CN101177670B - Novel strain sphingosine gingivalis ZUTE03 for producing coenzyme Q10 and uses thereof - Google Patents
Novel strain sphingosine gingivalis ZUTE03 for producing coenzyme Q10 and uses thereof Download PDFInfo
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- CN101177670B CN101177670B CN2007100708068A CN200710070806A CN101177670B CN 101177670 B CN101177670 B CN 101177670B CN 2007100708068 A CN2007100708068 A CN 2007100708068A CN 200710070806 A CN200710070806 A CN 200710070806A CN 101177670 B CN101177670 B CN 101177670B
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- ubiquinone
- zute03
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- saponification
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- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 title claims abstract description 74
- ACTIUHUUMQJHFO-UHFFFAOYSA-N Coenzym Q10 Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 235000017471 coenzyme Q10 Nutrition 0.000 title claims abstract description 67
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 title claims description 5
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 title claims description 5
- 229940110767 coenzyme Q10 Drugs 0.000 title abstract 4
- 238000007127 saponification reaction Methods 0.000 claims abstract description 28
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 241001135759 Sphingomonas sp. Species 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 3
- NPCOQXAVBJJZBQ-UHFFFAOYSA-N reduced coenzyme Q9 Natural products COC1=C(O)C(C)=C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)C(O)=C1OC NPCOQXAVBJJZBQ-UHFFFAOYSA-N 0.000 claims description 63
- 229940035936 ubiquinone Drugs 0.000 claims description 63
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 27
- 230000001580 bacterial effect Effects 0.000 claims description 21
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 18
- 239000008103 glucose Substances 0.000 claims description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 15
- 229910052799 carbon Inorganic materials 0.000 claims description 15
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000001888 Peptone Substances 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 230000012010 growth Effects 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 230000001360 synchronised effect Effects 0.000 claims description 10
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
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- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 238000013019 agitation Methods 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 235000012000 cholesterol Nutrition 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
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- 238000002156 mixing Methods 0.000 claims description 4
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- 229940079877 pyrogallol Drugs 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 3
- 241000345369 Edwardsiella hoshinae Species 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 241000726221 Gemma Species 0.000 claims description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 210000003495 flagella Anatomy 0.000 claims description 3
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- 235000015278 beef Nutrition 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 18
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- 238000000855 fermentation Methods 0.000 description 21
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- 108010080698 Peptones Proteins 0.000 description 11
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- 241001052560 Thallis Species 0.000 description 8
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- 125000001477 organic nitrogen group Chemical group 0.000 description 5
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- 239000000843 powder Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a new strain Sphingomonas sp.ZUTE03 that can produce coenzyme Q10 and is preserved in China Center for Type Culture Collection (CCTCC) on June 18, 2007, and the preservation number is CCTCC No. M207084. The strain leavening of the invention is used for preparing the coenzyme Q10, a new technology of synchronization saponification extraction is adopted, which can save manpower, reagent and time with convenience and can improve the efficiency of the extraction technique for the coenzyme Q10 under the condition of nearly no damage of products.
Description
(1) technical field
The present invention relates to a strain and produce ubiquinone
10New bacterial strain---Sphingol single-cell ZUTE03 and application thereof belong to the microbiological pharmacy field.
(2) background technology
Ubiquinone
10(CoenzymeQ
10, CoQ
10) extensively be present in animal, plant and the microbe, be hydrogen carrier important in the biomass cells respiratory chain, also be a kind of good biochemical drug.It has oxidation-resistance, eliminates functions such as free radical, enhance immunity power, has been widely used in treatment of diseases such as all kinds of heart troubles, diabetes, cancer, acute, chronic hepatitis, parkinsonism.The preparation ubiquinone
10Method mainly contain zooblast extraction method, tobacco leaf eggplant Buddhist nun alcohol semi-synthesis method, synthesis method, microbe fermentation method or culture plant cell method fully.Compare with additive method, microbe fermentation method has its lytic activity height, raw materials cost is low, easy to control and can realize characteristics such as scale operation.Obtaining the excellent fermentation bacterial strain is the Production by Microorganism Fermentation ubiquinone
10One of key.The ubiquinone of having reported at present
10Fermentation strain mainly contains Rhodopseudomonas, Saccharomycodes, photosynthetic bacterium genus and paracoccus etc.The fermentation thalline extracts ubiquinone
10Method mostly is pure alkali soap method for post extraction, and step is loaded down with trivial details, waste time and energy, and it is bigger to extract solvent loss.
