CN102212568A - Method for synthesizing medicament L-dopa by enzyme catalysis - Google Patents
Method for synthesizing medicament L-dopa by enzyme catalysis Download PDFInfo
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- CN102212568A CN102212568A CN2011100697888A CN201110069788A CN102212568A CN 102212568 A CN102212568 A CN 102212568A CN 2011100697888 A CN2011100697888 A CN 2011100697888A CN 201110069788 A CN201110069788 A CN 201110069788A CN 102212568 A CN102212568 A CN 102212568A
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Abstract
The invention belongs to the technical field of preparation of a medicament L-dopa. In order to solve the technical problem that the medicament L-dopa is difficultly synthesized by biological catalysis in the prior art, the invention provides a method for synthesizing a medicament L-dopa by enzyme catalysis. In the method, L-tyrosine is used as a raw material, and the L-dopa is generated by catalysis of carrier-free immobilized tyrosinase.
Description
[technical field]
The invention belongs to a kind of method, related in particular to synthetic method with a kind of carrier-free immobilization tyrosinase catalysis L-DOPA with enzyme catalysis synthetic drugs L-DOPA.
[background technology]
Enzyme is as a kind of biological catalyst, is widely used in fields such as foodstuff production and detection, green technology, bio-pharmaceuticals by people in recent years.But the development and application of enzyme preparation product that enzyme drawbacks limit such as has been difficult to reclaim after to environment sensitive, reaction.In this case, enzyme immobilization technology is arisen at the historic moment.The immobilization of enzyme is with solid material enzyme to be fettered or is limited in certain zone, but still can carry out its distinctive catalyzed reaction, and a class technology recyclable and recycling.Compare with resolvase, immobilized enzyme has overcome the deficiency of resolvase when keeping its efficient single-minded and gentle enzymic catalytic reaction characteristic, present series of advantages such as stability is high, Separation and Recovery is easy, can be repeatedly used.The optimization research that improves conventional fixed method and application art condition is the main trend of present enzyme immobilization research.
The process for fixation of tradition enzyme has absorption method, entrapping method, covalent cross-linking etc., and these methods all are need the enzyme molecule be fixed on the carrier, like this activity/the concentration of enzyme are subjected to " dilution ", and enzyme is come to harm.In calendar year 2001, method---the Cross-Linked Enzyme Aggregates (cross-linked enzyme aggregate) of a kind of novel carrier free enzyme immobilization that Roger professor Sheldon proposes, molecule crosslinked being fixed up of enzyme that is in the precipitation state, resulting immobilized enzyme has shown good catalytic activity to the use glutaraldehyde as linking agent.This technology is verified all having a wide range of applications aspect the immobilization of plurality of enzymes such as lytic enzyme, oxydo-reductase and lyase, is worth promoting in industrial application.
