CN116334057B - Enzyme assembly constructed by polyethylene glycol maleimide derivative, and preparation method and application thereof - Google Patents
Enzyme assembly constructed by polyethylene glycol maleimide derivative, and preparation method and application thereof Download PDFInfo
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- CN116334057B CN116334057B CN202210982067.4A CN202210982067A CN116334057B CN 116334057 B CN116334057 B CN 116334057B CN 202210982067 A CN202210982067 A CN 202210982067A CN 116334057 B CN116334057 B CN 116334057B
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- glycol maleimide
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 108
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 108
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 74
- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 66
- 125000005439 maleimidyl group Chemical class C1(C=CC(N1*)=O)=O 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 37
- 230000035772 mutation Effects 0.000 claims abstract description 20
- 238000004132 cross linking Methods 0.000 claims abstract description 10
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims abstract description 4
- 238000006467 substitution reaction Methods 0.000 claims abstract description 4
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 claims description 44
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical group SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 44
- 150000003923 2,5-pyrrolediones Chemical class 0.000 claims description 40
- 238000006243 chemical reaction Methods 0.000 claims description 36
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 claims description 33
- 235000018417 cysteine Nutrition 0.000 claims description 33
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 claims description 33
- 229960004949 glycyrrhizic acid Drugs 0.000 claims description 33
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 claims description 33
- 239000001685 glycyrrhizic acid Substances 0.000 claims description 33
- 235000019410 glycyrrhizin Nutrition 0.000 claims description 33
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 claims description 33
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 32
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 claims description 22
- 229960003720 enoxolone Drugs 0.000 claims description 22
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 16
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims description 16
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 9
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- 125000003827 glycol group Chemical group 0.000 claims description 9
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- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 6
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 3
- 239000004473 Threonine Substances 0.000 claims description 3
- 235000003704 aspartic acid Nutrition 0.000 claims description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 3
- 125000005647 linker group Chemical group 0.000 claims description 3
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims 3
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims 1
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- 241000588724 Escherichia coli Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
- 229920003192 poly(bis maleimide) Polymers 0.000 description 1
- 108010094020 polyglycine Proteins 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01031—Beta-glucuronidase (3.2.1.31)
-
- 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
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Engineering & Computer Science (AREA)
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- Wood Science & Technology (AREA)
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- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses an enzyme assembly constructed by polyethylene glycol maleimide derivative, a preparation method and application thereof, wherein the method comprises the following steps: crosslinking polyethylene glycol maleimide derivative with enzyme to obtain enzyme assembly; wherein: the enzyme consists of SEQ ID NO:1 by a single mutation of the amino acid sequence shown in (a) by substitution with a cysteine residue. The invention also relates to an enzyme assembly constructed by the method and application thereof. The invention realizes the assembly of protein at specific sites by a simple and efficient method, and successfully prepares the enzyme assembly with controllable relative spatial positioning. The enzyme assembly formed by the method can further improve the thermal stability of the enzyme on the basis of not affecting the activity of the enzyme basically.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to the field of enzyme assembly, and more particularly relates to an enzyme assembly constructed by using polyethylene glycol maleimide derivatives, and a preparation method and application thereof.
Background
In the related art, methods for improving enzyme stability are as follows: cross-linked enzyme aggregation technology or active inclusion body technology; the cross-linking enzyme aggregate technology is a carrier-free immobilization method in which enzyme protein is precipitated by using a precipitant such as salt, an organic solvent or nonionic polymer to obtain an enzyme aggregate, and then cross-linking is performed by using a cross-linking agent. Active inclusion body technology refers to the fusion expression of hydrophobic or amphiphilic polypeptides at the sequence ends of enzyme molecules, wherein nonspecific actions between polypeptides lead to the formation of active aggregates, i.e. active inclusion bodies, of the enzyme during expression. Both of the above techniques can improve the stability of the enzyme, but because they merely aggregate the enzyme molecules together in a disordered manner, the overall activity of the enzyme aggregate is reduced.
Therefore, how to accurately design the enzyme assembly, so that the enzyme assembly can improve the stability of the enzyme on the basis of not influencing the catalytic activity of the enzyme, and has great application value. At present, many design strategies related to protein assemblies have been developed, and acting force for driving protein assembly mainly comprises biological methods such as isopeptidic bond formed by SpyTag-SpyCatcher, connection of N-terminal polyglycine and C-terminal LPXTG label catalyzed by sortase, splicing reaction catalyzed by split intein, domain exchange and the like, however, the biological methods not only require a large amount of genetic manipulation, but also have the problems of low protein assembly rate, low assembly efficiency, incapacity of controlling relative spatial positioning of proteins in the assemblies and the like. In addition, chemical methods of interaction of a host and a guest, glutaraldehyde crosslinking and metal coordination are introduced, however, the chemical methods have the defects of low specificity of non-covalent interaction, low assembly efficiency, complex separation and purification and the like, and the practical application of enzyme assembly is hindered.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent.
