CN114540333B - Immobilized modified aspartase and application thereof - Google Patents
Immobilized modified aspartase and application thereof Download PDFInfo
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- CN114540333B CN114540333B CN202011339653.4A CN202011339653A CN114540333B CN 114540333 B CN114540333 B CN 114540333B CN 202011339653 A CN202011339653 A CN 202011339653A CN 114540333 B CN114540333 B CN 114540333B
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- ile
- ala
- leu
- glu
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- 108700016171 Aspartate ammonia-lyases Proteins 0.000 title claims abstract description 20
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 150000001576 beta-amino acids Chemical class 0.000 claims abstract description 7
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 6
- 239000003054 catalyst Substances 0.000 claims description 18
- 229920005989 resin Polymers 0.000 claims description 17
- 239000011347 resin Substances 0.000 claims description 17
- 125000000217 alkyl group Chemical group 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 7
- 230000009257 reactivity Effects 0.000 claims description 6
- 125000003545 alkoxy group Chemical group 0.000 claims description 4
- 125000004414 alkyl thio group Chemical group 0.000 claims description 4
- 125000004644 alkyl sulfinyl group Chemical group 0.000 claims description 3
- 125000004390 alkyl sulfonyl group Chemical group 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 2
- 125000003700 epoxy group Chemical group 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 2
- 125000000475 sulfinyl group Chemical group [*:2]S([*:1])=O 0.000 claims description 2
- 125000003396 thiol group Chemical class [H]S* 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 150000002431 hydrogen Chemical class 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- OQEBBZSWEGYTPG-GSVOUGTGSA-N (3r)-3-aminobutanoic acid Chemical compound C[C@@H](N)CC(O)=O OQEBBZSWEGYTPG-GSVOUGTGSA-N 0.000 description 6
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- -1 methoxy, ethoxy, n-propoxy Chemical group 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- AAHSHTLISQUZJL-QSFUFRPTSA-N Gly-Ile-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AAHSHTLISQUZJL-QSFUFRPTSA-N 0.000 description 4
- NWDOPHYLSORNEX-QXEWZRGKSA-N Val-Asn-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N NWDOPHYLSORNEX-QXEWZRGKSA-N 0.000 description 4
- 108010089804 glycyl-threonine Proteins 0.000 description 4
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- 108010009298 lysylglutamic acid Proteins 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical group O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 229940000635 beta-alanine Drugs 0.000 description 3
- 125000004432 carbon atom Chemical group C* 0.000 description 3
- LDHQCZJRKDOVOX-NSCUHMNNSA-N crotonic acid Chemical compound C\C=C\C(O)=O LDHQCZJRKDOVOX-NSCUHMNNSA-N 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- LDHQCZJRKDOVOX-UHFFFAOYSA-N trans-crotonic acid Natural products CC=CC(O)=O LDHQCZJRKDOVOX-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- JNTMAZFVYNDPLB-PEDHHIEDSA-N (2S,3S)-2-[[[(2S)-1-[(2S,3S)-2-amino-3-methyl-1-oxopentyl]-2-pyrrolidinyl]-oxomethyl]amino]-3-methylpentanoic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JNTMAZFVYNDPLB-PEDHHIEDSA-N 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- WOJJIRYPFAZEPF-YFKPBYRVSA-N 2-[[(2s)-2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]propanoyl]amino]acetate Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)CNC(=O)CN WOJJIRYPFAZEPF-YFKPBYRVSA-N 0.000 description 2
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- WMYJZJRILUVVRG-WDSKDSINSA-N Ala-Gly-Gln Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O WMYJZJRILUVVRG-WDSKDSINSA-N 0.000 description 2
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 2
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 2
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- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- OTOXOKCIIQLMFH-KZVJFYERSA-N Arg-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N OTOXOKCIIQLMFH-KZVJFYERSA-N 0.000 description 2
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- HPKSHFSEXICTLI-CIUDSAMLSA-N Arg-Glu-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HPKSHFSEXICTLI-CIUDSAMLSA-N 0.000 description 2
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011342 resin composition Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/091—Phenol resins; Amino resins
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
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Abstract
The invention provides immobilized modified aspartase and application thereof in catalyzing and synthesizing beta-amino acid. Compared with free enzyme, the immobilized enzyme has the advantages of improving the purity of the product, and reducing the use cost of the enzyme and the purification cost of subsequent reactions.
