CN114540333A - Immobilized modified aspartase and application thereof - Google Patents

Immobilized modified aspartase and application thereof Download PDF

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CN114540333A
CN114540333A CN202011339653.4A CN202011339653A CN114540333A CN 114540333 A CN114540333 A CN 114540333A CN 202011339653 A CN202011339653 A CN 202011339653A CN 114540333 A CN114540333 A CN 114540333A
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ala
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immobilized enzyme
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CN114540333B (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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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    • 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
    • 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
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract

The invention provides immobilized modified aspartase and application thereof in catalytic synthesis of beta-amino acid. Compared with free enzyme, the immobilized enzyme improves the purity of the product, and reduces the use cost of the enzyme and the purification cost of subsequent reaction.

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 application of immobilized modified aspartate enzyme to catalyze and synthesize beta-amino acid.
Background
The structural general formula of the beta-amino acid is shown as follows:
Figure BDA0002798248000000011
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 Lutelvir (Dolutegravir), and beta-alanine can be used as a key intermediate for synthesizing calcium pantothenate.
(R) -3-aminobutyric acid has the following structural formula:
Figure BDA0002798248000000012
the structural formula of beta-alanine is shown as follows:
Figure BDA0002798248000000013
at present, (R) -3-aminobutyric acid is mainly prepared by organic synthesis, and the method has the defects of multiple steps, low yield, poor stereoselectivity and the like.
Some attempts have been made by biologists to prepare such compounds, for example, Andrea Vogel et al (Andrea Vogel et al, Converting aspartic enzyme in a b-Amino Acid Lyase by Cluster Screening, ChemCatchem,6,965-968(2014)) have modified Aspartase, which originally cannot catalyze such reactions, to have the ability to synthesize (R) -3-aminobutyric Acid, and the reaction formula is shown below:
Figure BDA0002798248000000021
however, the free enzyme used in the reaction can bring the product into residual host protein and nucleic acid, which affects the purity of the product, and the enzyme cannot be recycled, which causes waste. Accordingly, there remains a need in the art to provide enzymatic catalysts that can be used to synthesize 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 catalytically 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-containing resin or an activated amino group-containing resin, and the activating agent is preferably a glutaraldehyde solution.
Further, the concentration of the glutaraldehyde solution is 1% to 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 load firmness of the mutated aspartase on the resin are greatly improved;
further, the immobilized enzyme catalyst still maintains more than 90% of reactivity after 30 times of reaction;
further, the immobilized enzyme catalyst still maintains more than 95% of reactivity after reacting for 30 times;
further, the immobilized enzyme catalyst still maintains more than 99% of reactivity after 30 times of reaction;
further, the mutation occurs at amino acid position 448 of aspartase;
further, the mutant is E448K, and the mutated aspartase has an 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 process for the synthesis of a β -amino acid, characterized in that an immobilized enzyme catalyst as described above is used, the reaction steps being as follows:
Figure BDA0002798248000000031
wherein R is selected from: hydrogen radicals, alkyl radicals, alkoxy radicals, alkylsulfonyl radicals, alkylsulfinyl radicals, alkylthio radicals, sulfonic acid radicals, sulfinic acid radicals, mercapto radicals, nitro radicals and halogens;
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" refers to 1 to 4 carbon atoms which may be straight or branched in the chain.
"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" means alkyl-SO2-a group wherein alkyl is as defined above. Exemplary groups are those in which alkyl is lower alkyl.
"Alkylsulfinyl" refers to an alkyl-SO-group, wherein alkyl is as defined above. Exemplary groups are those in which alkyl is lower alkyl.
"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 fixed on the resin, so that host nucleic acid and protein are prevented from remaining in the reaction solution, the product purity is improved, and the production cost is reduced.
Detailed Description
The present invention will be described in further detail with reference to specific 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 from blue dawn technologies;
DNA Polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa;
the plasmid extraction kit is purchased from Axygen corporation;
acrylic acid was purchased from alatin, cat # a103526, purity 99%;
crotonic acid was purchased from alatin under cat # C104149 with 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(DE3) purchased from Tiangen Biochemical technology, Inc. (Beijing).
iii) sequencing, primer synthesis and Gene Synthesis were performed by Suzhou Hongxin Biotechnology Ltd. Wherein the gene is constructed into a vector pET-30a after being synthesized.
iv) site-directed mutagenesis:
specific primer pairs are designed to introduce the desired substitution at the corresponding base at the amino acid position of the desired mutation. Mutants were prepared using the extracted pre-mutation plasmid (containing the wild-type aspartase (derived from Ureibacillus thermophilus) coding sequence, pET-30a (+) backbone) as a template using the method described by Packer and Liu (Methods for the directed evolution of proteins. nat. Rev Genet,2015,16(7): 379-394).
The mutagenesis strategy for site-directed mutagenesis is shown in Table 1:
Figure BDA0002798248000000051
TABLE 1
Note: the mutational modification of SEQ ID No.1 was carried out according to the teachings of Andrea Vogel et al (Andrea Vogel et al, Converting Aspartase inta b-Amino Acid Lyase by Cluster Screening, ChemCatchem,6,965-968(2014)) as described in the background, and the enzyme of this sequence was made to have the ability to synthesize (R) -3-aminobutyric Acid.
v) protein expression and preparation of enzyme solution:
escherichia 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, pH7.0) 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, potassium dihydrogenphosphate 2.31g/L, dipotassium hydrogenphosphate 12.54g/L), incubated at 37 ℃ with shaking until OD600 reached 0.6-0.8, and incubated overnight at 30 ℃ with the addition of IPTG (final concentration of 0.4mM) to induce protein expression.
After incubation, the culture was centrifuged at 4,000g at 4 ℃ for 10min, and the supernatant was discarded to collect E.coli cells. The collected E.coli cells were resuspended in pre-cooled 20mL Phosphate Buffered Saline (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 resulting supernatant was used as an enzyme solution containing the recombinant enzyme for catalytic reaction. Or freeze drying the enzyme solution to obtain enzyme powder, and storing at 4 deg.C.
vi) immobilized enzyme preparation
Amino resin:
the resin LX-1000NH was previously activated overnight with a 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 18 h. After the fixation is finished, the immobilized enzyme is filtered, washed with water for three times and stored at 4 ℃ for later use.
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 18 h. After the fixation is finished, the immobilized enzyme is filtered, washed with water for three times and stored at 4 ℃ for later use.
The reaction products are shown in Table 2:
numbering of immobilized enzymes Resin composition Enzyme sequences
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: crotonic acid 100g/L, adjusting pH to 8.5 with ammonia water, heating to 30 ℃, magnetically stirring uniformly, adding 30g of immobilized enzyme prepared in example 1, starting stirring reaction, reacting for 24 hours, sampling by HPLC to detect conversion rate, and the first and second 30 th conversion results are shown in Table 3.
Numbering of immobilized enzymes First conversion (%) Conversion (%). 30 th time of use
Immob-NH-AAL1 99 92
Immob-HFA-AAL1 97 78
Immob-NH-AAL2 99 99
Immob-HFA-AAL2 97 93
TABLE 3
Example 3: preparation of beta-alanine by immobilized enzyme
Preparing a 1L reaction system: acrylic acid 200g/L, ammonia water to adjust pH to 9.0, heating to 30 ℃, magnetic stirring evenly, adding immobilized enzyme 20g, starting stirring reaction, reacting for 24 hours, sampling and detecting conversion rate by HPLC, and the first and second 30 times conversion results are shown in Table 4.
Numbering of immobilized enzymes First conversion (%) Conversion (%). 30 th time of use
Immob-NH-AAL1 99 95
Immob-HFA-AAL1 99 91
Immob-NH-AAL2 99 99
Immob-HFA-AAL2 99 99
TABLE 4
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Sequence listing
<110> Hunan piloting Biotechnology Ltd
<120> immobilized modified aspartase and use thereof
<130> 2020
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Glu Tyr Ile Lys Ile Val Thr Glu His Leu Val Lys Leu Ser Gly His
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Gly Leu Ser Glu Ile Ile Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile
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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
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Tyr Val Glu Arg Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val
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Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly
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Glu Ser Ile Arg Asp Leu Cys Ile Lys Tyr Asp Val Leu Thr Glu Lys
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Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Thr His Pro Gly Ile
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Ala Gly Lys His
465