To this end, embodiments of the present invention provide a method of driving enzyme assembly with polyethylene glycol maleimide derivatives, the method comprising: crosslinking polyethylene glycol maleimide derivative with enzyme to obtain enzyme assembly; wherein:
the enzyme consists of SEQ ID NO:1, wherein the single mutation is a substitution with a cysteine residue;
the polyethylene glycol maleimide derivative contains at least two maleimide terminal groups.
The embodiment of the invention provides a method for driving self-assembly of Aspergillus oryzae-derived beta-glucuronidase (PGUS) (the amino acid sequence of which is shown as SEQ ID NO: 1) by using a polyethylene glycol maleimide derivative. The click addition reaction (sulfhydryl-maleimide addition) of the mutation-introduced cysteine and the polyethylene glycol maleimide derivative realizes the assembly of the protein at a specific site by a simple and efficient method, and successfully prepares the enzyme assembly with controllable relative spatial positioning. The enzyme assembly formed by the method can further improve the thermal stability of the enzyme on the basis of not affecting the activity of the enzyme basically.
Preferably, the polyethylene glycol maleimide derivative contains two or four maleimide end groups.
The structural formula of the polyethylene glycol maleimide derivative is as follows: PEG-X-MAL; wherein,
PEG is polyethylene glycol residue, MAL is maleimide group;
X is a linking group of PEG and MAL, selected from one or more of ：-(CH2)r-、-(CH2)rO-、-(CH2)rCO-、-(CH2)rNH-、-(CH2)rCONH-、-(CH2)rNHCO-、-(CH2)rS-、-(CH2)rCOO- and- (CH 2)r OCO) -and r is an integer of 0 to 10, preferably r is an integer of 0 to 5.
In some embodiments, the PEG is one of a linear polyethylene glycol residue, a four-arm branched polyethylene glycol residue, a six-arm branched polyethylene glycol residue.
In some embodiments, the single mutation is any one of the following:
(1) Asparagine (Asn) at position 28 to cysteine (Cys);
(2) Threonine (Thr) at position 34 is mutated to cysteine (Cys);
(3) Asparagine (Asn) at position 100 is mutated to cysteine (Cys);
(4) Glutamine (gin) at position 180 is mutated to cysteine (Cys);
(5) Glycine (Gly) at position 194 is mutated to cysteine (Cys);
(6) Aspartic acid (Asp) at position 220 to cysteine (Cys);
preferably, the single mutation is any one of the following:
mutation of asparagine (Asn) at position 28 to cysteine (Cys), or
Glycine (Gly) at position 194 is mutated to cysteine (Cys), or
Glutamine (Gln) at position 180 is mutated to cysteine (Cys).
More preferably, the single mutation is a glycine (Gly) to cysteine (Cys) mutation at position 194.
In some embodiments, the polyethylene glycol maleimide derivative is a two-arm polyethylene glycol maleimide (Mal 2), or a four-arm polyethylene glycol maleimide (Mal 4). Preferably, it is: compared with the double-arm polyethylene glycol maleimide, the four-arm polyethylene glycol maleimide can further improve the enzyme self-assembly efficiency.
In some embodiments, the polyethylene glycol maleimide derivative has a molecular weight of 2000-3400Da.
In some embodiments, the polyethylene glycol maleimide derivative to enzyme molar ratio is 1 (1.5-2.5), preferably a molar ratio of 1:2.
In some embodiments, the conditions of crosslinking are: the pH value is 6.8-7.4, the reaction temperature is 0-10 ℃, and the reaction time is 2-12 hours.
The embodiment of the invention also provides an enzyme assembly prepared by the method. The enzyme assembly prepared by the embodiment of the invention has the advantages of high catalytic activity, high thermal stability, mild reaction conditions, high efficiency, green and safe performance and the like.
The embodiment of the invention also provides application of the enzyme assembly in converting glycyrrhizic acid into glycyrrhetinic acid.
The invention has the following advantages and beneficial effects:
(1) The invention uses polyethylene glycol maleimide cross-linking agent to drive the self-assembly of Aspergillus oryzae-derived beta-glucuronidase (PGUS) (the amino acid sequence of which is shown as SEQ ID NO: 1). The click addition reaction (sulfhydryl-maleimide addition) of the mutation-introduced cysteine and the polyethylene glycol maleimide derivative (cross-linking agent) realizes the assembly of the protein at a specific site by a simple and efficient method, and successfully prepares the enzyme assembly with controllable relative spatial positioning. The enzyme assembly formed by the method can further improve the thermal stability of the enzyme on the basis of not affecting the activity of the enzyme basically.
(2) The enzyme assembly provided by the invention has the advantages of rapid reaction, quantitative conversion of sulfhydryl-maleimide reaction as driving force, simplicity in operation, high assembly efficiency and easiness in expanded production.
(3) The beta-glucuronidase assembly has the advantages of high catalytic activity, high thermal stability, mild reaction conditions, high efficiency, green safety and the like.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows the feasibility of assembling N28C, T, C, N, 100, C, Q, C, G, 194, C, D, 220C by double arm crosslinker Mal 2 by Native-PAGE.
