CN106636294A - Process for producing unnatural amino acid products through coupling reaction of immobilized bi-enzyme - Google Patents
Process for producing unnatural amino acid products through coupling reaction of immobilized bi-enzyme Download PDFInfo
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Abstract
The invention discloses a process for producing unnatural amino acid products through a coupling reaction of immobilized bi-enzyme. With polyvinyl alcohol, sodium alginate and carrageenan as immobilization materials and a boric acid-borax-calcium chloride-sodium carbonate system as a cross-linking agent, granular immobilized cells are prepared by mixing two genetically engineered bacteria which have acylating racemase and amino-acylase activities and then immobilizing a mixture. The unnatural amino acid series products can be effectively produced from acylated-DL-amino acid by conducting the bi-enzyme coupling reaction in a bioreactor.
Description
Technical field
The present invention relates to a kind of immobilized bi-enzyme coupled reaction produces the technique of non-natural amino acid product, belong to bioid
Technical field of enzyme engineering in.
Background technology
Alpha-non-natural amino acid, is numerous medicines especially core intermediate of numerous new drugs nearly ten years, and such as left second draws west
Smooth use C4H9NO2, Perindopril uses L- norvalines, clopidogrel to use L-2- o-chlorobenzene glycines, Ta Tana
Non-usage D-trp, Zomitriptan uses D-PROLINE, atazanavir, VX-960 to use L-2- Terleus, Na Gelie
How, Octreotide uses D-phenylalanine etc..
The main means of production of alpha-non-natural amino acid is to utilize biological enzyme technology, and the enzyme for utilizing has amino-acylase, mould
Plain acylase, hydantoin enzyme, dehydrogenase, amino acid oxidase etc..Wherein amino-acylase is one of main method, for a long time
For producing alpha-non-natural amino acid.Amino-acylase has D- amino-acylases and L- amino-acylases, wherein D- amino-acylases
Catalysis acetyl-D-amino acid generates D- amino acid, and L- aminoacylates enzymatic acetyl-l-amino acid generates l-amino acid.In work
In industry, the raw material of aminoacylates enzyme process production alpha-non-natural amino acid is acetylation-DL- amino acid, therefore conversion ratio only has 50%.
Patent report will acylated racemase mix with amino-acylase after coupling reaction, by DL- acetylation-DL- amino
A sour step is converted to chiral amino acid, and conversion ratio has reached 100%.The shortcoming of the method is to use free cell method, is not easy to weight
Use again, free cell is easily broken, product cost is high, unstable product quality；How biocatalyst is repeated to make
With, reduce catalyst cost be the technology can commercial Application key factor, wherein immobilised enzymes or immobilized cell skill
Art is main solution route.
Immobilized cell technology has the advantages that low cost, pollutes little, efficiency high as bioengineering new technology.At present
The material that immobilization technology is used has carragheen, sodium alginate, resin, glucan, polyacrylamide etc., immobilized cell material
For industrialization, require to have the characteristics that in theory：Easily quickly mix with cell or enzyme, and rapid solidification；It is as far as possible low
Temperature solidification, to avoid the loss of activity of enzyme；It is as far as possible graininess, is easy to load reactor, and obtains preferable response characteristic；
With mechanical performances such as preferable intensity hardness；With porous, with good mass-transfer performance；It is cheap.In order to prepare
Immobilized cell with above performance, researcher is by optimization immobilization material and preparation technology, the crosslinking of selection high-quality
The modes such as agent have released the different immobilized cell of all kinds of performances.
Polyvinyl alcohol as a kind of new biocatalyst entrapped immobilized carrier, with intensity is high, chemical stability
Good, Resistance to microbes performance is strong, it is cheap the advantages of, but still suffer from the drawback that：1. the molten of cross-linking polyvinyl alcohol is used for
Liquid is acid relatively strong, reduces fixation cell cytoactive；2. there is high viscosity, and it is slower with acid reaction, the instillation time can be made
Two drop adhesions being more or less the same, and agglomeration is agglomerating, is difficult gel balling-up；3. enzyme activity retention rate is low after immobilization, partly to acid
Sensitive enzyme can be inactivated gradually.
Therefore a kind of good compounding cross linker system is studied, it is that immobilization material Production by Enzymes is non-to have become polyvinyl alcohol
The important factor in order of natural amino acid.
The content of the invention
In order to overcome drawbacks described above, the invention provides a kind of immobilized bi-enzyme coupling one-step method reaction production non-natural ammonia
The technique of base acid product.