The L-DOPA, English L-3 by name, 4 dihydroxyphenylalanine.It is the precursor substance of synthetic norepinephrine, Dopamine HCL etc. in the body, enter maincenter by hemato encephalic barrier, change into Dopamine HCL and play a role through dopa decarboxylase, (a kind of back skeletal muscle of getting involved is difficult for loosening after contraction to improving myotony, alleviate after shrinking continuously or disappear, it is cold that to make sx be the muscle disease of feature) and effect such as bradykinesia obvious, to tremble, hydrostomia, posture shakiness and dysphagia have certain effect, being used for Parkinsonism (Parkinson's disease) clinically, is the Parkinsonian choice drug of treatment.To light, moderate state of an illness person effect is better, severe or the elderly's effect are relatively poor, patient is clear-headed, doing well,improving can to make hepatic coma (a kind of syndrome based on the central nervous system of metabolism disorder that is caused by serious hepatopathy).The L-DOPA of Chinese market sale at present mainly obtains by extracting from plant, and the L-DOPA that obtains is like this limited by raw material, and output is little, can't satisfy the growing market requirement.The L-DOPA also can be synthetic by chemical method.But L-DOPA and preparation thereof are mainly used in nervous system disease, required based on very high purity.The chemical synthesis poor selectivity can generate the DOPA (enantiomer each other) of D type and L type simultaneously, be difficult to D type and L type DOPA are separated, and the D-DOPA has bigger toxic action to human body, can not use as medicament.The DOPA by fermentative Production L-is abroad also arranged.Though fermentation method output is higher, its production cycle is longer, and fermented liquid composition relative complex, separates the cost height of purifying.Patent US5837504A uses the synthetic L-DOPA of Phenylalanine hydroxylase catalysis L-tyrosine, though L-DOPA yield is pretty good, and complicated operation, and also efficient is not high.Tyrosine oxidase can catalysis L-tyrosine be oxidized to the reaction of L-DOPA, but because the free tyrosine oxidase can quick catalysis L-DOPA continuation oxidation generate a series of downstream product and the final melanochrome that generates, cause its L-DOPA productive rate very low, and enzyme and product be difficult to separate, so can't put into production.Immobilized tyrosine oxidase has good stability, is convenient to advantages such as recycling.Also do not find at present relate to applying immobilized tyrosine oxidase particularly the tyrosine oxidase of carrier-free immobilization carry out the production of L-DOPA.
[summary of the invention]
For this reason, the technical problem to be solved in the present invention is how to use that the carrier-free immobilization zymotechnic carries out immobilization to tyrosine oxidase and with this catalysis synthetic drugs L-DOPA.
For solving the problems of the technologies described above, the present invention relates to a kind of method with enzyme catalysis synthetic drugs L-DOPA.This method is a raw material with L-tyrosine, generates the L-DOPA with the carrier-free immobilization tyrosinase catalysis.Wherein, the L-DOPA of generation can continue oxidation and generate corresponding DOPA quinone, and present method is further used the L-xitix, and the DOPA quinone of correspondence is reduced into the L-DOPA.
Wherein, described L-tyrosine and L-xitix are dissolvable in water in the damping fluid.Described pH of buffer value is 5.4-8.0.Described damping fluid can be phosphoric acid buffer.Described phosphate buffer density is 50-200mmol/L.
As preferably, the carrier-free immobilization tyrosine oxidase can be filled in the pillar, and the mixed solution with L-tyrosine and L-xitix slowly flows through this pillar then, to obtain product L-DOPA.
As preferably, the carrier-free immobilization tyrosine oxidase can be added to one and contain in the flow reactor of L-tyrosine and L-xitix, the reaction solution that continuous adding contains L-tyrosine and L-xitix is constantly derived this reactor to this reactor with the reaction solution that contains product L-DOPA that generates.
As preferably, the carrier-free immobilization tyrosine oxidase can be added to one and contain in the batch-type reactor of L-tyrosine and L-xitix, and stirring reaction for some time obtains product L-DOPA.
As preferably, this carrier-free immobilization tyrosine oxidase can make by the following method:
S1. fresh white mushroom is mixed with phosphoric acid buffer and smash to pieces, filter the removal residue, obtaining remaining the liquid cooling of tyrosine oxidase proenzyme, to freeze preservation frozen more than 1 month;
S2. frozen tyrosine oxidase proenzyme lyolysis is frozen, and frozen centrifugation go to precipitate enzyme liquid;
S3. the enzyme liquid among the step S2 adds ammonium sulfate solids in magnetic agitation, makes the ammonium sulfate saturation ratio of above-mentioned solution be about 50% at 0 ℃;
S4. after step S3 finished, the volume ratio of glutaraldehyde in 12.5% and enzyme liquid was 1: 50 a ratio, dropwise adds glutaraldehyde, and stir crosslinked suspension liquid;
S5. through the resulting water-fast particle in centrifugal back for several times, do not contain to the supernatant till the tyrosinase activity with buffer solution for cleaning used among the S1 with the crosslinked suspension liquid among the step S4;
S6. the water-fast particle that obtains among the step S5 obtains the tan Powdered carrier-free immobilization tyrosine oxidase that is after lyophilize makes its complete drying.
