CN117448391A - Method for synthesizing dihydroxyacetone by using immobilized carbon enzyme as catalyst - Google Patents
Method for synthesizing dihydroxyacetone by using immobilized carbon enzyme as catalyst Download PDFInfo
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- CN117448391A CN117448391A CN202311407443.8A CN202311407443A CN117448391A CN 117448391 A CN117448391 A CN 117448391A CN 202311407443 A CN202311407443 A CN 202311407443A CN 117448391 A CN117448391 A CN 117448391A
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- enzyme
- dihydroxyacetone
- microgel
- synthesizing
- methanol
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 113
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 113
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 title claims abstract description 111
- 229940120503 dihydroxyacetone Drugs 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 40
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 26
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims description 13
- 229910052799 carbon Inorganic materials 0.000 title claims description 13
- 239000003054 catalyst Substances 0.000 title claims description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 90
- 108010048916 alcohol dehydrogenase (acceptor) Proteins 0.000 claims abstract description 26
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- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 claims abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 92
- 239000000243 solution Substances 0.000 claims description 28
- 108090000623 proteins and genes Proteins 0.000 claims description 26
- 238000006555 catalytic reaction Methods 0.000 claims description 18
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
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- 241000588724 Escherichia coli Species 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2,2'-azo-bis-isobutyronitrile Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 3
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 3
- 239000012046 mixed solvent Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 claims description 3
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- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 6
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- 125000000539 amino acid group Chemical group 0.000 description 3
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- 229910052759 nickel Inorganic materials 0.000 description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 3
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- 101710192597 Protein map Proteins 0.000 description 2
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 2
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- FWTFCJFGBSEGFO-UHFFFAOYSA-N n-(methylideneamino)aniline Chemical compound C=NNC1=CC=CC=C1 FWTFCJFGBSEGFO-UHFFFAOYSA-N 0.000 description 2
- 239000002121 nanofiber Substances 0.000 description 2
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 102000003677 Aldehyde-Lyases Human genes 0.000 description 1
- 108090000072 Aldehyde-Lyases Proteins 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
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- WVVOBOZHTQJXPB-UHFFFAOYSA-N N-anilino-N-nitronitramide Chemical compound [N+](=O)([O-])N(NC1=CC=CC=C1)[N+](=O)[O-] WVVOBOZHTQJXPB-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
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- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 150000002584 ketoses Chemical class 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
-
- 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
- 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
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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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
- 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
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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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
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01244—Methanol dehydrogenase (1.1.1.244)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/02—Aldehyde-lyases (4.1.2)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention discloses a preparation method for synthesizing dihydroxyacetone by combining a methanol dehydrogenase MDHBs mutant from bacillus stearothermophilus with methanol dehydrogenase, and also discloses a method for synthesizing dihydroxyacetone by using microgel coated with the carbon-fixing enzyme, wherein the two methods can improve the catalytic efficiency, and the microgel has better biocompatibility and degradability. The microgel coated with the methanol dehydrogenase and the formaldehyde-dependent aldolase is environment-friendly, pollution-free and low in cost when the dihydroxyacetone is industrially produced. The catalytic effect of the preparation method of dihydroxyacetone provided by the invention is 2.67 times of that of the traditional wild type enzyme, and the conversion efficiency is greatly improved.
Description
Technical Field
The invention belongs to the technical fields of biological carbon fixation and biochemical engineering, and relates to a method for synthesizing dihydroxyacetone by using a carbon-fixing enzyme as a catalyst and a product thereof.
Background
1, 3-Dihydroxyacetone (DHA) is a naturally occurring ketose, has biodegradability, is harmless to human body and environment, and is widely applied to the fields of cosmetics, medicines, food additives and the like. DHA can be derived from CO 2 Is used as a carbon source and is prepared by methanol or formic acid as an intermediate through biocatalysis, and can be further converted into organic chemicals such as lactic acid and the like, and is CO 2 The biological transformation into important intermediate products in the process of high-value chemicals becomes a research hot spot of biological carbon fixation.
The dehydrogenase is an enzyme commonly used in the enzymatic regeneration of NADH system, and in vivo and in vitro enzyme activities derived from B.stearothermophilus are reported to be higher than those of MDH enzyme derived from B.methanolicus MGA3 (Metab Eng.,2017, 39:49-59), so that the invention uses MDH derived from B.stearothermophilus Bs The enzyme is used as a key enzyme for catalyzing methanol to synthesize formaldehyde. In recent years, formaldehyde-dependent aldolases (FLS) engineered by Siegel JB team (Proceedings of the NationaI Academy of sciences, 2015, 112 (12): 3704-3709) are capable of rapidly catalyzing formaldehyde synthesis to DHA. However, the two enzymes have poor enzymatic properties and insignificant catalytic effects, and have a great gap from industrial application. In particular, MDH enzyme is taken as an important carbon compound key metabolizing enzyme, is an important speed limiting enzyme for catalytic conversion of methanol, and the digging and modification of the enzymatic performance can greatly improve the conversion rate and the utilization rate of the methanol.
