CN104561152B - It is a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes - Google Patents
It is a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes Download PDFInfo
- Publication number
- CN104561152B CN104561152B CN201510003451.5A CN201510003451A CN104561152B CN 104561152 B CN104561152 B CN 104561152B CN 201510003451 A CN201510003451 A CN 201510003451A CN 104561152 B CN104561152 B CN 104561152B
- Authority
- CN
- China
- Prior art keywords
- lipase
- decarboxylation
- fatty
- fatty acids
- enzymes
- 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.)
- Expired - Fee Related
Links
Classifications
-
- 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
-
- 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
It is specifically a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes the invention belongs to the biological preparation method of fatty alkene in bioengineering and energy technology field.Using glyceride as raw material, by the coupling catalysed of lipase hydrolysis and P450 decarboxylations, i.e. lipase hydrolysis glyceride generates free fatty, and the aliphatic acid of generation generates fatty alkene using P450 decarboxylations.The external coupling living things catalysis process that the lipase of the present invention is mediated with P450 decarboxylation of fatty acids enzymes, only need two step enzyme-catalyzed change steps, reaction condition and enzyme ratio are easy regulation and control, substrate conversion efficiency is high, low energy consumption, it is pollution-free, it is a kind of process control, the emerging green catalysis system of low-cost fatty alkene, there is good application prospect.
Description
Technical field
It is specifically a kind of the invention belongs to the biological preparation method of fatty alkene in bioengineering and energy technology field
Based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes.
Background technology
It is increasingly prominent the problems such as energy resource safety strategy and environmental protection with the continuous consumption of the traditional fossil energies such as oil
Go out, countries in the world all attempt to produce bio-fuel and chemicals by raw material of renewable biological source positive.With short chain alcohol, fat
Fatty acid methyl esters (ethyl ester), aliphatic hydrocarbon (including alkene and alkane) etc. are the traditional and novel advanced biological liquid fuel of representative, just
Attract more and more concerns.In terms of bio-fuel, alcohol fuel and with fatty acid methyl ester (ethyl ester) be representative first
For the exploitation relative maturity of biodiesel, but there is respective deficiency.Substitute of the alcohol fuel as gasoline, there are water-soluble
Property high, high volatility, the disadvantages such as energy density is low.With short chain alcohol ate (such as ethyl alcohol) and first generation biodiesel fatty acid methyl esters
(ethyl ester) is compared, and the aliphatic hydrocarbon structure and property as advanced bio-fuel (Advanced biofuels) are closer to petrochemical industry
Diesel oil has energy density high, and hygroscopicity is low, the advantageous characteristics such as efficiency of combustion height, can be used as gasoline, diesel oil and aviation fuel
Substitute or additive.Therefore, aliphatic hydrocarbon, which catalyzes and synthesizes Study of way, becomes one big hot spot of advanced professor Eugene C. Koo field.
It is expensive that the preparation of aliphatic hydrocarbon at present depends on platinum palladium etc. under the conditions of high temperature (250-450 DEG C) high pressure (20-70bar)
The hydrogenation chemical technology that metallic catalyst mediates.Compared with traditional chemical process, living things catalysis has efficient, energy consumption with synthesis
It is low, it is environmentally friendly the advantages that.So far, some aliphatic hydrocarbon biosynthesis pathways have been reported successively：
(1) 2010 year, the Schirmer etc. of LS9 bioenergies company of the U.S. identified cyanobacteria Synechococcus
Acyl-ACP the reductases and fatty aldehyde decarbonylation base enzyme of elongates PCC 7942 by acyl-ACP be converted into fat alkane or
The biosynthesis pathway of fatty alkene.
(2) 2010 years, Bel ler etc., which are identified, was based on OleA enzymes in bacterium Micrococcus luteus ATCC 4698
Acyl-CoA can be condensed, ketone group hydrogenating reduction by the long-chain olefin biosynthesis pathway of catalytic decarboxylation condensation reaction by decarboxylation,
A series of biochemical reactions such as hydroxyls dehydrate generate the fatty alkene containing internal double bonds.
(3) 2011 years, Pfleger etc. is identified to be closed based on cyanobacteria Synechococcus sp.PCC 7002Ols polyketone
The fatty alkene biosynthesis pathway of end of tape double bond is generated at enzyme.Long chain fatty acids acyl carrier protein ACP1 is closed by ketone group
Being generated at enzyme, acyltransferase, keto reductase, sulfotransferase and a series of biochemical reactions of thioesters enzymatic has end
The fatty alkene of double bond.
