CN108516931A - A kind of preparation method of alpha-keto-leucine-calcium - Google Patents
A kind of preparation method of alpha-keto-leucine-calcium Download PDFInfo
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- CN108516931A CN108516931A CN201810497455.7A CN201810497455A CN108516931A CN 108516931 A CN108516931 A CN 108516931A CN 201810497455 A CN201810497455 A CN 201810497455A CN 108516931 A CN108516931 A CN 108516931A
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- leucine
- calcium
- keto
- alpha
- preparation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/41—Preparation of salts of carboxylic acids
- C07C51/412—Preparation of salts of carboxylic acids by conversion of the acids, their salts, esters or anhydrides with the same carboxylic acid part
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- 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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
Abstract
The present invention relates to the biosynthesis technology fields of drug, and in particular to a kind of preparation method of α keto-leucines calcium carries out leucine by using complex microorganism to convert obtained α keto-leucines, then α keto-leucine calcium is made at salt with calcium ion.The method of the present invention mild condition, step is simple, and high conversion rate.
Description
Technical field
The present invention relates to the biosynthesis technology fields of drug, and in particular to a kind of preparation method of alpha-keto-leucine-calcium.
Background technology
Alpha-keto-leucine-calcium is that a variety of aliphatic amino acid ketone acids react the calcium salt admixture generated, Neng Gougai with calcium ion
It is kind to become thin because of caused by malnutrition, loss of appetite and endocrine function.Synthetic method known to alpha-keto-leucine-calcium has：
CN101759553 obtains alpha-keto-leucine-calcium with 4- methyl -2- oxopentanoic acids and calcium carbonate reaction.CN101514154、
Isobutylidene glycolylurea is obtained by the reaction with butanone and glycolylurea in CN101607888, JP54095512, then with sodium hydrate aqueous solution water
Solution, alpha-keto-leucine-calcium is obtained by the reaction with calcium chloride.CN10203063 is obtained using glycine as raw material with isobutylaldehyde ring-closure reaction
Alpha-keto-leucine-calcium is obtained by the reaction in 4- isobutylidene -2- methyl dihydro-oxazole ketone, ring opening hydrolysis.Patent of invention
CN201210021183.6 discloses a kind of preparation method of keto-leucine calcium dihydrate crystal, i.e., will be obtained by chemical synthesis
Alpha-keto-leucine-calcium is dissolved in aqueous solvent, then by alpha-keto-leucine-calcium dihydrochloride dihydrate crystal under the conditions of 10~60 DEG C
It stirs and stands under the conditions of -10~25 DEG C, continue 1 hour to 1 day, by filtering, centrifuging or the like separation, washing,
It is air-dried or is dried under reduced pressure, obtain alpha-keto-leucine-calcium dihydrochloride dihydrate crystal.Process adds one or two or more
Solvent is used in combination.The mode that this patent uses a large amount of organic solvent conjunction secondary refining obtains the hydration of keto-leucine calcium two
Object.Patent of invention CN201410319464.9 discloses a kind of use chemical synthesis synthesis keto-leucine calcium, then by gained
Coarse powder flows back decoloration again, by the refined technique for obtaining keto-leucine calcium.Process above has been all made of chemical synthesis and two
Secondary refined scheme, process flow is longer, and energy consumption is larger, and production process has the discharge of a large amount of organic gas.Have at present
The report of alpha-keto-leucine-calcium is prepared with biological enzyme conversion leucine, but the conversion ratio of wild-type enzyme is relatively low, needs by multiple
Miscellaneous genetic engineering is transformed to improve conversion ratio enzyme.
Invention content
For the above state of the art, the present invention provides the preparation side of a kind of at low cost and high conversion rate alpha-keto-leucine-calcium
Method.
To achieve the above object of the invention, present invention employs the following technical solutions：
A kind of preparation method of alpha-keto-leucine-calcium, includes the following steps：
(1) complex microorganism is added in leucine solution, 4-6h is reacted at 5-65 DEG C, the complex microorganism is by wax
Shape bacillus CGMCC No.9697 and Lactococcus lactis CGMCC No：4496, the mass ratio of the two is 1:0.2-1.5；
(2) it is filtered to remove microorganism, is added into salt assitant after filtrate decoloration, obtains alpha-keto-leucine calcium precipitate；
(3) filtering is precipitated and is washed with deionized and gets product.
