CN112111477A - Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide - Google Patents
Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide Download PDFInfo
- Publication number
- CN112111477A CN112111477A CN202011059690.XA CN202011059690A CN112111477A CN 112111477 A CN112111477 A CN 112111477A CN 202011059690 A CN202011059690 A CN 202011059690A CN 112111477 A CN112111477 A CN 112111477A
- Authority
- CN
- China
- Prior art keywords
- fatty acid
- reaction
- p450bs
- hydrogen peroxide
- acid decarboxylase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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.)
-
- 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
- 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
-
- 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
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
Abstract
The invention discloses an artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide, belonging to the field of protein engineering. The biocatalyst constructed by the invention can utilize hydrogen peroxide as a direct electron donor and fatty acid as a substrate to directly synthesize 1-alkene. Catalytic substrate extension to C6‑C18Linear saturated fatty acids of chain length and unsaturated fatty acids of oleic and linoleic acids. Introduction of glucose oxidase supplyThe double-enzyme system of the hydrogen peroxide provides slow, continuous and mild hydrogen peroxide for the reaction, greatly reduces the generation of ketone substances in the reaction, and further improves the synthesis efficiency of the 1-alkene. The reaction system directly utilizes hydrogen peroxide as an electron donor, and does not need expensive coenzyme factors and chaperonin. The catalyst has higher efficiency, can make the reaction simpler, has mild reaction conditions, wide substrate range and lower cost, and has good industrial production prospect.
Description
Technical Field
The invention relates to an artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide, belonging to the field of protein engineering.
Background
1-olefins (1-alkenes) have physicochemical properties (low freezing point, high energy density, easy extraction, compatibility with existing engines and transportation systems, etc.) that are highly similar to those of gasoline, and are an ideal feedstock that can replace petroleum. Meanwhile, 1-olefin is also an important production and living raw material for synthesizing surfactants, lubricating oil, pesticides and various polymers. At present, olefins are mainly produced by polymerization of ethylene in the fossil energy industry, but this method can obtain only olefins of even carbon chain length, resulting in that olefins of odd carbon chain length are extremely expensive. In addition, the reduction of fossil energy and the severe environmental problems, the search for 1-olefins that can be produced continuously, are problems to be solved urgently.
The method for synthesizing 1-olefin by decarboxylation by using fatty acid which is abundant in nature and can be synthesized by organisms as a raw material is a more direct and convenient method. In nature, fatty acid mostly takes even carbon as the main component, and long olefin with odd carbon number can be directly obtained by decarboxylation, thereby making up for the defect of the polyethylene industry in producing long olefin with odd carbon number. The existing method for synthesizing corresponding 1-alkene by using fatty acid as a substrate is a chemical synthesis method utilizing metal catalytic decarboxylation, the same proportion of acid anhydride is required to be added for activating the substrate (generating industrial waste), and the selectivity of the product is required to be controlled by means of high pressure, distillation and the like under normal conditions. Therefore, the traditional chemical synthesis is not an ideal, environment-friendly and low-energy-consumption method for synthesizing the 1-olefin.
As shown in FIG. 1, other methods for biosynthesis of 1-olefin currently exist: (a) hydrogen peroxide drives a prototype reaction, which uses inexpensive hydrogen peroxide, but has the disadvantages of low catalytic efficiency and narrow substrate range. (b) The in situ hydrogen peroxide generation system uses a novel electron donor, but has the defects of narrow substrate range and high enzyme concentration requirement. (c) Fusion protein system, the reaction system is simple, but its use of NADPH is too expensive. (d) The redox partner system has a drawback that the reaction requires participation of a plurality of enzymes and the reaction time is long, although the synthesis efficiency of the reaction is high. The application prospect of the reaction systems is limited due to the narrow range of substrates, low catalytic efficiency, high price, complex reaction system and the like.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the technical defects in the prior art, provide an artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide, and prepare the fatty acid decarboxylase P450BS beta mutant for catalyzing and generating 1-olefin, and the artificial biocatalyst has the advantages of high reaction rate, wide substrate range, simple system, high synthesis efficiency, low cost and the like.
In order to solve the technical problems, the invention provides a fatty acid decarboxylase P450BS beta mutant, which comprises an amino acid sequence shown as SEQ ID NO. 1.
The invention also provides a nucleotide for coding the fatty acid decarboxylase P450BS beta mutant, and the nucleotide sequence is shown as SEQ ID NO. 2.
The invention also provides a vector containing the nucleotide of the fatty acid decarboxylase P450BS beta mutant.
The invention also provides a genetic engineering bacterium for expressing the fatty acid decarboxylase P450BS beta mutant.