(4) 2011 years, Ls9 Inc. Rude etc. was reported based on thin in bacterium Jeotagalicoccus sp.ATCC 8456
Born of the same parents' cytochrome p 450 decarboxylase (OleTJE) it is catalyzed the reaction that decarboxylation of fatty acids generates alkene.P450 (the OleTJE) in co-factor mistake
Hydrogen oxide (H2O2) it is existing under the conditions of aliphatic acid passed through into decarboxylic reaction generate the fatty alkene with terminal double bond.
(5) 2013 years, Akhtar etc. constructed the fat being made of thioesterase, carboxylate reductase and fatty aldehyde decarbonylation base enzyme
Hydrocarbon biosynthesis pathway converts acyl-ACP to aliphatic hydrocarbon.
Although the exploitation of bio-fuel solves the problems such as raw material is renewable and environmentally friendly, but it is to restrict it that cost is high
One of the main bottleneck of industrial scale production.These costs include cost of material, catalyst cost and production process cost,
Wherein cost of material is particularly critical.The above-mentioned aliphatic hydrocarbon biosynthesis pathway reported is to be based on fatty acid metabolism approach mostly,
Fatty acid form (acyl-ACP or acyl-CoA) with free aliphatic acid (Free fatty acid) or acyl is
Direct material.Aliphatic acid de novo formation (De novo biosynthesis) is related to the metabolism network of multistep enzymatic reaction composition,
The utilization rate that starting material (sugar etc.) converts aliphatic acid is relatively low.The key gene and knockout being overexpressed in fatty acid synthesis pathway
The metabolic engineerings such as the key gene in fatty acid oxidation pathway (Metabolic engineering) although means in certain journey
The accumulation of aliphatic acid can be improved on degree, but due to the accuracy and complexity of fatty acid metabolism regulated and control network, pass through overexpression
More key enzyme can increase the metabolism burden of host cell in fatty acid metabolism approach, while strengthen fat by genetic modification
Fat acid synthesizes the uncontrollable factor that can increase fatty acid metabolism network.Recognize to comprehensive system, utilization and fatty acid metabolism be transformed
Network difficulty is larger, keeps the space that aliphatic acid accumulation is further promoted limited.Therefore it is based on fatty acid metabolism approach, with fat
Acid is that the cost of the fatty alkene of direct material production is excessively high, constrains its commercial application.
Lipase (Lipase) is used as typical carboxylic acid hydrolase, can be catalyzed the hydrolysis of natural substrate glyceride, generate trip
From aliphatic acid and glycerine.Glyceride is widely present in the animal and plant fat and microbial grease in nature, resourceful,
Price is cheaper relative to free fatty.Through retrieval, using glyceride as raw material, the aliphatic hydrocarbon based on enzymatic catalyzes and synthesizes
Method is there is not yet any report.
Invention content
The purpose of the present invention is to provide a kind of based on lipase and the coupling catalysed fatty alkene of P450 decarboxylation of fatty acids enzymes
The method that hydrocarbon catalyzes and synthesizes
The technical solution adopted by the present invention is to achieve the above object：
It is a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes, with sweet
Grease is as raw material, and by the coupling catalysed of lipase hydrolysis and P450 decarboxylations, i.e., lipase hydrolysis glyceride generates the fat that dissociates
The aliphatic acid of fat acid, generation generates fatty alkene using P450 decarboxylations.
It is further, using the grease of external source addition as substrate, using the hydrogen peroxide of external source supply as co-factor, to urge
0.5-48h, the fatty alkene of coupling catalysed preparation are reacted under the action of agent under the conditions of 20-50 DEG C；
The catalyst is growth state cell culture fluid, the recombination of the lipase and P450 decarboxylation of fatty acids enzymes of recombinant expression
The lipase of expression and the full cell of tranquillization state of P450 decarboxylation of fatty acids enzymes or the lipase and P450 aliphatic acid of recombinant expression are de-
The pure enzyme of the acellular crude extract of carboxylic acid or the lipase of recombinant expression and P450 decarboxylation of fatty acids enzymes.
Further, using a concentration of 0.2-100mM greases as substrate, the hydrogen peroxide of a concentration of 0.2-300mM
(0.3%, v/v) is used as co-factor, and coupled catalytic reaction is anti-in the case where pH is the buffer solution system of 4-12 under the effect of the catalyst
It answers, ethyl acetate or n-hexane extraction containging interior traget are analyzed in equal volume with reactant for addition after reaction, and grease is converted into fat
The conversion ratio of alkene is 10-85%；
Lipase and P450 decarboxylation of fatty acids enzyme molar ratios are 1 in the catalyst：100-100：1.
The grease is grease, microbe-derived grease or the waste grease containing high acid value of plant and animal material；
The lipase is the lipase for having hydrolysis to grease；
The P450 decarboxylation of fatty acids enzyme is the enzyme for having decarboxylation activity to aliphatic acid.
