WO2011078648A2 - Method of producing reduced coenzyme q10 by enzymatic method - Google Patents
Method of producing reduced coenzyme q10 by enzymatic method Download PDFInfo
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- WO2011078648A2 WO2011078648A2 PCT/MY2010/000105 MY2010000105W WO2011078648A2 WO 2011078648 A2 WO2011078648 A2 WO 2011078648A2 MY 2010000105 W MY2010000105 W MY 2010000105W WO 2011078648 A2 WO2011078648 A2 WO 2011078648A2
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- Prior art keywords
- bacteria
- fungus
- reductases
- yeast
- supernatant
- Prior art date
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- 238000000034 method Methods 0.000 title claims abstract description 45
- QNTNKSLOFHEFPK-UPTCCGCDSA-N ubiquinol-10 Chemical compound COC1=C(O)C(C)=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(O)=C1OC QNTNKSLOFHEFPK-UPTCCGCDSA-N 0.000 title claims abstract description 24
- 238000006911 enzymatic reaction Methods 0.000 title description 2
- 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 claims abstract description 38
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 36
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 36
- 108091007187 Reductases Proteins 0.000 claims abstract description 26
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 52
- 239000012530 fluid Substances 0.000 claims description 37
- 241000894006 Bacteria Species 0.000 claims description 34
- 239000006228 supernatant Substances 0.000 claims description 33
- 241000233866 Fungi Species 0.000 claims description 31
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 30
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 26
- 239000005515 coenzyme Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 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 claims description 18
- 238000001035 drying Methods 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 15
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 15
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 15
- 235000017471 coenzyme Q10 Nutrition 0.000 claims description 15
- 238000001816 cooling Methods 0.000 claims description 15
- 239000000706 filtrate Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 11
- 229940110767 coenzyme Q10 Drugs 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 8
- 239000012267 brine Substances 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 5
- 238000000151 deposition Methods 0.000 claims description 4
- 239000010802 sludge Substances 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 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 3
- 241000191043 Rhodobacter sphaeroides Species 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 230000000865 phosphorylative effect Effects 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- 241000589516 Pseudomonas Species 0.000 claims description 2
- 241000235346 Schizosaccharomyces Species 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 230000026731 phosphorylation Effects 0.000 claims description 2
- 238000006366 phosphorylation reaction Methods 0.000 claims description 2
- 230000000243 photosynthetic effect Effects 0.000 claims description 2
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims 2
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims 2
- 102100022040 Coenzyme Q-binding protein COQ10 homolog B, mitochondrial Human genes 0.000 claims 1
- 101000896923 Homo sapiens Coenzyme Q-binding protein COQ10 homolog B, mitochondrial Proteins 0.000 claims 1
- YTAHJIFKAKIKAV-XNMGPUDCSA-N [(1R)-3-morpholin-4-yl-1-phenylpropyl] N-[(3S)-2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepin-3-yl]carbamate Chemical compound O=C1[C@H](N=C(C2=C(N1)C=CC=C2)C1=CC=CC=C1)NC(O[C@H](CCN1CCOCC1)C1=CC=CC=C1)=O YTAHJIFKAKIKAV-XNMGPUDCSA-N 0.000 claims 1
- 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 1
- 239000000126 substance Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- XREILSQAXUAAHP-NXGXIAAHSA-N 2,3-dimethoxy-5-methyl-6-[(2e,6e)-3,7,11-trimethyldodeca-2,6,10-trienyl]cyclohexa-2,5-diene-1,4-dione Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O XREILSQAXUAAHP-NXGXIAAHSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 235000015278 beef Nutrition 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical group O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000004098 cellular respiration Effects 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 235000015872 dietary supplement Nutrition 0.000 description 2
- 231100001261 hazardous Toxicity 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- -1 isoprenyl Chemical group 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 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 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003239 periodontal effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960004747 ubidecarenone Drugs 0.000 description 1
- 229940040064 ubiquinol Drugs 0.000 description 1
- 229940035936 ubiquinone Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/66—Preparation of oxygen-containing organic compounds containing the quinoid structure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
Definitions
- the present invention relates to a novel method of producing reduced coenzyme QIO which is particularly suitable for large-scale or mass industrial production of said substance.
- Coenzyme . ' QIO is a substance which is present in most eukaryotic cells of living organisms, primarily in the mitochondria.
