CN101117640B - Method for preparing D-amino acid by biological catalysis - Google Patents
Method for preparing D-amino acid by biological catalysis Download PDFInfo
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- CN101117640B CN101117640B CN2007100701088A CN200710070108A CN101117640B CN 101117640 B CN101117640 B CN 101117640B CN 2007100701088 A CN2007100701088 A CN 2007100701088A CN 200710070108 A CN200710070108 A CN 200710070108A CN 101117640 B CN101117640 B CN 101117640B
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Abstract
The present invention provides a biocatalysis preparation method of D-amino acid. Yeast (ATCC 20804) is centrifuged after being cultivated, and the clear liquid is poured out to be frozen, ultrasonic wall broken and homogenated, then the L-type in the racemate amino acid can be selectively oxidized to keep back the target product of D-type amino acid. The present invention can also use the livingcell as activator; when using the living cell, the freezing and the ultrasonic wall breaking are not required. The method provided by the present invention can be used to the preparation of D-type amino acid, and can be used to the elimination of L-adulterant in the D-type amino acid, and also can be used to the preparation of other D-type nonprotein amino acid. The method of the present invention needs not to adopt the commonly used steps of protection and protection again in the traditional method, and no metal catalyst and no organic solvent as well as additive are required in the preparation process, except the biomass, no chemical discharging exists, thus being a green environmental protecting production method. The present invention has the advantages that the design is reasonable, the preparation route is short, the step is simple, and the efficiency is high.
Description
Technical field
The invention belongs to the preparation method of compound, relate to the amino acid whose biocatalysis preparation method of D-, relate generally to the D-tryptophane, the biocatalysis preparation method of D-phenylalanine and D-tyrosine.
Background technology
Non-protein amino acid as D type amino acid and the amino acid derivative that contains different substituents, is the important tool and the basic material of modern new drug development.The various non-protein amino acid of structure costs an arm and a leg in world markets always, and supply falls short of demand.The preparative capacibility of non-protein amino acid etc. also is the symbolic index of a national new drug development ability, and compared with developed countries, China is bigger in the gap that this field falls behind.Because the added value of product height comes into the market easily the development dog-eat-dog of non-protein amino acid manufacturing technology.Current methods has chemical synthesis and biotransformation method.The former uses asymmetric catalyst, costs an arm and a leg and reclaims difficulty, still has protection to go steps such as protection in the process; The latter has the glycolylurea method, lytic enzyme Split Method etc., but every kind of method all needs polystep reaction, complex process, drawback such as route is long, and the discharge kind is many.
Summary of the invention
The purpose of this invention is to provide the amino acid whose biocatalysis preparation method of a kind of D-, be achieved through the following technical solutions:
(1) make catalyzer with yeast homogenate:
Shake in the bottle at 500mL, culturing yeast bacterium Rhodotorula graminis (ATCC 20804) (herbage rhodotorula) grew after 24 hours, and is centrifugal, inclining clear liquid, freezing, and ultrasonic broken wall is made homogenate, put back and shake in the bottle, add same amount raceme D in every bottle, L-amino acid 50mg; Transfer pH7.3, blowing air, ventilative sealing put back in the temperature control shaking table, the circumference formula is shaken, and reaction times 4-40 hour, ultrafiltration, dilution were injected HPLC and carried out follow-up analysis, with contest road crown ether chromatogram column analysis, time lengthening to 120 minute determines to obtain the purpose product.The yeast that the present invention is used, the general ATCC20804 that is numbered is available from the U.S..
(2) viable cell is made catalyzer:
The present invention can grow after 24 hours, through centrifugal in yeast Rhodotorula graminis (ATCC 20804), inclining after the clear liquid directly with viable cell as catalyzer, when using viable cell, does not need freezing, ultrasonic broken wall step is directly put back and is shaken in the bottle, enters next step (the same).