The present invention also provides a method for obtaining a mutant of fatty acid decarboxylase P450BS beta, in which glycine at position 290 of wild-type fatty acid decarboxylase P450BS beta is mutated into valine, glutamine at position 85 is mutated into histidine, phenylalanine at position 166 is mutated into methionine, and valine at position 74 is mutated into isoleucine.
The invention also provides a preparation method of the fatty acid decarboxylase P450BS beta mutant, which comprises the following steps:
(1) construction of a plasmid expressing wild-type fatty acid hydroxylase P450BS β: the nucleotide sequence of SEQ ID NO 3 is recombined on an expression vector through enzyme digestion;
(2) constructing a saturation mutation library through four-wheel fixed-point saturation mutation;
(3) transforming the plasmids which are constructed in the steps (1) and (2) and express the fatty acid decarboxylase P450BS beta and the mutant into escherichia coli;
(4) expression of the fatty acid decarboxylase P450BS β mutant: culturing the strain obtained in the step (3) in a TB liquid culture medium (Kan/Cam) overnight, adding the strain into the TB liquid culture medium (Kan/Cam) for propagation, adding a heme precursor-aminolevulinic acid and IPTG (isopropyl-beta-thiogalactoside) into a logarithmic phase for induction expression, and centrifuging to collect the strain;
(5) preparation of cell lysates containing the fatty acid decarboxylase P450BS β mutants: resuspending the thallus obtained in the step (4) by using a buffer solution, cracking cells by ultrasonic disruption, and centrifuging to obtain a supernatant;
(6) purification of fatty acid decarboxylase P450BS β mutant: and (3) adding the supernatant obtained in the step (5) into a nickel ion affinity column which is pretreated in advance, enabling the target protein with the His label to be combined with nickel ions, then removing impurity proteins by using imidazole with lower concentration, finally eluting the target protein by using a buffer solution of high-concentration imidazole, dialyzing the eluted target protein to remove the imidazole, and storing the target protein at-80 ℃ after concentrating.
The preparation method of the fatty acid decarboxylase P450BS beta mutant comprises the following specific steps:
(1) construction of fatty acid decarboxylase P450BS beta gene expression vector: the P450BS beta fragment (shown in SEQ ID NO:3, synthesized by Jinzhi organism, Suzhou) synthesized by the whole gene is cut by restriction enzymes NcoI and XhoI according to the instruction, and then is connected to an expression vector pET28a cut by the same enzyme by T4 ligase; transforming the ligation product into E.coli DH5 alpha competent cells; the colonies of the single colonies successfully transformed were picked from the solid LB medium plate containing kanamycin to a final concentration of 50. mu.g/ml, and cultured overnight in LB liquid medium containing kanamycin to the same concentration at 37 ℃ with a shaker speed of 220 rpm/min. Extracting recombinant plasmids from the overnight-cultured bacterial liquid by using a small plasmid extraction kit, and carrying out sequencing identification on the extracted plasmids;
(2) construction of fatty acid decarboxylase P450BS beta mutant plasmid: using the plasmid with successful sequencing as a PCR template, constructing a mutant plasmid by a Quickchange method, and sending the mutant plasmid to sequencing for identification;
(3) preparation of fatty acid decarboxylase P450BS β mutant: glycerol bacteria preserved at-80 deg.C in an ultra-low temperature refrigerator were selected and inoculated into 5ml TB liquid medium (Kan/Cam) for overnight culture at 37 deg.C. Inoculating the overnight cultured bacterial liquid to 500ml TB medium (Kan/Cam), and performing amplification culture at 37 ℃ and with the rotating speed of a shaking table of 220 rpm/min; to be OD600At a value of about 0.6, expression was induced by the addition of precursor ALA (-aminolevulinic acid) at a final concentration of 0.5mM and 1mM IPTG. Culturing at 22 deg.C and 220rpm/min of shaking table for 16 h. The cells were harvested by centrifugation at 5000rpm/min for l 0min, resuspended in 50ml buffer A (0.1M KPi, 0.3M KCl, 20% glycerol, pH 7.0) and disrupted using a sonicator. Centrifuging at 4 deg.C and 10000rpm/min for 45min to obtain supernatant. And (3) loading the supernatant containing the target protein into a nickel ion affinity column which is loaded in advance, and combining the target protein carrying the His label with nickel ions. The hetero-proteins were removed with buffer A containing 10mM, 35mM of imidazole, respectively, and then the objective protein was eluted with buffer B (0.1M KPi, 0.3M KCl, 20% glycerol,250mM of imidazole, pH 7.0). SDS-PAGE identifies the purity of the protein of interest. The eluted target protein was dialyzed against buffer A overnight at 4 ℃ to remove imidazole. Protein packaging and storing at-80 deg.C.
The invention also provides a process for the production of 1-olefins, in particular of C6-C18The linear fatty acid with chain length and the naturally-occurring unsaturated long-chain fatty acid are taken as substrates, hydrogen peroxide or a reaction system for generating hydrogen peroxide is added, reaction buffer solution is added, and the P450BS beta fatty acid decarboxylase mutant as the catalyst of claim 1 is used for catalytic reaction.
Further, the reaction system for generating hydrogen peroxide comprises glucose and glucose oxidase; the pH value of the reaction system of the catalytic reaction is 7.0, the reaction temperature is 10 ℃, the rotating speed is 120rpm/min, and the reaction time is 2 hours.
Further, the reaction temperature of the catalytic reaction is 10 ℃, the pH value of the reaction system is 7.0, the rotating speed is 120rpm/min, and the reaction time is 30 min.
The invention also provides application of the P450BS beta fatty acid decarboxylase mutant in producing 1-olefin.
The fatty acid decarboxylase P450BS beta mutant of the invention is obtained by mutating the amino acid sequence of wild-type fatty acid hydroxylase P450BS beta at positions 74, 85, 166 and 290. The biocatalyst constructed by the invention can utilize hydrogen peroxide as a direct electron donor and fatty acid as a substrate to directly synthesize 1-alkene. With C14Fatty acids for example, the reaction rate (TOF) is up to 3,744h-110 to 1000 times that of other known reaction systems, and is the highest enzyme-catalyzed 1-olefin production efficiency known at present. And the range of the substrate capable of catalyzing is expanded to C6-C18Linear saturated fatty acids of chain length and unsaturated fatty acids of oleic and linoleic acids. The double-enzyme system for providing hydrogen peroxide by introducing the glucose oxidase provides slow, continuous and mild hydrogen peroxide for the reaction, greatly reduces the generation of ketone substances in the reaction, and further improves the synthesis efficiency of 1-alkene. The reaction system directly utilizes hydrogen peroxide as an electron donor, and does not need expensive coenzyme factors (such as NADPH) and chaperonin. The reaction system catalyzed by the enzyme is simpler, the reaction condition is mild, the catalysis efficiency is high, the substrate range is wide, the cost is lower, and the enzyme has good biological energy and industrial production prospects in the aspect of synthesizing 1-olefin.
The invention achieves the following beneficial effects:
(1) the biocatalyst constructed by the invention directly utilizes cheap hydrogen peroxide as an electron donor C6-C18The linear chain saturated fatty acid with the chain length and the oleic acid and linoleic acid unsaturated fatty acid are taken as substrates, thereby widening the range of the substrates and reducing the price of a reaction system.
(2) The novel enzyme fatty acid decarboxylase P450BS beta mutant catalyzes fatty acid to synthesize 1-alkene without chaperonin and accessory factor, and the reaction system is simple.
(3) The reaction and the purification process are carried out in an environment-friendly aqueous solution at room temperature, the reaction condition is mild, the catalytic efficiency is high, and the synthesis rate is high.
Drawings
FIG. 1 is a comparison of the process for synthesizing 1-olefins.
Detailed Description
The invention is further described below. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Unless otherwise specified, all compounds and reagents below were purchased from Biotechnology engineering (Shanghai) Inc., Sigma-Aldrich, Tokyo Chemical Industry, EMD Millipore, New England Biolabs.
Example 1 construction of P450BS beta Gene expression vector
The fully-genetically-synthesized P450BS β fragment (sequence shown in SEQ ID NO:3, synthesized by Kinzonly organisms, Suzhou) was ligated to the NcoI and XhoI double-digested expression vector pET28a using T4 ligase (New England Biolabs) by performing double digestion using restriction enzymes NcoI and XhoI (New England Biolabs) according to the instructions. The ligation product was transformed into E.coli DH 5. alpha. competent cells (Tiangen Biochemical technology Co., Ltd.). The colonies of the single colonies successfully transformed were picked from a solid LB medium plate containing 50. mu.g/ml kanamycin, and cultured overnight in LB liquid medium containing the same concentration of kanamycin at 37 ℃ with a shaker speed of 220 rpm/min. Recombinant plasmids were extracted from overnight-cultured bacterial suspension using a plasmid miniprep kit (Tiangen Biochemical technology Co., Ltd.) according to the instructions, and the extracted plasmids were subjected to sequencing and identification (Suzhou Jinwei Zhi Biol., Ltd.).
Example 2 purification of P450BS beta mutant protein by Mass expression
Selecting glycerol strain stored in-80 deg.C ultra-low temperature refrigerator, and inoculating to 5ml TB liquid culture medium
(Kan/Cam) was cultured overnight at 37 ℃. Inoculating the overnight cultured bacterial liquid to 500ml TB medium (Kan/Cam), and performing amplification culture at 37 ℃ and with the rotating speed of a shaking table of 220 rpm/min; to be OD600About 0.6, precursor ALA was added to a final concentration of 0.5mMThe expression was induced with (-aminolevulinic acid) and 1mM IPTG and cultured for 16h at 22 ℃ with shaker speed 220 rpm/min. The cells were harvested by centrifugation at 5000rpm/min for l 0min, resuspended in 50ml buffer A (0.1M KPi, 0.3M KCl, 20% glycerol, pH 7.0) and disrupted by sonication. Centrifuging at 4 deg.C and 10000rpm/min for 45min to obtain supernatant. And (3) loading the supernatant containing the target protein into a nickel ion affinity column which is loaded in advance, and combining the target protein carrying the His tag with nickel ions. The heteroproteins were removed with buffer A containing 10mM, 35mM imidazole, respectively, and the desired protein was eluted with buffer B (0.1M KPi, 0.3M KCl, 20% glycerol,250mM imidazole, pH 7.0). SDS-PAGE identifies the purity of the protein of interest. The eluted target protein was dialyzed against buffer A to remove imidazole and incubated overnight at 4 ℃. Protein packaging and storing at-80 deg.C. The concentration of the protein was determined by the P450-CO method.
Example 3P450BS beta mutant Hydrogen peroxide reaction System
The reaction system of oxidative decarboxylation by using P450BS beta mutant. The decarboxylation reaction system contained buffer A, purified P450BS beta mutant 2.5mM, fatty acid substrate of different chain length 2.5mM, hydrogen peroxide 5mM, and final volume of 1 ml. The reaction was carried out at 10 ℃ and pH 7.0 at 120rpm/min for 30 min. The results are shown in table 1 below. P450BS beta mutant protein not only can catalyze C6-C18The linear fatty acids with different chain lengths can also catalyze naturally-existing unsaturated long-chain fatty acids such as oleic acid and linoleic acid, thereby successfully expanding the range of catalytic substrates. The application prospect of the reaction system in the synthesis field and the energy field is widened.
TABLE 1 Hydrogen peroxide reaction system with decarboxylation catalyzed by P450BS beta mutant under different substrates[a]
[a]Reaction system: purified P450BS beta mutant (2.5. mu.M), substrate (2.5mM), hydrogen peroxide (5mM), 10 ℃, 1ml system, 120rpm/min, 30 min.[b]Gas chromatography peak area ratio.[c]Head airAnd (4) performing phase chromatography.
Example 4 identification analysis of P450BS beta mutant product
And taking 500 mul of the extracted ethyl acetate extract, adding 200 mul of methanol, uniformly mixing, and adding 75 mul of 10% n-hexane solution of trimethylsilyldiazomethane for esterification. Sampling, detection and quantification by gas chromatography. A chromatographic column: DB-WAX. The procedure is as follows: keeping at 40 deg.C for 5min and at 20 deg.C for min-1To 170 ℃ for 30min-1Keeping the temperature at 300 ℃ for 20min, and keeping the total time for 40 min. The peak position of the 1-tridecene is 12.1min, the peak position of the alpha-hydroxylation product is 23.5min, and the peak position of the beta-hydroxylation product is 24.2 min. In the presence of C14-C20During the reaction in which fatty acid was used as a substrate, after completion of the reaction, the reaction was terminated with 50. mu.l of 5N HCl and extracted with an equal volume of ethyl acetate. Samples were taken and the corresponding 1-olefins were quantified by gas chromatography. A chromatographic column: DB-5 MS. The procedure is as follows: keeping at 60 deg.C for 1min and at 20 deg.C for min-1To 320 ℃ for a total time of 14 min. In the presence of C6-C12During the reaction process of the volatile product with fatty acid as the substrate, after the reaction is finished, quantifying the corresponding 1-alkene by headspace gas chromatography. A chromatographic column: DB-5 MS. The procedure is as follows: keeping at 40 deg.C for 5min and at 20 deg.C for min-1To 170 ℃ for 30min-1Keeping the temperature at 300 ℃ for 3min, and the total time is 18.8 min. The identification method of the ketone by-product in the reaction product comprises the following steps: after the reaction was complete, the reaction was quenched with 50. mu.l of 5N HCl and extracted with an equal volume of ethyl acetate. Samples were taken and detected by GC-MS. A chromatographic column: TG-5 MS. The procedure is as follows: keeping at 60 deg.C for 1min and at 20 deg.C for min-1To 320 ℃ for a total time of 14 min.
Example 5P450BS beta mutant Glucose Oxidase (GO) reaction System
In order to reduce the generation of ketone substances caused by excessive oxidation of excessive hydrogen peroxide in a reaction system, a GO reaction system is introduced into the reaction system. The reaction system can slowly and continuously generate hydrogen peroxide, and overcomes the defect caused by directly adding excessive hydrogen peroxide into the reaction system. And explores the range of substrates that the reaction system can catalyze. The decarboxylation reaction system contains buffer A and purified P450BS beta mutant 2.5mM,fatty acid substrates of different chain lengths 2.5mM, hydrogen peroxide 5mM, glucose oxidase 10U/ml and glucose 10mM, in a final volume of 1 ml. The reaction was carried out at 120rpm/min for 2h at 10 ℃. The results are shown in table 2 below. The mutant can still catalyze the straight chain fatty acid containing 6-18 carbon atoms, and compared with a hydrogen peroxide system, the mutant can remove C16And C18The conversion rate of 1-olefin of saturated fatty acid and other fatty acid is improved. Wherein C is6Fatty acids, almost complete conversion of the substrate is achieved.
TABLE 2 reaction system for decarboxylation catalyzed by P450BS beta mutant coupled with glucose oxidase under different substrates[a]
[a]Reaction system: purified P450BS beta mutant (2.5. mu.M), substrate (2.5mM), glucose (10mM), GO (10U/ml), 10 ℃, 1ml system, 120rpm/min, 2 hours.[b]Gas chromatography peak area ratio.[c]Headspace gas chromatography
The foregoing illustrates the general principles, principal features, and advantages of the invention. The biocatalyst constructed by the present invention can utilize C6-C18The method for synthesizing the 1-olefin in one step by using the linear chain saturated fatty acid with the chain length and the oleic acid and linoleic acid unsaturated fatty acid as the substrates and using the hydrogen peroxide as the electron donor has the advantages that: 1) broadens the range of the substrate and reduces the price of the reaction system. 2) The novel enzyme fatty acid decarboxylase P450BS beta mutant catalyzes fatty acid to synthesize 1-alkene without chaperonin and accessory factor, and the reaction system is simple. 3) The reaction and the purification process are carried out in an environment-friendly aqueous solution at room temperature, the reaction condition is mild, the catalytic efficiency is high, and the synthesis rate is high. Therefore, the method has good biological energy and industrial production prospects in the aspect of synthesizing 1-olefin.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Yangzhou university
<120> an artificial biocatalyst for converting fatty acids to olefins using hydrogen peroxide
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 417
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Asp Glu Gln Ile Pro His Asp Lys Ser Leu Asp Asn Ser Leu Thr
1 5 10 15
Leu Leu Lys Glu Gly Tyr Leu Phe Ile Lys Asn Arg Thr Glu Arg Tyr
20 25 30
Asn Ser Asp Leu Phe Gln Ala Arg Leu Leu Gly Lys Asn Phe Ile Cys
35 40 45
Met Thr Gly Ala Glu Ala Ala Lys Val Phe Tyr Asp Thr Asp Arg Phe
50 55 60
Gln Arg Gln Asn Ala Leu Pro Lys Arg Ile Gln Lys Ser Leu Phe Gly
65 70 75 80
Val Asn Ala Ile His Gly Met Asp Gly Ser Ala His Ile His Arg Lys
85 90 95
Met Leu Phe Leu Ser Leu Met Thr Pro Pro His Gln Lys Arg Leu Ala
100 105 110
Glu Leu Met Thr Glu Glu Trp Lys Ala Ala Val Thr Arg Trp Glu Lys
115 120 125
Ala Asp Glu Val Val Leu Phe Glu Glu Ala Lys Glu Ile Leu Cys Arg
130 135 140
Val Ala Cys Tyr Trp Ala Gly Val Pro Leu Lys Glu Thr Glu Val Lys
145 150 155 160
Glu Arg Ala Asp Asp Met Ile Asp Met Val Asp Ala Phe Gly Ala Val
165 170 175
Gly Pro Arg His Trp Lys Gly Arg Arg Ala Arg Pro Arg Ala Glu Glu
180 185 190
Trp Ile Glu Val Met Ile Glu Asp Ala Arg Ala Gly Leu Leu Lys Thr
195 200 205
Thr Ser Gly Thr Ala Leu His Glu Met Ala Phe His Thr Gln Glu Asp
210 215 220
Gly Ser Gln Leu Asp Ser Arg Met Ala Ala Ile Glu Leu Ile Asn Val
225 230 235 240
Leu Arg Pro Ile Val Ala Ile Ser Tyr Phe Leu Val Phe Ser Ala Leu
245 250 255
Ala Leu His Glu His Pro Lys Tyr Lys Glu Trp Leu Arg Ser Gly Asn
260 265 270
Ser Arg Glu Arg Glu Met Phe Val Gln Glu Val Arg Arg Tyr Tyr Pro
275 280 285
Phe Val Pro Phe Leu Gly Ala Leu Val Lys Lys Asp Phe Val Trp Asn
290 295 300
Asn Cys Glu Phe Lys Lys Gly Thr Ser Val Leu Leu Asp Leu Tyr Gly
305 310 315 320
Thr Asn His Asp Pro Arg Leu Trp Asp His Pro Asp Glu Phe Arg Pro
325 330 335
Glu Arg Phe Ala Glu Arg Glu Glu Asn Leu Phe Asp Met Ile Pro Gln
340 345 350
Gly Gly Gly His Ala Glu Lys Gly His Arg Cys Pro Gly Glu Gly Ile
355 360 365
Thr Ile Glu Val Met Lys Ala Ser Leu Asp Phe Leu Val His Gln Ile
370 375 380
Glu Tyr Asp Val Pro Glu Gln Ser Leu His Tyr Ser Leu Ala Arg Met
385 390 395 400
Pro Ser Leu Pro Glu Ser Gly Phe Val Met Ser Gly Ile Arg Arg Lys
405 410 415
Ser
<210> 2
<211> 1254
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggatgagc agattccaca tgacaaaagt ctcgataaca gtctgacact gctgaaggaa 60
gggtatttat ttattaaaaa cagaacagag cgctacaatt cagatctgtt tcaggcccgt 120
ttgttgggaa aaaactttat ttgcatgact ggcgctgagg cggcgaaggt gttttatgat 180
acggatcgat tccagcggca gaacgctttg cctaagcgga ttcagaaatc gctgtttggt 240
gttaatgcga ttcacggaat ggatggcagc gcgcatatcc atcggaagat gctttttctg 300
tcattgatga caccgccgca tcaaaaacgt ttggctgagt tgatgacaga ggagtggaaa 360
gcagcagtca caagatggga gaaggcagat gaggttgtgt tatttgaaga agcaaaagaa 420
atcctgtgcc gggtagcgtg ctattgggca ggtgttccgt tgaaggaaac ggaagtcaaa 480
gagagagcgg atgacatgat tgacatggtc gacgcgttcg gtgctgtggg accgcggcat 540
tggaaaggaa gaagagcaag gccgcgtgcg gaagagtgga ttgaagtcat gattgaagat 600
gctcgtgccg gcttgctgaa aacgacttcc ggaacagcgc tgcatgaaat ggcttttcac 660
acacaagaag atggaagcca gctggattcc cgcatggcag ccattgagct gattaatgta 720
ctgcggccta ttgtcgccat ttcttacttt ctggtgtttt cagctttggc gcttcatgag 780
catccgaagt ataaggaatg gctgcggtct ggaaacagcc gggaaagaga aatgtttgtg 840
caggaggtcc gcagatatta tccgttcgtc ccgtttttag gggcgcttgt caaaaaagat 900
tttgtatgga ataactgtga gtttaagaag ggcacatcgg tgctgcttga tttatatgga 960
acgaaccacg accctcgtct atgggatcat cccgatgaat tccggccgga acgatttgcg 1020
gagcgggaag aaaatctgtt tgatatgatt cctcaaggcg gggggcacgc cgagaaaggc 1080
caccgctgtc caggggaagg cattacaatt gaagtcatga aagcgagcct ggatttcctc 1140
gtccatcaga ttgaatacga tgttccggaa caatcactgc attacagtct cgccagaatg 1200
ccatcattgc ctgaaagcgg cttcgtaatg agcggaatca gacgaaaaag ttaa 1254
<210> 3
<211> 1254
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaatgagc agattccaca tgacaaaagt ctcgataaca gtctgacact gctgaaggaa 60
gggtatttat ttattaaaaa cagaacagag cgctacaatt cagatctgtt tcaggcccgt 120
ttgttgggaa aaaactttat ttgcatgact ggcgctgagg cggcgaaggt gttttatgat 180
acggatcgat tccagcggca gaacgctttg cctaagcggg tgcagaaatc gctgtttggt 240
gttaatgcga ttcagggaat ggatggcagc gcgcatatcc atcggaagat gctttttctg 300
tcattgatga caccgccgca tcaaaaacgt ttggctgagt tgatgacaga ggagtggaaa 360
gcagcagtca caagatggga gaaggcagat gaggttgtgt tatttgaaga agcaaaagaa 420
atcctgtgcc gggtagcgtg ctattgggca ggtgttccgt tgaaggaaac ggaagtcaaa 480
gagagagcgg atgacttcat tgacatggtc gacgcgttcg gtgctgtggg accgcggcat 540
tggaaaggaa gaagagcaag gccgcgtgcg gaagagtgga ttgaagtcat gattgaagat 600
gctcgtgccg gcttgctgaa aacgacttcc ggaacagcgc tgcatgaaat ggcttttcac 660
acacaagaag atggaagcca gctggattcc cgcatggcag ccattgagct gattaatgta 720
ctgcggccta ttgtcgccat ttcttacttt ctggtgtttt cagctttggc gcttcatgag 780
catccgaagt ataaggaatg gctgcggtct ggaaacagcc gggaaagaga aatgtttgtg 840
caggaggtcc gcagatatta tccgttcggc ccgtttttag gggcgcttgt caaaaaagat 900
tttgtatgga ataactgtga gtttaagaag ggcacatcgg tgctgcttga tttatatgga 960
acgaaccacg accctcgtct atgggatcat cccgatgaat tccggccgga acgatttgcg 1020
gagcgggaag aaaatctgtt tgatatgatt cctcaaggcg gggggcacgc cgagaaaggc 1080
caccgctgtc caggggaagg cattacaatt gaagtcatga aagcgagcct ggatttcctc 1140
gtccatcaga ttgaatacga tgttccggaa caatcactgc attacagtct cgccagaatg 1200
ccatcattgc ctgaaagcgg cttcgtaatg agcggaatca gacgaaaaag ttaa 1254
Claims (10)
1. A P450BS beta fatty acid decarboxylase mutant is characterized by comprising an amino acid sequence shown as SEQ ID NO. 1.
2. A nucleotide encoding the P450BS β fatty acid decarboxylase mutant according to claim 1, wherein the nucleotide sequence is as shown in SEQ ID No. 2.
3. A vector comprising the nucleotide of the P450BS β fatty acid decarboxylase mutant of claim 2.
4. A genetically engineered bacterium expressing the P450BS beta fatty acid decarboxylase mutant according to claim 1.
5. A method for producing a P450BS β fatty acid decarboxylase mutant according to claim 1, wherein the glycine at position 290 is mutated to valine, the glutamine at position 85 is mutated to histidine, the phenylalanine at position 166 is mutated to methionine, and the valine at position 74 is mutated to isoleucine.
6. A method for preparing a fatty acid decarboxylase P450BS beta mutant, which is characterized by comprising the following steps:
constructing a plasmid for expressing wild-type fatty acid hydroxylating enzyme P450BS beta;
constructing a saturation mutation library through four-wheel fixed-point saturation mutation;
transforming the constructed plasmid expressing the fatty acid decarboxylase mutant into escherichia coli;
expression and purification of fatty acid decarboxylase P450BS beta mutant.
7. A process for the production of 1-olefins, characterized in that C is used6-C18The linear fatty acid with chain length and the naturally-occurring unsaturated long-chain fatty acid are taken as substrates, hydrogen peroxide or a reaction system for generating hydrogen peroxide is added, reaction buffer solution is added, and the P450BS beta fatty acid decarboxylase mutant as the catalyst of claim 1 is used for catalytic reaction.
8. The process for producing 1-alkenes according to claim 7, wherein the reaction system for producing hydrogen peroxide comprises glucose and glucose oxidase; the pH value of the reaction system of the catalytic reaction is 7.0, the reaction temperature is 10 ℃, the rotating speed is 120rpm/min, and the reaction time is 2 hours.
9. The method for producing 1-olefin according to claim 7, wherein the reaction temperature of the catalytic reaction is 10 ℃, the pH of the reaction system is 7.0, the rotating speed is 120rpm/min, and the reaction time is 30 min.
10. Use of the P450BS β fatty acid decarboxylase mutant according to claim 1 for the production of 1-alkenes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011059690.XA CN112111477B (en) | 2020-09-30 | 2020-09-30 | Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011059690.XA CN112111477B (en) | 2020-09-30 | 2020-09-30 | Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112111477A true CN112111477A (en) | 2020-12-22 |
| CN112111477B CN112111477B (en) | 2022-08-19 |
Family
ID=73797472
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011059690.XA Active CN112111477B (en) | 2020-09-30 | 2020-09-30 | Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112111477B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278598A (en) * | 2021-04-25 | 2021-08-20 | 山东大学 | Biological enzyme system for preparing drug metabolites and application thereof |
| CN113774033A (en) * | 2021-06-11 | 2021-12-10 | 合肥生融生物科技有限公司 | Artificial biocatalyst for converting fatty acid into beta-hydroxy fatty acid |
| CN114058650A (en) * | 2021-11-16 | 2022-02-18 | 北京化工大学 | Method for preparing epoxy vegetable oil by double-enzyme coupling reaction system |
| CN114921427A (en) * | 2022-05-07 | 2022-08-19 | 扬州大学 | Artificial enzyme with wide substrate spectrum for specifically converting carboxylic acid into chiral beta-hydroxycarboxylic acid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779476A (en) * | 2015-06-30 | 2018-11-09 | 道达尔销售服务公司 | The production of alpha-olefin |
-
2020
- 2020-09-30 CN CN202011059690.XA patent/CN112111477B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779476A (en) * | 2015-06-30 | 2018-11-09 | 道达尔销售服务公司 | The production of alpha-olefin |
Non-Patent Citations (1)
| Title |
|---|
| SARAH MATTHEWS ET AL.,: ""Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE"", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278598A (en) * | 2021-04-25 | 2021-08-20 | 山东大学 | Biological enzyme system for preparing drug metabolites and application thereof |