The grease is soybean oil, rapeseed oil, corn oil, cottonseed oil, microalgae oil, yeast source grease, fish oil or trench
Oil；
The lipase is Thermomyces lanuginosus (Thermomyces lanuginosus) lipase Tl l, black song
Mould (Aspergillus niger) lipase, geotrichum candidum (Geotrichum candidum) lipase, fold candida
(Candida rugosa) lipase, antarctic candida (Candida antarctica) lipase, aspergillus oryzae
(Aspergillus oryzae) lipase, Rhizopus oryzae (Rhizopus oryzae) lipase, Yarrowia lipolytica
(Yarrowia lipolylica) lipase, conspicuous Mucor of rice (Mucor miehe) lipase, serratia marcescens (Serratia
Marcescens) lipase, pseudomonas (Pseudomonas sp.) lipase or bacillus (Bacillus sp.)
Lipase；
The P450 decarboxylation of fatty acids enzyme be derived from Jeotgalicoccus sp.) OleTJEOr derive from Bacillus
The P450 of subtilisBSβ。
The catalyst obtains as follows：
1) by building the recombinant plasmid containing purposeful lipase gene, conversion recombinant plasmid is thin to E. coli competent
Born of the same parents obtain the recombination bacillus coli engineering strain of expression lipase and P450 decarboxylation of fatty acids enzymes；The Escherichia coli of acquisition
Engineering strain is fermented as starting strain by two benches；
2) above-mentioned zymotic fluid is to grow state cell culture fluid as catalyst；
Above-mentioned zymotic fluid collects the recombination large intestine bar for being overexpressed lipase and P450 decarboxylation of fatty acids enzymes by centrifuging
Bacterium cell, the as full cell of tranquillization state is as catalyst；
Above-mentioned zymotic fluid through sonicated cells, make by the acellular crude extract for obtaining lipase and P450 decarboxylation of fatty acids enzymes
For catalyst；
Or, above-mentioned zymotic fluid is using pure enzyme obtained by the affinity chromatography of recombinase His-tag and Ni-NTA as catalyst.
It is above-mentioned using inducible plasmid respectively with lipase or P450 decarboxylation of fatty acids enzyme by way of digestion and enzyme company
The recombinant plasmid built respectively, the recombinant plasmid transformed competent escherichia coli cell that then will be obtained respectively, obtains structure respectively
The genetic engineering bacterium of the high efficient expression lipase or P450 decarboxylation of fatty acids enzymes built；
The genetic engineering bacterium of above-mentioned acquisition is accumulated by the Fungal biodiversity before induction, until cell density reaches OD600
=0.6-0.8, and after the two benches of the expression of enzymes through IPTG inductions ferment, and keep the further growth of thalline.
The inducible plasmid is the pET plasmid series controlled by T7 promoters；Such as pRSFDuet1 or pET28b or
PET22b, the Escherichia coli are Escherichia coli BL21 (DE3).
In 37 DEG C of cultures under the engineering bacteria of above-mentioned acquisition is shaken in LB culture mediums with the rotating speed of 200-250rpm respectively
To overnight, overnight after continue at a temperature of 37 DEG C, by culture solution according to the inoculum concentration of 1-3 (v/v) % in LB culture mediums with
Lower culture to the cell density of rotating speed concussion of 200-250rpm reaches OD600=0.6-0.8；And 0.1- is added in backward culture medium
For 1mM IPTG in 18-20 DEG C, the induced expression time is 18-20h, i.e., obtains the zymotic fluid of different engineering bacterias respectively；
The engineering bacteria stablizes matter for 25-100 μ g/ml are added in the LB culture mediums of lipase gene engineering bacterium fermentation culture
Antibiotic needed for grain；
25- is added in the LB culture mediums for the engineering bacteria fermentation culture of P450 decarboxylation of fatty acids enzymes in the engineering bacteria
Antibiotic needed for 100 μ g/ml stable plasmids and 1mM vitamin B12s.
Antibiotic needed for the stable plasmid is not all ampicillin, kanamycins, chloramphenicol or chain according to plasmid
Mycin.
The present invention is had the advantage that：
The present invention passes through the coupling catalysed of lipase hydrolysis and P450 decarboxylases using glyceride cheap and easy to get as raw material
Fatty alkene synthetic method, traditional hydrogenation chemical technology and from the beginning biosynthesis pathway relative to aliphatic hydrocarbon, it is only necessary to two steps
Enzyme-catalyzed change step, reaction condition and enzyme ratio are easy regulation and control, and substrate conversion efficiency is high, and low energy consumption, pollution-free, are that a kind of process can
Control, the emerging green catalysis system of low-cost fatty alkene, have good application prospect.