Enzyme catalysis has the problem of low stability of free enzyme, and is not easy to recycle for long-term reuse. Aiming at the problem, many researchers adopt nanofiber materials and MOF materials to adsorb and fix enzymes, and the enzyme activity is improved to a certain extent, but the preparation process of the nanofiber materials and the MOF materials consumes a large amount of energy and is not easy to degrade.
Disclosure of Invention
In view of the above, the invention provides a method for synthesizing dihydroxyacetone by using immobilized enzyme as a catalyst, and also provides a method for synthesizing dihydroxyacetone by using microgel to cover MDHBs and FLS enzymes at the same time.
In order to achieve the above purpose, the invention adopts the following technical scheme:
1.a method for synthesizing dihydroxyacetone by using immobilized carbon enzyme as catalyst comprises the steps of utilizing methanol dehydrogenase to convert methanol into formaldehyde, adding formaldehyde-dependent aldolase, and catalyzing formaldehyde to synthesize dihydroxyacetone.
Further, the amino acid sequence of the methanol dehydrogenase has more than 90% of identity with the amino acid sequence shown in SEQ ID NO. 4.
Further, in the method for synthesizing dihydroxyacetone by using the immobilized carbon enzyme as a catalyst, the preparation method of the methanol dehydrogenase comprises the following steps: the gene fragment shown in SEQ ID NO.3 of the encoding mutant is connected to an expression vector and is transformed into an expression host for expression.
Further, in the method for synthesizing dihydroxyacetone by using the immobilized carbon enzyme as a catalyst, the expression vector is pET-28a (+).
Further, the expression host is E.coli, and the E.coli is BL21 (DE 3), top10, DH 5. Alpha.
2. A method for synthesizing dihydroxyacetone by using microgel coated immobilized enzyme as catalyst comprises the steps of coating methanol dehydrogenase and formaldehyde dependent aldolase in microgel, and carrying out enzymatic reaction by taking methanol as a substrate to synthesize dihydroxyacetone, wherein the amino acid sequence of the methanol dehydrogenase is shown as SEQ ID NO. 4.
Further, in the method for synthesizing dihydroxyacetone by the catalysis of the microgel coated carbon-fixing enzyme, the preparation method of the methanol dehydrogenase comprises the following steps: the gene fragment shown in SEQ ID NO.3 of the encoding mutant is connected to an expression vector and is transformed into an expression host for expression.
More specifically, a recombinant plasmid containing the gene shown by the methanol dehydrogenase SEQ ID NO.3 was transferred into competent cells of E.coli, inoculated into LB containing 1%o kanamycin, and kept constant at 37℃and 200rpmCulturing in Wen Yaochuang incubator for 12-16 h to activate, inoculating the activated bacterial liquid into LB liquid medium containing 1%o kanamycin, culturing in 200rpm constant temperature shaking incubator at 20-40deg.C until OD600 = 0.6-0.8, adding appropriate amount of IPTG to final concentration of 0.1 mM-1 mM, inducing in 200rpm constant temperature shaking incubator at 30 deg.C for 6-24h to obtain MDH Bs Enzyme expression.
Further, the method for synthesizing dihydroxyacetone by the catalysis of the microgel coated carbohydrase also comprises MDH Bs And (3) preparing a crude enzyme solution. More specifically, MDH will be expressed Bs The enzyme fermentation broth is frozen and centrifuged in a refrigerated centrifuge, the supernatant is discarded to collect the thalli, and the thalli are suspended for 1-2 times by using PBS buffer solution with equal volume. Then the thalli are dissolved in buffer A (100mM Tris+150mM NaCl+20mM imidazole, pH=7.5) and placed in an ice-water bath, and are subjected to ultrasonic disruption for 10-15 min by an ultrasonic disruption instrument, and then the precipitate is removed by centrifugation at 4 ℃ to obtain crude enzyme liquid.
Further, the method for synthesizing dihydroxyacetone by the catalysis of the microgel coated carbohydrase also comprises MDH Bs And (5) purifying the enzyme. More specifically, the invention adopts a nickel column for purification, and the crude enzyme solution is filtered by 0.22 mu m to obtain supernatant. Next, the hetero-protein was eluted with buffer A, and after repeated washing for about 10mL, the effluent was collected. Eluting with buffer B (100 mM Tris, 150mM NaCl, 500mM imidazole, ph=7.5), and collecting the elution peaks to obtain the target protein.
Further, in the method, the gene shown in SEQ ID NO.2 is taken as a template, and the mutation is carried out by adopting an iterative saturation mutation method, so that a gene segment for coding the mutant is obtained.