The inventive method has following beneficial effect:
1, plant extract L-DOPA has great limitation, because plant origin is very limited, production cost is also very high.With respect to the plant extract method, the enzyme immobilization technology application prospect is wider, and cost can obviously reduce, and the most important thing is to satisfy the great demand amount of L-DOPA.
2, better with respect to chemical synthesis products therefrom specificity of the present invention, side reaction is few, and the reaction conditions gentleness more helps environmental protection.
3, fast with respect to the microbe fermentation method production cycle of the present invention, composition is more single, and separation costs is low.
4, owing to tyrosine oxidase self, its product L-DOPA has restraining effect to it.Directly carrying out the production of L-DOPA with the free tyrosine enzyme is caused its productive rate extremely low because the L-DOPA can continue oxidation.Find document utilization additive method fixed tyrosine oxidase and produced 170 hours transformation efficiencys about 60% of L-DOPA.And just can reach 60% with 2 hours transformation efficiencys of present method.
[embodiment]
Technical scheme of the present invention: with the method that the carrier-free immobilization tyrosine oxidase is produced the L-DOPA, be raw material, by the tyrosinase catalysis generation L-DOPA of carrier-free immobilization with L-tyrosine.Because the L-DOPA that generates can continue oxidized generation DOPA quinone, therefore in reaction solution, add DOPA quinone reduction the becoming L-DOPA that the L-xitix makes generation.
Reaction formula of the present invention can be expressed as following formula:
Wherein, R is-CH2CH (NH2) COOH
Illustrate further the present invention below in conjunction with specific embodiment.
At first, the invention provides a kind of application carrier-free immobilization zymotechnic tyrosine oxidase is carried out immobilized method.Should be noted that, the method of carrier-free immobilization tyrosine oxidase of the present invention is not limited only to the present embodiment method, use the method for other carrier-free immobilization tyrosine oxidases, and use the present invention to conceive the scheme of synthetic drugs L-DOPA, all should be considered as being contained by claim of the present invention.
In the present embodiment, the method for carrier-free immobilization tyrosine oxidase comprises that step is as follows:
The fresh white mushroom that S1 buys market with refiner according to mushroom: phosphoric acid buffer (50mM, ratio pH6.0)=1: 2 (kg/L) is smashed it to pieces, filters and removes residue ,-20 ℃ of refrigerators are frozen more than 1 month after the packing of residual enzyme liquid;
S2 under 4 ℃ of conditions with S1 in frozen tyrosine oxidase proenzyme lyolysis freeze and frozen centrifugation go to precipitate enzyme liquid;
Enzyme liquid among the S3 step S2 adds ammonium sulfate solids in magnetic agitation, making the ammonium sulfate saturation ratio (0 ℃) of above-mentioned solution is 50%;
After S4 step S3 finishes, 1: 50 by volume (12.5% glutaraldehyde: enzyme liquid) dropwise add glutaraldehyde (12.5%, v/v) and stir 16h in 4 ℃ and get crosslinked suspension liquid;
S5 does not contain to the supernatant till the tyrosinase activity with the suspension liquid among the step S4 through the resulting water-fast particle in centrifugal back with buffer solution for cleaning used among the S1 for several times;
The water-fast particle that obtains among the S6 step S5 obtains the tan Powdered carrier-free immobilization tyrosine oxidase that is after lyophilize makes its complete drying.
Embodiment one
The carrier-free immobilization tyrosine oxidase is loaded to a pillar, allowed the mixed solution of L-tyrosine and L-xitix the pillar of carrier-free immobilization tyrosine oxidase is housed, just can obtain the L-DOPA in the effusive product mixed solution by this.Transformation efficiency can reach more than 25% behind this method 0.5h.
