is the mixture of Butylated Hydroxytoluene and alpha-linolenic acid, and the Butylated Hydroxytoluene addition is that 20～40mg/L, alpha-linolenic acid addition are 60～100mg/L.
6. application as claimed in claim 5 is characterized in that: the Butylated Hydroxytoluene addition is that 30mg/L, alpha-linolenic acid addition are 70mg/L.
7. as the described application of one of claim 4～6, it is characterized in that it is Sphingol single-cell CCTCC No:M 207084 that described Coenzyme Q10 99.0 produces bacterium.
8. as the described application of one of claim 4～6, it is characterized in that described application is as follows: be inoculated in liquid nutrient medium with Sphingol single-cell CCTCC No:M 207084, add precursor and cultivate 24～36h down in 25～28 ℃, get fermented liquid, fermented liquid separates gets mycelium, obtains described Coenzyme Q10 99.0 in mycelium; Described liquid nutrient medium is pressed following formulated: every 1000mL water adds glucose 10～20g, (NH 4) 2SO 45～15g, KH 2PO 40.1～1.0g, Na 2HPO 4.05～2.0g and MgSO 40.1～1.0g, pH6.0～8.0.
CN 201010293424 2010-09-27 2010-09-27 Application of butylated hydroxytoluene and/or alpha-linolenic acid as precursors to preparing coenzyme Q10 Pending CN101955979A (en)

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CN101177670A (en) * 2007-08-16 2008-05-14 浙江工业大学 Novel strain sphingosine gingivalis ZUTE03 for producing coenzyme Q10 and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
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