CN104561152A - Method for catalytically synthesizing fatty olefin based on coupled catalysis of lipase and P450 fatty acid decarboxylase - Google Patents
Method for catalytically synthesizing fatty olefin based on coupled catalysis of lipase and P450 fatty acid decarboxylase Download PDFInfo
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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
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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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
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
Abstract
The invention belongs to a biological preparation method of fatty olefin in the technical fields of bioengineering and energy, and specifically discloses a method for catalytically synthesizing fatty olefin based on coupled catalysis of lipase and P450 fatty acid decarboxylase. Glyceride is used as a raw material, free fatty acid is generated by coupled catalysis of lipase hydrolysis and P450 decarboxylation, namely hydrolysis of the glyceride with lipase, and the generated fatty acid is subjected to P450 decarboxylation to produce the fatty olefin. The lipase and P450 fatty acid decarboxylase mediated in-vitro coupled bio-catalysis process only needs two enzymatic catalysis steps, so the reaction conditions and the enzyme proportion are easily controlled, the substrate conversion efficiency is high, the energy consumption is low, and the method is pollution-free, is a new green catalysis method for fatty olefin with controllable processes and low cost and has a good application prospect.
Description
Technical field
The invention belongs to the biological preparation method of fatty alkene in biotechnology and energy technology field, is a kind of method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme specifically.
Background technology
Along with the continuous consumption of the traditional fossil energy such as oil, becoming increasingly conspicuous of the problem such as energy resource safety strategy and environment protection, countries in the world are all raw material production biofuel and chemical actively attempting with renewable biological source.Be the tradition of representative and novel advanced biological liquid fuel with short chain alcohol, fatty acid methyl ester (ethyl ester), aliphatic hydrocarbon (comprising alkene and alkane) etc., just attract increasing concern.In biofuel, alcohol fuel and the exploitation relative maturity with fatty acid methyl ester (ethyl ester) first-generation biofuel that is representative, but all there is respective deficiency.As the substitute of gasoline, there is water-soluble height, high volatility in alcohol fuel, the shortcomings such as energy density is low.Compare with first-generation Biodiesel Fatty acid methyl esters (ethyl ester) with short chain alcohol fuel (as ethanol), as the aliphatic hydrocarbon structure of advanced biofuel (Advancedbiofuels) and character closer to petrifaction diesel, there is energy density high, water absorbability is low, the advantageous characteristic such as efficiency of combustion is high, can as the substitute of gasoline, diesel oil and aviation fuel or additive.Therefore, aliphatic hydrocarbon catalyzes and synthesizes Study of way becomes the large focus in advanced professor Eugene C. Koo field one.
The preparation of current aliphatic hydrocarbon depends on the hydrogenation chemical technology of the noble metal catalyst mediations such as platinum palladium under high temperature (250-450 DEG C) high pressure (20-70bar) condition.Compared with traditional chemical technique, biocatalysis and synthesis have efficiently, the advantages such as energy consumption is low, environmental protection.So far, some aliphatic hydrocarbon biosynthetic pathways are reported successively:
(1) 2010 year, the Schirmer etc. of LS9 bioenergy company of the U.S. identified the biosynthetic pathway that acyl-ACP is converted into fat alkane or fatty alkene by the acyl-ACP reductase enzyme of cyanobacteria Synechococcus elongates PCC 7942 and alkanoic decarbonylation base enzyme.
(2) 2010 years, Bel ler etc. identifies the long-chain olefin biosynthetic pathway based on OleA enzyme catalysis decarboxylation condensation reaction in bacterium Micrococcus luteus ATCC 4698, can by acyl-CoA by decarboxylation condensation, ketone group hydrogenating reduction, a series of biochemical reaction such as hydroxyls dehydrate generates the fatty alkene containing internal double bonds.
(3) 2011 years, Pfleger etc. identified the fatty alkene biosynthetic pathway based on cyanobacteria Synechococcus sp.PCC 7002Ols polyketide synthases generating strap terminal double link.Longer chain fatty acid acyl carrier protein ACP1 generates the fatty alkene with terminal double link by the enzymatic a series of biochemical reaction of ketosynthase, acyltransferase, keto reductase, sulfotransferase and thioesters.