The present invention's contest road crown ether chromatogram column analysis, Crownpak CR (+), D-Try, tr=65min, product is confirmed as indolylacetic acid through evaluation.
The indolylacetic acid English name: Indole Acetic Acid has another name called: Auxin is a topmost growth hormone in the plant.The discovery of this material and research have had century more than one, and its mechanism of action finds just that recently (Nature 446,640-644,2007). the inventive method shows the IAA pathways metabolism through L-Try, this pathways metabolism extensively is present in the plant, in some plant dependency microorganisms, but have never seen the report that in this bacterium, exists.
Usefulness of the present invention is: (1) contains yeast Rhodotorula graminis (ATCC20804) homogenate (or viable cell) of plurality of enzymes, and under the condition of blowing air, optionally L-type in the oxidation raceme amino acid stays unreacted D-type.This method can be used for the amino acid whose preparation of D-type, also can be used for the removing of D-amino acid L-hotchpotch.(2) the inventive method can directly optionally transform L-type amino acid in the raceme without the step of deriving, and stays unreacted highly purified D-amino acid.(3) the inventive method need not in the traditional method protection earlier commonly used, de-protected step again, and route is brief, efficient height, the enantiomeric purity of may command product.(4) the inventive method also can be used for the preparation of other classes D type non-protein amino acid.(5) the inventive method, preparation process are reflected at neutrallty condition and carry out without metal catalyst, organic solvent and auxiliary agent, except that biomass, do not have the chemistry discharging, belong to the environmental protection production process.(6) the inventive method is reasonable in design, and step is simple.
Description of drawings
Fig. 1 is the color atlas of reaction mixture under the tryptophane bio-transformation different time.
Fig. 2 is the color atlas of reaction mixture under the phenylalanine bio-transformation different time.
Fig. 3 is the color atlas of reaction mixture under the tyrosine bio-transformation different time.
Fig. 4 is the color atlas that tryptophane and phenylalanine mixture transform reaction mixture under the different time.
Fig. 5 is the color atlas of reaction mixture under the tryptophane bio-transformation different time.
Fig. 6 is the color atlas of reaction mixture under the phenylalanine bio-transformation different time.
The color atlas of reaction mixture under Fig. 7 tryptophane and the phenylalanine bio-transformation different time.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
The preparation of embodiment 1D-tryptophane and indolylacetic acid
Shake in the bottle at 500mL and (in 28 bottles, to carry out simultaneously), culturing yeast bacterium Rhodotorulagraminis (ATCC 20804) (herbage rhodotorula), grow after 24 hours, centrifugal, inclining clear liquid, freezing, ultrasonic broken wall is made homogenate, puts back and shakes in the bottle, add same amount raceme D in every bottle, L-tryptophane 50mg; Reconcile pH 7.3, blowing air, ventilative sealing, put back in the temperature control shaking table, the circumference formula was shaken, reaction 0 hour, the interim ml sample that takes out in 6 hours and 12 hours is analyzed following response process, ultrafiltration, dilution, inject HPLC and carry out follow-up analysis, with contest road crown ether chromatographic column, Crownpak CR (+), D-Try, tr=22min, L-Try, tr=30min. extends to 120 minutes analysis time.The result shows the change procedure of each amount in the tryptophane biotransformation referring to Fig. 1, wherein schemes A: t=0:L/D=1/1 when conversion reaction begins; B: conversion reaction is t=6:L/D=1/3 after 6 hours; C: 12 hours t=12:L=0 (L-consumes the only surplus D of light) of conversion reaction, the product indolylacetic acid increases in time and increases.L-Try (tr=28-31min) consumes light gradually, only remaining D-type (tr=22-24min) and product indolylacetic acid (tr=60-65min).
The same, in 28 bottles, carry out simultaneously, the interim ml sample that takes out is analyzed, the following response process, with contest road crown ether chromatographic column, Crownpak CR (+), D-Try, tr=20-23min, L-Try, tr=28-31min. extends to 90 minutes analysis time.L-Try consumes the light time, and the product that stopped reaction, spent ion exchange resin divide is with nucleus magnetic resonance, mass spectroscopy, identify that confirming as product is: indolylacetic acid.