(3) summary of the invention
The objective of the invention is in order to provide a strain to produce ubiquinone
10The new bacterial strain that output is preferable, and utilize this strain fermentating liquid to prepare ubiquinone
10Synchronous saponification of Shi Caiyong and extraction process are simplified extraction process, save manpower and reagent, are guaranteeing that product almost under the loss-free condition, improves extraction process efficient.
For reaching goal of the invention the technical solution used in the present invention be:
Produce ubiquinone
10New bacterial strain---Sphingol single-cell ZUTE03 (Sphingomonas sp.ZUTE03) is preserved in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University, preservation date on June 18th, 2007, deposit number CCTCC NO:M 207084.
Described Sphingol single-cell ZUTE03 feature is as follows: bacterial strain is bright orange moist, less after cultivating 1 day on the beef extract-peptone agar plate, circle, and the edge is neat, and smooth surface is easily provoked, and produces yellow pigment; It is shaft-like that thalline is, and size is (0.20～0.36) μ m * (0.40～0.80) μ m, and tool is extremely given birth to single flagellum, can move Gram-negative, no gemma; Aerobic, the hydrogen peroxide enzyme positive, the reduction anitratum utilizes glucose, lactose etc. to produce acid, edwardsiella hoshinae, the optimum growth temperature is 28～30 ℃.
The ZUTE03 bacterial strain, separation is littoral Hangzhou Jiu Bao section soil from the Qiantang River, through the Phylogenetic Analysis of morphology, Physiology and biochemistry, Biolog GN evaluation and 16S rRNA sequence, find that this bacterial strain belongs to Sphingol single-cell and belongs to called after Sphingomonas sp.ZUTE03.Do not see relevant Sphingol single-cell at present both at home and abroad as yet and be applied to ubiquinone
10The research report of producing.
Saponification synchronously and extraction ubiquinone in the fermentation thalline
10Technology do not appear in the newspapers equally.
Sphingol single-cell ZUTE03 is mainly used in the fermentative preparation ubiquinone
10
Concrete, described being applied as: be inoculated in Sphingol single-cell ZUTE03 and be applicable to that sphingosine belongs to the liquid nutrient medium of bacterial classification, cultivate 24～36h down for 25～28 ℃, the centrifugal collection wet thallus of gained fermented liquid, wet thallus extract through saponification and obtain described ubiquinone
10
Preferably, described saponification is extracted as synchronous saponification and extracts, i.e. saponification is carried out synchronously with extraction.
Concrete, describedly be applicable to that liquid nutrient medium that sphingosine belongs to bacterial classification is by following formulated: every adding 1000mL water adds carbon source 10～20g, nitrogenous source 5～15g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
40.5g, pH6.0～8.0.
Described nitrogenous source is preferably ammonium sulfate.
Described carbon source is preferably glucose.
Preferably, describedly be applicable to that liquid nutrient medium that sphingosine belongs to bacterial classification is by following formulated: every adding 1000mL water adds glucose 15g, ammonium sulfate 10g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
40.5g, pH8.0.
Concrete, described synchronous saponification extraction process is carried out as follows: wet thallus 2g is moved in the container, add 0.3～0.7g pyrogallol, 1～2.5g KOH, 10～20ml methyl alcohol, 3～7ml distilled water, mixing adds alkanes reagent 30～70ml of C5～C10 simultaneously, refluxes in 70～90 ℃ of water-baths, saponification and synchronous extraction product 30～60min, be cooled to room temperature rapidly, pour separating funnel into, standing demix is got upper organic phase behind the thermal agitation, under 50 ℃ of Rotary Evaporators, it is concentrated into 5～10ml, after to put into refrigerator freezing, separate out cholesterol, filter, get filtrate, promptly get ubiquinone
10
The inventive method has been saved ubiquinone
10The step that organic solvent repeatedly extracts after the saponification in the traditional extraction process, in the saponification removal of impurities, the fermentation thalline is extracted to extract solvent, this synchronous saponification extracting method has been saved extraction time, and, through repeatedly control experiment, this synchronous saponification extracting method of final certification is to ubiquinone in the fermentation thalline
10Do not obtain and can cause damage, and it is higher to extract solvent recovering rate, has reduced the consumption of extracting reagent, has improved ubiquinone in the fermentation thalline
10The efficient of extraction process.
Processing parameter of the present invention is this method optimal conditions through experiment of single factor, orthogonal experiment parallel verified repeatedly, and promptly saponification temperature is 70 ℃～90 ℃, saponification time 30～60min, and the thalline saponification adds simultaneously extracts solvent 30～70ml.
(4) description of drawings
Fig. 1 is the Electronic Speculum figure (amplifying 20000 times) of Sphingol single-cell (Sphingomonas sp.) ZUTE03;
(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: ubiquinone is produced in the separation of Sphingol single-cell (Sphingomonas sp.) ZUTE03, evaluation and fermentation thereof
10Characteristic
1, the separation of bacterial strain:
(1) collected specimens
After gathering littoral Hangzhou, Qiantang River Jiu Bao section pedotheque, get 10g, add sterilized water 90mL, make soil suspension.
(2) strains separation
Get bacteria suspension 1mL, dilution 10
5Doubly, rule on the selectivity flat board, selective medium (g/L) composition is: isoprene 0.5ml, (NH
4)
2SO
41.0g, KH
2PO
44.5g, Na
2HPO
412H
2O 21.6g, agar 15～20g, water 1000ml, pH 7.0.
According to the purebred isolating ordinary method of microorganism, above-mentioned separation and Culture is placed 2～3d based on 28 ℃.The a plurality of single bacterium colonies of picking are inoculated on the slant medium, and numbering is preserved.Carry out the fermentable check again in the liquid medium within, the result obtains a strain ubiquinone
10The bacterial strain ZUTE03 that output is higher.Used substratum is: glucose 20g, peptone 10g, yeast extract paste 10g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
47H
2O 0.5g, PH7.0.(, adding 20g/1000ml agar again) as being solid medium.
2, the evaluation of bacterial strain
(1) ubiquinone
10Produce thalline and the colony morphology characteristic of bacterium ZUTE03
Ubiquinone
10It is shaft-like producing bacterium ZUTE03, and size is (0.20～0.36) μ m * (0.40～0.80) μ m, Gram-negative, and no gemma, tool are extremely given birth to single flagellum; It is bright orange moist, less that bacterium colony is, circle, and the edge is neat, and smooth surface is easily provoked, and produces yellow pigment.
(2) ubiquinone
10Produce the physiological and biochemical property of bacterium ZUTE03
Physiological and biochemical property is: aerobic, the hydrogen peroxide enzyme positive utilizes glucose, lactose etc. to produce acid, edwardsiella hoshinae.
The Physiology and biochemistry proterties of the Sphingol single-cell that above-mentioned mycology feature and document (common bacteria identification handbook) are edited and recorded matches.In conjunction with the 16S rRNA sequence homology analysis and the Biolog systems analysis result of bacterial strain, this bacterium pearl is accredited as and belongs to the product ubiquinone that Sphingol single-cell belongs to
10New bacterium pearl (Sphingomonas sp.).
3, produce ubiquinone
10Performance
Sphingol single-cell (Sphingomonas sp.) ZUTE03 with 6% inoculum size, is inoculated in liquid nutrient medium (carbon source 20g, nitrogenous source 10g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
40.5g water complements to 1000mL pH 7.0), be nitrogenous source with ammonium sulfate, as the carbon source of fermenting, investigate its cell growth total amount and ubiquinone with glucose, sucrose, these several carbon sources commonly used of maltose
10Single factor research of carbon source is carried out in the influence of total amount.The result is as shown in table 1, and the utilization that shows carbon source is to bacteria growing amount and ubiquinone
10Yield effect is more inhomogeneous.Sucrose is the most favourable to bacteria growing, but ubiquinone
10Output is minimum, and glucose is to the minimum that influences of bacteria growing amount, but ubiquinone
10Output is the highest, from bacteria growing amount and ubiquinone
10Proportionlity between the output, glucose are optimum carbon source.In like manner, the result of nitrogenous source is as shown in table 2, and the result shows: in the organic nitrogen source be best with the peptone, its with inorganic nitrogen-sourced ammonium sulfate relatively, though the inorganic nitrogen-sourced growths that are unfavorable for that bacterium is enriched in a large number, the ubiquinone of unit thalline
10Output can reach the level of organic nitrogen source.