(4) 2011 years, Ls9 Inc. Rude etc. reported based on Cytochrome P450 decarboxylase (OleT in bacterium Jeotagalicoccus sp.ATCC 8456
jE) catalyze fatty acid decarboxylation generates the reaction of alkene.This P450 (OleT
jE) at cofactor hydrogen peroxide (H
2o
2) lipid acid is generated under existent condition the fatty alkene with terminal double link by decarboxylic reaction.
(5) 2013 years, Akhtar etc. constructed the aliphatic hydrocarbon biosynthetic pathway be made up of thioesterase, carboxylate reductase and alkanoic decarbonylation base enzyme, and acyl-ACP is converted into aliphatic hydrocarbon.
Although the exploitation of biofuel solves the problems such as the renewable and environmental protection of raw material, it is one of main bottleneck of producing of its industrial scale of restriction that cost remains high.These costs comprise raw materials cost, catalyzer cost and production process cost, and wherein raw materials cost is particularly crucial.The above-mentioned aliphatic hydrocarbon biosynthetic pathway reported is based on fatty acid metabolism approach mostly, with the fatty acid form (acyl-ACP or acyl-CoA) of free lipid acid (Free fattyacid) or acyl for direct material.Lipid acid de novo synthesis (De novo biosynthesis) relates to the metabolism network of multistep enzymatic reaction composition, and the utilization ratio of starting material (sugar etc.) conversions of fats acid is lower.Key gene in process LAN fatty acid synthesis pathway and knock out the metabolic engineerings (Metabolic engineering) such as key gene in fatty acid oxidation pathway although means can improve the accumulation volume of lipid acid to a certain extent, but due to accuracy and the complicacy of fatty acid metabolism regulated and control network, the metabolism burden of host cell can be increased by key enzyme more in process LAN fatty acid metabolism approach, strengthen by genetic modification the uncontrollable factor that lipid acid synthesis can increase fatty acid metabolism network simultaneously.Be familiar with comprehensive system, utilize and transform fatty acid metabolism network difficulty comparatively greatly, make the limited space that lipid acid accumulation volume promotes further.Therefore based on fatty acid metabolism approach, be the high cost that direct material produces fatty alkene with lipid acid, constrain its commercial application.
Lipase (Lipase), can the hydrolysis of catalysis natural substrate glyceride, the lipid acid that generation is free and glycerine as typical carboxylic acid lytic enzyme.In the animal-plant oil that glyceryl ester is extensively present in occurring in nature and microbial oil, aboundresources, price is cheaper relative to free fatty acids.Through retrieval, take glyceryl ester as raw material, there is not yet any report based on enzymatic aliphatic hydrocarbon process for catalytic synthesis.
Summary of the invention
The object of the present invention is to provide a kind of method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme
The technical solution used in the present invention is for achieving the above object:
A kind of method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme, using glyceryl ester as raw material, coupling catalysed by lipase hydrolysis and P450 decarboxylation, namely lipase hydrolysis glyceryl ester generates free fatty acids, and the lipid acid of generation produces fatty alkene through P450 decarboxylation again.
Further, the grease added with external source, for substrate, with the hydrogen peroxide of external source supply for cofactor, reacts 0.5-48h under the effect of catalyzer under 20-50 DEG C of condition, coupling catalysed preparation fat alkene;
Described catalyzer is the pure enzyme of the full cell of tranquillization state of the growth state cell culture fluid of recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme, recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme or the acellular crude extract of recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme or recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme.
Further, the hydrogen peroxide (0.3% take concentration as 0.2-100mM grease being 0.2-300mM as substrate, concentration, v/v) as cofactor, under the effect of catalyzer, coupled catalytic reaction is react under the buffer solution system of 4-12 at pH, add after reaction and contain interior target ethyl acetate or n-hexane extraction analysis with reactant equal-volume, it is 10-85% that grease is converted into fatty conversion of olefines rate;
In described catalyzer, lipase and P450 decarboxylation of fatty acids enzyme mol ratio are 1:100-100:1.
Described grease is the grease of plant and animal material, microbe-derived grease or the waste grease containing high acid value;
Described lipase is lipase grease to hydrolytic action;
Described P450 decarboxylation of fatty acids enzyme is enzyme lipid acid to decarboxylation activity.
Described grease is soybean oil, rapeseed oil, Semen Maydis oil, Oleum Gossypii semen, micro-algae oil, yeast source grease, fish oil, or sewer oil;
Described lipase is Thermomyces lanuginosus (Thermomyces lanuginosus) lipase Tl l, aspergillus niger (Aspergillus niger) lipase, geotrichum candidum (Geotrichum candidum) lipase, fold candida (Candida rugosa) lipase, antarctic candida (Candidaantarctica) lipase, aspergillus oryzae (Aspergillus oryzae) lipase, Rhizopus oryzae (Rhizopus oryzae) lipase, Yarrowia lipolytica (Yarrowia lipolylica) lipase, conspicuous Mucor (Mucor miehe) lipase of rice, serratia marcescens (Serratiamarcescens) lipase, Rhodopseudomonas (Pseudomonas sp.) lipase or bacillus (Bacillus sp.) lipase,
Described P450 decarboxylation of fatty acids enzyme is for coming from Jeotgalicoccus sp.) OleT
jEor derive from the P450 of Bacillus subtilis
bS β.
Described catalyzer obtains as follows:
1) by building the recombinant plasmid containing object lipase gene, transforming recombinant plasmid to competent escherichia coli cell, obtaining the recombination bacillus coli engineering strain of expressing lipase and P450 decarboxylation of fatty acids enzyme; The Recombinant organism strain obtained is fermented by two benches as starting strain;
2) above-mentioned fermented liquid is growth state cell culture fluid as catalyzer;
Above-mentioned fermented liquid collects the recombinant Bacillus coli cells of process LAN lipase and P450 decarboxylation of fatty acids enzyme by centrifugation, is the full cell of tranquillization state as catalyzer;
Above-mentioned fermentation liquor sonicated cells, obtains the acellular crude extract of lipase and P450 decarboxylation of fatty acids enzyme as catalyzer;
Or above-mentioned fermented liquid utilizes the pure enzyme of affinity chromatography gained of recombinase His-tag and Ni-NTA as catalyzer.
Above-mentioned employing inducible plasmid cuts with lipase or P450 decarboxylation of fatty acids enzyme the recombinant plasmid built respectively with enzyme mode even respectively by enzyme, then the recombinant plasmid transformed competent escherichia coli cell will obtained respectively, obtains the high expression lipase of structure or the genetic engineering bacterium of P450 decarboxylation of fatty acids enzyme respectively;
By the genetic engineering bacterium of above-mentioned acquisition by the Fungal biodiversity accumulation before induction, until cell density reaches OD
600=0.6-0.8, and after through the two benches fermentation of the expression of enzymes of IPTG induction, and keep the further growth of thalline.
Described inducible plasmid is the pET plasmid series controlled by T7 promotor; As pRSFDuet1 or pET28b or pET22b, described intestinal bacteria are Escherichia coli BL21 (DE3).
The engineering bacteria of above-mentioned acquisition is spent the night to be cultured in 37 DEG C under the concussion of the rotating speed of 200-250rpm respectively in LB substratum, continue after spending the night at the temperature of 37 DEG C, nutrient solution is reached OD to be cultured to cell density under the concussion of the rotating speed of 200-250rpm according to the inoculum size of 1-3 (v/v) % in LB substratum
600=0.6-0.8; And adding 0.1-1mM IPTG in backward substratum in 18-20 DEG C, the abduction delivering time is 18-20h, namely obtains the fermented liquid of different engineering bacteria respectively;
The microbiotic needed for 25-100 μ g/ml stable plasmid is added in the LB substratum that described engineering bacteria is cultivated for lipase gene engineering bacterium fermentation;
The microbiotic needed for 25-100 μ g/ml stable plasmid and 1mM vitamin B12 is added in the LB substratum that the engineering bacteria fermentation that described engineering bacteria is P450 decarboxylation of fatty acids enzyme is cultivated.
Microbiotic needed for described stable plasmid is not all penbritin, kantlex, paraxin or Streptomycin sulphate according to plasmid.
The present invention has advantage:
The present invention is using glyceryl ester cheap and easy to get as raw material, by the method for the coupling catalysed fatty alkene synthesis of lipase hydrolysis and P450 decarboxylase, relative to traditional hydrogenation chemical technology and the from the beginning biosynthetic pathway of aliphatic hydrocarbon, only need two step enzyme-catalyzed change steps, reaction conditions and enzyme ratio easily regulate and control, and substrate conversion efficiency is high, energy consumption is low, pollution-free, be a kind of process control, the emerging green catalysis system of fatty alkene with low cost, there is good application prospect.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1
The method of the present embodiment lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzyme is:
(1) structure of genetic engineering bacterium: by enzyme cut with enzyme connect mode by pRSFDuet1 plasmid respectively with Thermomyces lanuginosus (Thermomyces lanuginosus) lipase Tl l gene or the OleT deriving from Jeotgalicoccus sp.
jEgene constructed recombinant plasmid, gained transforms recombinant plasmid to competent escherichia coli cell Escherichia coli BL21 (DE3), adopt the LB resistant panel screening positive transformant containing 50 μ g/ml kantlex, obtain the recombination bacillus coli engineering strain of high expression lipase or P450 decarboxylation of fatty acids enzyme respectively.
(2) preparation of lipase and the pure enzyme of P450 decarboxylation of fatty acids enzyme: by the recombination engineering strain list colony inoculation of above-mentioned screening in the 250ml shaking flask that 20ml LB substratum is housed, in shaking speed 250rpm, 37 DEG C of overnight incubation, seed liquor of spending the night being seeded to according to 1% (v/v) inoculum size is equipped with in the 2L shaking flask of 500ml LB, in 37 DEG C, 250rpm shaking table is cultivated, and treats that cell density reaches OD
600during=0.6-0.8, add 0.2mM IPTG, start enzyme induction in 20 DEG C and express, and keep the further growth of thalline until 20h.
Described engineering bacteria is add 50 μ g/ml kantlex in the LB substratum of lipase gene engineering bacterium fermentation cultivation;
Described engineering bacteria is add 50 μ g/ml kantlex and 1mM vitamin B12 in the LB substratum of the engineering bacteria fermentation cultivation of P450 decarboxylation of fatty acids enzyme.
The fermented liquid of above-mentioned acquisition is collected respectively the recombinant Bacillus coli cells of intracellular expression lipase or P450 decarboxylation of fatty acids enzyme by centrifugation, then sonicated cells, obtain the acellular crude extract of lipase or P450 decarboxylation of fatty acids enzyme respectively, the acellular crude extract obtained respectively utilizes the affinity chromatography of recombinase His-tag and Ni-NTA to prepare respective pure enzyme, as catalyzer.
(3) lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzyme: the 0.5mM soya-bean oil added with external source is substrate, with 0.3% hydrogen peroxide (v/v) of the 1mM of external source supply for cofactor, using the pure enzyme of above-mentioned gained as catalyzer, wherein in pure enzyme, the lipase of purifying is 2 μMs, the P450 decarboxylation of fatty acids enzyme of purifying is 4 μMs, reaction medium to be pH be 7.8 phosphate buffered saline buffer, 6h is reacted under 30 DEG C of water bath condition, the decarboxylic reaction of the hydrolysis reaction coupling P450 decarboxylation of fatty acids enzyme mediation mediated by lipase, prepares fatty alkene.After linked reaction terminates, add and contain target extraction into ethyl acetate analysis in ten seven-carbon fatty acids with reaction system equal-volume, adopt marker method to calculate, the transformation efficiency that soya-bean oil is converted into fatty alkene (15 carbon and heptadecene hydrocarbon) is 31%.
Embodiment 2
The method of the present embodiment lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzyme is:
(1) structure of genetic engineering bacterium: cut the OleT that enzyme connects pRSFDuet1 plasmid and Thermomyces lanuginosus (Thermomyces lanuginosus) lipase Tl l gene or derive from Jeotgalicoccus sp. by enzyme
jEgene constructed recombinant plasmid, transform recombinant plasmid to competent escherichia coli cell Escherichia coli BL21 (DE3), adopt the LB resistant panel screening positive transformant containing 50 μ g/ml kantlex, obtain the recombination bacillus coli engineering strain of high expression lipase and P450 decarboxylation of fatty acids enzyme respectively.
(2) preparation of lipase and the acellular crude extract of P450 decarboxylation of fatty acids enzyme: by the recombination engineering strain list colony inoculation of above-mentioned screening in the 250ml shaking flask that 20ml LB substratum is housed, in shaking speed 250rpm, 37 DEG C of overnight incubation, seed liquor of spending the night being seeded to according to 1% (v/v) inoculum size is equipped with in the 2L shaking flask of 500ml LB, in 37 DEG C, 250rpm shaking table is cultivated, and treats that cell density reaches OD
600during=0.6-0.8, add 0.2mM IPTG, start enzyme induction in 20 DEG C and express, and keep the further growth of thalline until 20h.
Described engineering bacteria is add 50 μ g/ml kantlex in the LB substratum of lipase gene engineering bacterium fermentation cultivation;
Described engineering bacteria is add 50 μ g/ml kantlex and 1mM vitamin B12 in the LB substratum of the engineering bacteria fermentation cultivation of P450 decarboxylation of fatty acids enzyme.
The fermented liquid of above-mentioned acquisition is collected respectively the recombinant Bacillus coli cells of intracellular expression lipase or P450 decarboxylation of fatty acids enzyme by centrifugation, namely obtain the full cell of tranquillization state of two kinds of enzymes respectively as catalyzer.
(3) lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzyme: micro-algae oil (0.5mM) of adding with external source is for substrate, with 0.3% (v/v) hydrogen peroxide (1.5mM) of external source supply for cofactor, with tranquillization state Whole-cell lipase (20mg) and tranquillization state full cell P450 decarboxylation of fatty acids enzyme (30mg) for mixed catalyst, reaction medium to be pH be 7.8 phosphate buffered saline buffer, 12h is reacted under 30 DEG C of water bath condition, the decarboxylic reaction of the hydrolysis reaction coupling P450 decarboxylation of fatty acids enzyme mediation mediated by lipase, prepare fatty alkene.After linked reaction terminates, add equal-volume and contain target extraction into ethyl acetate analysis in ten seven-carbon fatty acids, adopt marker method to calculate, the transformation efficiency that micro-algae oil is converted into fatty alkene (15 carbon and heptadecene hydrocarbon) is 20%.
Embodiment 3
(1) structure of genetic engineering bacterium: cut enzyme by enzyme and connect pRSFDuet1 plasmid and Thermomyces lanuginosus (Thermomyces lanuginosus) lipase Tl l gene fragment builds pRSFDuet-tll recombinant plasmid, enzyme is cut enzyme and is connected pACYCDuet1 plasmid and the OleT deriving from Jeotgalicoccus sp.
jEgene fragment construction recombination plasmid pACYCDuet-oleT
jEby two recombinant plasmid cotransformation competent escherichia coli cells Escherichia coli BL21 (DE3), adopt the LB resistant panel containing 50 μ g/ml kantlex and 25 μ g/ml paraxin to screen positive transformant, obtain the recombination bacillus coli engineering strain of efficient coexpression lipase and P450 decarboxylation of fatty acids enzyme.
(2) preparation of lipase and the acellular crude extract of P450 decarboxylation of fatty acids enzyme: by the recombination engineering strain list colony inoculation of above-mentioned screening in the 250ml shaking flask that 20ml LB substratum is housed, in shaking speed 250rpm, 37 DEG C of overnight incubation, seed liquor of spending the night being seeded to according to 1% (v/v) inoculum size is equipped with in the 2L shaking flask of 500ml LB, in 37 DEG C, 250rpm shaking table is cultivated, and treats that cell density reaches OD
600during=0.6-0.8, add 0.2mM IPTG, start enzyme induction in 20 DEG C and express, and keep the further growth of thalline until 20h.
Described engineering bacteria is add 50 μ g/ml kantlex and 25 μ g/ml paraxin in the LB substratum of lipase gene engineering bacterium fermentation cultivation;
Described engineering bacteria is add 50 μ g/ml kantlex and 25 μ g/ml paraxin and 1mM vitamin B12 in the LB substratum cultivated of the engineering bacteria fermentation of P450 decarboxylation of fatty acids enzyme.
The fermented liquid of above-mentioned acquisition is collected respectively the recombinant Bacillus coli cells of intracellular expression lipase or P450 decarboxylation of fatty acids enzyme by centrifugation, namely obtain the full cell of tranquillization state of two kinds of enzymes respectively as catalyzer.
(3) lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzyme: micro-algae oil (0.5mM) of adding with external source is for substrate, with 0.3% (v/v) hydrogen peroxide (1.5mM) of external source supply for cofactor, with the full cell of tranquillization state (50mg) of two kinds of enzyme coexpressions for catalyzer, reaction medium to be pH be 7.8 phosphate buffered saline buffer, 12h is reacted under 30 DEG C of water bath condition, the decarboxylic reaction of the hydrolysis reaction coupling P450 decarboxylation of fatty acids enzyme mediation mediated by lipase, prepares fatty alkene.After linked reaction terminates, add equal-volume and contain target extraction into ethyl acetate analysis in ten seven-carbon fatty acids, adopt marker method to calculate, the transformation efficiency that micro-algae oil is converted into fatty alkene (15 carbon and heptadecene hydrocarbon) is 30%.
Embodiment 4
(1) structure of genetic engineering bacterium: cut enzyme by enzyme and connect pET22b plasmid and Thermomyces lanuginosus (Thermomyces lanuginosus) lipase Tl l gene fragment builds pET22b-tll recombinant plasmid, enzyme is cut enzyme and is connected pACYCDuet1 plasmid and the OleT deriving from Jeotgalicoccus sp.
jEgene fragment construction recombination plasmid pACYCDuet-oleT
jEby two recombinant plasmid cotransformation competent escherichia coli cells Escherichia coli BL21 (DE3), adopt the LB resistant panel containing 100 μ g/ml penbritins and 25 μ g/ml paraxin to screen positive transformant, obtain the recombination bacillus coli engineering strain of efficient coexpression lipase and P450 decarboxylation of fatty acids enzyme.
(2) preparation of lipase and the acellular crude extract of P450 decarboxylation of fatty acids enzyme: by the recombination engineering strain list colony inoculation of above-mentioned screening in the 250ml shaking flask that 20ml LB substratum is housed, in shaking speed 250rpm, 37 DEG C of overnight incubation, seed liquor of spending the night being seeded to according to 1% (v/v) inoculum size is equipped with in the 2L shaking flask of 500ml LB, in 37 DEG C, 250rpm shaking table is cultivated, and treats that cell density reaches OD
600during=0.6-0.8, add 0.2mM IPTG, start enzyme induction in 20 DEG C and express, and keep the further growth of thalline until 12h.
Described engineering bacteria is add 100 μ g/ml penbritins and 25 μ g/ml paraxin in the LB substratum of lipase gene engineering bacterium fermentation cultivation;
Described engineering bacteria is add 100 μ g/ml penbritins and 25 μ g/ml paraxin and 1mM vitamin B12 in the LB substratum cultivated of the engineering bacteria fermentation of P450 decarboxylation of fatty acids enzyme.
By the fermented liquid of the growth state cell culture fluid of the above-mentioned coexpression obtained respectively, as situ catalytic agent.
(3) lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzyme: the sweet oil (0.5mM) added with external source is substrate, with 0.3% (v/v) hydrogen peroxide (1.5mM) of external source supply for cofactor, with above-mentioned growth in situ state cell culture fluid for catalyzer, 36h is reacted under 28 DEG C of water bath condition, the decarboxylic reaction of the hydrolysis reaction coupling P450 decarboxylation of fatty acids enzyme mediation mediated by lipase, prepares fatty alkene.After linked reaction terminates, add equal-volume and contain target extraction into ethyl acetate analysis in ten seven-carbon fatty acids, adopt marker method to calculate, the transformation efficiency that sweet oil is converted into fatty alkene (15 carbon and heptadecene hydrocarbon) is 23%.
Claims (10)
1. a method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme, it is characterized in that: using glyceryl ester as raw material, coupling catalysed by lipase hydrolysis and P450 decarboxylation, namely lipase hydrolysis glyceryl ester generates free fatty acids, and the lipid acid of generation produces fatty alkene through P450 decarboxylation again.
2. by method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme according to claim 1, it is characterized in that: the grease added with external source is for substrate, with the hydrogen peroxide of external source supply for cofactor, under 20-50 DEG C of condition, 0.5-48h is reacted, coupling catalysed preparation fat alkene under the effect of catalyzer;
Described catalyzer is the pure enzyme of the full cell of tranquillization state of the growth state cell culture fluid of recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme, recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme or the acellular crude extract of recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme or recombinant expressed lipase and P450 decarboxylation of fatty acids enzyme.
3. by method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme according to claim 2, it is characterized in that: the hydrogen peroxide (0.3% take concentration as 0.2-100mM grease being 0.2-300mM as substrate, concentration, v/v) as cofactor, under the effect of catalyzer, coupled catalytic reaction is react under the buffer solution system of 4-12 at pH, add after reaction and contain interior target ethyl acetate or n-hexane extraction analysis with reactant equal-volume, it is 10-85% that grease is converted into fatty conversion of olefines rate;
In described catalyzer, lipase and P450 decarboxylation of fatty acids enzyme mol ratio are 1:100-100:1.
4. by the method that the fatty olefin catalytic coupling catalysed based on lipase and P450 decarboxylation of fatty acids enzyme described in claim 1,2 or 3 synthesizes, it is characterized in that: described grease is the grease of plant and animal material, microbe-derived grease or contain the waste grease of high acid value;
Described lipase is lipase grease to hydrolytic action;
Described P450 decarboxylation of fatty acids enzyme is enzyme lipid acid to decarboxylation activity.
5. by method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme according to claim 4, it is characterized in that: described grease is soybean oil, rapeseed oil, Semen Maydis oil, Oleum Gossypii semen, micro-algae oil, yeast source grease, fish oil, or sewer oil;
Described lipase is Thermomyces lanuginosus (Thermomyces lanuginosus) lipase Tll, aspergillus niger (Aspergillus niger) lipase, geotrichum candidum (Geotrichum candidum) lipase, fold candida (Candida rugosa) lipase, antarctic candida (Candidaantarctica) lipase, aspergillus oryzae (Aspergillus oryzae) lipase, Rhizopus oryzae (Rhizopus oryzae) lipase, Yarrowia lipolytica (Yarrowia lipolylica) lipase, conspicuous Mucor (Mucor miehe) lipase of rice, serratia marcescens (Serratiamarcescens) lipase, Rhodopseudomonas (Pseudomonas sp.) lipase or bacillus (Bacillus sp.) lipase,
Described P450 decarboxylation of fatty acids enzyme is for coming from Jeotgalicoccus sp.) OleT
jEor derive from the P450 of Bacillus subtilis
bS β.
6., by the method for synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme described in claim 1,2 or 3, it is characterized in that: described catalyzer obtains as follows:
1) by building the recombinant plasmid containing object lipase gene, transforming recombinant plasmid to competent escherichia coli cell, obtaining the recombination bacillus coli engineering strain of expressing lipase and P450 decarboxylation of fatty acids enzyme; The Recombinant organism strain obtained is fermented by two benches as starting strain;
2) above-mentioned fermented liquid is growth state cell culture fluid as catalyzer;
Above-mentioned fermented liquid collects the recombinant Bacillus coli cells of process LAN lipase and P450 decarboxylation of fatty acids enzyme by centrifugation, is the full cell of tranquillization state as catalyzer;
Above-mentioned fermentation liquor sonicated cells, obtains the acellular crude extract of lipase and P450 decarboxylation of fatty acids enzyme as catalyzer;
Or above-mentioned fermented liquid utilizes the pure enzyme of affinity chromatography gained of recombinase His-tag and Ni-NTA as catalyzer.
7., by method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme according to claim 6, it is characterized in that:
Inducible plasmid is adopted to be cut with lipase or P450 decarboxylation of fatty acids enzyme the recombinant plasmid built respectively with enzyme mode even by enzyme respectively, then the recombinant plasmid transformed competent escherichia coli cell will obtained respectively, obtains the high expression lipase of structure or the genetic engineering bacterium of P450 decarboxylation of fatty acids enzyme respectively;
By the genetic engineering bacterium of above-mentioned acquisition by the Fungal biodiversity accumulation before induction, until cell density reaches OD
600=0.6-0.8, and after through the two benches fermentation of the expression of enzymes of IPTG induction, and keep the further growth of thalline.
8., by method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme according to claim 7, it is characterized in that:
Described inducible plasmid is the pET plasmid series controlled by T7 promotor; Described intestinal bacteria are Escherichia coli BL21 (DE3).
9., by method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme according to claim 7, it is characterized in that:
The engineering bacteria of above-mentioned acquisition is spent the night to be cultured in 37 DEG C under the concussion of the rotating speed of 200-250rpm respectively in LB substratum, continue after spending the night at the temperature of 37 DEG C, nutrient solution is reached OD to be cultured to cell density under the concussion of the rotating speed of 200-250rpm according to the inoculum size of 1-3 (v/v) % in LB substratum
600=0.6-0.8; And adding 0.1-1mM IPTG in backward substratum in 18-20 DEG C, the abduction delivering time is 18-20h, namely obtains the fermented liquid of different engineering bacteria respectively;
The microbiotic needed for 25-100 μ g/ml stable plasmid is added in the LB substratum that described engineering bacteria is cultivated for lipase gene engineering bacterium fermentation;
The microbiotic needed for 25-100 μ g/ml stable plasmid and 1mM vitamin B12 is added in the LB substratum that the engineering bacteria fermentation that described engineering bacteria is P450 decarboxylation of fatty acids enzyme is cultivated.
10., by method of synthesizing based on lipase and the coupling catalysed fatty olefin catalytic of P450 decarboxylation of fatty acids enzyme according to claim 9, it is characterized in that: the microbiotic needed for described stable plasmid is penbritin, kantlex, paraxin or Streptomycin sulphate.
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| CN106318607A (en) * | 2016-08-31 | 2017-01-11 | 中国科学院青岛生物能源与过程研究所 | Preparation method and application of microalgal acid oil |
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| CN108779476A (en) * | 2015-06-30 | 2018-11-09 | 道达尔销售服务公司 | The production of alpha-olefin |
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| CN106318607B (en) * | 2016-08-31 | 2020-03-17 | 中国科学院青岛生物能源与过程研究所 | Preparation method and application of microalgae acidified oil |
| US10220378B2 (en) | 2017-06-01 | 2019-03-05 | Uchicago Argonne, Llc | Semiconductor-metal nanoparticle hybrids with natural and artificial proton pump for hydrogen production |
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| CN112877347A (en) * | 2021-01-28 | 2021-06-01 | 华南理工大学 | Multi-enzyme complex and construction method and application thereof |
| CN112877347B (en) * | 2021-01-28 | 2023-02-17 | 华南理工大学 | Multi-enzyme complex and construction method and application thereof |
| CN113621657A (en) * | 2021-07-27 | 2021-11-09 | 华南理工大学 | Method for catalyzing grease hydrolysis decarboxylation by cell-like multiphase emulsion optical enzyme system |
| CN113621657B (en) * | 2021-07-27 | 2022-06-14 | 华南理工大学 | Method for catalyzing grease hydrolysis decarboxylation by cell-like multiphase emulsion optical enzyme system |
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