- - Coenzyme QIO (abbreviated as CoQ 10, CoQ, Q 10, or Q) is also known as ubiquinone, ubidecarenone or coenzyme Q. It is a benzoquinone, where Q refers to the quinone chemical group, and 10 refers to the isoprenyl chemical subunits.
- Coenzyme Q10 is a component of the electron transport chain and participates in aerobic cellular respiration, generating ATP (adenosine triphosphate) as energy. Ninety-five percent of the human's body energy is generated in this way. Thus organs such as the heart and the liver,- which have the highest enejgy requirements, have the highest coenzyme QIO concentrations.
- coenzyme Q10 is its reduced form which is sometimes known as ubiquinol or CoQ3 ⁇ 4. It is a potent antioxidant in this form. CoQ3 ⁇ 4 has been shown to recharge its active, potent form. Healthy human Coenzyme Q levels are 93% CoQ3 ⁇ 4 (reduced form) and 7% CoQlO (oxidized form). Substantially lower levels of CoQH 2 (reduced form) is a slow as 40% in individuals with liver, brain, heart and kidney diseases. Due to its ability to transfer electrons and therefore to act as an antioxidant, Coenzyme Q is used in dietary supplements, medicines and cosmetics etc. It is a vitamin-like substance. Supplemental coenzyme Q10 raises serum and mitochondrial levels of coenzyme Q10, thereby supporting cellular respiration. It can thus support diverse human health systems, including the brain, heart, immune and periodontal systems.
- Reduced coenzyme Q10 is a white crystal. It has been known that reduced coenzyme Q10 can be obtained by using chemical reducing agents to reduce oxidized coenzyme Q10. Such methods have been disclosed in publications such as WO01/52822A1, JPA , No. 2002- 114875 and ' WO2006/075502. Up to now, however, there have been. problems in producing reduced coenzyme Q10 on a commercial scale. Production of reduced coenzyme Q10 for use in health foods, beverages, cosmetics and drugs by using chemical reducing agents, chemical solvents, additives and protectants brings with it the risk of contamination of said products as well as the environment with hazardous or toxic chemicals. There is a risk that such dangers posed override the beneficial effects of coenzyme Q10. Another problem is the protection and stabilization of reduced coenzyme Q10 which is water-soluble. The present invention attempts to solve at least some of the aforesaid problems.
- biological reductases which are present naturally in the cells of the bacteria, yeast and or fungus, enables enzymatic conversion of oxidized coenzyme Q10 without the use of hazardous or polluting chemicals. This enables production of coenzyme Q10 which is safe and reliable for human and ariimal consumption and non-contamination of ,the environment.
- the reduced coenzyme Q 10 obtained by the method of the second aspect of the invention which uses biological reductases extracted by the method of the first aspect of the invention, is stable, completely natural and can be used on injections for human.
- the method of extraction of the first aspect of the invention and the method of production of the second aspect of the invention are suitable for large-scale industrial . production of coenzyme Q10 without the need to use special protective environment or atmosphere which would be required in large-scale industrial production of reduced coenzyme Q 10 involving the use of chemicals.
- the first aspect of the present invention provides a method of extracting biological -reductases from bacteria, fungus and/or yeast involving selection of bacteria, fungus and/or- yeast which are known to. contain coenzyme Q 10, culturing said bacteria, fungus and/or yeast in a zymotic fluid, disrupting the cells or mycelium of said bacteria, fungus and/or yeast and depositing the supernatant derived therefrom with 30%-70% by weight of ammonium sulfate, centrifuging the mixture of supernatant and ammonium sulfate and then isolating the sludge from the supernatant, dialyzing the supernatant, and drying the dialysed fluid to obtain the biological reductases.
- disruption of the cells or mycelium of the bacteria, fungus and/or yeast preferably involves the use of an ultrasonic cell disruptor. It is further recommended that the supernatant obtained as a result of the disruption of the cells or mycelium be deposited with the ammonium sulfate then washed at 35-45°C with cold water for 1-2 hours, the subsequent centrifugation be carried out for 10-30 minutes under 1-4°C at 10,000-15,000 rpm/min atmosphere, and adding phosphoric acid to the supernatant obtained from the centrifugation before dialyzing the supernatant.
- Sepharos 4B is added to the dialysed fluid, the fluid is then washed three times with phosphoric acid solution, mixed with sodium hydroxide to adjust the pH of said fluid to pH8.5 and then the fluid is dried, preferably freeze dried, with a cooling drying machine to obtain the biological red ⁇ ctases.