Table 1: carbon source is to the ZUTE03 ubiquinone
10The influence of output
| Carbon source | Thalli growth amount (g/L) | Ubiquinone 10Output (mg/L) | The ubiquinone of unit thalline 10Output (mg/g) |
| Glucose | 5.60? | 2.79? | 0.50? |
| Sucrose | 8.30? | 1.60? | 0.19? |
| Maltose | 6.10 | 2.30 | 0.38 |
Table 2: different organic nitrogen sources are to the ZUTE03 ubiquinone
10The influence of output
| Nitrogenous source | Thalli growth amount (g/L) | Ubiquinone 10Output (mg/L) | The ubiquinone of unit thalline 10Output (mg/g) |
| Corn steep liquor | 10.83 | 1.82 | 0.17 |
| Peptone | 11.83 | 4.43 | 0.37 |
| Yeast powder | 9.33 | 1.26 | 0.14 |
On above basis, carry out influence research that glucose concn adds and with the comparative studies of the combination nitrogenous source of ammonium sulfate and peptone and various inorganic and organic nitrogen source.The result is shown in table 3, table 4 and table 5, and the result shows: 1. very few carbon source add-on influences the increment of thalline, so makes ubiquinone
10Output descend thereupon.And too much carbon source amount can influence its biological metabolism approach, both wastes raw material, and output is increased, and has influenced the growth of thalline to a certain extent.Therefore the add-on of determining carbon source glucose is 15g/L.2. the nitrogenous source result of study shows, thalli growth amount and ubiquinone
10Total amount all do not have the promoter action of organic nitrogen source obvious, but calculate with regard to unit output value, ammonium sulfate has surpassed the level of the single nitrogenous source of peptone and other combination nitrogenous sources.Consider that from economic angle the cost of ammonium sulfate is also lower, therefore, selection ammonium sulfate is optimum nitrogen source, and add-on is 10g/L.
Table 3: glucose concn is to the E03 ubiquinone
10The influence of output
| Glucose (g/L) | Thalli growth amount (g/L) | Ubiquinone 10Output (mg/L) | The ubiquinone of unit thalline 10Output (mg/g) |
| 10 | 1.37 | 1.31 | 0.96 |
| 15 | 2.39 | 7.04 | 2.95 |
| 20? | 1.39? | 1.82? | 1.32? |
Table 4: inorganic and organonitrogen are to the ZUTE03 ubiquinone
10The influence of output
| Nitrogenous source | Thalli growth amount (g/L) | Ubiquinone 10Output (mg/L) | The ubiquinone of unit thalline 10Output (mg/g) |
| (NH 4) 2SO 4 | 5.20 | 9.21 | 1.77 |
| Peptone | 10.53 | 17.31 | 1.34 |
| Peptone+yeast powder | 12.93 | 23.73 | 1.83 |
| (each 5g/L) | ? | ? | ? |
Table 5: the combination nitrogenous source is to the ZUTE03 ubiquinone
10The influence of output
| Nitrogenous source/(g/L) | Thalli growth amount (g/L) | Ubiquinone 10Output (mg/L) | The ubiquinone of unit thalline 10Output (mg/g) |
| (NH 4) 2SO 410 | 2.89? | 1.10? | 0.40? |
| Peptone 10 | 8.12? | 1.79? | 0.22? |
| (NH 4) 2SO 45+ peptone 5 | 4.26? | 1.07? | 0.25? |
| (NH 4) 2SO 45+ peptone 5+ yeast powder 0.5 | 5.20? | 1.54? | 0.30? |
| (NH
4)
2SO
45+ peptone 5+ |
4.37? | 1.10? | 0.25? |
Prepare Optimal compositions of fermentation medium (glucose the 15g, (NH of different pH values
4)
2SO
410g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
47H
2O 0.5g, water 1000ml), investigate its cell growth total amount and ubiquinone
10The influence of total amount.The result is as shown in table 6, shows that initial pH is 8.0 o'clock fermentation preparation of cozymase Q
10Best.