Description
Technical Field
The invention relates to the field of enzyme engineering and immobilized enzymes. In particular, the invention relates to the use of immobilized modified aspartase for catalytic synthesis of beta-amino acids.
Background
The structural general formula of the beta-amino acid is shown as follows:
beta-amino acids are an important class of chiral intermediates for organic synthesis, which are widely used in pharmaceutical synthesis. Such as: (R) -3-aminobutyric acid is a key intermediate for synthesizing anti-HIV drug Dolutegravir (dolutegradvir), and beta-alanine can be used as a key intermediate for synthesizing calcium pantothenate.
The structural formula of the (R) -3-aminobutyric acid is shown as follows:
the structural formula of the beta-alanine is shown as follows:
at present, the (R) -3-aminobutyric acid is mainly prepared by organic synthesis, and the method has the defects of more steps, low yield, poor stereoselectivity and the like.
Biological workers have also attempted to prepare such compounds, for example, andreas Vogel et al (Andreas Vogel et al Converting Aspartase into a b-Amino Acid Lyase by Cluster Screening, chemCatChem,6,965-968 (2014)) have engineered aspartases to have the ability to synthesize (R) -3-aminobutyric acid from aspartases that would otherwise not catalyze such reactions, the reaction formulae being as follows:
however, the free enzymes used in this reaction can bring the product into the residual host proteins and nucleic acids, affecting the purity of the product, and waste due to the inability of the enzymes to be recycled. Thus, there remains a need in the art to provide enzymatic catalysts that are synthesized in high purity products and that can be recycled.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for synthesizing beta-amino acid by an immobilized enzyme method.
In order to achieve the above object, according to one aspect of the present invention, there is provided an immobilized enzyme catalyst comprising a resin and an enzyme covalently bound to the resin; the enzyme is aspartase (AAL).
Further, the resin is an epoxy group-bearing resin or an activated amino group-bearing resin, and the activator is preferably glutaraldehyde solution.
Further, the glutaraldehyde solution has a concentration of 1% -2%.
Further, the aspartase is derived from Ureibacillus thermophilus.
The invention also discovers that site-directed mutagenesis can be carried out on the aspartase, and the loading capacity and the loading firmness of the mutated aspartase on the resin are greatly improved;
further, the immobilized enzyme catalyst still maintains more than 90% of the reactivity after 30 times of reaction;
further, the immobilized enzyme catalyst still maintains more than 95% of the reactivity after 30 times of reaction;
further, the immobilized enzyme catalyst still maintains more than 99% of the reactivity after 30 times of reaction;
further, the mutation occurs at amino acid position 448 of the aspartase;
further, the mutant is E448K, and the mutated aspartase has the amino acid sequence of SEQ ID No.2.
Further, the mass ratio of the enzyme protein to the resin is 1:15 to 1:8.
According to another aspect of the present invention there is provided a method of synthesizing a β -amino acid, characterised in that an immobilized enzyme catalyst as hereinbefore described is used, the reaction steps being as follows:
wherein R is selected from: hydrogen, alkyl, alkoxy, alkylsulfonyl, alkylsulfinyl, alkylthio, sulfonic, sulfinyl, mercapto, nitro and halogen;
n is 0, 1, 2 or 3;
wherein:
"alkyl" refers to a straight or branched aliphatic hydrocarbon group having 1 to 20 carbon atoms in the chain. Preferred alkyl groups have 1 to 12 carbon atoms in the chain, more preferred are lower alkyl groups as defined herein. "branched" means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkyl chain. "lower alkyl" means 1 to 4 carbon atoms in the chain which may be straight or branched.
"alkoxy" refers to an alkyl-O-group, wherein alkyl is as described herein. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, z-propoxy, n-butoxy, heptoxy, and the like.
"alkylsulfonyl" refers to alkyl-SO 2 -a group wherein alkyl is as defined above. Exemplary groups are those in which the alkyl group is a lower alkyl group.
"Alkylsulfinyl" refers to an alkyl-SO-group, where alkyl is as defined above. Exemplary groups are those in which the alkyl group is a lower alkyl group.
"Alkylthio" refers to an alkyl-S-group, wherein alkyl is as described herein. Exemplary alkylthio groups include methylthio, ethylthio, z-propylthio and heptylthio.
By applying the technical scheme of the invention, the enzyme is efficiently immobilized on the resin, so that the residual host nucleic acid and protein in the reaction liquid are avoided, the purity of the product is improved, and the production cost is reduced.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
Example 1: preparation of immobilized enzyme catalyst
Unless otherwise specified, the experimental methods used in the present invention are all conventional methods.
i) Reagents and instrumentation:
LX-1000NH, LX-1000HFA are available from blue-known technology;
DNA polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonucleases were purchased from TaKaRa;
plasmid extraction kits were purchased from Axygen company;
acrylic acid was purchased from aladine, cat No. a103526, 99% purity;
crotonic acid was purchased from aladine, cat No. C104149, 98% purity.
ii) vectors and strains: the expression vector used was pET-30a (+), the plasmid was purchased from Novagen, and the host cell used was E.coli BL21 (DE 3), purchased from Tiangen Biochemical technology (Beijing) Co.
iii) Sequencing, primer synthesis and gene synthesis were performed by hong biosciences, inc. of Suzhou. Wherein, after gene synthesis, the gene is constructed into a vector pET-30 a.
iv) site-directed mutagenesis:
specific primer pairs are designed, and required substitutions are introduced at amino acid positions of required mutation corresponding to bases. Mutants were prepared using the extracted pre-mutation plasmid (containing the wild-type aspartase (derived from Ureibacillus thermophilus) coding sequence, pET-30a (+) scaffold) as a template using the methods described by Packer and Liu (Methods for the directed evolution of proteins. Nat Rev Genet,2015,16 (7): 379-394).
Mutation strategies for site-directed mutagenesis are shown in table 1:
TABLE 1
Note that: the mutant modification of SEQ ID No.1 was performed according to the teachings of Andreas Vogel et al (Andreas Vogel et al Converting Aspartase into a b-Amino Acid Lyase by Cluster Screening, chemCatchem,6,965-968 (2014)) described in the background to confer the ability of the enzyme of this sequence to synthesize (R) -3-aminobutyric acid.
v) protein expression and preparation of enzyme solution:
coli cells transformed with a plasmid containing the gene of interest were inoculated into LB liquid medium (peptone 10g/L, yeast powder 5g/L, naCl 10g/L, pH 7.0) containing 50mg/L kanamycin, and incubated overnight with shaking at 37 ℃. The culture was transferred to TB liquid medium (peptone 12g/L, yeast extract 24g/L, glycerol 4mL/L, monopotassium phosphate 2.31g/L, dipotassium phosphate 12.54 g/L), incubated with shaking at 37℃until OD600 reached 0.6-0.8, and IPTG (final concentration 0.4 mM) was added and incubated overnight at 30℃to induce protein expression.
After incubation, the culture was centrifuged at 4,000g for 10min at 4℃and the supernatant was discarded and E.coli cells were collected. The collected E.coli cells were resuspended in pre-chilled 20mL of Phosphate Buffer (PBS) pH7.0 and the E.coli cells were sonicated at 4 ℃. The cell disruption solution was centrifuged at 6,000g at 4℃for 15min to remove the precipitate, and the obtained supernatant was an enzyme solution containing a recombinase for catalyzing the reaction. Or lyophilizing the enzyme solution to obtain enzyme powder, and storing at 4deg.C.
vi) preparation of immobilized enzyme
Amino resin:
resin LX-1000NH was pre-activated overnight with 2% glutaraldehyde solution and washed clean with water.
