CN114540333B - Immobilized modified aspartase and application thereof - Google Patents

Immobilized modified aspartase and application thereof Download PDF

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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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ala
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glu
val
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CN114540333A (en
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谢新开
梁晓亮
杜好勉
李林林
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Hunan Yinhang Biological Technology Co ltd
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    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089Enzymes 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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    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089Enzymes 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
    • C12N11/091Phenol resins; Amino resins
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    • C12Y403/00Carbon-nitrogen lyases (4.3)
    • C12Y403/01Ammonia-lyases (4.3.1)
    • C12Y403/01001Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
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    • Y02P20/584Recycling 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

Immobilized modified aspartase and application thereof
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
<110> Hunan pilot biotechnology Co., ltd
<120> immobilized modified aspartase and use thereof
<130> 2020
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 468
<212> PRT
<213> Synthesis of the product
<400> 1
Met Glu Lys Asn Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Ile Asp Ala Tyr Tyr Gly Val Gln Thr Met Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ala Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Thr Ile Gly Glu Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asp Asp Gln Phe Ile Val Asp Pro Ile Gln
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Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Ala Glu Lys Gly Asn Tyr Ser Leu
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Asp Thr Thr Lys Thr Met Gln Gln Val Phe Leu Asn Lys Ala Asp
165 170 175
Glu Phe Ala Gly Ile Ile Lys Met Gly Arg Cys His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
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Ala Arg Asp Val Glu Arg Ile Ser Asn Thr Lys Asn Asn Leu Tyr Glu
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Val Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Glu Pro
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Glu Tyr Ile Lys Ile Val Thr Glu His Leu Val Lys Leu Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Lys His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ala Ala Leu Lys Ile Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Ile Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Ile Pro Gly Leu Val Ala Pro Val Met Pro Glu Val Val Asn Gln Val
325 330 335
Ala Phe Gln Val Ile Gly Asn Asp Leu Thr Ile Ser Ala Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Asn Asn Val Phe Lys Thr Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Gln Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Arg Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Asp Leu Cys Ile Lys Tyr Asp Val Leu Thr Glu Glu
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Thr His Pro Gly Ile
450 455 460
Ala Gly Lys His
465
<210> 2
<211> 468
<212> PRT
<213> Synthesis of the product
<400> 2
Met Glu Lys Asn Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu
1 5 10 15
Ile Pro Ile Asp Ala Tyr Tyr Gly Val Gln Thr Met Arg Ala Thr Glu
20 25 30
Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser
35 40 45
Leu Gly Ile Val Lys Lys Ala Ala Ala Leu Ala Asn Met Glu Val Gly
50 55 60
Leu Leu Asp Lys Thr Ile Gly Glu Tyr Ile Val Lys Ala Ala Asp Glu
65 70 75 80
Val Ile Glu Gly Lys Trp Asp Asp Gln Phe Ile Val Asp Pro Ile Gln
85 90 95
Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala
100 105 110
Asn Arg Ala Leu Glu Leu Met Gly Ala Glu Lys Gly Asn Tyr Ser Leu
115 120 125
Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Asn Asp Ala
130 135 140
Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu
145 150 155 160
Ile Asp Thr Thr Lys Thr Met Gln Gln Val Phe Leu Asn Lys Ala Asp
165 170 175
Glu Phe Ala Gly Ile Ile Lys Met Gly Arg Cys His Leu Gln Asp Ala
180 185 190
Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile
195 200 205
Ala Arg Asp Val Glu Arg Ile Ser Asn Thr Lys Asn Asn Leu Tyr Glu
210 215 220
Val Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Glu Pro
225 230 235 240
Glu Tyr Ile Lys Ile Val Thr Glu His Leu Val Lys Leu Ser Gly His
245 250 255
Pro Leu Arg Ser Ala Lys His Leu Val Asp Ala Thr Gln Asn Thr Asp
260 265 270
Cys Tyr Thr Glu Val Ser Ala Ala Leu Lys Ile Cys Met Ile Asn Met
275 280 285
Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala
290 295 300
Gly Leu Ser Glu Ile Ile Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
305 310 315 320
Ile Pro Gly Leu Val Ala Pro Val Met Pro Glu Val Val Asn Gln Val
325 330 335
Ala Phe Gln Val Ile Gly Asn Asp Leu Thr Ile Ser Ala Ala Ser Glu
340 345 350
Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn
355 360 365
Leu Ile Gln Ser Ile Ser Ile Met Asn Asn Val Phe Lys Thr Phe Thr
370 375 380
Glu Asn Cys Leu Lys Gly Ile Gln Ala Asn Glu Glu Arg Met Lys Glu
385 390 395 400
Tyr Val Glu Arg Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
405 410 415
Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
420 425 430
Glu Ser Ile Arg Asp Leu Cys Ile Lys Tyr Asp Val Leu Thr Glu Lys
435 440 445
Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Thr His Pro Gly Ile
450 455 460
Ala Gly Lys His
465

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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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1557944A (en) * 1978-01-30 1979-12-19 Sumitomo Chemical Co Enzyme immobilization carrier and preparation thereof
JPS62155089A (en) * 1985-12-27 1987-07-10 Miyoshi Oil & Fat Co Ltd Immobilized enzyme
CN109385415A (en) * 2017-08-04 2019-02-26 秦皇岛华恒生物工程有限公司 Aspartic acid enzyme variants and the preparation method and application thereof
CN112195171A (en) * 2019-07-08 2021-01-08 秦皇岛华恒生物工程有限公司 Method for preparing beta-alanine by using immobilized enzyme

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1557944A (en) * 1978-01-30 1979-12-19 Sumitomo Chemical Co Enzyme immobilization carrier and preparation thereof
JPS62155089A (en) * 1985-12-27 1987-07-10 Miyoshi Oil & Fat Co Ltd Immobilized enzyme
CN109385415A (en) * 2017-08-04 2019-02-26 秦皇岛华恒生物工程有限公司 Aspartic acid enzyme variants and the preparation method and application thereof
CN112195171A (en) * 2019-07-08 2021-01-08 秦皇岛华恒生物工程有限公司 Method for preparing beta-alanine by using immobilized enzyme

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Co-immobilised aspartase and transaminase for high-yield synthesis of l-phenylalanine;Max Cárdenas-Fernández 等;Biochemical Engineering Journal;第93卷;第174页右栏2.3,第176-177页,Fig.1 *
Converting Aspartase into a β-Amino Acid Lyase by Cluster Screening;Andreas Vogel 等;ChemCatChem;第6卷;摘要,第966-967页,表1 *
Max Cárdenas-Fernández 等.Co-immobilised aspartase and transaminase for high-yield synthesis of l-phenylalanine.Biochemical Engineering Journal.2014,第93卷第174页右栏2.3,第176-177页,Fig.1. *

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