Embodiment two
After the mixed solution of carrier-free immobilization tyrosine oxidase and L-tyrosine and L-xitix added a batch-type reactor reaction for some time, remove the carrier-free immobilization tyrosine oxidase after, obtain the L-DOPA.This method reaction 2h can obtain the transformation efficiency more than 60%.The carrier-free immobilization tyrosine oxidase can adopt the centrifugal major part of removing earlier of 6000r/min normal temperature earlier, removes remaining small part enzyme powder with filtering method again.
Embodiment three
The tyrosine oxidase of carrier-free immobilization is added in the flow reactor of the mixed solution that L-tyrosine and L-xitix are arranged, the magnetic agitation reaction, and constantly the product L-DOPA that generates is derived, and import the fresh L-tyrosine and the reaction solution of L-xitix with identical speed.Transformation efficiency can reach more than 50% behind the 1h.
It is to be noted; above-mentioned explanation only is the detailed description to preferred embodiment of the present invention; narration only is explanation realizability of the present invention and outstanding effect thereof; concrete feature can not be used as the restriction to technical scheme of the present invention, and protection scope of the present invention should be as the criterion with appended claims of the present invention.
Claims (9)
1. method with enzyme catalysis synthetic drugs L-DOPA, it is characterized in that, with L-tyrosine is raw material, generate the L-DOPA with the carrier-free immobilization tyrosinase catalysis, wherein, the L-DOPA that generates can further be generated corresponding DOPA quinone by the reaction of carrier-free immobilization tyrosinase catalysis, and DOPA quinone that should correspondence with the L-xitix is reduced into the L-DOPA.
2. the method for claim 1 is characterized in that, described L-tyrosine and L-dissolution of ascorbic acid are in a damping fluid.
3. method according to claim 2 is characterized in that, described pH of buffer value is 5.4-8.0.
4. method according to claim 3 is characterized in that, described damping fluid is a phosphoric acid buffer.
5. method according to claim 4 is characterized in that, described phosphate buffer density is 50-200mmol/L.
6. method according to claim 1 is characterized in that, is the carrier-free immobilization tyrosine oxidase is filled in the pillar, and the mixed solution with L-tyrosine and L-xitix slowly flows through this pillar then, to obtain product L-DOPA.
7. method according to claim 1, it is characterized in that, the carrier-free immobilization tyrosine oxidase is added to one to be contained in the flow reactor of L-tyrosine and L-xitix, the reaction solution that continuous adding contains L-tyrosine and L-xitix is constantly derived this reactor to this reactor with the product L-DOPA that generates.
8. method according to claim 1 is characterized in that, the carrier-free immobilization tyrosine oxidase is added to one contains in the batch-type reactor of L-tyrosine and L-xitix, and stirring reaction for some time obtains product L-DOPA.
9. method according to claim 1 is characterized in that, this carrier-free immobilization tyrosine oxidase makes by the following method:
S1. fresh white mushroom is mixed with phosphoric acid buffer and smash to pieces, filter the removal residue, obtaining remaining the liquid cooling of tyrosine oxidase proenzyme, to freeze preservation frozen more than 1 month;
S2. frozen tyrosine oxidase proenzyme lyolysis is frozen, and frozen centrifugation go to precipitate enzyme liquid;
S3. the enzyme liquid among the step S2 adds ammonium sulfate solids in magnetic agitation, makes the ammonium sulfate saturation ratio of above-mentioned solution be about 50% at 0 ℃;
S4. after step S3 finished, the volume ratio of glutaraldehyde in 12.5% and enzyme liquid was 1: 50 a ratio, dropwise adds glutaraldehyde, and stir crosslinked suspension liquid;
S5. through the resulting water-fast particle in centrifugal back for several times, do not contain to the supernatant till the tyrosinase activity with buffer solution for cleaning used among the S1 with the crosslinked suspension liquid among the step S4;
S6. the water-fast particle that obtains among the step S5 obtains the tan Powdered carrier-free immobilization tyrosine oxidase that is after lyophilize makes its complete drying.