Specific implementation mode
With reference to embodiment, the present invention will be further described.
Embodiment 1
The present embodiment lipase and the method for the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzymes are：
(1) structure of genetic engineering bacterium：It is in such a way that digestion connects with enzyme that pRSFDuet1 plasmids is thermophilic with thin continuous shape respectively
Hot hyphomycete (Thermomyces lanuginosus) lipase Tl l genes or from Jeotgalicoccus sp.'s
OleTJEGene constructed recombinant plasmid, gained convert recombinant plasmid to competent escherichia coli cell Escherichia coli
BL21 (DE3) screens positive transformant using the LB resistant panels containing 50 μ g/ml kanamycins, obtains high efficient expression respectively
The recombination bacillus coli engineering strain of lipase or P450 decarboxylation of fatty acids enzymes.
(2) preparation of lipase and the pure enzyme of P450 decarboxylation of fatty acids enzymes：By the recombination engineered strain list of above-mentioned screening
In colony inoculation to the 250ml shaking flasks equipped with 20ml LB culture mediums, in shaking speed 250rpm, 37 DEG C of overnight incubations, according to
Overnight seed liquor is seeded in the 2L shaking flasks equipped with 500ml LB by 1% (v/v) inoculum concentration, in 37 DEG C, 250rpm shaking table cultures,
Wait for that cell density reaches OD600When=0.6-0.8,0.2mM IPTG are added, start enzyme induction expression in 20 DEG C, and keep thalline
Further growth until 20h.
The engineering bacteria be lipase gene engineering bacterium fermentation culture LB culture mediums in 50 μ g/ml kanamycins are added；
50 μ g/ are added in the LB culture mediums for the engineering bacteria fermentation culture of P450 decarboxylation of fatty acids enzymes in the engineering bacteria
Ml kanamycins and 1mM vitamin B12s.
The zymotic fluid of above-mentioned acquisition is collected into intracellular expression lipase or P450 decarboxylation of fatty acids respectively by centrifuging
The recombinant Bacillus coli cells of enzyme, then sonicated cells, obtain respectively lipase or P450 decarboxylation of fatty acids enzymes without thin
Born of the same parents' crude extract, the acellular crude extract obtained respectively are prepared respective using the affinity chromatography of recombinase His-tag and Ni-NTA
Pure enzyme, as catalyst.
(3) lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzymes：The 0.5mM soya-bean oil added with external source
For substrate, 0.3% hydrogen peroxide (v/v) of the 1mM supplied with external source is co-factor, using the pure enzyme of above-mentioned gained as catalyst,
The lipase purified in wherein pure enzyme is 2 μM, and the P450 decarboxylation of fatty acids enzymes of purifying are 4 μM, and reaction medium is the phosphorus that pH is 7.8
Phthalate buffer reacts 6h under 30 DEG C of water bath conditions, passes through the hydrolysis that lipase mediates and is coupled P450 decarboxylation of fatty acids enzymes
The decarboxylic reaction of mediation prepares fatty alkene.After waiting for coupling reaction, it is added and contains 17 carbon fat in equal volume with reaction system
Target ethyl acetate extractive analysis, is calculated using internal standard method in fat acid, and soya-bean oil is converted into fatty alkene (15 carbon and heptadecene
Hydrocarbon) conversion ratio be 31%.
Embodiment 2
The present embodiment lipase and the method for the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzymes are：
(1) structure of genetic engineering bacterium：Connect pRSFDuet1 plasmids and Thermomyces lanuginosus by digestion enzyme
(Thermomyces lanuginosus) lipase Tl l genes or OleT from Jeotgalicoccus sp.JEGene
Construction recombination plasmid, conversion recombinant plasmid to competent escherichia coli cell Escherichia coli BL21 (DE3) use
LB resistant panels containing 50 μ g/ml kanamycins screen positive transformant, obtain high efficient expression lipase and P450 fat respectively
The recombination bacillus coli engineering strain of fat acid decarboxylase.
(2) preparation of lipase and the acellular crude extract of P450 decarboxylation of fatty acids enzymes：By the recombination work of above-mentioned screening
Journey bacterial strain single bacterium colony is seeded in the 250ml shaking flasks equipped with 20ml LB culture mediums, and in shaking speed 250rpm, 37 DEG C were cultivated
Overnight seed liquor is seeded in the 2L shaking flasks equipped with 500ml LB by night according to 1% (v/v) inoculum concentration, and in 37 DEG C, 250rpm shakes
Bed culture, waits for that cell density reaches OD600When=0.6-0.8,0.2mM IPTG are added, start enzyme induction expression in 20 DEG C, and protect
The further growth of thalline is held until 20h.
The engineering bacteria be lipase gene engineering bacterium fermentation culture LB culture mediums in 50 μ g/ml kanamycins are added；
50 μ g/ are added in the LB culture mediums for the engineering bacteria fermentation culture of P450 decarboxylation of fatty acids enzymes in the engineering bacteria
Ml kanamycins and 1mM vitamin B12s.
The zymotic fluid of above-mentioned acquisition is collected into intracellular expression lipase or P450 decarboxylation of fatty acids respectively by centrifuging
The recombinant Bacillus coli cells of enzyme obtain the full cell of tranquillization state of two kinds of enzymes as catalyst respectively.
(3) lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzymes：With the microalgae oil of external source addition
(0.5mM) is substrate, with 0.3% (v/v) hydrogen peroxide (1.5mM) of external source supply for co-factor, with the full cytolipin of tranquillization state
Fat enzyme (20mg) is mixed catalyst with the full cell P450 decarboxylation of fatty acids enzyme (30mg) of tranquillization state, and reaction medium is that pH is 7.8
Phosphate buffer, react 12h under 30 DEG C of water bath conditions, it is de- to be coupled P450 aliphatic acid by the hydrolysis that lipase mediates
The decarboxylic reaction that carboxylic acid mediates prepares fatty alkene.After waiting for coupling reaction, it is added in equal volume containing in ten seven-carbon fatty acids
Target ethyl acetate extractive analysis, is calculated using internal standard method, and microalgae oil is converted into fatty alkene (15 carbon and heptadecene hydrocarbon)
Conversion ratio be 20%.
Embodiment 3
(1) structure of genetic engineering bacterium：Connect pRSFDuet1 plasmids and Thermomyces lanuginosus by digestion enzyme
(Thermomyces lanuginosus) lipase Tl l genetic fragments build pRSFDuet-tll recombinant plasmids, and digestion enzyme connects
PACYCDuet1 plasmids and the OleT for deriving from Jeotgalicoccus sp.JEGenetic fragment construction recombination plasmid pACYCDuet-
oleTJE, by two recombinant plasmid cotransformation competent escherichia coli cell Escherichia coli BL21 (DE3), using containing
The LB resistant panels of 50 μ g/ml kanamycins and 25 μ g/ml chloramphenicol screen positive transformant, obtain efficiently coexpression lipase
With the recombination bacillus coli engineering strain of P450 decarboxylation of fatty acids enzymes.
(2) preparation of lipase and the acellular crude extract of P450 decarboxylation of fatty acids enzymes：By the recombination work of above-mentioned screening
Journey bacterial strain single bacterium colony is seeded in the 250ml shaking flasks equipped with 20ml LB culture mediums, and in shaking speed 250rpm, 37 DEG C were cultivated
Overnight seed liquor is seeded in the 2L shaking flasks equipped with 500ml LB by night according to 1% (v/v) inoculum concentration, and in 37 DEG C, 250rpm shakes
Bed culture, waits for that cell density reaches OD600When=0.6-0.8,0.2mM IPTG are added, start enzyme induction expression in 20 DEG C, and protect
The further growth of thalline is held until 20h.
The engineering bacteria be lipase gene engineering bacterium fermentation culture LB culture mediums in be added 50 μ g/ml kanamycins with
25 μ g/ml chloramphenicol；
50 μ g/ are added in the LB culture mediums for the engineering bacteria fermentation culture of P450 decarboxylation of fatty acids enzymes in the engineering bacteria
Ml kanamycins and 25 μ g/ml chloramphenicol and 1mM vitamin B12s.
The zymotic fluid of above-mentioned acquisition is collected into intracellular expression lipase or P450 decarboxylation of fatty acids respectively by centrifuging
The recombinant Bacillus coli cells of enzyme obtain the full cell of tranquillization state of two kinds of enzymes as catalyst respectively.
(3) lipase and the fatty alkene of the coupling catalysed preparation of P450 decarboxylation of fatty acids enzymes：With the microalgae oil of external source addition
(0.5mM) is substrate, with 0.3% (v/v) hydrogen peroxide (1.5mM) of external source supply for co-factor, is co-expressed with two kinds of enzymes
The full cell of tranquillization state (50mg) is catalyst, and reaction medium is the phosphate buffer that pH is 7.8, is reacted under 30 DEG C of water bath conditions
12h is coupled the decarboxylic reaction that P450 decarboxylation of fatty acids enzymes mediate by the hydrolysis that lipase mediates, prepares fatty alkene.
After waiting for coupling reaction, it is added in equal volume containing target ethyl acetate extractive analysis in ten seven-carbon fatty acids, using internal standard method
It calculates, the conversion ratio that microalgae oil is converted into fatty alkene (15 carbon and heptadecene hydrocarbon) is 30%.