| CN113278598B (en) * | 2021-04-25 | 2022-08-30 | 山东大学 | Biological enzyme system for preparing drug metabolites and application thereof |
| CN113774033A (en) * | 2021-06-11 | 2021-12-10 | 合肥生融生物科技有限公司 | Artificial biocatalyst for converting fatty acid into beta-hydroxy fatty acid |
| CN113774033B (en) * | 2021-06-11 | 2023-10-24 | 合肥生融生物科技有限公司 | Artificial biocatalyst for converting fatty acid into beta-hydroxy fatty acid |
| CN114058650A (en) * | 2021-11-16 | 2022-02-18 | 北京化工大学 | Method for preparing epoxy vegetable oil by double-enzyme coupling reaction system |
| CN114921427A (en) * | 2022-05-07 | 2022-08-19 | 扬州大学 | Artificial enzyme with wide substrate spectrum for specifically converting carboxylic acid into chiral beta-hydroxycarboxylic acid |
| CN114921427B (en) * | 2022-05-07 | 2023-08-01 | 扬州大学 | Artificial enzyme with wide substrate spectrum for converting carboxylic acid into chiral beta-hydroxy carboxylic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112111477B (en) | 2022-08-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112111477B (en) | Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide | |
| US10053713B2 (en) | Biological alkane oxidation | |
| EP2922963B1 (en) | Process for producing alpha,omega-diols from c4-alkanes or c4-1-alkanols employing a cyp153 alkane hydroxylase | |
| CN108467861B (en) | Sequence for catalytically synthesizing linear alpha-olefin biocatalyst OleT-BM3R, preparation method and application thereof | |
| JP2011526489A (en) | Process for producing alkenes by enzymatic decarboxylation of 3-hydroxyalkanoic acids | |
| EP3169789B1 (en) | Process for producing alkanes using microorganisms combined with kolbe synthesis | |
| JPWO2007142210A1 (en) | Method for producing optically active alcohol | |
| Guo et al. | Efficient (3R)-acetoin production from meso-2, 3-butanediol using a new whole-cell biocatalyst with co-expression of meso-2, 3-butanediol dehydrogenase, NADH oxidase, and Vitreoscilla hemoglobin | |
| EP3061827A1 (en) | Alkene production | |
| Schneider et al. | Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM‐3 monooxygenase under varied dissolved oxygen concentrations | |
| EP3230462B1 (en) | Genetically modified phenylpyruvate decarboxylase, processes to prepare, and uses thereof | |
| CN110551771B (en) | Synthesis method of chiral 3-amino-1-butanol | |
| Sudheer et al. | Screening, expression, and characterization of Baeyer-Villiger monooxygenases for the production of 9-(nonanoyloxy) nonanoic acid from oleic acid | |
| CN113151230A (en) | Mutant protein of formaldehyde lyase and application thereof | |
| Cao et al. | Metabolic engineering of Escherichia coli for the production of hydroxy fatty acids from glucose | |
| Buathong et al. | Biotransformation of lauric acid into 1, 12-dodecanedioic acid using CYP52A17 expressed in Saccharomyces cerevisiae and its application in refining coconut factory wastewater | |
| CN108410831B (en) | Ketoacid reductase, gene, engineering bacterium and application in synthesis of chiral aromatic 2-hydroxy acid | |
| KR101808381B1 (en) | An Enzymatic production method of methanol from carbon dioxide by polyenzymatic cascade reaction and a composition therefor | |
| Lee et al. | Whole cell bioconversion of ricinoleic acid to 12-ketooleic acid by recombinant Corynebacterium glutamicum-based biocatalyst | |
| CN111411128A (en) | Whole cell biocatalysis method for producing α omega-dicarboxylic acid and application thereof | |
| Jang et al. | Whole cell biotransformation of 1-dodecanol by Escherichia coli by soluble expression of ADH enzyme from Yarrowia lipolytica | |
| CN113774033B (en) | Artificial biocatalyst for converting fatty acid into beta-hydroxy fatty acid | |
| CN111944774B (en) | Alcohol dehydrogenase, encoding gene thereof and application of alcohol dehydrogenase in catalytic synthesis of (R) -styrene glycol | |
| US9909154B2 (en) | Methods for producing dicarboxylic acids | |
| CN114921427B (en) | Artificial enzyme with wide substrate spectrum for converting carboxylic acid into chiral beta-hydroxy carboxylic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |