CN108315367A - Method for producing creatine phosphate by two-step enzymolysis method - Google Patents
Method for producing creatine phosphate by two-step enzymolysis method Download PDFInfo
- Publication number
- CN108315367A CN108315367A CN201810224210.7A CN201810224210A CN108315367A CN 108315367 A CN108315367 A CN 108315367A CN 201810224210 A CN201810224210 A CN 201810224210A CN 108315367 A CN108315367 A CN 108315367A
- Authority
- CN
- China
- Prior art keywords
- creatine
- phospholipase
- limited
- biological enzyme
- production method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
- C12P13/00—Preparation of nitrogen-containing organic compounds
Abstract
The invention belongs to the technical field of sports supplements, and particularly discloses a method for producing creatine phosphate by a two-step enzymolysis method. Taking natural phospholipid as a raw material, adding water, a creatine compound and biological enzyme, and carrying out primary enzymolysis reaction at 40-80 ℃ for 4-72 h; and after the primary enzymolysis reaction is finished, adding phospholipase C, carrying out secondary enzymolysis reaction at 40-80 ℃ for 4-8 h, and purifying to obtain the phosphocreatine. The preparation method does not use any organic solvent such as normal hexane, ethanol, ethyl acetate, acetone and the like, the used raw materials are natural products, and the product has higher physiological activity.
Description
Technical field
The invention belongs to sports tonica technical fields, and in particular to a kind of method of two steps enzymatic isolation method production phosphocreatine.
Background technology
Phosphocreatine is in muscle or other excitable tissues（Such as brain and nerve）In a kind of high energy phosphate compound phosphorus
Fat takes on supplement atriphos（ATP）Energy reserve effect.
Phosphocreatine is usually synthesized with phosphoenolpyruvate and creatine.Figure specific as follows：
But the raw material phosphoenolpyruvate that the synthetic method uses is chemical industry synthesis product, and needs to use when synthesis organic
Solvent greatly limits large-scale production and application of the phosphocreatine as sports tonica.
Invention content
In place of overcoming the shortcomings of the prior art, it is original that the purpose of the present invention is to provide one kind with natural phospholipid
The method of material, two step enzymatic isolation methods production phosphocreatine.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of method of two steps enzymatic isolation method production phosphocreatine：Using natural phospholipid as raw material, water, flesh acid compounds and life is added
Object enzyme, in 40-80 DEG C of primary enzymolysis reaction 4-72 h；After reaction, phospholipase C is added, at 40-80 DEG C two in primary enzymolysis
Secondary enzyme digestion reaction 4-8 h purify to get phosphocreatine；Wherein, natural phospholipid and water, flesh acid compounds, biological enzyme, phosphatide
The mass ratio of enzyme C is 1: 0.1-20: 0.1-4: 0.001-0.1: 0.01-0.1；The U/g of the enzyme activity of the biological enzyme >=1000, phosphorus
The U/g of the enzyme activity of lipase C >=10000.
The natural phospholipid includes but not limited to phosphatidyl choline (PC), phosphatidyl-ethanolamine (PE), phosphatidylinositols
(PI), one or more of phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl glycerol (PG) and lysophosphatide (LP)
Mixture.
The natural phospholipid is originated from following sources (1), (2) or (3)：
(1), plant or its extract, the plant include but not limited to soybean, vegetable seed, sunflower seeds；
(2), animal or its extract, the animal include but not limited to shrimps, fish, birds, mammal；
(3), two or more of mixtures in (1) and (2).
The flesh acid compounds include but not limited to one kind in the salt of anhydrous creatine, the hydrate of creatine, creatine
Or several mixture.
The biological enzyme includes but not limited to the mixture of one or both of phospholipase D, creatine kinase.
The method of the purification is centrifugal filtration process, organic solvent extractionprocess or chromatographic column partition method.
The route schematic diagram of production method of the present invention is as follows：
Wherein, R1, R2 in natural phospholipid represent H or fatty acid chain, and X represents micromolecular compound, such as：X=H, it is described natural
Phosphatide is phosphatidic acid；X=choline, the natural phospholipid are phosphatidyl choline（PC）；X=ethanol amine, the natural phospholipid are
For phosphatidyl-ethanolamine（PE）.
Advantageous effect：Preparation method of the present invention is any organic molten without using such as n-hexane, ethyl alcohol, ethyl acetate, acetone
Agent, the raw material used are natural products, and product has higher physiological activity.
Description of the drawings
Fig. 1：The process flow chart of production method of the present invention.
Specific implementation mode
To keep the present invention clearer, clear, the present invention is described in more detail below.It should be appreciated that this place is retouched
The specific embodiment stated is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
Process flow chart as shown in Figure 1, the specific steps are：
Rapeseed phosphatide（Powder）And egg yolk lecithin（Powder）Mixture（After testing, the content of phospholipid in mixture is 95.6
wt%）Middle addition water, creatine and biological enzyme react 16 h in 45 DEG C of primary enzymolysis；After reaction, phosphatide is added in primary enzymolysis
Enzyme C, in 55 DEG C of 6 h of enzyme reaction, by low temperature（5 ℃）It centrifuges, obtained solid phase was cleaned through 10 times of weight distilled water
Filter 3 times, obtains target product, and through high performance liquid chromatography detection, the content of phosphocreatine is 90.8% in target product；
Wherein, rapeseed phosphatide and yolk phospholipid mixture（In terms of content of phospholipid therein）With water, creatine, biological enzyme, phospholipase C
Mass ratio be 1: 10: 1: 0.01: 0.01；The biological enzyme is phospholipase D（1000 U/g of enzyme activity）And creatine kinase（Enzyme activity
1000 U/g）With the mixture of mass ratio 1: 1；The enzyme activity of the phospholipase C is 10000 U/g.
Embodiment 2
Process flow chart as shown in Figure 1, the specific steps are：
Concentrated soybean phospholipid（It is extracted in soybean oil, after testing, content of phospholipid therein is 60 wt%）Middle addition water, creatine and life
Object enzyme reacts 72 h in 55 DEG C of primary enzymolysis；After reaction, phospholipase C is added in primary enzymolysis, in 60 DEG C of enzyme reaction 4h,
By low temperature（5 ℃）It centrifuges, obtained solid phase obtains target product, through height through 10 times of weight distilled water cleaning filter 23
Effect liquid phase chromatogram detects, and the content of phosphocreatine is 91.2% in target product；
Wherein, concentrated soybean phospholipid（In terms of content of phospholipid therein）Mass ratio with water, creatine, biological enzyme, phospholipase C is 1:
0.1∶0.1∶0.01∶0.01；The biological enzyme is phospholipase D（1000 U/g of enzyme activity）；The enzyme activity of the phospholipase C is 10000
U/g。
Embodiment 3
Process flow chart as shown in Figure 1, the specific steps are：
Medulla Bovis seu Bubali extract（After testing, content of phospholipid therein is 15.6 wt%）Middle addition water, creatine and biological enzyme, at 40 DEG C
Primary enzymolysis reacts 72 h；After reaction, phospholipase C is added, in 55 DEG C of enzyme reaction 4h, by low temperature in primary enzymolysis（5
℃）It centrifuges, obtained solid phase obtains target product, examined through high performance liquid chromatography through 10 times of weight distilled water cleaning filter 23
It surveys, the content of phosphocreatine is 90.2% in target product；
Wherein, Medulla Bovis seu Bubali extract（In terms of content of phospholipid therein）Mass ratio with water, creatine, biological enzyme, phospholipase C is 1: 18
∶2∶0.01∶0.01；The biological enzyme is phospholipase D（1000 U/g of enzyme activity）；The enzyme activity of the phospholipase C is 10000 U/g.
Claims (6)
1. a kind of method of two steps enzymatic isolation method production phosphocreatine, it is characterised in that：Using natural phospholipid as raw material, water, flesh is added
Acid compounds and biological enzyme, in 40-80 DEG C of primary enzymolysis reaction 4-72 h；After reaction, phosphatidase is added in primary enzymolysis
C reacts 4-8 h in 40-80 DEG C of secondary enzymolysis, purifies to get phosphocreatine；Wherein, natural phospholipid and water, creatine class chemical combination
Object, biological enzyme, phospholipase C mass ratio be 1: 0.1-20: 0.1-4: 0.001-0.1: 0.01-0.1；The enzyme activity of the biological enzyme
>=1000 U/g, the U/g of the enzyme activity of phospholipase C >=10000.
2. production method as described in claim 1, it is characterised in that：The natural phospholipid includes but not limited to phosphatidyl courage
One kind in alkali, phosphatidyl-ethanolamine, phosphatidylinositols, phosphatidylserine, phosphatidic acid, phosphatidyl glycerol, lysophosphatide or
Several mixtures.
3. production method as claimed in claim 1 or 2, it is characterised in that：The natural phospholipid is originated from following sources (1), (2)
Or (3)：
(1), plant or its extract, the plant include but not limited to soybean, vegetable seed, sunflower seeds；
(2), animal or its extract, the animal include but not limited to shrimps, fish, birds, mammal；
(3), two or more of mixtures in (1) and (2).
4. production method as described in claim 1, it is characterised in that：The flesh acid compounds include but not limited to anhydrous flesh
Acid, the hydrate of creatine, creatine one or more of salt mixture.
5. production method as described in claim 1, it is characterised in that：The biological enzyme includes but not limited to phospholipase D, creatine
The mixture of one or both of kinases.
6. production method as described in claim 1, it is characterised in that：The method of the purification is centrifugal filtration process, You Jirong
Agent extraction or chromatographic column partition method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810224210.7A CN108315367B (en) | 2018-03-19 | 2018-03-19 | Method for producing creatine phosphate by two-step enzymolysis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810224210.7A CN108315367B (en) | 2018-03-19 | 2018-03-19 | Method for producing creatine phosphate by two-step enzymolysis method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108315367A true CN108315367A (en) | 2018-07-24 |
| CN108315367B CN108315367B (en) | 2021-05-11 |
Family
ID=62898680
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810224210.7A Active CN108315367B (en) | 2018-03-19 | 2018-03-19 | Method for producing creatine phosphate by two-step enzymolysis method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108315367B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3956069A (en) * | 1974-04-29 | 1976-05-11 | Abbott Laboratories | Enzymatic assays for glucose, creatine phosphokinase or plasma ammonia |
| CN1478899A (en) * | 2002-08-27 | 2004-03-03 | 中国科学院生物物理研究所 | Production method of creatine phosphate |
| CN102533880A (en) * | 2010-12-31 | 2012-07-04 | 北京百川飞虹生物科技有限公司 | Bioengineering method for synthesizing sodium phosphocreatine |
| CN102653778A (en) * | 2012-05-08 | 2012-09-05 | 广东省微生物研究所 | Enzymolysis preparation method of phosphatidylinositol |
| CN102808006A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for producing phosphocreatine by microbial enzyme method |
| CN103014083A (en) * | 2012-12-31 | 2013-04-03 | 湖南宝利士生物技术有限公司 | Method of preparing creatine phosphate sodium |
| CN103266126A (en) * | 2013-06-06 | 2013-08-28 | 上海兆维科技发展有限公司 | Method for producing creatine phosphate by using enzyme method |
| US20150335718A1 (en) * | 2014-05-21 | 2015-11-26 | The Johns Hopkins University | Preservation and reconstitution of cell-free protein expression systems |
| CN105647985A (en) * | 2015-04-23 | 2016-06-08 | 邦泰生物工程(深圳)有限公司 | Method for producing phosphocreatine by biological catalysis |
-
2018
- 2018-03-19 CN CN201810224210.7A patent/CN108315367B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3956069A (en) * | 1974-04-29 | 1976-05-11 | Abbott Laboratories | Enzymatic assays for glucose, creatine phosphokinase or plasma ammonia |
| CN1478899A (en) * | 2002-08-27 | 2004-03-03 | 中国科学院生物物理研究所 | Production method of creatine phosphate |
| CN102533880A (en) * | 2010-12-31 | 2012-07-04 | 北京百川飞虹生物科技有限公司 | Bioengineering method for synthesizing sodium phosphocreatine |
| CN102653778A (en) * | 2012-05-08 | 2012-09-05 | 广东省微生物研究所 | Enzymolysis preparation method of phosphatidylinositol |
| CN102808006A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for producing phosphocreatine by microbial enzyme method |
| CN103014083A (en) * | 2012-12-31 | 2013-04-03 | 湖南宝利士生物技术有限公司 | Method of preparing creatine phosphate sodium |
| CN103266126A (en) * | 2013-06-06 | 2013-08-28 | 上海兆维科技发展有限公司 | Method for producing creatine phosphate by using enzyme method |
| US20150335718A1 (en) * | 2014-05-21 | 2015-11-26 | The Johns Hopkins University | Preservation and reconstitution of cell-free protein expression systems |
| CN105647985A (en) * | 2015-04-23 | 2016-06-08 | 邦泰生物工程(深圳)有限公司 | Method for producing phosphocreatine by biological catalysis |
Non-Patent Citations (4)
| Title |
|---|
| YOKO DOHKE 等: "Involvement of phospholipase D in the cAMP-regulated exocytosis of rat parotid acinar cells", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 杨志雄 等: "固定化肌酸激酶柱连续合成磷酸肌酸工艺", 《药物生物技术》 * |
| 陈临溪 等: "《细胞信号转导药理与临床》", 31 October 2014, 人民军医出版社 * |
| 陈石良 等: "磷脂酶D的研究进展", 《工业微生物》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108315367B (en) | 2021-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103555783B (en) | A kind of method preparing phosphatidylserine | |
| Dembitsky et al. | Lipid compounds of freshwater sponges: family Spongillidae, class Demospongiae | |
| CN102099453B (en) | Method for production of highly pure phospholipid, and highly pure sphingomyelin and plasmalogen-type glycerophospholipid produced by the method | |
| US20080044487A1 (en) | Anti-inflammatory properties of marine lipid compositions | |
| JPH0515719B2 (en) | ||
| DE19917249C2 (en) | Process for the preparation of phosphatidylserine products | |
| CN104327114A (en) | Preparation method of phosphatidyl serine | |
| RU2289625C2 (en) | Method for production of pure phosphides and uses thereof in cosmetic, pharmaceutics, and food | |
| CN102676600A (en) | Method for preparing phosphatidylserine | |
| CN104059949B (en) | Preparation method of phosphatidyl serine | |
| CN103554174A (en) | Extracting method of egg yolk lecithin | |
| CN108315367A (en) | Method for producing creatine phosphate by two-step enzymolysis method | |
| BR0113132B1 (en) | Method for Producing Transphosphatidylation Phospholipid. | |
| Yunusova et al. | Polyunsaturated fatty acids from several plant species of the family Boraginaceae | |
| JP2001186898A (en) | Method for producing phosphatidyl serine having polyvalent unsaturated fatty acid residue | |
| JP5041790B2 (en) | Process for producing phosphatidylserine having polyunsaturated fatty acid as a constituent | |
| CN109438508A (en) | A kind of preparation method of phosphatidyl-ethanolamine | |
| JP4036595B2 (en) | Lipids containing n-4 and / or n-7 polyunsaturated fatty acids and method for producing the same | |
| EP0288255B1 (en) | Method of producing phospholipid derivatives | |
| CN106282251A (en) | A kind of preparation method of PHOSPHATIDYL ETHANOLAMINE | |
| JPS63123389A (en) | Production of phospholipid-d-serine derivative by enzymatic method | |
| KR102433277B1 (en) | Choline alfoscerate composition manufacturing method using fatty oil | |
| JP2010159383A (en) | Method for separating complex lipid | |
| JPH06228170A (en) | Phosphatidylchromanol derivative, its production, antioxidizing agent and emulsifying agent | |
| JPH0586796B2 (en) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220428 Address after: 450000 F3 building, No. 1 plant, No. 11, Changchun Road, high tech Industrial Development Zone, Zhengzhou City, Henan Province Patentee after: Zhengzhou Siwei Biotechnology Co.,Ltd. Address before: 450000 F3 building, No. 1 plant, No. 11, Changchun Road, high tech Industrial Development Zone, Zhengzhou City, Henan Province Patentee before: ZHENGZHOU SIWEI HEALTH MANAGEMENT Co.,Ltd. |
|
| TR01 | Transfer of patent right |