Fig. 2 is a Native-PAGE verification of the feasibility of assembling N28C, T, C, N100C, Q180C, G194C, D220C by four-arm crosslinker Mal 4.
FIG. 3 shows the degree and efficiency of assembly of the gel filtration chromatograms comparing G194C-Mal 2、G194C-Mal4 and PGUS.
FIG. 4 is a TEM image of PGUS free enzymes.
FIG. 5 is a TEM image of G194C-Mal 2 assembly enzymes.
FIG. 6 is a TEM image of G194C-Mal 4 assembly enzymes.
FIG. 7 shows the relative activities of mutant N28C, T, C, N, C, Q, 180, C, G, 194, C, D, 220C and corresponding assemblies.
Fig. 8 shows the thermal stability of the assembly N28C-Mal2、T34C-Mal2、N100C-Mal2、Q180C-Mal2、G194C-Mal2、D220C-Mal2 at 70 ℃.
Fig. 9 is a graph showing the thermal stability of the assembly N28C-Mal4、T34C-Mal4、N100C-Mal4、Q180C-Mal4、G194C-Mal4、D220C-Mal4 at 70 ℃.
FIG. 10 shows the change in glycyrrhetinic acid concentration of assemblies G194C-Mal 2、G194C-Mal4 and PGUS for catalyzing the conversion of glycyrrhizic acid at different temperatures.
FIG. 11 shows the conversion of glycyrrhizic acid by assemblies G194C-Mal 2、G194C-Mal4 and PGUS at different temperatures.
FIG. 12 shows the variation in glycyrrhetinic acid yield of assemblies G194C-Mal 2、G194C-Mal4 and PGUS for catalyzing the conversion of glycyrrhizic acid at various temperatures.
Detailed Description
The following detailed description of embodiments of the invention is exemplary and intended to be illustrative of the invention and not to be construed as limiting the invention. The specific conditions were not specified in the examples and the procedure was carried out under conventional conditions.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
The materials, reagents, devices and the like used in the following examples, unless otherwise specified, were prepared commercially or according to the methods of the publications.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The embodiment of the invention provides a method for driving enzyme assembly by using polyethylene glycol maleimide derivatives, which comprises the following steps: crosslinking polyethylene glycol maleimide derivative with enzyme to obtain enzyme assembly; wherein:
the enzyme consists of SEQ ID NO:1, wherein the single mutation is a substitution with a cysteine residue;
the polyethylene glycol maleimide derivative contains at least two maleimide terminal groups.
The embodiment of the invention provides a method for driving self-assembly of Aspergillus oryzae-derived beta-glucuronidase (PGUS) (the amino acid sequence of which is shown as SEQ ID NO: 1) by using a polyethylene glycol maleimide derivative. The click addition reaction (sulfhydryl-maleimide addition) of the mutation-introduced cysteine and the polyethylene glycol maleimide derivative realizes the assembly of the protein at a specific site by a simple and efficient method, and successfully prepares the enzyme assembly with controllable relative spatial positioning. The enzyme assembly formed by the method can further improve the thermal stability of the enzyme on the basis of not affecting the activity of the enzyme basically.
Preferably, the polyethylene glycol maleimide derivative contains two or four maleimide end groups.
The structural formula of the polyethylene glycol maleimide derivative is as follows: PEG-X-MAL; wherein,
PEG is polyethylene glycol residue, MAL is maleimide group;
X is a PEG-MAL linking group selected from one or a combination of more than two of ：-(CH2)r-、-(CH2)rO-、-(CH2)rCO-、-(CH2)rNH-、-(CH2)rCONH-、-(CH2)rNHCO-、-(CH2)rS-、-(CH2)rCOO- and- (CH 2)r OCO) -and r is an integer of 0 to 10.
By way of non-limiting example, the X is selected from: one or a combination of two or more single bonds 、-CH2-、-CH2CH2-、-CH2CH2CH2-、-CH2O-、-CH2CH2O-、-CO-、-CH2CO-、-CH2CH2CO-、-CH2CH2CH2CO-、-NH-、-CH2NH-、-CH2CH2NH-、-CH2CH2CH2NH-、-CONH-、-CH2CONH-、-CH2CH2CONH-、-CH2CH2CH2CONH-、-CH2S-、-CH2CH2S-、-COO-、-CH2COO-、-CH2CH2COO-、-NHCO-、-CH2NHCO-、-CH2CH2NHCO-、-CH2OCO-、-CH2CH2OCO-、-CH2CH2CH2OCO-.
In some embodiments, the PEG is one of a linear polyethylene glycol residue, a four-arm branched polyethylene glycol residue, a six-arm branched polyethylene glycol residue.