- bacteria examples include photosynthetic bacteria, red
- the method of the first aspect of the invention preferably comprises the following steps: a. selecting bacteria, fungus and/or yeast which are known to contain
- the second aspect of the present invention provides a method of producing reduced coenzyme QIO involving phosphorylation of oxidized coenzyme QIO by dissolving the oxidized coenzyme QIO in phosphoric acid solution, adding a phosphonolipide salt of 2- 10% weight of the oxidized coenzyme Q 10 and then stirring the mixture for 10-20 minutes under 28-45 atmosphere, prior to reducing the oxidized coenzyme Q10 with one or more biological reductases extracted by a method according to a first aspect of the invention.
- the pH of the phosphoric acid solution is preferably in the range of 6.0-8.2.
- the method of the second aspect of the invention preferably comprises the following steps:
- the pressure used for the film filtering in step d above is preferably 0.1 - 0.8 MP.
- Example 4 Further possible embodiments and optional features of the second aspect of the invention are illustrated by Example 4.
- Rhodobacter sphaeroides a healthy sporty bateria seed of 0.5-0.9 micron wideth, 1.2-2. Omicron length, gammae, no capsule, single polar flagellum.
- zymotic fluid 5.5g monometallic sodium orthophosphate', 1.5g dimalic acid, 2.0g sodium acetate, 2.0g sodium hydroxide, l.Og ammonium chloride, 0.25g magnesium chloride, 0.05g calcium chloride, 3.5g glucose dissolved in 1.01 distilled water, stirring, pH range is between 6.5-7.0.
- Method of using microzyme to produce biolgical reductase 1. Seed selection: Schizosaccharomyces Promb.
- zymotic fluid beef extract 0.3g, peptone l.Og, NaCl 0.5g, water 100ml, adjust pH to 7.0-7.2, adding water into breaker, weigh up beef extract, peptone and NaCl, warm them up to be melted, adjust the pH to 7.0-7.2 packing each with cotton tampon, then high pressure steaming sterilizing to finish.
- zymotic fluid beef extract 0.3g, peptone l.Og, NaCl 0.5g, agar 1.5g, water 100ml.
- oxidized coenzyme Q10 to react with reductase, converting into natural reduced coenzyme Q10 phosphorylating the oxidized coenzyme Q10 then dissolve 250g oxidized coenzyme QIO, in 1000ml phosphoric acid solution (pH 7.4), then adding 12.5g phosphonolipide in an amount of 5% weight of oxidized coenzyme Q10, with
- Film filterririg with a pressure of 0.5mp, making filtering at 30°C, and leave the filtrate in the air for above 5 hours at 2°C.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Analytical Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a method of extracting biological reductases, and a method of producing reduced coenzyme Q10 by converting oxidized coenzyme Q10 to reduced coenzyme Q10 using one or more of the said biological reductases.
Description
METHOD- OF PRODUCING REDUCED COENZYME QIO BY
ENZYMATIC METHOD
TECHNICAL FIELD
The present invention relates to a novel method of producing reduced coenzyme QIO which is particularly suitable for large-scale or mass industrial production of said substance. BACKGROUND ART
Coenzyme. ' QIO is a substance which is present in most eukaryotic cells of living organisms, primarily in the mitochondria. - Coenzyme QIO (abbreviated as CoQ 10, CoQ, Q 10, or Q) is also known as ubiquinone, ubidecarenone or coenzyme Q. It is a benzoquinone, where Q refers to the quinone chemical group, and 10 refers to the isoprenyl chemical subunits.
Coenzyme Q10 is a component of the electron transport chain and participates in aerobic cellular respiration, generating ATP (adenosine triphosphate) as energy. Ninety-five percent of the human's body energy is generated in this way. Thus organs such as the heart and the liver,- which have the highest enejgy requirements, have the highest coenzyme QIO concentrations.
. i
The active form of coenzyme Q10 is its reduced form which is sometimes known as ubiquinol or CoQ¾. It is a potent antioxidant in this form. CoQ¾ has been shown to recharge its active, potent form. Healthy human Coenzyme Q levels are 93% CoQ¾ (reduced form) and 7% CoQlO (oxidized form). Substantially lower levels of CoQH2 (reduced form) is a slow as 40% in individuals with liver, brain, heart and kidney diseases.
Due to its ability to transfer electrons and therefore to act as an antioxidant, Coenzyme Q is used in dietary supplements, medicines and cosmetics etc. It is a vitamin-like substance. Supplemental coenzyme Q10 raises serum and mitochondrial levels of coenzyme Q10, thereby supporting cellular respiration. It can thus support diverse human health systems, including the brain, heart, immune and periodontal systems.
In light of the above, a number of methods of producing reduced coenzyme QI O, to be used as an ingredient in dietary supplements, medicines, cosmetics etc., from oxidized coenzyme Q10 have been developed. However, there are problems with these methods which have yet to be resolved.
Reduced coenzyme Q10 is a white crystal. It has been known that reduced coenzyme Q10 can be obtained by using chemical reducing agents to reduce oxidized coenzyme Q10. Such methods have been disclosed in publications such as WO01/52822A1, JPA , No. 2002- 114875 and ' WO2006/075502. Up to now, however, there have been. problems in producing reduced coenzyme Q10 on a commercial scale. Production of reduced coenzyme Q10 for use in health foods, beverages, cosmetics and drugs by using chemical reducing agents, chemical solvents, additives and protectants brings with it the risk of contamination of said products as well as the environment with hazardous or toxic chemicals. There is a risk that such dangers posed override the beneficial effects of coenzyme Q10. Another problem is the protection and stabilization of reduced coenzyme Q10 which is water-soluble. The present invention attempts to solve at least some of the aforesaid problems. SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a method of extracting biological reductases from bacteria, fungus and/or yeast which can reduce oxidized coenzyme Q10.
According to a second aspect of the invention, there is provided a method of producing reduced coenzyme Q10 by using biological reductases, obtained from bacteria, fungus and/or yeast to reduce oxidized coenzyme Q10. The use of biological reductases, which are present naturally in the cells of the bacteria, yeast and or fungus, enables enzymatic conversion of oxidized coenzyme Q10 without the use of hazardous or polluting chemicals. This enables production of coenzyme Q10 which is safe and reliable for human and ariimal consumption and non-contamination of ,the environment. The reduced coenzyme Q 10 obtained by the method of the second aspect of the invention, which uses biological reductases extracted by the method of the first aspect of the invention, is stable, completely natural and can be used on injections for human. The method of extraction of the first aspect of the invention and the method of production of the second aspect of the invention are suitable for large-scale industrial . production of coenzyme Q10 without the need to use special protective environment or atmosphere which would be required in large-scale industrial production of reduced coenzyme Q 10 involving the use of chemicals.
The first aspect of the present invention provides a method of extracting biological -reductases from bacteria, fungus and/or yeast involving selection of bacteria, fungus and/or- yeast which are known to. contain coenzyme Q 10, culturing said bacteria, fungus and/or yeast in a zymotic fluid, disrupting the cells or mycelium of said bacteria, fungus and/or yeast and depositing the supernatant derived therefrom with 30%-70% by weight of ammonium sulfate, centrifuging the mixture of supernatant and ammonium sulfate and then isolating the sludge from the supernatant, dialyzing the supernatant, and drying the dialysed fluid to obtain the biological reductases.
In the first aspect of the invention, disruption of the cells or mycelium of the bacteria, fungus and/or yeast preferably involves the use of an ultrasonic cell disruptor. It is further recommended that the supernatant obtained as a result of the disruption of the cells or mycelium be deposited with the ammonium sulfate then washed at 35-45°C with cold water for 1-2 hours, the subsequent centrifugation be carried out for 10-30 minutes under
1-4°C at 10,000-15,000 rpm/min atmosphere, and adding phosphoric acid to the supernatant obtained from the centrifugation before dialyzing the supernatant. More preferably, Sepharos 4B is added to the dialysed fluid, the fluid is then washed three times with phosphoric acid solution, mixed with sodium hydroxide to adjust the pH of said fluid to pH8.5 and then the fluid is dried, preferably freeze dried, with a cooling drying machine to obtain the biological redμctases.
Examples of bacteria which may be selected are photosynthetic bacteria, red
Pseudomonas or Rhodobacter sphaeroides.