Preferably, the mass concentration of leucine solution described in step (1) is 2-30%.
It is further preferred that the mass concentration of leucine solution described in step (1) is 2-20%.
It is further preferred that the addition of microorganism described in step (1) is the 0.1-50% of leucine quality.
It is further preferred that the addition of microorganism described in step (1) is the 2-30% of leucine quality.
It is further preferred that the microorganism to filtering and removing in step (2) carries out recovery, it is 2-200 to apply mechanically number
It is secondary.
It is further preferred that described in step (2) at salt assitant be calcium oxide, calcium hydroxide, calcium chloride, calcium carbonate or carbon
Sour hydrogen calcium.
It is further preferred that being at the addition of salt assitant described in step (2)：It is calculated as leucine mole with calcium ion
0.4-0.7 times.
The present invention is had been surprisingly found that in the course of the research by having to leucine after being combined to two plants of microbial strains
The amino of leucine efficiently can be converted to carbonyl by high selectivity, and then transformation efficiency is significant lower for other bacterial strains of the same race.
Compared with the existing technology, the present invention prepares alpha-keto-leucine-calcium using microorganism catalysis leucine, avoids chemistry
A large amount of solvents are used in synthetic method, process is complicated for operation, and yield is low, the big defect of energy consumption, more wide compared with the condition of enzymatic conversion method
Hold, cost significantly reduces, and has higher conversion ratio.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Each microorganism is bought from China General Microbiological culture presevation administrative center in the present invention.
Microorganism described in following embodiment be the culture medium of respectively specifying each microorganism preservation through seed culture and
Centrifugation obtains wet thallus after fermented and cultured, and after loss on drying is given money as a gift, thalline is mixed to obtain required micro- life according to the ratio
Object.
Embodiment 1
40kg leucines are dissolved in be configured in a certain amount of water 4% aqueous solution 1000L, process temperature control temperature of charge
Between 5-30 DEG C, into feed liquid, addition 12kg microorganisms are reacted.The group of microorganism becomes 1:0.2 Bacillus cercus
CGMCC No.9697 and Lactococcus lactis CGMCC No：4496.Process tracking substrate residue and product formation, after reacting 4h,
Substrate residue 0.4g/L, while product formation no longer increases, then reaction terminates, and conversion ratio reaches 99% or more.Reaction terminates
Filtering removal complex microorganism afterwards, and after being washed with a small amount, put into next batch.
Activated carbon 1.5kg is added under the conditions of 5-30 DEG C and carries out decoloration half an hour for gained feed liquid, the activity of filtering removal later
The calcium oxide of 0.7 times of mole is added in charcoal, gained feed liquid, and adition process has precipitation to generate, and after being added completely, stirs half an hour,
5 DEG C are cooled to, filtering obtains wet-milling.Finished product is obtained after forced air drying, efficient liquid phase detects purity 99.8%.
Embodiment 2
60kg leucines are dissolved in be configured in a certain amount of water 20% aqueous solution 300L, process temperature control temperature of charge
Between 31-60 DEG C, into feed liquid, addition 2.4kg complex microorganisms are reacted.The group of complex microorganism becomes 1:1.5 wax
Shape bacillus CGMCC No.9697 and Lactococcus lactis CGMCC No：4496.Process tracking substrate residue and product generate
Amount reacts substrate residue 0.4g/L after 6h, while product formation no longer increases, then reaction terminates, conversion ratio reach 99% with
On.Filtering removal complex microorganism after reaction, and after being washed with a small amount, put into next batch.
0.4 times mole of calcium chloride is added in gained feed liquid under the conditions of 31-60 DEG C, and adition process has precipitation to generate, and is added
After completely, half an hour is stirred, is cooled to 5 DEG C, filtering obtains wet-milling.Finished product is obtained after forced air drying, efficient liquid phase detects purity
99.7%.
Embodiment 3
30kg leucines are dissolved in be configured in a certain amount of water 30% aqueous solution 100L, process temperature control temperature of charge
Between 31-60 DEG C, into feed liquid, addition 15kg complex microorganisms are reacted.The group of complex microorganism becomes 1:1 it is wax-like
Bacillus CGMCC No.9697 and Lactococcus lactis CGMCC No：4496.Process tracking substrate residue and product formation,
Substrate residue 1g/L after reaction 6h, while product formation no longer increases, then reaction terminates, and conversion ratio reaches 99% or more.Instead
Filtering removal complex microorganism after answering, and after being washed with a small amount, put into next batch.