Table 6:pH value is to ubiquinone
10The influence of output
| Initial pH | Thalli growth amount (g/L) | Ubiquinone 10Output (mg/L) | The ubiquinone of unit thalline 10Output (mg/g) |
| 5.0? | 9.94? | 3.65? | 0.34? |
| 6.0? | 13.10? | 17.75? | 1.36? |
| 7.0? | 11.02? | 10.55? | 0.96? |
| 8.0? | 10.43? | 14.86? | 1.42? |
Temperature can influence the physical properties of speed of reaction and fermented liquid, also can change the compound direction of meta-bolites, and then influences the fermentation kinetics characteristic.The optimum result such as the table 7 of temperature show that optimum fermentation temp is 25 ℃.
With this understanding, produce ubiquinone by bacterial strain Sphingol single-cell (Sphingomonas sp.) ZUTE03 fermentation
10Output the highest, reached 36.09mg/L, improved 483% than before optimizing.
Table 7: temperature is to ubiquinone
10The influence of output
| T(℃)? | The thalli growth amount | Ubiquinone 10Output | The ubiquinone of unit thalline 10Output (mg/g) |
| ? | (g/L)? | (mg/L)? | ? |
| 25? | 8.167? | 36.09? | 4.42? |
| 30? | 12.67? | 31.92? | 2.52? |
| 37? | 12.67? | 30.06? | 2.37? |
Embodiment 2: Sphingol single-cell (Sphingomonas sp.) ZUTE03 fermentation thalline is extracted ubiquinone
10The optimization of technology and simplification
Sphingol single-cell (Sphingomonas sp.) ZUTE03 with 6% inoculum size, is inoculated in and optimizes post-fermentation and culture base (glucose 15g, (NH
4)
2SO
410g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
40.5g, water 1000ml pH 7.0), 25 ℃ of culture temperature are got the fermentation thalline behind the 200r/min shaking table incubation time 36h, collect fermented liquid, the centrifugal 15min of 15000r/min, the supernatant liquor that inclines is got thalline, uses the distilled water thorough washing, the centrifugal thalline that must ferment.
Thalline is moved in the 150ml round-bottomed flask, respectively with traditional saponification extraction method (concrete grammar: wet thallus 2g is moved in the container, add the 0.7g pyrogallol, 2.5g KOH, 19ml methyl alcohol, 7ml distilled water, mixing.The 30min that refluxes in 90 ℃ of water-baths is cooled to room temperature rapidly with tap water, pours separating funnel into, adds normal hexane 40ml, thermal agitation 5min, extraction ubiquinone
10, continuous extraction 2 times, combining extraction liquid.It is concentrated under 50 ℃ with Rotary Evaporators.Add the 5ml dehydrated alcohol, it is freezing to put into refrigerator, separates out cholesterol, filters, and gets filtrate, promptly gets ubiquinone
10Solution, be settled to 100ml, to be measured) and synchronous saponification extracting method (concrete grammar: wet thallus 2g is moved in the container, add 0.3～0.7g pyrogallol, 1～2.5g KOH, 10～20ml methyl alcohol, 3～7ml distilled water, mixing, add normal hexane 30～70ml simultaneously, reflux in 70～90 ℃ of water-baths, saponification and synchronous extraction product 30～60min are cooled to room temperature rapidly, pour separating funnel into, standing demix is got upper organic phase behind the thermal agitation, under 50 ℃ of Rotary Evaporators it is concentrated into 5～10ml, after to put into refrigerator freezing, separate out cholesterol, filter, get filtrate, promptly get ubiquinone
10Solution) ubiquinone in the extraction fermentation thalline
10, every group establish 3 groups parallel.Product detects by HPLC, shown in the table 8, shows that synchronous saponification extracting method is to the ubiquinone in the fermentation thalline as a result
10Product can not cause damage.
Table 8: different method for saponification are to ubiquinone
10The influence of extracting
| Method for saponification | Ubiquinone 10Output (mg/L) | The ubiquinone of unit thalline 10Output (mg/g) |
| Traditional method | 16.82 | 11.06 |
| Synchronous saponification extraction method | 16.98 | 13.91 |
Claims (10)
1. product ubiquinone
10New bacterial strain---Sphingol single-cell ZUTE03 (Sphingomonas sp.ZUTE03) is preserved in Chinese typical culture collection center, preservation date on June 18th, 2007, deposit number CCTCC NO:M 207084.
2. Sphingol single-cell ZUTE03 as claimed in claim 1 is characterized in that described Sphingol single-cell ZUTE03 feature is as follows: bacterial strain is bright orange moist, less after cultivating 1 day on the beef extract-peptone agar plate, circle, the edge is neat, smooth surface, easily provoke, produce yellow pigment; It is shaft-like that thalline is, and size is (0.20～0.36) μ m * (0.40～0.80) μ m, and tool is extremely given birth to single flagellum, can move Gram-negative, no gemma; Aerobic, the hydrogen peroxide enzyme positive, the reduction anitratum utilizes glucose, lactose to produce acid, edwardsiella hoshinae, the optimum growth temperature is 28～30 ℃.
3. Sphingol single-cell ZUTE03 as claimed in claim 1 is at the fermentative preparation ubiquinone
10In application.
4. application as claimed in claim 3, it is characterized in that described being applied as: be inoculated in Sphingol single-cell ZUTE03 and be applicable to that Sphingol single-cell belongs to the liquid nutrient medium of bacterial classification, cultivate 24～36h down for 25～28 ℃, the centrifugal collection wet thallus of gained fermented liquid, wet thallus extract through saponification and obtain described ubiquinone
10
5. application as claimed in claim 4 is characterized in that described saponification is extracted as saponification and extraction is carried out synchronously.
6. as claim 4 or 5 described application, it is characterized in that describedly being applicable to that liquid nutrient medium that sphingosine belongs to bacterial classification is by following formulated: every adding 1000mL water adds carbon source 10～20g, nitrogenous source 5～15g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
40.5g, pH6.0～8.0.
7. application as claimed in claim 6 is characterized in that described nitrogenous source is an ammonium sulfate.
8. application as claimed in claim 6 is characterized in that described carbon source is a glucose.
9. application as claimed in claim 6, it is characterized in that describedly being applicable to that liquid nutrient medium that Sphingol single-cell belongs to bacterial classification is by following formulated: every adding 1000mL water adds glucose 15g, ammonium sulfate 10g, KH
2PO
40.5g, Na
2HPO
41.5g, MgSO
40.5g, pH8.0.
10. application as claimed in claim 5, it is characterized in that described saponification is extracted carries out as follows: wet thallus 2g is moved in the container, add 0.3～0.7g pyrogallol, 1～2.5g KOH, 10～20mL methyl alcohol, 3～7mL distilled water, mixing adds alkanes reagent 30～70mL of C5～C10 simultaneously, refluxes in 70～90 ℃ of water-baths, saponification and synchronous extraction product 30～60min, be cooled to room temperature rapidly, pour separating funnel into, standing demix is got upper organic phase behind the thermal agitation, under 50 ℃ of Rotary Evaporators, it is concentrated into 5～10mL, after to put into refrigerator freezing, separate out cholesterol, filter, get filtrate, promptly get ubiquinone
10Solution.
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Assignee: Hangzhou Huadong Pharmaceutical Group Kangrun Pharmaceutical Co., Ltd. Assignor: Zhejiang University of Technology Contract record no.: 2011330000908 Denomination of invention: Production of coenzyme Q[sub]10[/sub], a new strain of ZUTE03, and its application Granted publication date: 20110525 License type: Exclusive License Open date: 20080514 Record date: 20110706 |
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