The method of the first aspect of the invention preferably comprises the following steps: a. selecting bacteria, fungus and/or yeast which are known to contain
coenzyme Q10
b. preparing zymotic fluid and inoculating said zymotic fluid with an
inoculum of the selected bacteria, fungus and/or yeast
c. incubating said inoculated zymotic fluid at a suitable temperature for a suitable period of time
d. centrifuging the inoculated zymotic fluid at a suitable speed for a suitable period of time
e. disrupting the cells or mycelium of the bacteria, fungus and/or yeast, adding brine and thereafter collecting the supernatant
f. depositing 30%-70% by weight of ammonium sulfate into said supernatant g. washing the mixture of ammonium sulfate and supernatant with water h. centrifuging the mixture with cooling centrifuge for a suitable period of time at a suitable speed
i. isolating the sludge from the supernatant
j. adding phosphoric acid to the supernatant
k. dialyzing the mixture of supernatant and phosphoric acid
1. adding Sepharos 4B to the dialysed fluid
m. washing the dialysed fluid with phosphoric acid
n. adding sodium hydroxide to adjust the pH of said fluid to pH8.5 o. freeze drying the fluid with a cooling drying machine to obtain the
biological reductases.
Further possible embodiments and optional features of the first aspect of the invention are illustrated by Examples 1,2 and 3.
The second aspect of the present invention provides a method of producing reduced coenzyme QIO involving phosphorylation of oxidized coenzyme QIO by dissolving the oxidized coenzyme QIO in phosphoric acid solution, adding a phosphonolipide salt of 2- 10% weight of the oxidized coenzyme Q 10 and then stirring the mixture for 10-20 minutes under 28-45 atmosphere, prior to reducing the oxidized coenzyme Q10 with one or more biological reductases extracted by a method according to a first aspect of the invention. The pH of the phosphoric acid solution is preferably in the range of 6.0-8.2.
The method of the second aspect of the invention preferably comprises the following steps:
a. phosphorylating the oxidized coenzyme Q10 by dissolving the .oxidized coenzyme Q10 in phosphoric acid solution, adding a phosphonolipide salt of 2- 10% weight of the oxidized coenzyme Q10 and then stirring the mixture for 10-20 minutes under 28-45 atmosphere
. b. putting the phosphorylated oxidized coenzyme Q10 into a reactor and adding into said reactor one or more biological reductases obtained from a method according to the first aspect of the invention
c. stirring the mixture in the reactor for a suitable period of time at a suitable pressure
d. filtering off the reduced coenzyme QIO from the reaction mixture obtained from step c by film filtering at a suitable pressure and temperature and then leaving the filtrate in the air for a suitable period of time at a suitable temperature
e. concentrating the filtrate containing the reduced coenzyme Q 10 at vacuum atmosphere
f. washing the concentrated filtrate with water
" g. crystallizing the concentrated filtrate under 2-10°C
h.dr ying the crystallized concentrated filtrate preferably at a temperature of
15-50°C or in a cooling drying machine to obtain the reduced coenzyme Q10.
The pressure used for the film filtering in step d above is preferably 0.1 - 0.8 MP.
-
Further possible embodiments and optional features of the second aspect of the invention are illustrated by Example 4.
EXAMPLES
EXAMPLE 1
Method of using bacteria to produce biolgical reductase:
Seed selection: Rhodobacter sphaeroides, a healthy sporty bateria seed of 0.5-0.9 micron wideth, 1.2-2. Omicron length, gammae, no capsule, single polar flagellum.
Preparing zymotic fluid: 5.5g monometallic sodium orthophosphate', 1.5g dimalic acid, 2.0g sodium acetate, 2.0g sodium hydroxide, l.Og ammonium chloride, 0.25g magnesium chloride, 0.05g calcium chloride, 3.5g glucose dissolved in 1.01 distilled water, stirring, pH range is between 6.5-7.0.
Prepare 10 triangular flasks of 250ml, pouring 100ml inoculum into each flask after sterilization. Seal them after innoculation. Put them into incubator for , 72 hours in
sunlight of 32°C, then get them out of the incubator. Collecting inoculum, centrifuging it with centrifuge for 20 minutes, 6000r/min, then disrupt the mycelium after centrifugation, adding 200ml brine of 3% weight percentage in still for 1 hour. Then collect supernatant, adding 500ml ammonium sulfate, washed by 40ml water for 1.5 hour, next centrifuging with cooling centrifuge for 20 minutes, 12000r/min at 4°C. Then cast supernatant, adding 300ml phosphoric acid solution to dialysis, stirring with 100ml Sepharos 4B, washed three times with 200ml phosphoric acid solution. Adding sodium hydroxide to adjust to pH 8.5, breakdowning, cool drying the filtered liquid with cooling drying machine to obtain 80mg biological reductase.
EXAMPLE 2
Method of using microzyme to produce biolgical reductase: 1. Seed selection: Schizosaccharomyces Promb.
2. Preparing zymotic fluid: beef extract 0.3g, peptone l.Og, NaCl 0.5g, water 100ml, adjust pH to 7.0-7.2, adding water into breaker, weigh up beef extract, peptone and NaCl, warm them up to be melted, adjust the pH to 7.0-7.2 packing each with cotton tampon, then high pressure steaming sterilizing to finish.
3. Preparing 10 triangular flasks of 250ml, pouring 100ml inoculum into each flask after sterilization. Seal them after innoculation. Put them into incubator for 72 hours in sunlight of 35°C, then get them out of the incubator.
4. Collecting inoculum, centrifuging it with centrifuge for 20 minutes, 6000r/min, then disrupt the mycelium after centrifugation, adding 200ml brine of 3% weight percentage in still for 1 hour. Then collect supernatant, adding 500ml ammonium sulfate, washed by 40ml water for 1.5 hour, next centrifuging with cooling centrifuge for 20 minutes, 12000r/min at 5°C. Then cast supernatant, adding 300ml phosphoric
acid solution to dialysis, stirring with 100ml Sepharos 4B, washed three times with 200ml phosphoric acid solution. Adding sodium hydroxide to adjust to pH 8.5, breakdowning, cool drying the filtered liquid with cooling drying machine to obtain 80mg biological reductase.
EXAMPLE 3
Method of using fungus to produce biological reductase:
1. Seed selection: Enophytic
2. Preparing zymotic fluid: beef extract 0.3g, peptone l.Og, NaCl 0.5g, agar 1.5g, water 100ml.
3. Preparing 10 triangular flasks of 250ml, pouring 100ml inoculum into each flask after sterilization. Seal them after innoculation. Put them into incubator .for 72 hours in sunlight of 36°C, then get them out of the incubator.
4. Collecting inoculum, centrifuging it with centrifuge for 20 minutes, 6000r/min, then disrupt the mycelium after centrifugation, adding 200ml brine of 3% weight percentage in still for 1 hour. Then collect supernatant, adding 500ml ammonium sulfate, washed by 40ml water for 1.5 hour, next centrifuging with cooling centrifuge for 20 rninutes, 12000r/min at 2°C. Then cast supernatant, adding 300ml phosphoric acid solution to dialysis, stirring with 100ml Sepharos 4B, washed three times wit 200ml phosphoric acid solution. Adding sodium hydroxide to adjust to pH 8.5, breakdowning, cool drying the filtered liquid with cooling drying machine to obtain 80mg biological reductase.
EXAMPLE 4
Using oxidized coenzyme Q10 to react with reductase, converting into natural reduced coenzyme Q10 phosphorylating the oxidized coenzyme Q10 then dissolve 250g oxidized
coenzyme QIO, in 1000ml phosphoric acid solution (pH 7.4), then adding 12.5g phosphonolipide in an amount of 5% weight of oxidized coenzyme Q10, with
10-120 minutes stirring under 37 atmosphere.
Prepare biological reductase according to Examples 1-3.
Put the phosphorylated oxidized coenzyme Q10 into reactor, adding 80g biological reductase, strirring for 10-30 minutes at 37- 60 atmosphere
Film filterririg: with a pressure of 0.5mp, making filtering at 30°C, and leave the filtrate in the air for above 5 hours at 2°C.
Concentrating the filtrate at 35 °Q 200Pa vacuum atmosphere.
Washing the concentration for 3 times with water, then cystallizing, at 2°C.
Obtaining 248.2g crystal in the cooling drying machine, the purity reaches 99.4% as tested, no oxidized coenzyme Q10 exists.
COMPARATIVE EXAMPLE 1
Method to test presence of reduced coenzyme Q10
Prepare reduced coenzyme Q10 according to example 4, test it with HPLC.
Equipments: Shimazu wave length: 275nm
Mobile phase: enthanol: methanol =(v:V)
Column: octadecyl silica gel column; length : 180mm inner diamester: 3.2mm
Weigh up reduced coenzyme QIO.
20mg, dissolved in ethanol to 100ml, sample size 20μ1, using three-point external standard method to calculate assay.
Appearance time 8.974min: .