0.4 times mole of calcium chloride is added in gained feed liquid under the conditions of 31-60 DEG C, and adition process has precipitation to generate, and is added
After completely, half an hour is stirred, is cooled to 5 DEG C, filtering obtains wet-milling.Finished product is obtained after forced air drying, efficient liquid phase detects purity
99.5%.
Example the above is only the implementation of the present invention is not intended to limit the invention, it is all the present invention spirit and
Any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention made by within principle.
Claims (8)
1. a kind of preparation method of alpha-keto-leucine-calcium, it is characterised in that：Include the following steps：
(1) complex microorganism is added in leucine solution, 4-6h is reacted at 5-65 DEG C, the complex microorganism is by wax-like bud
Spore bacillus CGMCC No.9697 and Lactococcus lactis CGMCC No：4496, the mass ratio of the two is 1:0.2-1.5；
(2) it is filtered to remove microorganism, is added into salt assitant after filtrate decoloration, obtains alpha-keto-leucine calcium precipitate；
(3) filtering is precipitated and is washed with deionized and gets product.
2. the preparation method of alpha-keto-leucine-calcium as described in claim 1, it is characterised in that：Leucine described in step (1)
The mass concentration of solution is 2-30%.
3. the preparation method of alpha-keto-leucine-calcium as claimed in claim 2, it is characterised in that：Leucine described in step (1)
The mass concentration of solution is 2-20%.
4. the preparation method of alpha-keto-leucine-calcium as described in claim 1, it is characterised in that：Microorganism described in step (1)
Addition be leucine quality 0.1-50%.
5. the preparation method of alpha-keto-leucine-calcium as claimed in claim 4, it is characterised in that：Microorganism described in step (1)
Addition be leucine quality 2-30%.
6. the preparation method of alpha-keto-leucine-calcium as described in claim 1, it is characterised in that：To filtering and removing in step (2)
Complex microorganism carry out recovery, apply mechanically number be 2-200 times.
7. the preparation method of alpha-keto-leucine-calcium as described in claim 1, it is characterised in that：Step helps described in (2) at salt
Agent is calcium oxide, calcium hydroxide, calcium chloride, calcium carbonate or calcium bicarbonate.
8. the preparation method of alpha-keto-leucine-calcium as claimed in claim 7, it is characterised in that：Step helps described in (2) at salt
The addition of agent is：Be calculated as leucine mole with calcium ion 0.4-0.7 times.
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| CN201810497455.7A CN108516931B (en) | 2018-05-22 | 2018-05-22 | Preparation method of alpha-ketoleucine calcium |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514154A (en) * | 2009-04-03 | 2009-08-26 | 南京化学试剂有限公司 | Synthetic method for aliphatic alpha-calcium picrolonate |
| WO2013055667A2 (en) * | 2011-10-10 | 2013-04-18 | Regents Of The University Of Minnesota | Biocatalysis cells and methods |
-
2018
- 2018-05-22 CN CN201810497455.7A patent/CN108516931B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514154A (en) * | 2009-04-03 | 2009-08-26 | 南京化学试剂有限公司 | Synthetic method for aliphatic alpha-calcium picrolonate |
| WO2013055667A2 (en) * | 2011-10-10 | 2013-04-18 | Regents Of The University Of Minnesota | Biocatalysis cells and methods |
Non-Patent Citations (4)
| Title |
|---|
| LIU, L: "Process modeling and optimization of whole-cell biotransformation synthesis of alpha-ketoisocaproate by Bacillus cereus producing branched-chain amino acid aminotransferase with artificial neural network coupling genetic algorithm", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| MYRTA W. ATILES: "Gene Cloning, Sequencing, and Inactivation of the Branched-Chain Aminotransferase of Lactococcus lactis LM0230", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 刘松梅: "《生物化学》", 30 June 2000, 哈尔滨工业大学出版社 * |
| 李良铸: "《最新生化药物制备技术》", 31 March 2001, 中国医药科技出版社 * |
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