1g of aspartase (SEQ ID No.1 or mutant SEQ ID No. 2) was mixed with 10g of resin LX-1000NH and stirred at 25℃for 18h. After the fixation, the immobilized enzyme is filtered, washed three times with water and stored at 4 ℃ for standby.
Epoxy resin:
1g of aspartase (SEQ ID No.1 or mutant SEQ ID No. 2) was mixed with 10g of resin LX-1000HFA and stirred at 25℃for 18h. After the fixation, the immobilized enzyme is filtered, washed three times with water and stored at 4 ℃ for standby.
The reaction products are shown in Table 2:
immobilized enzyme numbering | Resin composition | Enzyme sequence |
Immob-NH-AAL1 | LX-1000NH | SEQ ID No.1 |
Immob-HFA-AAL1 | LX-1000HFA | SEQ ID No.1 |
Immob-NH-AAL2 | LX-1000NH | SEQ ID No.2 |
Immob-HFA-AAL2 | LX-1000HFA | SEQ ID No.2 |
TABLE 2
Example 2: preparation of (R) -3-aminobutyric acid by immobilized enzyme
Preparing a 1L reaction system: the pH of crotonic acid is adjusted to 8.5 by ammonia water at 100g/L, the crotonic acid is heated to 30 ℃ and stirred magnetically uniformly, 30g of the immobilized enzyme prepared in the example 1 is added, stirring reaction is started, the conversion rate is detected by sampling HPLC after 24 hours, and the conversion results of the first conversion and the second conversion are shown in Table 3.
Immobilized enzyme numbering | First conversion (%) | Conversion by 30 th time (%) |
Immob-NH-AAL1 | 99 | 92 |
Immob-HFA-AAL1 | 97 | 78 |
Immob-NH-AAL2 | 99 | 99 |
Immob-HFA-AAL2 | 97 | 93 |
TABLE 3 Table 3
Example 3: preparation of beta-alanine by immobilized enzyme
Preparing a 1L reaction system: the pH of the acrylic acid is regulated to 9.0 by ammonia water at 200g/L, the mixture is heated to 30 ℃ and stirred evenly by magnetic force, 20g of immobilized enzyme is added, stirring reaction is started, the reaction is started for 24 hours, HPLC is used for detecting the conversion rate, and the conversion results of the first and the second application are shown in Table 4.
Immobilized enzyme numbering | First conversion (%) | Conversion by 30 th time (%) |
Immob-NH-AAL1 | 99 | 95 |
Immob-HFA-AAL1 | 99 | 91 |
Immob-NH-AAL2 | 99 | 99 |
Immob-HFA-AAL2 | 99 | 99 |
TABLE 4 Table 4
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Sequence listing
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Claims (7)
1. An immobilized enzyme catalyst, characterized in that the immobilized enzyme catalyst comprises a resin and an enzyme covalently bound to the resin; the enzyme is aspartase, and the amino acid sequence of the aspartase is SEQ ID No.2.
2. The immobilized enzyme catalyst of claim 1, wherein the resin is an epoxy group-bearing resin or an activated amino group-bearing resin.
3. The immobilized enzyme catalyst of claim 1, wherein the immobilized enzyme catalyst retains more than 90% of its reactivity after 30 reactions.
4. The immobilized enzyme catalyst of claim 3, wherein the immobilized enzyme catalyst retains more than 95% of its reactivity after 30 reactions.
5. The immobilized enzyme catalyst of claim 4, wherein the immobilized enzyme catalyst retains more than 99% of its reactivity after 30 reactions.
6. The immobilized enzyme catalyst of any one of claims 1-5, wherein the enzyme to resin mass ratio is from 1:15 to 1:8.
7. A method for synthesizing a β -amino acid, characterized in that the immobilized enzyme catalyst according to any one of claims 1 to 6 is used, the reaction steps being as follows:
wherein R is selected from: hydrogen, alkyl, alkoxy, alkylsulfonyl, alkylsulfinyl, alkylthio, sulfonic, sulfinyl, mercapto, nitro and halogen.
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CN114540333A (en) | 2022-05-27 |
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