It is characterized in that：With polyvinyl alcohol, sodium alginate, carragheen mixture as immobilization material, with boric acid-boron
Sand-calcium chloride-sodium carbonate system is crosslinking agent, will be common after the genetic engineering bacterium mixing with amino-acylase and acylated racemase
Immobilization, prepares graininess immobilized cell.With acetylation-DL- amino acid as raw material, using double enzymes in bioreactor
Coupled reaction, step production alpha-non-natural amino acid series of products.Concrete operations technique is as follows：
The preparing process of cross-linking agent solution：
Water is added in stirring container, adds a certain proportion of boric acid, borax, calcium chloride, sodium carbonate, adjustment pH to be crosslinked
Agent solution.Part material and proportioning can as requested be increased and decreased.
Cell co-immobilization technique：
Take a certain amount of polyvinyl alcohol and add water-swellable 24h, be subsequently adding sodium alginate and carragheen, autoclave heating makes its abundant
Dissolve, stir while hot, cool to 40～50 DEG C, the L-type amino-acylase that 50 DEG C of insulation in advance is added in liquid is stated then up
With acylated racemase plastc ring, stir, sucked with syringe, be then added drop-wise to above-mentioned boric acid-borax-calcium chloride-
In the cross-linking agent solution of sodium carbonate system, the bead of certain diameter is formed, 3～12h is soaked in crosslinking agent.Washed with physiology salt
Wash, L-type amino acid immobilized cell is obtained, for converting.It is D- amino-acylases that above-mentioned L- amino-acylases are substituted, then
Prepare D- type amino acid immobilized cells.
Living things catalysis technique：
Acetylation-DL- amino acid is added in distilled water, 0.1-0.2M solubility solution is typically made into, with piece alkali PH=7.5 is adjusted,
Above-mentioned L-type amino acid Cell of Anmrobe is added, 37 DEG C, is determined after 5-10 hours, acetylation-DL- amino acid is less than
0.001%, it is totally converted（The conversion for referring to more than more than 99.9% is totally converted, similarly hereinafter）.For ALANINE, through concentration and
Purification process, obtains l-amino acid.
Acetylation-DL- amino acid is added in distilled water, 0.1-0.2M solution is typically made into, with piece alkali PH=7.5 is adjusted,
Above-mentioned D- types amino acid Cell of Anmrobe is added, 37 DEG C, is determined after 5-10 hours, acetylation-DL- amino acid is less than
0.001%, D- amino acid is all converted to, through concentration and purification process, obtain l-amino acid.
Above-mentioned immobilization material is polyvinyl alcohol, sodium alginate, the mixture of carragheen, and the concentration of polyvinyl alcohol material is
5-15%, preferred 8-12%.
The weight proportion of major ingredient and auxiliary material is, polyvinyl alcohol：Sodium alginate：Carragheen=100:0.1-0.5:0.2-1.0.
Above-mentioned crosslinking agent is boric acid-borax-calcium chloride-sodium carbonate system, and crosslinked at low temperature is taken in crosslinking, crosslinking temperature for-
10～20 DEG C.
The immobilized cell for preparing according to the method described above is graininess.With amino-acylase and acylated Racemase activity
Thalline prepares graininess immobilized cell according to immobilization after the mixing of enzyme activity ratio, using double enzyme coupled reactions, step production
Alpha-non-natural amino acid series of products.
Invention beneficial effect
The present invention adopts Novel polyvinyl alcohol immobilization complex material, preferably solves the granularity of immobilized cell, stablizes
Property and quick setting rate；
The present invention adopts novel crosslinker, is crosslinked under weakly acidic pH environmental condition, it is to avoid destruction to enzyme.This technique is used successfully to
Immobilized aminoacylase and acylated racemase mixing thalline, and by coupled reaction technology, a step produces non-natural amino
Acid.
Specific embodiment
Analysis method used in the present embodiment is as follows：
Immobilized cell intensity：Immobilized spherule is put in 100mL syringes, applies certain pressure, observe the broken of bead
Damage situation.
Immobilized cell mass transfer degree：Peek particle fixing bead, in being put into the 250mL conical flasks for filling 200mL water, adds
One drop methylene blue, timing observation color penetrates into the situation of immobilized spherule
Immobilized cell swellbility (%)：100mL immobilized cells are taken, 25 DEG C preserve 24 hours in 0.05M phosphate buffers, body
Long-pending incrementss.
Immobilized cell enzyme activity storage rate：Immobilized cell is in 0.05M phosphate buffers, 0.1M acetyl-DL-phenylalanine
After reacting 5 hours under concentration, the ratio for determining immobilized cell is lived, and is compared than work with initial, is enzyme activity storage rate
Immobilized cell particle serviceability rate：Immobilized cell is in 0.05M phosphate buffers, 0.1M acetyl-DL-phenylalanine concentration
After lower reaction 5 hours, to reject and retain percentage after the damaged particle more than a quarter volume.
The balling-up time：Immobilized cell drips from syringe, the fully hardened time in cross-linking agent solution.
It is adhesive：Visually observe, immobilized cell drips from syringe, whether be adhered mutually in cross-linking agent solution.
Embodiment 1
(A) preparation of cross-linking agent solution
800mL water is added in flask, adds 15 grams of boric acid, 15 grams of borax, mixing to be subsequently adding 10 grams of calcium chloride, sodium carbonate
50 grams, pH value is determined, with sodium carbonate or boric acid adjustment pH to 7.0, constant volume is well mixed stand-by to 1000mL.Can basis
Require increase and decrease part material and proportioning.
(B) method for immobilizing cell
Take 10 grams of polyvinyl alcohol and add the water-swellable 24h of 90mL, add 0.2 gram of sodium alginate and 0.2 gram of carragheen, autoclave heating
It is fully dissolved, stir while hot, cool to 40～50 DEG C, the aminoacyl that 50 DEG C of insulation in advance is added in liquid is stated then up
Change enzyme and acylated racemase plastc ring takes（L- amino-acylase specific activity of enzyme 40umol/g.min, acylated racemase enzyme ratio
Vigor 80umol/g.min）100mL, stirs.Sucked with the syringe of 50 mL, be then added drop-wise to above-mentioned boric acid-borax-
In the cross-linking agent solution of calcium chloride-sodium carbonate system, the bead of certain diameter is formed, 3～12 h are soaked in crosslinking agent.With life
Reason salt water washing, obtains L-type amino acid immobilized cell, and the Rate activity of immobilized cell is 15.5umol/g.min, and refrigerator is protected
Deposit, for conversion and performance measurement.
Above-mentioned preparation process and immobilized cell are further determined, data is obtained as follows：Immobilization particle is about
3mm, intensity is higher, and the balling-up time is 12 hours, and mass transfer effect is good, and the swellbility of immobilized cell is 12%, without adhesive,
Particle serviceability rate 95%.Immobilized cell enzyme activity storage rate 91%.
(C) conversion test method：
27 grams of acetylation-DL-Alanine is added in 1L distilled water, 0.2M strength solutions are made into, with piece alkali pH=7.5 is adjusted, plus
Enter the 50 grams of conversions of above-mentioned L-type amino acid immobilized cell, 37 DEG C, determine after 10 hours, acetylation-DL-Alanine is less than
0.001%, almost all is converted to ALANINE, through concentration and purification process, obtains 18 grams of ALANINE.
With DL- acetyl-leucine, DL- acetyl-valine, DL- acetylphenylalanines, DL- acetyl nor-leucine is original
Material, is equipped with 0.2M strength solutions, and with piece alkali pH=7.5 is adjusted, and adds 50 grams of above-mentioned immobilized cell to convert, 37 DEG C, 10 hours
After determine, acetylation-DL- amino acid be less than 0.001%, be all converted to l-amino acid.Above-mentioned raw materials respectively obtain the bright ammonia of L-
Acid, Valine, L-phenylalanine, L- nor-leucines.
Comparative example 1：
Taking 10 grams of polyvinyl alcohol adds water-swellable 24 h of 90mL, autoclave heating it is fully dissolved, stir while hot, cools to 40
～50 DEG C, it is subsequently adding the amino-acylase of 50 DEG C of insulation in advance and acylated racemase plastc ring takes（L- amino-acylases
Specific activity of enzyme 40umol/g.min, acylated racemase specific activity of enzyme 80umol/g.min）100mL, stirs.With 50 mL's
Syringe is sucked, and is then added drop-wise to cross-linking agent solution (the boric acid 15g/L, calcium chloride 10g/ of boric acid-calcium chloride-sodium carbonate system
L, sodium carbonate 50g/L) in, the bead of certain diameter is formed, 3～12 h are soaked in crosslinking agent.With brine, obtain
L-type amino acid immobilized cell, determines performance.
2%, 5%, 8%, 10%, 15% is respectively configured, 20% polyvinyl alcohol concentration repeats to test above, and other conditions are constant, as a result such as
Under：
Go out from the experimental results, independent polyvinyl alcohol immobilized cell swelling ratio is high, balling-up speed is slow, and ball is adhered mutually, while
Increase with polyvinyl alcohol concentration, viscosity is increased, even being capable of not balling-up.Polyvinyl alcohol concentration is suitable with 10%.
Comparative example 2
Take 10 grams of polyvinyl alcohol and add water-swellable 24 h of 90mL, add different proportion sodium alginate to repeat to test, other conditions are same
Comparative example 1, as a result as follows：
Experimental result shows：After adding sodium alginate, bead intensity increases, and swelling ratio is reduced, and easy balling-up is not adhered, with
0.2% is most suitable.
Comparative example 3
Take 10 grams of polyvinyl alcohol and add water-swellable 24 h of 90mL, add different proportion carragheen to repeat to test, other conditions are with right
It is as a result as follows than embodiment 1：
Experimental result shows：After adding carragheen, bead intensity increases, and swelling ratio is reduced, and is still difficult balling-up, but immobilised enzymes
The rate of recovery is improved, most suitable with 0.5%.
Comparative example 4
Taking 10 grams of polyvinyl alcohol adds water-swellable 24 h of 90mL, sodium alginate 0.2% to add different proportion carragheen to repeat to test,
Other conditions are as a result as follows with comparative example 1：
Compared by experiment, preferred polyvinyl alcohol 10%, sodium alginate 0.2%, 0.5% best proportion of carragheen.
Comparative example 5
Change the proportioning of crosslinking agent and add borax, experimental condition is as a result as follows with comparative example 4：
By comparing, preferred boric acid 15g/L, borax 15g/L, calcium chloride 10g/L, sodium carbonate 50g/L is used as optimal cross-linking system.
Embodiment 2
(A) preparation of cross-linking agent solution
With embodiment 1
(B) method for immobilizing cell
Take 10 grams of polyvinyl alcohol and add water-swellable 24 h of 90mL, add 0.2 gram of sodium alginate and 0.2 gram of carragheen, autoclave to add
Heat makes it fully dissolve, and stirs while hot, cools to 40～50 DEG C, is subsequently adding the D- amino-acylases and acyl of 50 DEG C of insulation in advance
Change racemase plastc ring（D- amino-acylase specific activity of enzyme 30umol/g.min, acylated racemase specific activity of enzyme 98umol/
g.min）100mL, stirs.Sucked with the syringe of 50mL, be then added drop-wise to above-mentioned boric acid-borax-calcium chloride-sodium carbonate
In the cross-linking agent solution of system, the bead of certain diameter is formed, 3～12 h are soaked in crosslinking agent.With brine, obtain
To D- type amino acid immobilized cells, through determining, the Rate activity of immobilized cell is 12.2umol/g.min, Refrigerator store,
For converting.
(C) conversion test method：
27 grams of acetylation-DL-Alanine is added in 1L distilled water, 0.2M strength solutions are made into, with piece alkali pH=7.5 is adjusted, plus
Enter the 50 grams of conversions of above-mentioned D- types amino acid immobilized cell, 37 DEG C, determine after 10 hours, acetylation-DL-Alanine is less than
0.001%, D-alanine is all converted to, through concentration and purification process, obtain 18 grams of D-alanine.
With DL- acetyl-leucine, DL- acetyl-valine, DL- acetylphenylalanines, DL- acetyl nor-leucine is original
Material, is equipped with 0.2M strength solutions, and with piece alkali pH=7.5 is adjusted, and adds 50 grams of above-mentioned immobilized cell to convert, 37 DEG C, 10 hours
After determine, acetylation-DL- amino acid be less than 0.001%, be all converted to D- amino acid.Above-mentioned raw materials respectively obtain the bright ammonia of D-
Acid, D-Val, D-phenylalanine, D- nor-leucines.
Embodiment 3
(A) preparation of cross-linking agent solution
With embodiment 1
(B) method for immobilizing cell
Take 10 grams of polyvinyl alcohol and add water-swellable 24 h of 90mL, add 0.2 gram of sodium alginate and 0.2 gram of carragheen, autoclave to add
Heat makes it fully dissolve, and stirs while hot, cools to 40-50 degree, is subsequently adding the amino-acylase bacteria suspension of 50 DEG C of insulation in advance
Take（L- amino-acylase specific activity of enzyme 80umol/g.min）100mL, stirs.Sucked with the syringe of 50 mL, then dripped
In being added to the cross-linking agent solution of above-mentioned boric acid-borax-calcium chloride-sodium carbonate system, the bead of certain diameter is formed, in crosslinking agent
3～12 h of middle immersion.With brine, L-type amino acid immobilized cell is obtained, through determining, the ratio of immobilized cell
Vigor is 36.5umol/g.min, Refrigerator store, for converting.
(C) conversion test method：
27 grams of acetylation-DL-Alanine is added in 1L distilled water, 0.2M solubility solution is made into, with piece alkali pH=7.5 is adjusted, plus
Enter the 50 grams of conversions of above-mentioned L-type amino acid immobilized cell, 37 DEG C, determine after 10 hours, ALANINE conversion ratio 50%, Jing Guonong
Contracting and purification process, obtain 6.8 grams of ALANINE.
With DL- acetyl-leucine, DL- acetyl-valine, DL- acetylphenylalanines, DL- acetyl nor-leucine is original
Material, respectively obtains L-Leu, Valine, L-phenylalanine, L- nor-leucines.Raw material is tested：It is above-mentioned that to be made into 0.2M molten
Liquid, with piece alkali pH=7.5 is adjusted, and adds the 50 grams of conversions of above-mentioned immobilized cell, 37 DEG C, is determined after 10 hours, acetylation-DL- ammonia
Base acid is converted to l-amino acid with 50% conversion ratio.
Embodiment 4
(A) preparation of cross-linking agent solution
With embodiment 1
(B) method for immobilizing cell
Take 10 grams of polyvinyl alcohol and add water-swellable 24 h of 90mL, add 0.2 gram of sodium alginate and 0.2 gram of carragheen, autoclave to add
Heat makes it fully dissolve, and stirs while hot, cools to 40～50 DEG C, and the D- amino-acylases bacterium for being subsequently adding 50 DEG C of insulation in advance is hanged
Liquid takes（D- amino-acylase specific activity of enzyme 65umol/g.min）100mL, stirs.Sucked with the syringe of 50 mL, then
In being added drop-wise to the cross-linking agent solution of above-mentioned boric acid-borax-calcium chloride-sodium carbonate system, the bead of certain diameter is formed, in crosslinking
3～12 h are soaked in agent.With brine, D- type amino acid immobilized cells are obtained, through determining, immobilized cell
Rate activity is 28.5umol/g.min, Refrigerator store, for converting.
(C) conversion test method：
27 grams of acetylation-DL-Alanine is added in 1L distilled water, 0.2M solubility solution is made into, with piece alkali PH=7.5 is adjusted, plus
Enter the 50 grams of conversions of above-mentioned D- types amino acid immobilized cell, 37 DEG C, determine after 10 hours, D-alanine conversion ratio 50%, Jing Guonong
Contracting and purification process, obtain 7.0 grams of D-alanine.
With DL- acetyl-leucine, DL- acetyl-valine, DL- acetylphenylalanines, DL- acetyl nor-leucine is original
Material, respectively obtains D-Leu, D-Val, D-phenylalanine, D- nor-leucines.Raw material is tested：It is above-mentioned that to be made into 0.2M molten
Liquid, with piece alkali pH=7.5 is adjusted, and adds the 50 grams of conversions of above-mentioned immobilized cell, 37 DEG C, is determined after 10 hours, acetylation-DL- ammonia
Base acid is converted to D- amino acid with 50% conversion ratio.
Claims (6)
1. the technique that a kind of immobilized bi-enzyme coupled reaction produces non-natural amino acid product, it is characterised in that：In immobilization material
In the presence of material and crosslinking agent, co-immobilization after the two kinds of genetic engineering bacteriums mixing with amino-acylase and acylated racemase is obtained
To immobilized cell, with acetylation-DL- amino acid as raw material, non-natural amino acid product is obtained after biocatalytic reaction.
2. the technique for non-natural amino acid product being produced according to a kind of immobilized bi-enzyme coupled reaction of claim 1：The fixation
Change material is the mixture of polyvinyl alcohol, sodium alginate and carragheen, and wherein polyvinyl alcohol concentration is 5-15%；Weight proportion is,
Polyvinyl alcohol：Sodium alginate：Carragheen=100:0.1-0.5:0.2-1.0.
3. the technique for non-natural amino acid product being produced according to a kind of immobilized bi-enzyme coupled reaction of claim 1：The crosslinking
Agent is boric acid-borax-calcium chloride-sodium carbonate system.
4. the technique for non-natural amino acid product being produced according to a kind of immobilized bi-enzyme coupled reaction of claim 1：The fixation
Change cell is graininess.
5. the technique for non-natural amino acid product being produced according to a kind of immobilized bi-enzyme coupled reaction of claim 1：It is described solid altogether
Fixedization operation is that polyvinyl alcohol is added into water-swellable 24h, is subsequently adding sodium alginate and carragheen, and heating makes it fully dissolve,
Stir while hot, cool to 40～50 DEG C, state add the amino-acylase for being incubated 50 DEG C in advance and acylation to disappear in liquid then up
Rotation enzyme plastc ring, stirs, and is then dripped in cross-linking agent solution in the way of being added dropwise, and bead is formed, in crosslinking agent
3～12h of immersion；With brine, amino acid immobilized cell is obtained.
6. the technique for non-natural amino acid product being produced according to a kind of immobilized bi-enzyme coupled reaction of claim 1：The biology
Catalytic reaction operation is that acetylation-DL- amino acid is added in water, is made into 0.1-0.5M solution, and with piece alkali pH=7.5 is adjusted,
Above-mentioned D- types or L-type amino acid Cell of Anmrobe are added, 37 DEG C, is determined after 5-10 hours, acetylation-DL- amino acid is little
In 0.001%, D- types or ALANINE are converted into more than more than 99.9%, through concentration and purification process, obtain D- types or L-
Amino acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710114172.5A CN106636294B (en) | 2017-02-28 | 2017-02-28 | Process for producing unnatural amino acid product by immobilized double-enzyme coupling reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710114172.5A CN106636294B (en) | 2017-02-28 | 2017-02-28 | Process for producing unnatural amino acid product by immobilized double-enzyme coupling reaction |
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| Publication Number | Publication Date |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136298A (en) * | 2018-08-10 | 2019-01-04 | 浙江正硕生物科技有限公司 | A kind of preparation method of D- amino acid |
| CN109270056A (en) * | 2018-10-12 | 2019-01-25 | 中国肉类食品综合研究中心 | A kind of method and microorganism colour reagent box detecting high-throughput antibiotic residue |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020102662A1 (en) * | 2000-03-01 | 2002-08-01 | Akinobu Matsuyama | Methods for racemizing N-acylamino acids and producing optically active amino acids |
| CN101603063A (en) * | 2009-07-23 | 2009-12-16 | 南京大学 | The fixation of microbial cell enzyme process prepares the method for the amino lipid acid of L-2- |
| CN102174632A (en) * | 2009-05-19 | 2011-09-07 | 重庆邮电大学 | Bio-catalytic deracemization preparation method of non-natural L-amino acid |
| CN105368913A (en) * | 2015-12-22 | 2016-03-02 | 滨海瀚鸿生化有限公司 | Bi-enzyme preparation method for industrial production of chiral unnatural amino acid |
-
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- 2017-02-28 CN CN201710114172.5A patent/CN106636294B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020102662A1 (en) * | 2000-03-01 | 2002-08-01 | Akinobu Matsuyama | Methods for racemizing N-acylamino acids and producing optically active amino acids |
| CN102174632A (en) * | 2009-05-19 | 2011-09-07 | 重庆邮电大学 | Bio-catalytic deracemization preparation method of non-natural L-amino acid |
| CN101603063A (en) * | 2009-07-23 | 2009-12-16 | 南京大学 | The fixation of microbial cell enzyme process prepares the method for the amino lipid acid of L-2- |
| CN105368913A (en) * | 2015-12-22 | 2016-03-02 | 滨海瀚鸿生化有限公司 | Bi-enzyme preparation method for industrial production of chiral unnatural amino acid |
Non-Patent Citations (1)
| Title |
|---|
| 苏志鹏等: "嗜热脂肪芽孢杆菌氨基酰化酶在大肠杆菌中的克隆、表达及细胞固定化研究", 《工业微生物》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136298A (en) * | 2018-08-10 | 2019-01-04 | 浙江正硕生物科技有限公司 | A kind of preparation method of D- amino acid |
| CN109270056A (en) * | 2018-10-12 | 2019-01-25 | 中国肉类食品综合研究中心 | A kind of method and microorganism colour reagent box detecting high-throughput antibiotic residue |
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