In some embodiments, the single mutation is any one of the following:
(1) Asparagine (Asn) at position 28 to cysteine (Cys);
(2) Threonine (Thr) at position 34 is mutated to cysteine (Cys);
(3) Asparagine (Asn) at position 100 is mutated to cysteine (Cys);
(4) Glutamine (gin) at position 180 is mutated to cysteine (Cys);
(5) Glycine (Gly) at position 194 is mutated to cysteine (Cys);
(6) Aspartic acid (Asp) at position 220 to cysteine (Cys);
preferably, the single mutation is any one of the following:
mutation of asparagine (Asn) at position 28 to cysteine (Cys), or
Glycine (Gly) at position 194 is mutated to cysteine (Cys), or
Glutamine (Gln) at position 180 is mutated to cysteine (Cys).
More preferably, the single mutation is a glycine (Gly) to cysteine (Cys) mutation at position 194.
In some embodiments, the polyethylene glycol maleimide derivative is a two-arm polyethylene glycol maleimide (Mal 2), or a four-arm polyethylene glycol maleimide (Mal 4). Preferably, it is: compared with the double-arm polyethylene glycol maleimide, the four-arm polyethylene glycol maleimide can further improve the enzyme self-assembly efficiency.
In some embodiments, the polyethylene glycol maleimide derivative has a molecular weight of 2000-3400Da.
In some embodiments, the polyethylene glycol maleimide derivative to enzyme molar ratio is 1 (1.5-2.5), such as, by way of non-limiting example, a molar ratio of 1:1.5, 1:1.7, 1:1.8, 1:2, 1:2.2, 1:2.5. Preferably, the molar ratio is 1:2.
In some embodiments, the conditions of crosslinking are: the pH value is 6.8-7.4, the reaction temperature is 0-10 ℃, and the reaction time is 2-12 hours.
The embodiment of the invention also provides an enzyme assembly prepared by the method. The enzyme assembly prepared by the embodiment of the invention has the advantages of high catalytic activity, high thermal stability, mild reaction conditions, high efficiency, green and safe performance and the like.
The embodiment of the invention also provides application of the enzyme assembly in converting glycyrrhizic acid into glycyrrhetinic acid.
Embodiments of the present invention are described in further detail below.
Materials: primer synthesis: the primers used in the examples of the present invention were all prepared synthetically by Jin Weizhi Biotechnology Co. FastPfu high-fidelity polymerase was purchased from holo gold company; restriction enzymes were purchased from Thermo Fisher company; the plasmid miniprep kit and the yeast genome kit used were purchased from Tiangen corporation.
In the specific embodiment of the invention, the double-arm polyethylene glycol maleimide (Mal 2) is purchased from major biosystems, bismaleimide PEG, product number PS2-M-2K, and the molecular weight is 2K.
In the specific embodiment of the invention, four-arm polyethylene glycol maleimide (Mal 4) is purchased from major biosystems, four-arm-PEG-maleimide, cat# PS4-M-2K, and has a molecular weight of 2K.
Example 1 plasmid construction
The vector is pET28a (+) -PGUS stored in a laboratory, and the amino acid sequence of the PGUS is shown as SEQ ID NO:1 (gene PGUS: genbank accession number EU 095019), 28, 34, 100, 180, 194, 220 were mutated into cysteines to construct single point mutant N28C, T34C, N100C, Q180C, G194C, D220C, respectively, and primers for the mutations are shown in Table 1. The vector was sequenced by the airlida company using Bomaide TOP10 competent enrichment plasmid and Bomaide BL21 competent as expression host.
The amplification system is as follows: fastPfu Buffer (5X) 10.0 μ L, dNTP (2.5 mM) 4 μ L, pET28a (+) -PGUS template 1 μL, upstream and downstream primers (5 μM) 1 μ L, fastPfu each high fidelity DNA polymerase 1 μL, supplemented with double distilled water to 50 μL. The amplification conditions were 95℃for 3 min of pre-denaturation; denaturation at 95℃for 20 sec, annealing at 60℃for 20 sec, extension at 72℃for 3 min (30 cycles); extension was carried out at 72℃for 10 minutes.
Restriction endonuclease Dpn I digests the methylation template: 5. Mu.L of the point mutation amplification product, 10x digest buffer1. Mu.L of restriction endonuclease Dpn I1. Mu.L, and a 10. Mu.L cleavage system was supplemented with double distilled water under the conditions of 37℃for 1 hour, followed by reaction at 80℃for 20 minutes to inactivate the enzyme.
The digested product was transformed into E.coli TOP 10 competent cells by heat shock, plated on solid LB medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agarose 20 g/L) containing 50mg/L kanamycin, and cultured at 37℃for about 16 hours.
Transformants were identified by colony PCR and sequencing methods. Colony PCR system: template LB plates were single-colony, 1. Mu.L each of T7 promoter and terminator universal primer, 10. Mu.L of 2 XTaq mix (America Biolabs.), and 20. Mu.L of double distilled water was made up. PCR conditions: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 1min for 30s, circulation for 30 times, 10min at 72℃and preservation at 4 ℃. The transformants containing the target bands were confirmed by colony PCR to be subjected to DNA sequencing, and the success of plasmid construction was confirmed.
TABLE 1 primers for mutation
| Primer | Sequence(5’--3’) |
| N28C-F | ATCCGACGACTGCAATACGCAACCATGGACAAG |
| N28C-R | GTCGTCGGATGCTAGGGCAAATTTC |
| T34C-F | GCAACCATGGTGCAGCCAACTAAAAACGTCCCTG |
| T34C-R | CCATGGTTGCGTATTGTTGTCG |
| N100C-F | CGTCAATGGATGCCTGGTCGCGGACCATGTG |
| N100C-R | TCCATTGACGTAGATCCGGCC |
| Q180C-F | ATTCTGTGCCATGCCAGCACATTCAGGATATCACTGTTCG |
| Q180C-R | TGGCACAGAATACAGCCACACC |
| G194C-F | GGATGTGCAGTGCACCACCGGGCTG |
| G194C-R | CTGCACATCCGTCCGAACAGTG |
| D220C-F | GATAGATGAGTGTGGCACAACCGTAGCGACAAG |
| D220C-R | CTCATCTATCACGGCAACCTG |
PGUS the amino acid sequence is as follows:
MLKPQQTTTRDLISLDGLWKFALASDDNNTQPWTSQLKTSLECPVPASYNDIFADSKIHDHVGWVYYQRDVIVPKGWSEERYLVRCEAATHHGRIYVNGNLVADHVGGYTPFEADITDLVAAGEQFRLTIAVDNELTYQTIPPGKVEILEATGKKVQTYQHDFYNYAGLARSVWLYSVPQQHIQDITVRTDVQGTTGLIDYNVVASTTQGTIQVAVIDEDGTTVATSSGSNGTIHIPSVHLWQPGAAYLYQLHASIIDSSKKTIDTYKLATGIRTVKVQGTQFLINDKPFYFTGFGKHEDTNIRGKGHDDAYMVHDFQLLHWMGANSFRTSHYPYAEEVMEYADRQGIVVIDETPAVGLAFSIGAGAQTSNPPATFSPDRINNKTREAHAQAIRELIHRDKNHPSVVMWSIANEPASNEDGAREYFAPLPKLARQLDPTRPVTFANVGLATYKADRIADLFDVLCLNRYFGWYTQTAELDEAEAALEEELRGWTEKYDKPIVMTEYGADTVAGLHSVMVTPWSEEFQVEMLDMYHRVFDRFEAMAGEQVWNFADFQTAVGVSRVDGNKKGVFTRDRKPKAAAHLLRKRWTNLHNGTAEGGKTFQ
(SEQ ID NO：1)
EXAMPLE 2 protein expression and purification
400ML of LB medium was added with 50. Mu.g/mL kanamycin at a final concentration, 1% of inoculation amount, cultured at 37℃for 5h until OD600 = 0.8, added with IPTG at a final concentration of 1mM, and induced overnight at 16℃for 16h. The cells were collected by centrifugation at 9000rpm, resuspended in 50mM PBS (pH 7.0,0.15M NaCl), broken at 1200bar under low temperature and ultra high pressure, the supernatant and the pellet were separated at 13000rpm, the pellet was discarded, and the supernatant was subjected to a 0.45 μm water film. Purified by AKTA system and HISTRAP HP affinity chromatography column, the loading buffer was 50mM PBS (pH 7.0,0.15M NaCl), the elution buffer was 50mM PBS (pH 7.0,0.15M NaCl,200mM imidazole), the target component protein was collected, and desalted by Millipore (30 kDa) ultrafiltration concentration tube.
Example 3 verification of Assembly and Structure
Cross-linking agent Mal 2、Mal4 was dissolved in sterile deionized water to give a final concentration of 20mM stock solution, and diluted to 10mM and 1mM for ready use.
Mal 2、Mal4 was mixed with the protein (mutant) in a molar ratio of 1:2 according to Nanodrop measurement of protein concentration, the protein being in excess such that assembly between the cross-linker and the protein was as much as possible.
Reaction conditions: 50mM PBS buffer, pH 7.0, reaction temperature 4℃and reaction time 10 hours.
Constructed to obtain N28C-Mal2、T34C-Mal2、N100C-Mal2、Q180C-Mal2、G194C-Mal2、D220C-Mal2、N28C-Mal4、T34C-Mal4、N100C-Mal4、Q180C-Mal4、G194C-Mal4、D220C-Mal4.
Wherein PGUS (PDB ID:5C 71) was used as a control, PGUS was a homotetrameric structure consisting of two planes of symmetry forming a tetrameric interface of PGUS by the TIM barrel domain. (see the literature for specific structures) ：Bo Lv,et al.Structure-guided engineering of the substrate specificity of a fungalβ-glucuronidase toward triterpenoid saponins[J].Journal of Biological Chemistry.2018;293(2):433-443)
(1) Native-PAGE results (10% concentration of separator and 5% concentration of concentrate) as shown in FIGS. 1 and 2, using PGUS tetramer as a control, bands with slower migration rates than tetramer were observed for all assemblies, and the molecular weights of the bands were heterogeneous, indicating that all mutants were able to cross-link assemble with Mal 2、Mal4.