Claims (10)

1. An immobilized enzyme catalyst comprising a resin and an enzyme covalently bound to said resin; the enzyme is aspartase.
2. The immobilized enzyme catalyst according to claim 1, wherein the resin is an epoxy-group-containing resin or an activated amino-group-containing resin.
3. The immobilized enzyme catalyst of claim 1, wherein the aspartase is site-directed mutated.
4. The immobilized enzyme catalyst of claim 1, wherein the site-directed mutation occurs at amino acid position 448 of threonine transaldolase.
5. The immobilized enzyme catalyst of claim 1, wherein the immobilized enzyme catalyst retains 90% or more of reactivity after 30 reactions.
6. The immobilized enzyme catalyst of claim 5, which retains a reactivity of 95% or more after 30 reactions.
7. The immobilized enzyme catalyst according to any one of claims 1 to 6, wherein the immobilized enzyme catalyst retains a reactivity of 99% or more after 30 times of reactions.
8. The immobilized enzyme catalyst according to claim 5, wherein the mutant is E448K, and the mutated aspartase has the amino acid sequence of SEQ ID No. 2.
9. The immobilized enzyme catalyst according to any one of claims 1 to 6, wherein the enzyme to resin mass ratio is from 1:15 to 1:8
10. A process for the synthesis of β -amino acids, characterized in that an immobilized enzyme catalyst as described above is used, the reaction steps being as follows:
Figure FDA0002798247990000021
wherein R is selected from: hydrogen radicals, alkyl radicals, alkoxy radicals, alkylsulfonyl radicals, alkylsulfinyl radicals, alkylthio radicals, sulfonic acid radicals, sulfinic acid radicals, mercapto radicals, nitro radicals and halogens.
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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 (2)

* Cited by examiner, † Cited by third party
Title
ANDREAS VOGEL 等: "Converting Aspartase into a β-Amino Acid Lyase by Cluster Screening", CHEMCATCHEM, vol. 6, pages 966 - 967 *
MAX CÁRDENAS-FERNÁNDEZ 等: "Co-immobilised aspartase and transaminase for high-yield synthesis of l-phenylalanine", BIOCHEMICAL ENGINEERING JOURNAL, vol. 93, pages 176 - 177 *

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