CN111951898B - Method for screening enzyme capable of converting L-amino acid into 2-keto acid - Google Patents
Method for screening enzyme capable of converting L-amino acid into 2-keto acid Download PDFInfo
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- 150000008575 L-amino acids Chemical class 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 10
- 238000012216 screening Methods 0.000 title claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 102100027841 Acyl-CoA wax alcohol acyltransferase 2 Human genes 0.000 claims description 14
- 101710092506 Aspartate aminotransferase Proteins 0.000 claims description 14
- 101710101107 Probable aspartate aminotransferase Proteins 0.000 claims description 14
- DOEADYYICZVJDD-UHFFFAOYSA-N [4-[(4-aminophenyl)diazenyl]phenyl]arsonic acid Chemical compound C1=CC(N)=CC=C1N=NC1=CC=C([As](O)(O)=O)C=C1 DOEADYYICZVJDD-UHFFFAOYSA-N 0.000 claims description 14
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 108010024239 aromatic amino acid aminotransferase Proteins 0.000 claims description 14
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 claims description 6
- 238000010523 cascade reaction Methods 0.000 claims description 5
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- 102000016938 Catalase Human genes 0.000 claims description 3
- 108010053835 Catalase Proteins 0.000 claims description 3
- 150000001412 amines Chemical class 0.000 claims description 3
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 3
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- 239000008103 glucose Substances 0.000 claims description 2
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- 239000000203 mixture Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000000855 fermentation Methods 0.000 abstract description 2
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- 108090000340 Transaminases Proteins 0.000 description 19
- 102000003929 Transaminases Human genes 0.000 description 19
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- 150000008574 D-amino acids Chemical class 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108010008292 L-Amino Acid Oxidase Proteins 0.000 description 2
- 108030000198 L-amino-acid dehydrogenases Proteins 0.000 description 2
- 102000007070 L-amino-acid oxidase Human genes 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000005580 one pot reaction Methods 0.000 description 2
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
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- 239000012488 sample solution Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 101100492781 Arabidopsis thaliana ATI2 gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- PUCGSAXUXFZALQ-UHFFFAOYSA-N CC(=O)C.[N+](=O)([O-])C1=C(C=CC(=C1)[N+](=O)[O-])F Chemical compound CC(=O)C.[N+](=O)([O-])C1=C(C=CC(=C1)[N+](=O)[O-])F PUCGSAXUXFZALQ-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
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- 230000001419 dependent effect Effects 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
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- 230000007935 neutral effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
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- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
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- G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
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Abstract
The invention belongs to the technical field of L-amino acid fermentation. The invention provides a method for screening an enzyme capable of converting L-amino acid into 2-keto acid, which comprises the following steps: scoring the sequences in the final database with each subfamily HMM profile to obtain sequences with E values below 0.001; resulting in 15 ATs belonging to class I and II. The invention has the beneficial effects that: the AT has wide sources, is easy to heterologously express, and does not need to additionally add expensive cofactors; high activity, high stereoselectivity, etc.
Description
Technical Field
The invention relates to the technical field of 2-keto acid and D-amino acid biocatalysis synthesis.
Background
In recent years, the development of metabolic engineering and synthetic biology has led to great success in the fermentation of L-amino acids. Global amino acid production in 2017 was about 850 ten thousand tons, which is expected to reach 1100 ten thousand tons in 2022. With the further development of modern biotechnology, the production cost of L-amino acids is continually decreasing. China is used as a large country for producing and consuming amino acids, and large-scale amino acid products are in a state of being supplied and required for a long time. Therefore, the production of high value-added products using L-amino acids as the initial raw material is a problem to be solved urgently.
2-keto acid plays an important role in metabolism of humans and animals as a direct precursor substance for L-amino acid synthesis, and is widely used in the fields of medicine, food, agriculture, and the like. However, the synthesis of 2-keto acid is currently mainly dependent on chemical methods, which require expensive catalysts or special starting compounds, and thus have the disadvantages of high production cost, unfriendly environment and the like.
The biological enzyme method for catalyzing the conversion of the natural L-amino acid into the 2-keto acid has outstanding advantages in terms of sustainability: renewable resources are used as starting materials, mild reaction conditions and high atomic economy. Based on this, four classes of enzymes have been found to catalyze the production of 2-keto acids from L-amino acids, namely L-amino acid dehydrogenase (LADH), L-amino acid oxidase (LAAO), L-amino acid deaminase (LAAD) and L-amino acid Aminotransferase (AT).
While high efficiency and high enantioselectivity and regioselectivity give ATs the ability to participate in enzymatic cascades and 2-keto acid synthesis, ATs generally accept only a set of similar amino acids as substrates, and this substrate tolerance has prevented widespread use of the technology. In addition, some amino acids (e.g., threonine, lysine, and arginine) do not have the corresponding transaminases or some transaminases have low activity. Therefore, how to find a novel AT compatible with the above reaction system by bioinformatics means to expand the substrate acceptance range has become a difficulty in the research of the art.
Since chiral unnatural amino acids have received increasing attention in the pharmaceutical and agrochemical industries in recent years, there is a great need for an economical and efficient method for synthesizing D-amino acids and N-methylated amino acids from produced 2-keto acids.
Disclosure of Invention
The invention aims at providing a method for screening an enzyme capable of converting L-amino acid into 2-keto acid, which comprises the following steps: scoring the sequences in the final database with each subfamily HMM profile to obtain sequences with E values below 0.001; resulting in 15 ATs belonging to class I and II.
Further, the method for obtaining the sequence in the final database comprises the following steps: the non-studied AspAT/ARAT and AlaAT sequences from bacterial and archaeal sources in the UniprotKB database were downloaded as a dataset, and the redundant sequences were removed from the dataset to give the sequence of the final database.
Further, the method for establishing the HMM subfamily map comprises the following steps: retrieving the confirmed AspAT/ARAT and AlaAT sequences from Swiss-Port; redundancy was removed from these sequences and HMMER software packages were used to build HMM subfamily maps.
Still further, the method for removing redundant sequences is to remove sequences with the number of amino acids more than 200 and the similarity with AspAT/ARAT or AlaAT sequences less than 90%.
The invention has the beneficial effects that:
1, AT sources are wide, heterologous expression is easy, and expensive cofactors are not required to be added additionally; high activity, high stereoselectivity, etc.
2. The cheap and renewable substrate and cofactor are used, no other cofactor regeneration system is added, the unfavorable chemical balance is transferred to the synthesis direction of the product, and the inhibition effect of 2-ketoglutarate on aminotransferase is weakened.
3. The reaction condition is mild (pH 8.0-8.5, 30 ℃), the substrate conversion rate is high, the HPLC yield of the target compound is high (> 92.7%), and the product has high stereoselectivity (ee value of product > 99%).
Drawings
FIG. 1 shows a diagram of a multi-enzyme cascade for converting an L-amino acid into a 2-keto acid.
FIG. 2 shows the overall analysis scheme and results of screening for transaminase sequences by subfamily HMM patterns.
Wherein "i n "and" j n "score for subfamily profile analysis of AspAT/ATAR and AlaAT for unknown sequence n, respectively.
FIG. 3 shows a scheme of enzymatic cascade synthesis of chiral unnatural amino acids.
Detailed Description
1. In AT families I and II, aspAT and ARAT are classified into the same subfamily (AspAT/ARAT) according to protein structure and sequence characteristics; alaAT is classified as another subfamily in classes I and II due to its large sequence difference from AspAT/ARAT.
TABLE 1 class of transaminases and their substrate profile
2. AspAT/ARAT and AlaAT sequences, which have been confirmed by experimental studies, were retrieved from Swiss-Port for use in constructing family sequence maps.
3. These sequences were de-redundant, removing sequences with more than 90% similarity. The remaining sequences were aligned in subfamilies using the MAFFT, V7.419 software package, and HMM subfamily patterns were created using HMMER3.2.1 software package.
4. Downloading non-researched AspAT/ARAT and AlaAT sequences from bacteria and archaea in the UniprotKB database as a data set, and removing redundant sequences from the data set to obtain the sequence of a final database;
the method for removing the redundant sequence is to remove the sequence with the number of amino acids less than 200 and the similarity with the AspAT/ARAT or AlaAT sequence more than 90% by using a CD-HIT software package.
5. Sequences in the final database were scored (parameters set to default values, sequences with E values below 0.001 were retained) using each subfamily HMM profile, and plotted in a 2-dimensional plane according to the final scoring values (fig. 2).
The number of sequences drawn together in two-dimensional space is 2667; such as scoring of sequences that are typically AspAT/ARAT or AlaAT, are clearly biased towards AspAT/ARAT or AlaAT.
Based on the distribution of sequences, 15 ATs belonging to class I and II were selected as candidate sequences.
TABLE 2 information of 15 AT's selected from families I and II
The related protein sequence can be obtained by searching an Access ID on Uniprot.
6. The candidate sequence was cloned into pET-28a vector and expressed in E.coli BL21 (DE 3), and then affinity purified using His tag. Two proteins AlaAT7 and ATI2, which are not expressed sufficiently in E.coli, were removed. The remaining 13 ATs were expressed in soluble form and purified to obtain higher purity proteins.
7. The activity and specificity of the 13 ATs for 18L-amino acids other than L-Pro and L-Glu were measured (Table 3).
It can be seen that ATs of different origins exhibit complementary substrate specificities, and that these aminotransferases are able to tolerate most L-amino acid substrates (14).
Table 3. Activity of the screened AT on 18L-amino acids.
TABLE 3 continuity
Activity unit: u/mg (1U equals the amount of enzyme required to catalyze the production of 1. Mu. Mol of product per minute)
Activity measurement conditions: the reaction system consisted of 0.2M phosphate buffer (pH 7.5), 20mM L-amino acid, 10mM 2-ketoglutarate, 10. Mu.g/ml ATs and 20. Mu.MPLP, measured in 1mL reaction system at 30℃at 200 rpm.
8. Catalytic ability of enzymes in 2-keto acid synthesis
An AT having a higher activity on a single substrate is selected as a candidate enzyme. The 9L-amino acids shown in Table 4 were tested and all enzymatic reactions were performed in a shaker to ensure oxygen supplementation. Almost all of the L-amino acids tested can be efficiently converted to the corresponding 2-keto acids by enzymatic cascade reactions with higher conversion (> 99%) within 24 hours (Table 4).
Notably, since glyoxylic acid, oxalacetic acid and B-indolylpyruvic acid readily undergo β -decarboxylation side reaction spontaneously at neutral pH, the maximum yields of the above 2-keto acids obtained by kinetic control reach 31.9%, 40% and 78.9%, respectively.
TABLE 4 enzymatic cascade oxidation of L-amino acids to the corresponding 2-keto acids.
The L-amino acid is oxidized to form 2-keto acid through AT-based cascade reaction, the reaction is carried out AT 30 ℃ and 200rpm in 1mL reaction system, and the main components of the reaction system are as follows: 0.2M phosphate buffer (pH 7.5), 30mM L-amino acid, 1mM 2-oxoglutarate, 0.25mg/ml ATs,0.1mg/ml LGOX,0.1mg/ml catalase and 20. Mu.M PLP.
[a] Product yield by HPLC analysis;
[b] no quantification of product yields was performed due to the lack of commercial standards.
9. The one-pot two-step enzymatic cascade reaction is used for synthesizing chiral unnatural amino acid.
The method comprises the steps of firstly, oxidizing L-amino acid into 2-keto acid through an AT-based cascade reaction system to obtain 2-keto acid reaction solution;
wherein the conditions of the reaction system are as follows: 30 ℃,200rpm,1mL reaction volume, reaction system components include: 0.2M phosphate buffer (pH 7.5), 30mM L-amino acid, 1mM 2-oxoglutarate, 0.25mg/ml ATs,0.1mg/ml LGOX,0.1mg/ml catalase and 20. Mu.M PLP.
Second, 0.3mg StDAPDH W121L/H227I or PfNMAADH,0.2mg GDH, and 1mM final NADP+ were added to the 2-keto acid reaction solution; 1.3 equivalents of glucose and 5 equivalents of amine are reacted at a pH of 8.0 to 8.5. During the reaction, the pH of the second reaction step was adjusted to 8.0-8.5 with NaOH.
The determination method of the product yield and the three-dimensional configuration comprises the following steps: the sample is derived by using 2, 4-dinitrofluorobenzene (the derivation method is that the total concentration of ammonia water and amino acid is lower than 50mM after a sample solution is diluted, 25 microliter of the sample solution is absorbed, 10 microliter of 1M sodium bicarbonate and 40 microliter of 36.7mM of 2, 4-dinitrofluorobenzene acetone solution are added, the temperature is kept at 60 ℃ for 30 minutes, 20 microliter of 1M hydrochloric acid is immediately added for stopping the reaction after taking out), and the peak area of the product is measured by HPLC (high performance liquid chromatography) of the derived solution, and the yield and ee value are calculated.
The reaction flow is as follows:
specific yields and product steric configuration results are shown in the following figures:
the beneficial effects are that:
1, AT sources are wide, heterologous expression is easy, and expensive cofactors are not required to be added additionally; high activity, high stereoselectivity and the like;
2. the cheap and renewable substrate and cofactor are used, no other cofactor regeneration system is added, the unfavorable chemical balance is transferred to the synthesis direction of the product, and the inhibition effect of 2-ketoglutarate on aminotransferase is weakened.
3. Under mild reaction conditions (pH 8.0-8.5, 30 ℃), a one-pot two-step sequential synthesis process is adopted to prepare 2-keto acid from L-amino acid and carry out two-step reaction with 5 times equivalent of ammonia or methylamine, so that the synthesis of chiral amine with high added value is gradually realized. From the results, it can be seen that the substrate was almost completely converted to the desired product, the HPLC yield of each target compound was high (> 92.7%) and high stereoselectivity (ee value of product > 99%).
Claims (4)
1.A method for screening an enzyme capable of converting an L-amino acid into a 2-keto acid, comprising the steps of:
downloading non-studied AspAT/ARAT and AlaAT sequences from bacteria and archaea in the UniprotKB database as a dataset, and then removing redundant sequences from the dataset to obtain the sequence of the final database; scoring sequences in the final database with each subfamily HMM profile to obtain sequences with E values below 0.001; obtaining 15 ATs belonging to class I and class II;
the method for removing the redundant sequence is to remove a sequence with the number of amino acids being more than 200 and the similarity with the AspAT/ARAT or AlaAT sequence being less than 90%;
the method for establishing the subfamily HMM map comprises the following steps:
retrieving from Swiss-Port the sequence of AspAT/ARAT and AlaAT that have been confirmed by the study;
redundancy was removed from these sequences and the subfamily HMM profile was built using the HMMER software package.
2. The method of claim 1, wherein the AT is used in a method of synthesizing a chiral unnatural amino acid comprising:
oxidizing L-amino acid into 2-keto acid through an AT-based cascade reaction system to obtain 2-keto acid reaction solution;
to the 2-keto acid reaction solution, 0.3mg StDAPDHW121L/H227I or PfNMAADH,0.2mg GDH,1.3 equivalents of glucose and 5 equivalents of amine were added, and the mixture was reacted at pH 8.0 to 8.5.
3. The method according to claim 2, wherein the reaction system conditions are: 30 ℃,200rpm,1ml reaction volume.
4. The method of claim 2, wherein the reaction system components comprise: 0.2M phosphate buffer pH7.5, 30mM L-amino acid, 1mM 2-ketoglutarate, 0.25mg/ml ATs,0.1mg/ml LGOX,0.1mg/ml catalase and 20. Mu.M PLP.
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Citations (2)
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|---|---|---|---|---|
| EP2072622A1 (en) * | 2006-10-12 | 2009-06-24 | Kaneka Corporation | Method for production of l-amino acid |
| CN104630171A (en) * | 2013-11-08 | 2015-05-20 | 中国科学院天津工业生物技术研究所 | New (R)-transaminase from Fusarium oxysporum and application thereof |
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| US9777301B2 (en) * | 2012-12-13 | 2017-10-03 | Industry-Academic Cooperation Foundation, Yonsei University | Method for producing optically active amine compounds by deracemization |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2072622A1 (en) * | 2006-10-12 | 2009-06-24 | Kaneka Corporation | Method for production of l-amino acid |
| CN104630171A (en) * | 2013-11-08 | 2015-05-20 | 中国科学院天津工业生物技术研究所 | New (R)-transaminase from Fusarium oxysporum and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Exploration of Transaminase Diversity for the Oxidative Conversion of Natural Amino Acids into 2‑Ketoacids and High-Value Chemicals;Tao Li等;ACS Catal.(第10期);7950−7957 * |
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