(2) Gel filtration chromatography: purification and analysis were performed using a Superdex 200Increase 10/300GL (GE Healthcare) column using PBS buffer as eluent for gel filtration chromatography at a flow rate and detection wavelength of 0.65mL/min and 280nm, respectively.
FIG. 3 shows the retention times of the various assembled degree assemblies of PGUS, G194C-Mal 2 and G194C-Mal 4, the retention time of the PGUS tetramer was 12min, two distinct peaks were observed for the G194C-Mal 2 assembly, corresponding to an assembled and unassembled tetramer, respectively, and the retention time of the assembly was about 8.5min. Two distinct peaks were also observed for G194C-Mal 4, with an assembly retention time of about 9min, which was close to that of the G194C-Mal 2 assembly. Assembly efficiency = S a/(Sa+Sf) x 100, where S a represents the peak area of assembled enzyme and S f represents the peak area of unassembled free enzyme. The peak areas of the assembled body and the tetramer are compared to obtain the assembly efficiencies of 13.4% and 71.5% of G194C-Mal 2 and G194C-Mal 4 respectively, so that the use of the four-arm crosslinking agent Mal 4 is beneficial to improving the assembly efficiency.
Fig. 4 is a TEM image of PGUS free enzyme, and as can be seen from fig. 4, PGUS is in a homogeneously distributed free state.
FIG. 5 is a TEM image of the assembled enzyme of G194C-Mal 2, and it can be seen from FIG. 5 that the assembled enzyme of G194C-Mal 2 has a one-dimensional linear structure, which can increase the rigidity of the enzyme structure, avoid activity loss caused by subunit dissociation, and thus improve the thermal stability.
FIG. 6 is a TEM image of G194C-Mal 4 assembly enzymes. As can be seen from fig. 6, G194C-Mal 4 presents a one-dimensional linear and branched mixed structure, and the maleimide group added in the four-arm crosslinking agent Mal 4 can crosslink with a plurality of enzyme molecules, so that the assembly efficiency and the assembly molecular weight are improved, and the structural rigidity and the thermal stability of the assembled enzyme are further improved.
Example 4 enzyme activity assay
The reverse phase high performance chromatography establishes a quantitative determination bioconversion glycyrrhizic acid analysis method. The main parameters of the instrument are as follows: shimadzu LC-10A, chromatographic column: ASC Accurasil (4.6X250 mm,5 μm), detector: SPD-10AVP deuterium lamp detector, detection wavelength: 254nm, mobile phase A is methanol, mobile phase B is acetic acid solution of 6 per mill, and the sample injection amount is as follows: 10 μl, flow rate: 1mL/min, column incubator: 40 ℃, workstation: LCsolution. Beta-glucuronidase activity is defined as: each activity unit consumes 1nmol of glycyrrhizic acid per minute under the above conditions.
40. Mu.g of the assembled enzyme was added to 400. Mu.L of 2g/L glycyrrhizic acid solution prepared in 50mM pH 5.0 acetic acid-sodium acetate buffer. After reacting for 10min at 40 ℃, adding 1000 mu L of methanol, mixing uniformly, filtering by a 0.22 mu m water-based filter membrane, and detecting the content of the substrate glycyrrhizic acid and the product glycyrrhetinic acid by HPLC.
Fig. 7 shows the relative activities of mutant N28C, T34C, N100C, Q180C, G194C, D220C and corresponding assemblies. As can be seen from FIG. 7, the activity of both the mutant and the assembly was substantially improved relative to PGUS, e.g., G194C-Mal 4 was approximately 45% higher than PGUS, thus, it was seen that Mal 2、Mal4 did not affect enzyme activity using the present invention.
Example 5 thermal stability determination
400. Mu.L of the assembly enzyme at a concentration of 1mg/mL was incubated in a metal bath at 70℃and 5. Mu.L of the enzyme was added to 100. Mu.L of 1.25mM pNPG in 50mM pH 5.0 acetic acid-sodium acetate buffer at regular intervals. After 5min of reaction in a metal bath at 40 ℃, 200 μl of 0.4M Na 2CO3 was added to terminate the reaction, and absorbance of pNP at 405nm was detected by an enzyme-labeled instrument. The enzyme activity was measured by the pNPG method, and the residual enzyme activity at different incubation times was calculated with the enzyme activity at time zero as 100% reference, and was determined as the temperature stability of the beta-glucuronidase. 1 enzyme activity unit is defined as the amount of enzyme required to release 1. Mu. Mol of pNP under the above reaction conditions.
Upon incubation at 70 ℃, the residual activity of the assembly enzyme gradually decreased with incubation time, and figure 8 shows the thermal stability of the assembly N28C-Mal2、T34C-Mal2、N100C-Mal2、Q180C-Mal2、G194C-Mal2、D220C-Mal2 at 70 ℃. As can be seen from FIG. 8, the half-life of the free PGUS is 43.2min, after Mal 2 assembly, the enzyme activities of N28C-Mal 2、T34C-Mal2、Q180C-Mal2 and G194C-Mal 2 are almost not lost after heat treatment for more than 1h, and after the heat treatment time is prolonged, the results show that more than 80% of enzyme activities of four assemblies still exist after the heat treatment for 2h, and the half-lives of 155.2min, 133.5min, 169.2min and 163.7min are finally obtained respectively, and the heat stability is improved by 2.6 times, 2.1 times, 2.9 times and 2.8 times compared with the free enzymes respectively. N100C-Mal 2 and D220C-Mal 2 also significantly improved thermal stability, half-lives 2.7 and 2.4 times that of free PGUS, respectively.
Figure 9 shows the thermal stability of the assembly N28C-Mal4、T34C-Mal4、N100C-Mal4、Q180C-Mal4、G194C-Mal4、D220C-Mal4 at 70 ℃. As can be seen from FIG. 9, after Mal 4 is assembled, the enzyme activities of N28C-Mal 4、Q180C-Mal4 and G194C-Mal 4 are hardly lost after heat treatment for more than 2 hours, and the half lives of 158.7min, 198min and 200.8min are finally obtained, so that the heat stability is improved by 2.67 times, 3.58 times and 3.65 times compared with that of free enzyme. T34C-Mal 4、N100C-Mal4 and D220C-Mal 4 also significantly improved thermal stability with half-lives of 2.6, 3.0 and 2.7 times that of free PGUS, respectively.
EXAMPLE 6 hydrolysis Process of catalytic glycyrrhizic acid
Catalytic glycyrrhizic acid conversion at 40 ℃): 70mL of 2g/L glycyrrhizic acid is placed on a heating magnetic stirrer to be heated in a water bath, so that the temperature is kept constant to 40 ℃. 8.51mL of the enzyme solution at a concentration of 2.35mg/mL was aspirated, diluted to 10mL with PBS buffer (50 mM PBS, 150mM NaCl), the whole enzyme solution was added to glycyrrhizic acid, 97. Mu.L was sampled at regular intervals, and the reaction was stopped with 3. Mu.L of 1M NaOH. Adding 0.14g glycyrrhizic acid at 40min, 140min, 240 min. 1mL of methanol was added to the reaction-terminated system, and after shaking, the mixture was subjected to an organic filter membrane of 0.22. Mu.m, and the amounts of GAMG and GA were measured by HPLC.
Catalytic glycyrrhizic acid conversion at 50 ℃): 70mL of 2g/L glycyrrhizic acid is placed on a heating magnetic stirrer to be heated in a water bath, so that the temperature is kept constant to 50 ℃. 8.51mL of the enzyme solution at a concentration of 2.35mg/mL was aspirated, diluted to 10mL with PBS buffer (50 mM PBS, 150mM NaCl), the whole enzyme solution was added to glycyrrhizic acid, 97. Mu.L was sampled at regular intervals, and the reaction was stopped with 3. Mu.L of 1M NaOH. Adding 0.14g glycyrrhizic acid at 40min, 140min, 240 min. 1mL of methanol is added into a reaction terminating system, the mixture is uniformly shaken and then is subjected to sample preparation through a 0.22 mu m organic filter membrane, and the contents of a substrate glycyrrhizic acid and a product glycyrrhetinic acid are detected by HPLC.
Glycyrrhizic acid conversion= (S 0-St)/St ×100, where S 0 represents the total glycyrrhizic acid concentration provided, S t represents the glycyrrhizic acid concentration at time t. Glycyrrhetinic acid yield = S GA/SGL ×100, where S GA and S GL represent the molar concentrations of glycyrrhetinic acid and glycyrrhizic acid, respectively.
FIG. 10 shows the change in glycyrrhetinic acid concentration of assemblies G194C-Mal 2、G194C-Mal4 and PGUS for catalyzing the conversion of glycyrrhizic acid at different temperatures. As can be seen from FIG. 10, at a reaction temperature of 40 ℃, the concentration of PGUS glycyrrhetinic acid hardly increased after 80min, while G194C-Mal 2 and G194C-Mal 4 still showed higher catalytic activity after 140 min. The final concentration of glycyrrhetinic acid obtained by G194C-Mal 2 is 2.24G/L, and the final concentration of glycyrrhetinic acid obtained by G194C-Mal 4 is 1.9G/L. When the reaction temperature is 50 ℃, PGUS has lower stability, the concentration of glycyrrhetinic acid is hardly increased after 10 minutes, and the concentration of the obtained glycyrrhetinic acid is 0.25g/L. And G194C-Mal 4 shows higher catalytic activity and stability at 50 ℃, the yield of glycyrrhetinic acid increases slowly after 80min, and the final concentration of the glycyrrhetinic acid is 1.1G/L.
FIG. 11 shows the conversion of glycyrrhizic acid by assemblies G194C-Mal 2、G194C-Mal4 and PGUS at different temperatures. As can be seen from FIG. 11, PGUS (40 ℃ C.) and G194C-Mal 2(40℃)、G194C-Mal4(40℃)、G194C-Mal4 (50 ℃ C.) were kept substantially uniform in the change of the glycyrrhizic acid conversion rate within 140 minutes from the start, the glycyrrhizic acid conversion rate was kept at about 60% at 10 to 40 minutes, and the glycyrrhizic acid conversion rate was kept at about 80% at 60 to 140 minutes. As more glycyrrhizic acid is added to the reaction system, the conversion of PGUS (40 ℃) gradually decreases, possibly with a decrease in stability. While G194C-Mal 2 and G194C-Mal 4 at different temperatures still maintain higher conversion at higher glycyrrhizic acid concentrations. PGUS is substantially inactive at 50 ℃.
FIG. 12 shows the variation in glycyrrhetinic acid yield of assemblies G194C-Mal 2、G194C-Mal4 and PGUS for catalyzing the conversion of glycyrrhizic acid at various temperatures. As can be seen from FIG. 12, the yield of glycyrrhetinic acid of 0-40min, PGUS (40 ℃) and G194C-Mal 2(40℃)、G194C-Mal4(40℃)、G194C-Mal4 (50 ℃) was close, the yield of glycyrrhetinic acid of G194C-Mal 2(40℃)、G194C-Mal4 (40 ℃) was kept around 60% after 40min, and the yield of glycyrrhetinic acid could be significantly increased after 260 min. Whereas the yield of PGUS (40 ℃) glycyrrhizic acid stabilized at 40%, the yield of glycyrrhetinic acid gradually decreased after 140 min.
For purposes of this disclosure, the terms "one embodiment," "some embodiments," "example," "a particular example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (11)
1. A method of driving enzyme assembly with polyethylene glycol maleimide derivatives, the method comprising: crosslinking polyethylene glycol maleimide derivative with enzyme to obtain enzyme assembly; wherein:
the enzyme consists of SEQ ID NO:1, wherein the single mutation is a substitution with a cysteine residue;
The polyethylene glycol maleimide derivative contains at least two maleimide end groups;
the single mutation is any one of the following:
(1) Asparagine at position 28 is mutated to cysteine;
(2) Threonine at position 34 is mutated to cysteine;
(3) Asparagine at position 100 is mutated to cysteine;
(4) Glutamine at position 180 is mutated to cysteine;
(5) Glycine at position 194 is mutated to cysteine;
(6) Aspartic acid at position 220 is mutated to cysteine;
The structural formula of the polyethylene glycol maleimide derivative is as follows: PEG-X-MAL; wherein,
PEG is polyethylene glycol residue, MAL is maleimide group;
X is a linking group of PEG and MAL, which is selected from one or more than two of ：-(CH2)r-、-(CH2)rO-、-(CH2)rCO-、-(CH2)rNH-、-(CH2)rCONH-、-(CH2)rNHCO-、-(CH2)rS-、-(CH2)rCOO- and- (CH 2)r OCO-, and r is an integer of 0-10.
2. A method of driving enzyme assembly with polyethylene glycol maleimide derivatives according to claim 1, wherein the single mutation is any of the following:
mutation of asparagine at position 28 to cysteine, or
The glycine at position 194 is mutated to cysteine, or
Glutamine at position 180 is mutated to cysteine.
3. The method of driving enzyme assembly using polyethylene glycol maleimide derivatives according to claim 1, wherein r is an integer of 0 to 5.
4. A method of driving enzyme assembly with a polyethylene glycol maleimide derivative according to any of claims 1-3, wherein said polyethylene glycol maleimide derivative is a double arm polyethylene glycol maleimide, or a four arm polyethylene glycol maleimide.
5. The method of driving enzyme assembly with a polyethylene glycol maleimide derivative according to claim 4, wherein said polyethylene glycol maleimide derivative is a four-arm polyethylene glycol maleimide.
6. The method of driving enzyme assembly with polyethylene glycol maleimide derivatives according to claim 4, wherein the polyethylene glycol maleimide derivatives have a molecular weight of 2000-3400Da.
7. The method of driving enzyme assembly using polyethylene glycol maleimide derivatives according to claim 4, wherein the molar ratio of polyethylene glycol maleimide derivatives to enzyme is 1 (1.5-2.5).
8. The method of driving enzyme assembly with polyethylene glycol maleimide derivatives according to claim 7, wherein the molar ratio of polyethylene glycol maleimide derivatives to enzyme is 1:2.
9. The method of driving enzyme assembly with polyethylene glycol maleimide derivatives according to claim 4, wherein the crosslinking conditions are: the pH value is 6.8-7.4, the reaction temperature is 0-10 ℃, and the reaction time is 2-12 hours.
10. An enzyme assembly prepared by the method of any one of claims 1-9.
11. Use of the enzyme assembly of claim 10 for converting glycyrrhizic acid to glycyrrhetinic acid.
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