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| CN2011100697888A CN102212568A (en) | 2011-03-22 | 2011-03-22 | Method for synthesizing medicament L-dopa by enzyme catalysis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102942248A (en) * | 2012-10-19 | 2013-02-27 | 深圳大学 | Method for removing phenol pollutants with carrier-free immobilized enzyme |
| CN106755133A (en) * | 2017-01-20 | 2017-05-31 | 深圳大学 | A kind of method that enzyme process prepares adjacent phenol compound |
| CN110055288A (en) * | 2019-03-15 | 2019-07-26 | 深圳大学 | A method of polyphenol compound is synthesized with novel enzyme immobilization technology |
Citations (4)
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|---|---|---|---|---|
| EP0242656A2 (en) * | 1986-04-25 | 1987-10-28 | Bio-Polymers, Inc. | Method for making dopa-containing bioadhesive proteins from tyroshine-containing proteins |
| JPS62259598A (en) * | 1986-04-22 | 1987-11-11 | ザ・ソーク・インステチュート・フォー・バイオロジカル・スタディーズ | Fibroblast growth factor antagonist |
| CN1004008B (en) * | 1986-07-19 | 1989-04-26 | 武汉大学 | Method for quickly and conveniently isolating and screeing of the l-dopa-producing bacterial strain |
| KR20100110691A (en) * | 2009-04-04 | 2010-10-13 | 서울대학교산학협력단 | Electroenzymatic process for manufacturing l-dopa |
-
2011
- 2011-03-22 CN CN2011100697888A patent/CN102212568A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62259598A (en) * | 1986-04-22 | 1987-11-11 | ザ・ソーク・インステチュート・フォー・バイオロジカル・スタディーズ | Fibroblast growth factor antagonist |
| EP0242656A2 (en) * | 1986-04-25 | 1987-10-28 | Bio-Polymers, Inc. | Method for making dopa-containing bioadhesive proteins from tyroshine-containing proteins |
| CN1004008B (en) * | 1986-07-19 | 1989-04-26 | 武汉大学 | Method for quickly and conveniently isolating and screeing of the l-dopa-producing bacterial strain |
| KR20100110691A (en) * | 2009-04-04 | 2010-10-13 | 서울대학교산학협력단 | Electroenzymatic process for manufacturing l-dopa |
Non-Patent Citations (3)
| Title |
|---|
| BURCU SELIN AYTAR, UFUK BAKIR: "Preparation of cross-linked tyrosinase aggregates", 《PROCESS BIOCHEMISTRY》, vol. 43, 31 December 2008 (2008-12-31), pages 125 - 131, XP 022420313, DOI: doi:10.1016/j.procbio.2007.11.001 * |
| GAYATHRI SEETHARAM, BRADLEY A. SAVILLE: "L-DOPA production from tyrosinase immobilized on zeolite", 《ENZYME AND MICROBIAL TECHNOLOGY》, vol. 31, 31 December 2002 (2002-12-31), pages 747 - 753 * |
| 魏甲乾: "无载体固定化酶的研究进展", 《甘肃科学学报》, vol. 18, no. 1, 31 March 2006 (2006-03-31), pages 66 - 70 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102942248A (en) * | 2012-10-19 | 2013-02-27 | 深圳大学 | Method for removing phenol pollutants with carrier-free immobilized enzyme |
| CN106755133A (en) * | 2017-01-20 | 2017-05-31 | 深圳大学 | A kind of method that enzyme process prepares adjacent phenol compound |
| CN106755133B (en) * | 2017-01-20 | 2021-05-25 | 深圳大学 | Method for preparing o-phenol compound by enzyme method |
| CN110055288A (en) * | 2019-03-15 | 2019-07-26 | 深圳大学 | A method of polyphenol compound is synthesized with novel enzyme immobilization technology |
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Application publication date: 20111012 |