The preparation of embodiment 2D-phenylalanine
Shake in the bottle at 500mL, culturing yeast bacterium Rhodotorula graminis (ATCC 20804) (herbage rhodotorula) grew after 24 hours, and is centrifugal, inclining clear liquid, freezing, and ultrasonic broken wall is made homogenate, divide equally rearmounted returning and shake in the bottle, add same amount raceme D, L-phenylalanine 50mg in every bottle; Transfer pH 7.3, blowing air is put back in the temperature control shaking table, the circumference formula is shaken, and the interim ml sample that takes out is analyzed the following response process, in reaction 4h, 6h and 40h, ultrafiltration, dilution, inject HPLC and carry out follow-up analysis, with contest road crown ether chromatographic column, Crownpak CR (+), D-Phe, tr=7.5min, L-Phe, tr=11min. extends to 60 minutes analysis time.Do not find other detectable product.Spent ion exchange resin separates, and obtains pure D-phenylalanine (D-Phe).
Referring to Fig. 2, be the change procedure (for the purpose of clear, every figure is an independent color atlas) of each amount in the phenylalanine biotransformation, illustrate that product increases in time and increases.Among the figure: when A begins for reaction: L/D=1/1; B is reaction 2 hours: L/D=2/5; C is reaction 4 hours, and L is near 0 (L-consumes the only surplus D of light).L-Phe(tr＝14.5min)。Consume light gradually, only remaining D-type (tr=7-9min).
Originally discover yeast, the L-Phe in the raceme can be converted into unknown product, kept the D-Phe of enantiomer-pure.Become one and prepare this raceme L at the same time, among the D-Phe or contain in the mixture of L-Phe, selective clearing L-Phe keeps the bioconversion method of pure D-Phe.
The preparation of embodiment 3D-tyrosine
Shake in the bottle at 500mL, culturing yeast bacterium (herbage rhodotorula) Rhodotorula graminis (ATCC 20804) grew after 24 hours, and centrifugal, inclining clear liquid, freezing, and ultrasonic broken wall is made homogenate, put back and shook in the bottle, added raceme D, L-tyrosinase 15 0mg; Transfer pH7.3, blowing air is put back in the temperature control shaking table, the circumference formula is shaken, and the interim ml sample that takes out is analyzed the following response process, in reaction 4h, 6h and 40h, ultrafiltration, dilution, inject HPLC and carry out follow-up analysis, with contest road crown ether chromatographic column, Crownpak CR (+), D-Tyr, tr=6min, L-Tyr, tr=9min. extends to 60 minutes analysis time.Spent ion exchange resin separates, and obtains pure D-tyrosine (D-Tyr).
Referring to Fig. 3, be the change procedure of each amount in the tyrosine biotransformation.Illustrate product to increase in time and increase.Among the figure: A is reaction 10 minutes, (t=10 minute): L/D=2/5; B is 4 hours (t=4 hour) of reaction, and L is near 0 (L-consumes the only surplus D of light).L-Tyr (tr=9min) consumes light gradually, only remaining D-type (tr=6min).
Originally discover yeast, the L-Tyr in the raceme can be converted into unknown product, kept the D-Tyr of enantiomer-pure.Become one and prepare this raceme L at the same time, among the D-Tyr or contain in the mixture of L-Phe, selective clearing L-Tyr keeps the bioconversion method of pure D-Tyr.
The preparation of embodiment 4D-tryptophane and D-phenylalanine mixture.
500mL shakes in the bottle, culturing yeast bacterium Rhodotorula graminis (ATCC 20804) grew after 24 hours, and is centrifugal, incline and clear liquid, freezing, ultrasonic broken wall is made homogenate, dividing equally rearmounted returning shakes in the bottle, add same amount raceme D in every bottle, L-tryptophane and D, L-phenylalanine (each 50mg); Transfer pH7.3, blowing air is put back in the temperature control shaking table, and the circumference formula is shaken, the interim ml sample that takes out is analyzed, and the following response process is at reaction 4h, 6h and 40h, ultrafiltration, dilution are injected HPLC and are carried out follow-up analysis, with contest road crown ether chromatographic column, Crownpak CR (+), D-Phe, tr=9min, L-Phe, tr=13min, D-Try, tr=20min, L-Try, tr=30min extends to 120 minutes analysis time.
Originally discover yeast, can be with raceme L, D-Phe and raceme L, the L type transforms in two racemic mixtures of D-Try, has only kept the mixture of two pure D-Phe of enantiomorph and D-Try.Become a L-selective clearing method.
The result is referring to Fig. 4, A: t=0 when conversion reaction begins: phenylalanine L/D=1/1; Tryptophane L/D=1/1; During B:t=40, the most of light that consumes of L type phenylpropyl alcohol and L-tryptophane, only surplus D-phenylalanine and D-tryptophane.
The preparation of embodiment 5D-tryptophane and indolylacetic acid
Present embodiment is to be catalyzer with viable cell, shakes in the bottle at 500mL and (carries out simultaneously in 28 bottles), culturing yeast bacterium Rhodotorula graminis (ATCC 20804) (herbage rhodotorula), grow after 24 hours, centrifugal, inclining clear liquid, put back and shake in the bottle, all the other processes are with embodiment 1.
Fig. 5-Fig. 7
The result shows the change procedure of each amount in the tryptophane biotransformation, wherein A referring to Fig. 5: the color atlas of (during t=0) mixture when conversion reaction begins, during the reaction beginning: L/D=1/1; B: conversion reaction 21 hours: L=0 (L-consumes the only surplus D of light) illustrates that product increases in time and increases.L-Try (tr=28-31min) consumes light gradually, only remaining D-type (tr=22-24min) and product indolylacetic acid (not showing tr=60-65min among this figure).
The preparation of embodiment 6D-phenylalanine (present embodiment be catalyzer with viable cell)
With embodiment 2, directly put back to behind the cell washing and shake bottle, add substrate raceme D, the L-phenylalanine, all the other processes are with embodiment 2.
The result is referring to Fig. 6, and the change procedure of each amount in the phenylalanine biotransformation (for the purpose of clear, every figure is an independent color atlas) illustrates that product increases in time and increases.Among the figure: A reaction 2 hours: L/D=2/5; B reaction 5 hours, L is near 0 (L-consumes the only surplus D of light).L-Phe (tr=14.5min) consumes light gradually, only remaining D-type (tr=7-9min)
The preparation of embodiment 7D-tryptophane and D-phenylalanine mixture (present embodiment be catalyzer with viable cell)
With embodiment 4, cell is put back and is shaken bottle after washing, adds substrate raceme D, L-tryptophane and D, and the L-phenylalanine, all the other processes are with embodiment 4.
The result is referring to Fig. 7, and A is that conversion reaction is when just having begun (t=10 minute); B is conversion reaction 6 hours (during t=640), when reacting 21 hours, and the most of light that consumes of L type phenylpropyl alcohol and L-tryptophane, only surplus D-phenylalanine and D-tryptophane.In the viable cell test, speed of response is fast than homogenate.
In present method, without organic reagent, be the process of an environmental protection in the sepn process of whole conversion process and product.The yeast Rhodotorula graminis (ATCC 20804) that the present invention selects for use is the optimum strain of selecting after screening.