Further, in the method for synthesizing dihydroxyacetone by the catalysis of the microgel coated with the carbohydrase, the preparation method of the microgel comprises the following steps: 1.212g of methacrylic acid, 0.736g of divinylbenzene and 0.03896g of 2, 2-azobisisobutyronitrile are added into each 40mL of acetonitrile system, and the mixture is uniformly dispersed by ultrasonic treatment; reflux-reacting at 90-100 deg.c for 2-4 hr, centrifuging to discard supernatant, adding absolute alcohol, ultrasonic dispersing, centrifuging to discard supernatant, and repeating for 2-3 times to obtain the microgel.
Further, in the method for synthesizing dihydroxyacetone by the catalysis of the microgel coated carbon-fixing enzyme, the specific method for coating the microgel with the methanol dehydrogenase and the formaldehyde-dependent aldolase comprises the following steps: placing the microgel and enzyme solution into PBS buffer solution, and stirring for 2-4 h at 30-40 ℃; centrifuging to remove supernatant, dispersing the lower layer solid with a mixed solvent of water and isopropanol, centrifuging again to remove supernatant, and repeating for 2-3 times; finally, the mixture solution of isopropanol and normal hexane is used for dispersing to form a microgel-enzyme coating.
Further, in the method for synthesizing dihydroxyacetone by the catalysis of the microgel coated carbon-fixing enzyme, the enzymatic reaction system is as follows: 0.5 mM PBS buffer pH7.5, 100mM methanol, 0.2mM anhydrous magnesium sulfate, 0.1mM NAD+,0.1mM TPP, final concentration 10g/L microgel-enzyme coating solution.
3. Dihydroxyacetone synthesized by any of the methods described above is also within the scope of the present invention.
The invention has the beneficial effects that: according to the invention, from the analysis of a crystal structure model of MDH enzyme, site-directed saturation mutation and iterative saturation mutation are adopted to construct a methanol dehydrogenase MDHBs mutant from bacillus stearothermophilus, and compared with the methanol dehydrogenase MDHBs from bacillus stearothermophilus shown in SEQ ID NO.1, the amino acid sequence of the methanol dehydrogenase MDHBs is mutated into glutamine at position 69, threonine at position 22 and isoleucine at position 20. The MDHBs mutant is used for catalyzing methanol to prepare formaldehyde, so that the enzyme activity of the mutant is improved, the conversion efficiency of the methanol is improved, and dihydroxyacetone can be prepared by further reacting with formaldehyde-dependent aldolase. The conversion efficiency was 2.67 times that of the wild type compared with the wild type methanol dehydrogenase. Furthermore, the invention also adopts microgel to simultaneously cover the methanol dehydrogenase and formaldehyde dependent aldolase with mutants, which can separate methanol and formaldehyde molecules from enzyme, improve the stability of the enzyme and promote the production of DHA. The microgel is a micron-sized hydrogel structure, is used as an independent biological material unit and an assembled bracket structure unit, and has good biocompatibility and degradability. The microgel coated with the methanol dehydrogenase and the formaldehyde-dependent aldolase is environment-friendly, pollution-free and low in cost when the dihydroxyacetone is industrially produced.
In the technical proposal provided by the invention, the CO can also be combined and utilized by electrocatalysis 2 And (3) catalyzing and synthesizing methanol, and further catalyzing the methanol to obtain DHA, so as to realize carbon neutralization and obtain higher economic value.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a technical scheme showing that microgel stabilized carbohydrase catalyzes the production of dihydroxyacetone from methanol.
FIG. 2 is an MDH Bs PCR validation plots after transfer of FLS into competent cells.
FIG. 3 is a SDS-PAGE map of FLS enzyme and MDH enzyme.
FIG. 4 is a schematic diagram of amino acid residues within the range of 6A centered on a methanol molecule.
FIG. 5 is a SDS-PAGE protein map of various mutation sites of MDH.
FIG. 6 shows the detection of MDH enzyme activity by detecting formaldehyde production using high performance liquid phase.
FIG. 7 is an MDH Bs With MDH Bs -enzymatic performance profile of E69Q/K22T/R20I mutant. (a) is enzyme activity at different temperatures; (b) is the enzyme activity at different pH.
FIG. 8 shows no microgel at various methanol concentrations-wild MDH Bs And FLS catalytic reaction, no microgel-mutant MDH Bs -E69Q/K22T/R20I enzyme and FLS catalyzed reaction with microgel-mutant MDH Bs -dihydroxyacetone yield map of E69Q/K22T/R20I enzyme and FLS catalyzed reaction.
Detailed Description
The following description of the preferred embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Example 1
In an embodiment of the present invention, an in vitro enzyme-catalyzed pathway from methanol to Dihydroxyacetone (DHA) is constructed by introducing Methanol Dehydrogenase (MDHBs) and formaldehyde-dependent aldolase (FLS) derived from Bacillus stearothermophilus (B.stearothermophilus) into host cells, respectively, to induce expression, extract crude enzyme and purify. This metabolic pathway involves a two-step enzyme-catalyzed reaction: (1) MDHBs enzyme catalyzes methanol and coenzyme factor NAD+ to generate formaldehyde and NADH; (2) FLS enzyme catalyzes formaldehyde to generate DHA. In addition, the invention adopts iterative site-directed saturation mutation to improve the activity of the rate-limiting enzyme MDHBs. In order to improve the stability of the enzyme, the invention adopts a method of coating the enzyme by microgel to separate the enzyme from a substrate, thereby reducing the toxic action of the enzyme. Therefore, the method can be applied to the fields of preparing dihydroxyacetone and derivative chemicals by biological carbon fixation conversion, and has important value and application prospect. The general technical route of the present invention is shown in fig. 1.
Expression and purification of MDHBs and FLS enzymes
(1) Gene MDH Bs And cloning of FLS and construction of expression vectors:
methanol Dehydrogenase (MDH) Bs ) Derived from Bacillus stearothermophilus (B.stearothermophilus), the gene coding sequence is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 1; the formaldehyde-dependent aldolase (FLS) is modified by Siegel J B team (Proceedings of the NationaI Academy of sciences, 2015, 112 (12): 3704-3709), the gene coding sequence is shown as SEQ ID NO.6, and the amino acid coding sequence is shown as SEQ ID NO. 3. The two sections of gene fragments are synthesized by the Kirschner Biotechnology Co., ltd, cloned to a vector pET-28a (+) and the cloning site is HindIII and XhoI; the C-terminal is fused to a 6XHis6X-His tag. The successfully cloned gene expression vector was named pET-28a (+) -MDH Bs And pET-28a (+) -FLS. Then respectively transferring them into different competent cells BL21 (DE 3) of colibacillus, culturing them for 12-16 hr overnight, picking single colony for PCR verification the next day, and making PCR verification result as shown in FIG. 2, and making lane 1 be MDH Bs Enzyme(s)The PCR product, the gene fragment length is about 1020bp, proves that pET-28a (+) -MDH is successfully transferred into competent cells of escherichia coli BL21 (DE 3); lane 2 is the PCR product of FLS enzyme, the gene fragment length is 1725bp, and the pET-28a (+) -FLS is proved to be successfully transferred into competent cells of escherichia coli BL21 (DE 3). The single colony which is successfully transformed is respectively inoculated into 4mL LB liquid culture medium containing 1%o of kana antibiotics for overnight culture for 12-16 h, then 700 mu L of bacterial liquid is respectively absorbed and mixed with 300 mu L of 50% glycerol, and the mixture is placed in a refrigerator at the temperature of minus 80 ℃ for preservation.
In some embodiments, the E.coli competent cells include, but are not limited to BL21 (DE 3), top10, DH 5. Alpha.
(2)MDH Bs And induced expression of FLS enzyme
The pET-28a (+) -MDH was separately aspirated Bs pET-28a (+) -FLS bacterial liquid is inoculated into a 4mL LB test tube containing 1%o kanamycin, and cultured for 12-16 hours in a constant temperature shaking incubator at 37 ℃ and 200rpm to activate. Followed by 1% activated pET-28a (+) -MDH was pipetted Bs The pET-28a (+) -FLS bacterial liquid is inoculated into a sealed conical flask containing 50mL LB liquid culture medium of 1%o kanamycin, and cultured to OD in a constant temperature shaking incubator at 37 ℃ and 200rpm 600 When the concentration is=0.6 to 0.8, adding a proper amount of IPTG to the final concentration of 0.1mM, and inducing the mixture for 12 hours in a constant temperature shaking incubator at 30 ℃ and 200rpm to lead the MDH to be formed Bs FLS enzyme expression.
In some embodiments, the final concentration of IPTG is 100-1000. Mu.M, specifically 100. Mu.M, 200. Mu.M, 400. Mu.M, 500. Mu.M, 800. Mu.M, 1000. Mu.M, etc., which are not specifically mentioned here for brevity.
In some embodiments, the induction time is from 6 to 24 hours, specifically 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, and the like.
In some embodiments, the induction temperature is 20-40 ℃, specifically 20 ℃, 25 ℃,30 ℃, 35 ℃,37 ℃, 40 ℃, and the like.
(3)MDH Bs And preparation of FLS crude enzyme solution
The fermentation broth is frozen and centrifuged in a refrigerated centrifuge at 6000rpm at 4 ℃ for 10min, and then the supernatant is discarded to collect the thalli, and the thalli are suspended for 1 to 2 times by using PBS buffer solution with the equal volume of pH of 7.5. Then the thalli are dissolved in buffer A (100mM Tris+150mM NaCl+20mM imidazole, pH=7.5) and placed in an ice-water bath, and are subjected to ultrasonic disruption for 10-15 min (working for 2s and interval for 3 s) by an ultrasonic disruption instrument, and then the thalli are centrifuged at 6000rpm for 15min at 4 ℃ to remove the precipitate, thus obtaining crude enzyme solution.
(4)MDH Bs And purification of FLS enzyme
The invention adopts nickel column purification, and adopts PPS-100-A protein chromatography system as the instrument. Filtering and sterilizing the enzyme crude liquid by using an organic filter membrane with the diameter of 0.22 mu m to obtain supernatant, balancing a nickel column by using a buffer solution A, and then loading the enzyme crude liquid; next, the hetero-protein was eluted with buffer A, and after repeated washing for about 10mL, the effluent was collected. Elution was performed with buffer B (100 mM Tris, 150mM NaCl, 500mM imidazole, ph=7.5), and the elution peak of 500mM imidazole was collected to obtain the target protein. And then placing the collected purified enzyme solution in a dialysis bag of 30kDa, soaking in a phosphate buffer solution, standing at 4 ℃ for 12h, and precipitating imidazole thoroughly. Finally, the desalted purified enzyme solution is centrifuged for 15min at 6000rpm at 4 ℃ by an ultrafiltration tube to obtain concentrated MDH Bs And FLS purified enzyme solution. The protein of interest was analyzed by SDS-PAGE protein gel electrophoresis, and the protein concentration was determined by Coomassie Brilliant blue (Journal of Molecular Catalysis B Enzymatic,2015, 115:105-112.). FIG. 3 is a SDS-PAGE of FLS and MDH enzymes, as shown in FIG. 3 (a), and the molecular weight of the FLS protein was substantially identical to the theoretical value of 66kDa, thus indicating successful expression of FLS, as shown in FIG. 3 (b), and the molecular weight of the MDH enzyme protein was substantially identical to the theoretical value of 36 kDa.
Example 2
Preparation of methanol dehydrogenase mutant
(1)MDH Bs Selection of enzyme mutation sites
Since amino acid mutations located near the enzyme substrate binding pocket generally affect the spatial structure, charge distribution, and hydrophobicity of the enzyme to some extent, catalytic properties such as catalytic activity, substrate specificity, and thermal stability of the enzyme are affected. MDH (MDH) Bs The amino acid sequence of the enzyme is shown as SEQ ID NO.4, and the crystal PDB ID is selected: 6IQD.1.A is used as a template, and is subjected to homologous construction by SWISS-MODEL on-line website to obtain a crystal MODEL of MDH enzyme derived from B.stearothermophilus, aminoThe acid homology was 87%. The cofactor nad+, methanol small molecules were obtained from the pubhem database. Then semi-flexible molecular docking simulation is carried out in Autodock 4.0 software by taking MDHBs as a receptor and NAD+ and methanol molecules as ligands and adopting a Lamarckian GA (4.2) genetic algorithm to generate a plurality of docking conformations. The docking conformation was then analysed in the crystal structure analysis software Pymol. One of the docking conformations is shown in FIG. 4, in the Pymol visualization software, the methanol molecule is taken as the center, and the amino acid residues within the range of 6A are LEU-23, LEU-29, LYS-22, GLU-69, PRO-21 and ARG-20, wherein the amino acid residues LEU-23 and GLU-69 form hydrogen bonding with the methanol molecule. In this example, LEU-23, LEU-29, LYS-22, GLU-69, PRO-21, ARG-20 were selected as site-directed saturation mutation sites.
(2) Containing MDH Bs Construction of enzyme saturation mutation library
Targeting MDH using pET-28a (+) -MDHBs constructed in step (1) of example 1 as a template Bs The single point amino acid sites at positions 20, 21, 22, 23, 29, 69 of the enzyme were designed to contain degenerate primers for degenerate codon NNN (N=A/T/C/G) (Table 1), and site-directed saturation mutagenesis was performed by full-plasmid Polymerase Chain Reaction (PCR), respectively, to construct a plasmid containing MDH Bs Recombinant plasmid of enzyme mutant.
TABLE 1 mutant primer sequences
The 50. Mu.L PCR reaction system was: 10X Pfu Buffer with MgSO 4 (5. Mu.L), 10mM dNTP Mix (1. Mu.L), forward primer (1. Mu.L), reverse primer (1. Mu.L), template DNA (1. Mu.L), pfu DNA Polymerase, 2.5U/. Mu.L (1.2. Mu.L), sterilized ddH 2 O (50. Mu.L added to the total reaction system).
The PCR amplification procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 1min with the Tm value of the primer minus 5℃as annealing temperature, extension at 68℃for 15min,18 cycles; fully extending for 10min at 68 ℃; preserving at 4 ℃.
mu.L of Dpn I restriction enzyme (10U/. Mu.L) was added to each amplification reaction, gently mixed, incubated at 37℃for 1h, and the parental templates digested.
Will contain MDH Bs Transferring the digested product of the enzyme mutant into competent cells of escherichia coli BL21 (DE 3) at 37 ℃ for culturing for 12-16 hours, selecting different mutant transformants and 1/8 wild transformants, fermenting and culturing for 8 hours in a 96 shallow well plate containing 200 mu L of LB liquid culture medium, transferring into a 96 deep well plate containing 1mL of LB liquid culture medium, and continuously culturing for 3 hours to OD under the conditions of 37 ℃ and 220rmp 600 0.6-0.8, adding IPTG with the final concentration of 0.1mM, inducing and culturing for 12h at 30 ℃ and 220rmp, adding glycerol with the final volume fraction of 15% into the transferred 96 shallow hole plates, and preserving at-80 ℃ for later use.
(3)MDH Bs High throughput screening of enzyme saturated mutation libraries
The 96 deep well plate in the above (2) was centrifuged at 4500rmp for 10min at 4℃to collect the cells, and washed 2 times with an equal amount of PBS buffer having a pH of 7.5, and 500. Mu.L of a solution containing 100mM methanol and 0.1mM NAD as final concentration was added to the 96 deep well plate + PBS buffer solution with pH of 7.5, and reacting for 2 hours at 30 ℃ and 220 r/min; centrifuging the reaction solution at 4deg.C and 4500r/min for 10min, collecting 125 μl supernatant, placing in 96-well cell culture plate, adding 125 μl Nash reagent (5M ammonium acetate, 50mM acetylacetone) and mixing at 37deg.C for 1 hr for color development, measuring absorbance of the color development solution at 412nm by ultraviolet spectrophotometer, and collecting absorbance corresponding to wild MDH Bs Sequencing and vitality identification are carried out on mutant strains with high transformants.
(4)MDH Bs Enzyme activity assay for enzymes and mutant enzymes
Will induce success and absorbance relative to wild MDH Bs The transformant-high mutant strain was prepared as a crude enzyme solution by the method of steps (3) and (4) in example 1 and purified to obtain a pure enzyme solution.
FIG. 5 is a SDS-PAGE protein map of different mutation sites of MDH, and Table 2 shows protein concentration tables of different mutation sites of MDH.
TABLE 2 protein concentration at the various mutation sites of MDH
The total volume of the enzyme activity assay system is 500. Mu.L, and the enzyme activity assay system comprises 100mM substrate methanol, 100mM PBS buffer solution and 0.1mM NAD + And 2-5 mg/ml of pure enzyme solution, 500 mu L of acetonitrile is added to terminate the reaction, and the filtered product is detected by adopting a high-efficiency liquid phase.
The method for detecting formaldehyde by high performance liquid phase comprises the following steps: formaldehyde contains carbonyl groups and can be derivatized with Dinitrophenylhydrazine (DNPH) to form formaldehyde phenylhydrazone, which is quantitatively detected using HPLC. 0.0201g of DNPH was dissolved in 50mL of a phosphoric acid-acetonitrile mixed solution having a volume ratio of 1.5% to obtain a derivative solution. 800. Mu.L of the derivative liquid was mixed with 200. Mu.L of formaldehyde standard liquid and sample liquid, and the mixture was subjected to metal bath derivatization at 60℃for 1 hour. After cooling it was filtered through a 0.22 μm filter for HPLC analysis. The chromatographic conditions are as follows: HPLC (ultraviolet detector), C18 chromatographic column (ShimNex CS, 4.6X250 mm,5 μm), double flow mobile phase (acetonitrile: water=70:30 (V: V)), column temperature 30 ℃, wavelength 338nm, flow rate 0.8 mL. Min -1 The sample injection amount is 10 mu L, and the detection is carried out for 15 minutes. Definition of enzyme Activity Unit (U): under the conditions described, the amount of enzyme required to convert 1. Mu. Mol of product formaldehyde per minute was converted. The enzyme activity calculation formula is as follows:
wherein, MDH Bs The unit of enzyme activity is mU/mg; formaldehyde formation units are mM; the reaction time unit is min; the volume unit of the reaction liquid is mL; the protein content unit is g/mL.
Example 3: application of iterative saturation mutation strategy
(1) Optimal MDH Bs Acquisition of mutants
Because the single-point mutant strain has unobvious improvement of enzyme activity, the combination mutation is further carried out on different key sites influencing the enzyme activity. If the optimal mutation of each site is simply carried outThe direct combination of plants cannot take the mutual influence among all the sites into consideration; if random combinatorial mutation is performed on four sites, more than 60000 transformants need to be selected even if NDT codons are selected. Therefore, the present study uses iterative saturation mutation strategy (ISM) (Molecular Biosystems,2009,5 (2): 115-122.) to combine mutations at key loci found previously, introducing saturation mutations at one locus on the optimal mutant at another locus in a certain iterative order, avoiding the negative effects that direct combination may bring and greatly reducing the screening workload. In this example, in the mutant pET28a (+) -MDH Bs And (3) sequentially carrying out saturation mutation on the rest forward mutation sites 20, 21, 22, 23 and 29 again on the basis of E69Q to gradually form a more stable structure, screening the enzyme activity of each mutation, and determining partial enzyme activity according to the results shown in tables 3 and 4 so as to obtain the optimal mutant. After several rounds of saturated mutant enzyme activity screening, the recombinant plasmid pET28a (+) -MDH is finally obtained Bs -E69Q/K22T/R20I，MDH Bs The coding sequence of the E69Q/K22T/R20I gene is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 2. The PCR reaction system and amplification procedure for mutation and the enzyme activity assay method used were the same as those in example 2.
TABLE 3 partial mutation site MDH Bs Enzyme activity data of mutant strain
TABLE 4 enzymatic Activity of partial iterative saturation mutant site MDHBs mutant strains
FIG. 6 shows the detection of MDH enzyme activity by detecting formaldehyde production by high performance liquid phase, and FIG. 6 (a) shows the high performance liquid phase diagram of formaldehyde standard, from which it can be found that formaldehyde phenylhydrazone shows a peak at 5.637min and an absorption peak at 4.075min is that of DNPH; in FIG. 6, (b) is a high-efficiency liquid phase diagram of the formaldehyde sample, from which it was judged that the peak diagram of the peak at 5.646min was formaldehyde.
(2) Enzymatic Property testing of optimal mutants
Testing of optimum temperature: enzymatic reactions were performed at pH7.5 in 100mM PBS buffer at different temperatures (25-60 ℃) for 1h, and the enzyme activities were measured separately to determine the optimum temperatures for the purified wild-type enzyme MDH and the mutant enzyme. The temperature at which the enzyme activity was highest was defined as the optimum reaction temperature, and the relative enzyme activities at the respective temperatures were calculated with the highest enzyme activity being 100%. As can be seen from FIG. 7 (a), the optimal reaction temperature of the mutant E69Q/K22T/R20I was 55℃as compared with the wild-type enzyme MDH Bs The temperature was raised by 10 ℃.
Testing of optimum pH: the enzyme solution is subjected to enzymatic reaction in buffers with different pH values of 2.5-9.5 at the optimal temperature for 1h to determine the optimal pH value. The pH at the highest enzyme activity was defined as the optimum reaction pH, and the relative enzyme activities at each pH were calculated with the highest enzyme activity being 100%. As can be seen from FIG. 7 (b), the mutant E69Q/K22T/R20I had an optimal reaction pH of about 8.5, which is higher than that of the wild-type enzyme MDH Bs The alkali resistance is enhanced.
Example 4: microgel-enzyme coating applied to dihydroxyacetone production
(1) Preparation of microgels
Into a 100mL round bottom flask was added 1.212g methacrylic acid, 0.736g divinylbenzene, 0.03896g 2, 2-azobisisobutyronitrile and 40mL acetonitrile and sonicated for 30min to disperse uniformly. Reflux is carried out at a constant temperature of between 85 and 90 ℃ for reaction, and the reaction is stopped after 1 to 2 hours. Centrifuging at 8000r/min for 10min, pouring out supernatant, adding absolute ethyl alcohol, performing ultrasonic dispersion, centrifuging at 8000r/min for 5min, and repeating for 2-3 times to obtain milky microgel pellets for later use. Preferably, the reaction is carried out under reflux at a constant temperature of 90℃and stopped after 2 hours.
(2) Preparation of microgel-enzyme coating
For immobilization of the enzyme, 5mg microgel pellets and 0.1mL of pure enzyme solution were dissolved in Phosphate (PBS) buffer (0.5M, pH=7.5) (final concentration wherein MDHBs enzyme protein content was 5 to 10mg/mL, FLS enzyme protein content was 2 to 5 mg/mL), stirred at 37℃for 2 hours, and then centrifuged at 8000r/min for 20min to pour out the supernatant, and the lower residue was redispersed in waterIn a mixed solvent of isopropanol (the volume ratio of water to isopropanol is 1:1-1:3), centrifuging again, discarding supernatant, and repeatedly rinsing for 2-3 times; finally, the mixed solution of normal hexane and isopropanol (the volume ratio of normal hexane to isopropanol is 1:1-1:2) is used for dispersing, and MDH is used Bs The molecular weight of the protein is 36.2kDa, the molecular weight of the FLS protein is 66kDa, and the molecular weight is smaller, so that the protein molecule is coated in the microgel to form a microgel-enzyme coating body, and the enzyme protein and the substrate micromolecule can be distinguished, thereby playing a role in stabilizing and protecting the enzyme protein.
(3) Catalytic production of dihydroxyacetone
The reaction system was 5mL, and the final concentration of each substance was: PBS buffer of 0.5M, pH 7.0-pH 7.5, 100mM methanol, 0.2mM anhydrous magnesium sulfate, 0.1mM NAD + 0.1mM TPP, final concentration 10mg/mL microgel-enzyme coating.
In comparison, in the test of dihydroxyacetone production without microgel-mutant MDHBs catalysis, the same final concentrations of MDHBs enzyme and FLS enzyme were directly added.
In comparison, in the test for dihydroxyacetone production without microgel-wild MDH catalysis, the same final concentrations of MDH enzyme and FLS enzyme were directly added.
(4) Detection of dihydroxyacetone content
In order to verify the technical effect of the microgel-carbohydrase coating, the invention adopts high performance liquid chromatography to measure the dihydroxyacetone content of the reaction liquid by taking the yield of dihydroxyacetone production as an index. The chromatographic conditions are as follows: HPLC (ultraviolet detector), C18 chromatographic column (ShimNex CS, 4.6X250 mm,5 μm), double flow mobile phase (methanol: 0.05% phosphoric acid in ultrapure water=5:95 (V: V)), column temperature 25 ℃, wavelength 272nm, flow rate 1.0mL min -1 The sample injection amount is 10 mu L, and the detection is carried out for 15 minutes.
(5) Determination of optimum substrate concentration
A reaction system in step (3) of this example was subjected to an enzymatic reaction by preparing a methanol solution having a concentration gradient of 50 to 300mM in a reaction solution containing a microgel-enzyme coating at a final concentration of 10mg/mL, and the dihydroxyacetone yields at different substrate concentrations were measured by using the reaction solution containing no free enzyme having a microgel coating effect as a control experiment and the yield/methanol input content of dihydroxyacetone as an evaluation index, and the results are shown in Table 5 and FIG. 8. As shown in FIG. 8, the optimal substrate concentration of the reaction solution without the microgel-immobilized enzyme coating was 150mM, and the optimal substrate concentration of the reaction solution containing the microgel coating was 250mM, and the yield was increased to 2.67 times as high as the original, indicating that the methanol tolerance was enhanced.
TABLE 5 dihydroxyacetone yields from enzymatic reactions of different substrates
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1.A method for synthesizing dihydroxyacetone by using immobilized enzyme as catalyst is characterized by that the amino acid sequence of methanol dehydrogenase shown in SEQ ID No.2 is used to convert methanol into formaldehyde, then formaldehyde-dependent aldolase is added to catalyze formaldehyde to synthesize dihydroxyacetone.
2. The method for synthesizing dihydroxyacetone by using the carbon-fixing enzyme as claimed in claim 1, wherein the preparation method of the methanol dehydrogenase is as follows: the gene fragment shown in SEQ ID NO.5 of the encoding mutant is connected to an expression vector and is transformed into an expression host for expression.
3. The method for the catalytic synthesis of dihydroxyacetone according to claim 2, wherein said expression vector is pET-28a (+).
4. The method for synthesizing dihydroxyacetone by using the carbon-fixing enzyme as claimed in claim 2, wherein the expression host is escherichia coli, and the escherichia coli is BL21 (DE 3), top10 and DH5 alpha.
5. A method for synthesizing dihydroxyacetone by using microgel coated immobilized enzyme as catalyst is characterized in that methanol dehydrogenase and formaldehyde dependent aldolase are coated in microgel, and then methanol is used as a substrate for enzymatic reaction to synthesize dihydroxyacetone, wherein the amino acid sequence of the methanol dehydrogenase is shown as SEQ ID NO. 2.
6. The method for synthesizing dihydroxyacetone by catalysis of microgel coated carbon fixation enzyme as claimed in claim 5, wherein the preparation method of the methanol dehydrogenase is as follows: the gene fragment shown in SEQ ID NO.5 of the encoding mutant is connected to an expression vector and is transformed into an expression host for expression.
7. The method for synthesizing dihydroxyacetone by catalysis of microgel coated carbon sequestration enzyme according to claim 5, wherein the preparation method of the microgel is as follows: 1.212g of methacrylic acid, 0.736g of divinylbenzene and 0.03896g of 2, 2-azobisisobutyronitrile are added into each 40mL of acetonitrile system, and the mixture is uniformly dispersed by ultrasonic treatment; reflux-reacting at 90-100deg.C for 2-4 h, centrifuging to remove supernatant, adding absolute ethanol, ultrasonic dispersing, centrifuging to remove supernatant, and repeating for 2-3 times to obtain microgel.
8. The method for synthesizing dihydroxyacetone by the catalysis of the microgel coated carbon fixation enzyme according to claim 5, wherein the specific method for coating the microgel with the methanol dehydrogenase and the formaldehyde-dependent aldolase is as follows: placing the microgel and enzyme solution into PBS buffer solution, and stirring for 2-4 h at 30-40 ℃; centrifuging to remove supernatant, dispersing the lower layer solid with a mixed solvent of water and isopropanol, centrifuging again to remove supernatant, and repeating for 2-3 times; finally, the mixture solution of isopropanol and normal hexane is used for dispersing to form a microgel-enzyme coating.
9. According to claimThe method for synthesizing dihydroxyacetone by the catalysis of the microgel coated carbon-fixing enzyme, which is characterized in that the enzymatic reaction system is as follows: PBS buffer, 100mM methanol, 0.2mM anhydrous magnesium sulfate, 0.1mM NAD, pH7.5, 0.5M + 0.1mM TPP, final concentration 10g/L microgel-enzyme coating solution.
10. Dihydroxyacetone synthesized by the process of any one of claims 1-4, and the process of any one of claims 5-9.
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