Embodiment 4
(1) structure of genetic engineering bacterium：Connect pET22b plasmids and Thermomyces lanuginosus by digestion enzyme
(Thermomyces lanuginosus) lipase Tl l genetic fragments build pET22b-tll recombinant plasmids, and digestion enzyme connects
PACYCDuet1 plasmids and the OleT for deriving from Jeotgalicoccus sp.JEGenetic fragment construction recombination plasmid pACYCDuet-
oleTJE, by two recombinant plasmid cotransformation competent escherichia coli cell Escherichia coli BL21 (DE3), using containing
The LB resistant panels of 100 μ g/ml ampicillins and 25 μ g/ml chloramphenicol screen positive transformant, obtain efficiently coexpression fat
The recombination bacillus coli engineering strain of fat enzyme and P450 decarboxylation of fatty acids enzymes.
(2) preparation of lipase and the acellular crude extract of P450 decarboxylation of fatty acids enzymes：By the recombination work of above-mentioned screening
Journey bacterial strain single bacterium colony is seeded in the 250ml shaking flasks equipped with 20ml LB culture mediums, and in shaking speed 250rpm, 37 DEG C were cultivated
Overnight seed liquor is seeded in the 2L shaking flasks equipped with 500ml LB by night according to 1% (v/v) inoculum concentration, and in 37 DEG C, 250rpm shakes
Bed culture, waits for that cell density reaches OD600When=0.6-0.8,0.2mM IPTG are added, start enzyme induction expression in 20 DEG C, and protect
The further growth of thalline is held until 12h.
The engineering bacteria be lipase gene engineering bacterium fermentation culture LB culture mediums in 100 μ g/ml ammonia benzyl moulds are added
Element and 25 μ g/ml chloramphenicol；
100 μ are added in the LB culture mediums for the engineering bacteria fermentation culture of P450 decarboxylation of fatty acids enzymes in the engineering bacteria
G/ml ampicillins and 25 μ g/ml chloramphenicol and 1mM vitamin B12s.
By the zymotic fluid 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 enzymes：The olive oil added with external source
(0.5mM) is substrate, with 0.3% (v/v) hydrogen peroxide (1.5mM) of external source supply for co-factor, with above-mentioned growth in situ state
Cell culture fluid is catalyst, reacts 36h under 28 DEG C of water bath conditions, and P450 fat is coupled by the hydrolysis that lipase mediates
The decarboxylic reaction that acid decarboxylase mediates prepares fatty alkene.After waiting for coupling reaction, it is added and contains 17 carbocyclic aliphatics in equal volume
Target ethyl acetate extractive analysis, is calculated using internal standard method in acid, and olive oil is converted into fatty alkene (15 carbon and heptadecene
Hydrocarbon) conversion ratio be 23%.
Claims (10)
1. a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes, feature
It is：Using glyceride as raw material, pass through coupling catalysed, the i.e. lipase hydrolysis glyceride life of lipase hydrolysis and P450 decarboxylations
At free fatty, the aliphatic acid of generation generates fatty alkene using P450 decarboxylations.
2. the fatty olefin catalytic coupling catalysed with P450 decarboxylation of fatty acids enzymes based on lipase as described in claim 1 synthesizes
Method, it is characterised in that：Using the grease of external source addition as substrate, using the hydrogen peroxide of external source supply as co-factor, it is being catalyzed
0.5-48h, the fatty alkene of coupling catalysed preparation are reacted under the action of agent under the conditions of 20-50 DEG C；
The catalyst is selected from the growth state cell culture fluid of the lipase and P450 decarboxylation of fatty acids enzymes of recombinant expression；Recombinate table
The full cell of tranquillization state of the lipase and P450 decarboxylation of fatty acids enzymes that reach；The lipase and P450 decarboxylation of fatty acids enzymes of recombinant expression
Acellular crude extract；One kind in the lipase of recombinant expression and the pure enzyme of P450 decarboxylation of fatty acids enzymes.
3. the fatty olefin catalytic coupling catalysed with P450 decarboxylation of fatty acids enzymes based on lipase as described in claim 2 synthesizes
Method, it is characterised in that：Using 0.2-100mM greases as substrate, 0.2-300mM, the hydrogen peroxide that volumetric concentration is 0.3%
Solution is reacted as co-factor, under the effect of the catalyst coupled catalytic reaction in the case where pH is the buffer solution system of 4-12, is reacted
Ethyl acetate or n-hexane extraction containging interior traget are analyzed in equal volume with reactant for addition afterwards, and grease is converted into turning for fatty alkene
Rate is 10-85%；
Lipase and P450 decarboxylation of fatty acids enzyme molar ratios are 1 in the catalyst：100-100：1.