Originally discover a yeast with L-Try IAA pathways metabolism, it can be converted into IAA with the L-Try in the raceme, keeps D-Try.D-Try and IAA performance difference are very big, are easy to separate, thereby become a bioconversion method for preparing these two compounds simultaneously.Present method successfully is used for the D-tryptophane, the preparation of D-phenylalanine and D-tyrosine, but method is not limited to the listed amino acid of present embodiment.The inventive method has been used for the removing of raceme tryptophane and the corresponding body of phenylalanine mixture L-type, but method is not limited to the mixing of this two seed amino acid, should be suitable for more different types of amino acid and may constitute various may mixtures in the removing of L-type enantiomorph.
Claims (5)
1. amino acid whose biocatalysis preparation method of D-, it is characterized in that being achieved through the following technical solutions: shake in the bottle at 500mL, cultivate herbage rhodotorula bacterium, grow after 24 hours, centrifugal, inclining clear liquid, freezing, ultrasonic broken wall, make homogenate, put back and shake in the bottle, add same amount raceme D in every bottle, L-amino acid 50mg; Transfer pH 7.3, blowing air, ventilative sealing, put back in the temperature control shaking table, the circumference formula is shaken, reaction times 4-40 hour, ultrafiltration, dilution are injected HPLC and are carried out follow-up analysis, analyze with contest road crown ether chromatographic column Crownpak CR (+), time lengthening to 120 minute, determine to obtain purpose product, above-mentioned used yeast, the general ATCC 20804 that is numbered, available from the U.S., described D-amino acid is D-phenylalanine, D-tryptophane or D-tyrosine.
2. the amino acid whose biocatalysis preparation method of D-is characterized in that being achieved through the following technical solutions: cultivate herbage rhodotorula bacterium, grow after 24 hours, through centrifugal, inclining clear liquid, directly viable cell is put back and is shaken in the bottle, add same amount raceme D in every bottle, L-amino acid 50mg; Transfer p H 7.3, blowing air, ventilative sealing, put back in the temperature control shaking table, the circumference formula is shaken, reaction times 4-40 hour, ultrafiltration, dilution are injected HPLC and are carried out follow-up analysis, analyze with contest road crown ether chromatographic column Crownpak CR (+), time lengthening to 120 minute, determine to obtain purpose product, above-mentioned used yeast, the general ATCC 20804 that is numbered, available from the U.S., described D-amino acid is D-phenylalanine, D-tryptophane or D-tyrosine.
3. the amino acid whose biocatalysis preparation method of D-according to claim 1 and 2, it is characterized in that: substrate is selected D for use in the method, and the L-tryptophane obtains purpose product D-tryptophane and by product indolylacetic acid.
4. the amino acid whose biocatalysis preparation method of D-according to claim 1 and 2, it is characterized in that: substrate is selected D for use in the method, the L-phenylalanine, obtaining the purpose product is the D-phenylalanine.
5. the amino acid whose biocatalysis preparation method of D-according to claim 1 and 2, it is characterized in that: substrate is selected D for use in the method, L-tyrosine, obtaining the purpose product is D-tyrosine.
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| CN110982858A (en) * | 2019-11-13 | 2020-04-10 | 上海星酶生物科技有限公司 | Production process of D-tryptophan |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5981239A (en) * | 1997-09-24 | 1999-11-09 | Great Lakes Chemical Corp. | Synthesis of optically active phenylalanine analogs using Rhodotorula graminis |
| CN1420935A (en) * | 2000-03-28 | 2003-05-28 | 第一精密化学股份有限公司 | Process for production of optically active beta-amino alcohols |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5981239A (en) * | 1997-09-24 | 1999-11-09 | Great Lakes Chemical Corp. | Synthesis of optically active phenylalanine analogs using Rhodotorula graminis |
| CN1420935A (en) * | 2000-03-28 | 2003-05-28 | 第一精密化学股份有限公司 | Process for production of optically active beta-amino alcohols |
Non-Patent Citations (2)
| Title |
|---|
| 徐金库等.光学纯氨基酸的工业化生产及展望.化学与生物工程 6.2004,(6),1-3. |
| 徐金库等.光学纯氨基酸的工业化生产及展望.化学与生物工程 6.2004,(6),1-3. * |
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