Claims
1. A method of extracting biological reductases from bacteria, fungus and/or yeast involving selection of bacteria, fungus and/or yeast which are known to contain coenzyme Q10, culturing said bacteria, fungus and/or yeast in a zymotic fluid, disrupting the cells or mycelium of said bacteria, fungus and/or yeast and depositing the supernatant derived therefrom with 30%-70% by weight of ammonium sulfate, centrifuging the mixture of supernatant and ammonium sulfate and then isolating the sludge from the supernatant, dialyzing the supernatant, and drying the dialysed fluid to obtain the biological reductases.
2. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 1 wherein the cells or mycelium of the bacteria, fungus and/or yeast are disrupted by an ultrasonic cell disruptor.
3. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 1 or claim 2 wherein the supernatant obtained as a result of the disruption of the cells or mycelium is deposited with the ammonium sulfate then washed with water at 35-45°C with cold water for 1-2 hours, the subsequent centrifugation is carried out for 10-30 minutes under 1-4°C at 10,000-15,000 rpm/min atmosphere, and adding phosphoric acid to the supernatant obtained from the centrifugation before dialyzing the supernatant.
4. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 3 wherein Sepharos 4B is added to the dialysed fluid, the fluid is then washed three times with phosphoric acid solution, mixed with sodium hydroxide to adjust the pH of said fluid to pH8.5 and then drying the fluid, preferably freeze drying, with a cooling drying machine to obtain the biological reductases.
5. A method of extracting biological reductases from bacteria, fungus and/or yeast according to any one of claims 1-4 comprising the following steps:
a. selecting bacteria, fungus and/or yeast which are known to contain
coenzyme Q10
b. preparing zymotic fluid and inoculating said zymotic fluid with an
inoculum of the selected bacteria, fungus and/or yeast
c. incubating said inoculated zymotic fluid at a suitable temperature for a suitable period of time
d. centrifuging the inoculated zymotic fluid at a suitable speed for a suitable : period of time
e. disrupting the cells or mycelium of the bacteria, fungus and/or yeast, adding brine and thereafter collecting the supernatant
f. depositing 30%-70% by weight of ammonium sulfate into said "supernatant g. washing the mixture of ammonium sulfate and supernatant with water h. centrifuging the mixture with cooling centrifuge for a suitable period of time at a suitable speed
i. isolating the sludge from the supernatant
j. adding phosphoric acid to the supernatant
k. dialyzing the mixture of supernatant and phosphoric acid
1. adding Sepharos 4B to the dialysed fluid
m. washing the dialysed fluid with phosphoric acid
n. adding sodium hydroxide to adjust the pH of said fluid to pH8.5 o. drying, preferably freeze drying, the fluid with a cooling drying machine to obtain the biological reductases. -
6. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 1 wherein the bacteria selected is photosynthetic bacteria, red Pseudomonas or Rhodobacter sphaeroides.
7. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 1 wherein the fungus selected is Enophytic.
8. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 1 wherein the yeast selected is Schizosaccharomyces Promb.
9. A method of extracting biological reductases from bacteria, fungus and/or yeast according to any one of claims 1-5 wherein the zymotic fluid comprises monometallic sodium orthophosphate, dimalic acid, sodium acetate, sodium hydroxide, ammonium chloride, magnesium chloride, calcium chloride and glucose dissolved in distilled water.
10. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 9 wherein the zymotic fluid comprises 5.5g monometallic sodium orthophosphate, 1.5g dimalic acid, 2.0g sodium acetate, 2.0g sodium hydroxide, l.Og ammonium chloride, 0.25g magnesium chloride, 0.05g calcium chloride and 3.5g glucose dissolved in 1.0 1 distilled water and the pH is between 6.5-7.0.
11. A method of extracting biological reductases from bacteria, fungus and/or yeast according to claim 5 wherein
(i) the inoculated zymotic fluid is incubated in sunlight at 32- 36°C for 72 hours in step c
(ii) the inoculated zymotic fluid is centrifuged at 6000r/min for 20 minutes in step d
(iii) in step e, the brine added is 200ml in volume and 3% weight percentage and the zymotic fluid containing the disrupted cells or mycelium is left in the brine for one (1) hour
(iv) the volume of ammonium sulfate used in step f is 500ml
(v) the mixture of ammonium sulfate and supernatant is washed with 40°C cold water for 1.5 hours in step g
(vi) the mixture is centrifuged for 20 minutes at 12000r/min at 4°C in step h
(vii) the volume of phosphoric acid used in step j is 300ml
(viii) 100ml of Sepharos 4B is used in step k
(ix) the dialysed fluid is washed three times with 200ml
phosphoric acid in step 1
12. A method of producing reduced coenzyme Q10 involving phosphorylation of oxidized coenzyme Q10 by dissolving the oxidized coenzyme Q10 in phosphoric acid solution, adding a phosphonolipide salt of 2-10% weight of the oxidized coenzyme Q10 and then stirring the mixture for 10-20 minutes under 28-45 atmosphere, prior to reducing the oxidized coenzyme Q10 with one or more biological reductases obtained from a method according to any one of claims 1- 11.
13. A method of producing reduced coenzyme Q10 according to claim 12 wherein the pH of the phosphoric acid solution is in the range of 6.0-8.2.
14. A method of producing reduced coenzyme Q10 according to claim 12
comprising: a. phosphorylating the oxidized coenzyme Q10 by dissolving the oxidized coenzyme Q10 in phosphoric acid solution, adding a phosphonolipide salt of 2- 10% weight of the oxidized coenzyme Q 10 and then stirring the mixture for 10-20 minutes under 28-45 atmosphere
b. putting the phosphorylated oxidized coenzyme Q10 into a reactor and adding into said reactor one or more biological reductases obtained from a method according to any one of claims 1-11
c. stirring the mixture in the reactor for a suitable period of time at a suitable pressure
d. filtering off the reduced coenzyme QIO from the reaction mixture obtained from step c by film filtering at a suitable pressure and temperature and then leaving the filtrate in the air for a suitable period of time at a suitable temperature
e. concentrating the filtrate containing the reduced coenzyme QIO at vacuum atmosphere
f. washing the concentrated filtrate with water ' g. crystallizing the concentrated filtrate under 2-10°C
h. drying the crystallized concentrated filtrate preferably at a temperature ofl 5-50°C or in a cooling drying machine to obtain the reduced coenzyme QIO.
15. A method of producing reduced coenzyme QIO according to claim 14 wherein in step a, 250g oxidized coenzyme QIO is dissolved in 1000ml phosphoric acid solution of pH 7.4 and 12.5g of phosphonolipide salt in an amount of 5% weight of the oxidized coenzyme Q10 is added and the mixture is stirred under 37 atmosphere, before allowing the phosphorylated oxidized coenzyme Q10 to react with 80g of the biological reductase in step b to produce reduced coenzyme Q10.
16. A method of producing reduced coenzyme Q10 according to claim J 4 wherein in step c, the mixture in the reactor is stirred for 10-30 minutes at 37-60 atmosphere.
17. A method of producing reduced coenzyme Q10 according to claim 14 wherein the pressure used for me film filtermg in step d is 0.1 - 0.8 MP.
18. A method of producing reduced coenzyme Q10 according to claim 14 wherein in step d, the reaction mixture is filtered at 0.5MP at 30°C and the filtrate is then left in the air for above 5 hours at 2°C.
19. A.method of producing reduced coenzyme QIO according to claim 14 wherein in step e, the filtrate is concentrated at 35°C at 200Pa vacuum atmosphere.
20. A method of producing reduced coenzyme QIO according to claim 14 wherein in step f, the concentrated filtrate is washed with water 3 times.
21. A method of producing reduced coenzyme QIO according to claims 15, 16, 17, 18, 19 and 20.
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| MYPI20092629 | 2009-12-22 | ||
| MYPI20092629 | 2009-12-22 |
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| WO2011078648A2 true WO2011078648A2 (en) | 2011-06-30 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2314709A1 (en) * | 2008-07-17 | 2011-04-27 | Ren, Lei | Method for preparing reduced type coenzyme q10 |
| CN102586211A (en) * | 2012-01-09 | 2012-07-18 | 天津大学 | Method for recycling hydrolytic enzyme from surplus sludge and application of method |
-
2010
- 2010-06-22 WO PCT/MY2010/000105 patent/WO2011078648A2/en not_active Application Discontinuation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2314709A1 (en) * | 2008-07-17 | 2011-04-27 | Ren, Lei | Method for preparing reduced type coenzyme q10 |
| EP2314709A4 (en) * | 2008-07-17 | 2012-02-22 | Zhejiang Zhongning Business Co Ltd | Method for preparing reduced type coenzyme q10 |
| CN102586211A (en) * | 2012-01-09 | 2012-07-18 | 天津大学 | Method for recycling hydrolytic enzyme from surplus sludge and application of method |
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