4. by claim 1-3 any one of them based on lipase and the coupling catalysed fatty alkene of P450 decarboxylation of fatty acids enzymes
The method catalyzed and synthesized, it is characterised in that：The grease is the grease of plant and animal material, microbe-derived grease or contains peracid
The waste grease of valence；
The lipase is the lipase for having hydrolysis to grease.
5. the fatty olefin catalytic coupling catalysed with P450 decarboxylation of fatty acids enzymes based on lipase as described in claim 4 synthesizes
Method, it is characterised in that：The grease is soybean oil, rapeseed oil, corn oil, cottonseed oil, microalgae oil, yeast source grease, fish
Oil or gutter oil；
The 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（Candida antarctica）Lipase, aspergillus oryzae
（Aspergillus oryzae）Lipase, Rhizopus oryzae（Rhizopus oryzae）Lipase, Yarrowia lipolytica
（Yarrowia lipolylica）Lipase, conspicuous Mucor of rice（Mucor miehe）Lipase, serratia marcescens（Serratia
marcescens）Lipase, pseudomonas（Pseudomonas sp.）Lipase or bacillus（Bacillus sp.）
Lipase；
The P450 decarboxylation of fatty acids enzyme is the OleT derived from Jeotgalicoccus sp.JEOr derive from Bacillus
The P450 of subtilisBSβ。
6. by claim 1-3 any one of them based on lipase and the coupling catalysed fatty alkene of P450 decarboxylation of fatty acids enzymes
The method catalyzed and synthesized, it is characterised in that：The catalyst obtains as follows：
1）By building the recombinant plasmid containing purposeful lipase gene Yu P450 decarboxylation of fatty acids enzyme genes respectively, will obtain respectively
The recombinant plasmid transformed obtained obtains the weight of expression lipase and P450 decarboxylation of fatty acids enzymes respectively to competent escherichia coli cell
Group Recombinant organism strain；The Recombinant organism strain of acquisition is fermented as starting strain by two benches；
2）Zymotic fluid is to grow state cell culture fluid as catalyst；
Zymotic fluid collects the recombinant Bacillus coli cells for being overexpressed lipase and P450 decarboxylation of fatty acids enzymes by centrifuging, i.e.,
It is the full cell of tranquillization state as catalyst；
Zymotic fluid obtains lipase with the acellular crude extract of P450 decarboxylation of fatty acids enzymes as catalysis through sonicated cells
Agent；
Or, zymotic fluid is using pure enzyme obtained by the affinity chromatography of recombinase His-tag and Ni-NTA as catalyst.
7. the fatty olefin catalytic coupling catalysed with P450 decarboxylation of fatty acids enzymes based on lipase as described in claim 6 synthesizes
Method, it is characterised in that：
It is built respectively by way of digestion and enzyme company with lipase or P450 decarboxylation of fatty acids enzyme respectively using inducible plasmid
Recombinant plasmid, the recombinant plasmid transformed competent escherichia coli cell that then will be obtained respectively obtains the efficient of structure respectively
Express the genetic engineering bacterium of lipase or P450 decarboxylation of fatty acids enzymes；
The genetic engineering bacterium of above-mentioned acquisition is accumulated by the Fungal biodiversity before induction, until cell density reaches OD600=
0.6-0.8, and after the two benches of the expression of enzymes through IPTG inductions ferment, and keep the further growth of thalline.
8. the fatty olefin catalytic coupling catalysed with P450 decarboxylation of fatty acids enzymes based on lipase as described in claim 7 synthesizes
Method, it is characterised in that：
The inducible plasmid is the pET plasmid series controlled by T7 promoters；The Escherichia coli are Escherichia
coli BL21 (DE3)。
9. the fatty olefin catalytic coupling catalysed with P450 decarboxylation of fatty acids enzymes based on lipase as described in claim 7 synthesizes
Method, it is characterised in that：
In 37 DEG C of cultures to mistake under the engineering bacteria of above-mentioned acquisition is shaken in LB culture mediums with the rotating speed of 200-250rpm respectively
Night, overnight after continue at a temperature of 37 DEG C, by culture solution according to the inoculum concentration of 1-3%v/v in LB culture mediums with 200-
Lower culture to the cell density of rotating speed concussion of 250rpm reaches OD600=0.6-0.8；And 0.1-1mM is added in backward culture medium
For IPTG in 18-20 DEG C, the induced expression time is 18-20h, i.e., obtains the zymotic fluid of different engineering bacterias respectively；
When the engineering bacteria is the genetic engineering bacterium of high efficient expression lipase, 25-100 μ are added in the LB culture mediums of fermented and cultured
Antibiotic needed for g/ml stable plasmids；
When the engineering bacteria is the genetic engineering bacterium of P450 decarboxylation of fatty acids enzymes, 25-100 is added in the LB culture mediums of fermented and cultured
Antibiotic needed for μ g/ml stable plasmids and 1mM vitamin B12s.
10. the fatty olefin catalytic coupling catalysed based on lipase and P450 decarboxylation of fatty acids enzymes as described in claim 9 closes
At method, it is characterised in that：Antibiotic needed for the stable plasmid is ampicillin, kanamycins, chloramphenicol or chain
Mycin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510003451.5A CN104561152B (en) | 2015-01-05 | 2015-01-05 | It is a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510003451.5A CN104561152B (en) | 2015-01-05 | 2015-01-05 | It is a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104561152A CN104561152A (en) | 2015-04-29 |
| CN104561152B true CN104561152B (en) | 2018-07-13 |
Family
ID=53078229
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510003451.5A Expired - Fee Related CN104561152B (en) | 2015-01-05 | 2015-01-05 | It is a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104561152B (en) |
Families Citing this family (7)
| 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 |
| CN107201356A (en) * | 2016-03-17 | 2017-09-26 | 中国科学院青岛生物能源与过程研究所 | Support the reduction chaperone combination of P450 decarboxylation of fatty acids enzymatic activitys and its apply |
| 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 |
| CN108467861B (en) * | 2018-04-27 | 2021-10-22 | 扬州大学 | Sequence for catalytically synthesizing linear alpha-olefin biocatalyst OleT-BM3R, preparation 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 |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8143034B2 (en) * | 2007-08-21 | 2012-03-27 | Polytechnic Institute Of New York University | Method for preparing long-chain hydroxyacids, diacids and oligomers and polymers thereof |
-
2015
- 2015-01-05 CN CN201510003451.5A patent/CN104561152B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104561152A (en) | 2015-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104561152B (en) | It is a kind of based on lipase and the coupling catalysed fatty olefin catalytic synthetic method of P450 decarboxylation of fatty acids enzymes | |
| Pradima et al. | Review on enzymatic synthesis of value added products of glycerol, a by-product derived from biodiesel production | |
| Seo et al. | Enzyme/whole-cell biotransformation of plant oils, yeast derived oils, and microalgae fatty acid methyl esters into n-nonanoic acid, 9-hydroxynonanoic acid, and 1, 9-nonanedioic acid | |
| Lo et al. | Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogen-producing bacteria | |
| Yan et al. | Waste molasses alone displaces glucose-based medium for microalgal fermentation towards cost-saving biodiesel production | |
| JP5067888B2 (en) | Method for producing 1,3-propanediol using crude glycerol as a by-product from biodiesel production | |
| Du et al. | Perspectives for biotechnological production of biodiesel and impacts | |
| Zhang et al. | Current progress on butyric acid production by fermentation | |
| Qin et al. | Biodiesel production catalyzed by whole-cell lipase from Rhizopus chinensis | |
| CN101802208B (en) | Reduce the toxic action of impurity in raw material by the fermentation of extraction formula | |
| Jiang et al. | Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor | |
| Li et al. | Whole-cell based solvent-free system for one-pot production of biodiesel from waste grease | |
| Xin et al. | Strategies for production of butanol and butyl-butyrate through lipase-catalyzed esterification | |
| CN102482676A (en) | Product of fatty acid esters from biomass polymers | |
| Ugur et al. | New lipase for biodiesel production: partial purification and characterization of LipSB 25-4 | |
| Yan et al. | Assembly of lipase and P450 fatty acid decarboxylase to constitute a novel biosynthetic pathway for production of 1-alkenes from renewable triacylglycerols and oils | |
| CN104955956A (en) | A fermentation process | |
| Kaur et al. | Advances in microbial bioresources for sustainable biofuels production: current research and future challenges | |
| Yang et al. | Microbial recycling of glycerol to biodiesel | |
| Fan | Enzymatic biodiesel production–the way of the Future | |
| Xu et al. | Integrated production for biodiesel and 1, 3-propanediol with lipase-catalyzed transesterification and fermentation | |
| Li et al. | Co-production of 1, 3-Propanediol and 2, 3-Butanediol from Waste Lard by Co-cultivation of Pseudomonas alcaligenes and Klebsiella pneumoniae | |
| Robles-Iglesias et al. | Sequential bioconversion of C1-gases (CO, CO2, syngas) into lipids, through the carboxylic acid platform, with Clostridium aceticum and Rhodosporidium toruloides | |
| Sun et al. | Exploring the effects of oil inducer on whole cell-mediated methanolysis for biodiesel production | |
| CN103882069A (en) | Preparation method of long chain fatty acid ester |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180713 Termination date: 20210105 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |