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

Abstract

The invention belongs to the biological preparation method of aliphatic olefins in the field of bioengineering and energy technology, and specifically relates to a method for catalyzing the synthesis of aliphatic olefins based on the coupling catalysis of lipase and P450 fatty acid decarboxylase. Glycerides are used as raw materials to catalyze the coupling of lipase hydrolysis and P450 decarboxylation, that is, lipase hydrolyzes glycerides to generate free fatty acids, and the generated fatty acids are decarboxylated by P450 to generate aliphatic olefins. The in vitro coupling biocatalysis process mediated by lipase and P450 fatty acid decarboxylase of the present invention only needs two steps of enzymatic catalysis, the reaction conditions and enzyme ratio are easy to control, the substrate conversion rate is high, the energy consumption is low, and there is no pollution. It is an emerging green catalytic system for aliphatic olefins with controllable process and low cost, and has a good application prospect.

Description

A kind of aliphatic olefin catalyst based on the coupling catalysis of lipase and P450 fatty acid decarboxylase Synthetic method

technical field

The invention belongs to the biological preparation method of aliphatic olefins in the field of bioengineering and energy technology, and specifically relates to a method for catalyzing the synthesis of aliphatic olefins based on the coupling catalysis of lipase and P450 fatty acid decarboxylase.

Background technique

With the continuous consumption of traditional fossil energy such as petroleum, and the increasingly prominent issues of energy security strategy and environmental protection, countries all over the world are actively trying to produce biofuels and chemicals from renewable biological resources. Traditional and new advanced bio-liquid fuels, represented by short-chain alcohols, fatty acid methyl esters (ethyl esters), aliphatic hydrocarbons (including olefins and alkanes), etc., are attracting more and more attention. In terms of biofuels, the development of fuel ethanol and the first generation of biodiesel represented by fatty acid methyl ester (ethyl ester) is relatively mature, but both have their own shortcomings. As a substitute for gasoline, fuel ethanol has the disadvantages of high water solubility, strong volatility, and low energy density. Compared with short-chain alcohol fuels (such as ethanol) and first-generation biodiesel fatty acid methyl esters (ethyl esters), the structure and properties of aliphatic hydrocarbons as advanced biofuels (Advanced biofuels) are closer to petrochemical diesel, with high energy density, With low hygroscopicity and high combustion efficiency, it can be used as a substitute or additive for gasoline, diesel and aviation fuel. Therefore, the research on the catalytic synthesis pathway of aliphatic hydrocarbons has become a hot spot in the field of advanced biofuel research.

At present, the preparation of aliphatic hydrocarbons mainly relies on the hydrogenation chemical process mediated by noble metal catalysts such as platinum and palladium under the conditions of high temperature (250-450°C) and high pressure (20-70bar). Compared with traditional chemical processes, biocatalysis and synthesis have the advantages of high efficiency, low energy consumption, and environmental protection. Up to now, some aliphatic hydrocarbon biosynthetic pathways have been reported successively:

(1) In 2010, Schirmer et al. of LS9 Bioenergy Company in the United States identified the biosynthetic pathway of fatty acyl-ACP reductase and fatty aldehyde decarbonylase in cyanobacteria Synechococcuselongates PCC 7942 to convert fatty acyl-ACP into fatty alkanes or fatty alkenes .

(2) In 2010, Beller et al. identified a long-chain olefin biosynthesis pathway based on OleA enzyme-catalyzed decarboxylation condensation reaction in the bacterium Micrococcus luteus ATCC 4698, which can decarboxylate fatty acyl-CoA, reduce ketone group hydrogenation, and dehydrate hydroxyl groups A series of biochemical reactions produce aliphatic olefins containing internal double bonds.

(3) In 2011, Pfleger et al. identified a biosynthetic pathway for aliphatic olefins with terminal double bonds based on the polyketide synthase of cyanobacteria Synechococcus sp. PCC 7002Ols. The long-chain fatty acid acyl carrier protein ACP1 generates fatty alkenes with terminal double bonds through a series of biochemical reactions catalyzed by ketosynthase, acyltransferase, ketoreductase, sulfotransferase and thioesterase.

(4) In 2011, Rude et al. from LS9 Company reported the reaction of cytochrome P450 decarboxylase (OleT _JE ) in bacterium Jeotagalicoccus sp.ATCC 8456 to catalyze the decarboxylation of fatty acids to form alkenes. The P450 (OleT _JE ) decarboxylates fatty acids to aliphatic olefins with terminal double bonds in the presence of the cofactor hydrogen peroxide (H ₂ O ₂ ).

(5) In 2013, Akhtar et al. constructed an aliphatic hydrocarbon biosynthetic pathway consisting of thioesterase, carboxylic acid reductase and fatty aldehyde decarbonylase to convert fatty acyl-ACP into aliphatic hydrocarbons.

Although the development of biofuels has solved the problems of renewable raw materials and environmental protection, the high cost is one of the most important bottlenecks restricting its industrial scale production. These costs include raw material cost, catalyst cost and production process cost, among which raw material cost is particularly critical. Most of the reported aliphatic hydrocarbon biosynthetic pathways are based on fatty acid metabolism pathways, using free fatty acid (Free fatty acid) or fatty acid acylated fatty acid form (acyl-ACP or acyl-CoA) as the direct raw material. De novo fatty acid synthesis (De novo biosynthesis) involves a metabolic network composed of multi-step enzymatic reactions, and the utilization rate of initial substrates (sugar, etc.) into fatty acids is low. Metabolic engineering methods such as overexpression of key enzyme genes in the fatty acid synthesis pathway and knockout of key enzyme genes in the fatty acid oxidation pathway can increase the accumulation of fatty acids to a certain extent, but due to the precision of the regulatory network of fatty acid metabolism With the complexity, overexpression of more key enzymes in the fatty acid metabolic pathway will increase the metabolic burden of the host cell, while enhancing fatty acid synthesis through genetic modification will increase the uncontrollable factors of the fatty acid metabolic network. It is difficult to comprehensively and systematically understand, utilize and transform fatty acid metabolic network, and there is limited room for further improvement of fatty acid accumulation. Therefore, based on the pathway of fatty acid metabolism, the cost of producing aliphatic olefins from fatty acids as direct raw materials is too high, which restricts its industrial application.

Lipase, as a typical carboxylic acid hydrolase, can catalyze the hydrolysis of natural substrate glycerolipids to generate free fatty acids and glycerol. Glycerides are widely found in animal and vegetable oils and microbial oils in nature. They are rich in resources and cheaper than free fatty acids. After retrieval, there is no report on the catalytic synthesis method of aliphatic hydrocarbons based on enzyme catalysis using glyceride as raw material.

Contents of the invention

The object of the present invention is to provide a method for the catalytic synthesis of aliphatic olefins based on lipase and P450 fatty acid decarboxylase coupling catalysis

The technical scheme that the present invention adopts for realizing the above object is:

A method for the catalytic synthesis of fatty olefins based on the coupling catalysis of lipase and P450 fatty acid decarboxylase, using glycerides as raw materials, through the coupling catalysis of lipase hydrolysis and P450 decarboxylation, that is, lipase hydrolyzes glycerides to generate free fatty acids, and generates The fatty acids are then decarboxylated by P450 to produce aliphatic olefins.

Furthermore, the oil added from an external source is used as a substrate, and the hydrogen peroxide supplied from an external source is used as a cofactor, and is reacted at 20-50°C for 0.5-48 hours under the action of a catalyst, and aliphatic olefins are prepared by coupling catalysis;

The catalyst is the growth state cell culture fluid of recombinantly expressed lipase and P450 fatty acid decarboxylase, the quiescent whole cell of recombinantly expressed lipase and P450 fatty acid decarboxylase or the absence of recombinantly expressed lipase and P450 fatty acid decarboxylase. Pure enzymes of crude cell extract or recombinantly expressed lipase and P450 fatty acid decarboxylase.

Furthermore, with a concentration of 0.2-100mM oil as a substrate and a concentration of 0.2-300mM hydrogen peroxide (0.3%, v/v) as a cofactor, the coupling catalytic reaction under the action of a catalyst is at pH 4 React under the buffer system of -12. After the reaction, add ethyl acetate or n-hexane with an internal standard equal to the volume of the reactant for extraction and analysis. The conversion rate of oil into aliphatic olefins is 10-85%;

The molar ratio of lipase to P450 fatty acid decarboxylase in the catalyst is 1:100-100:1.

The oils and esters are animal and plant derived oils, microbial derived oils or waste oils with high acid value;

The lipase is a lipase that hydrolyzes oils and esters;

The P450 fatty acid decarboxylase is an enzyme with decarboxylation activity on fatty acids.

The oil ester is soybean oil, rapeseed oil, corn oil, cottonseed oil, microalgae oil, yeast oil, fish oil, or waste oil;

The lipase is Thermomyces lanuginosus lipase T11, Aspergillus niger lipase, Geotrichum candidum lipase, Candida rugosa lipase Enzymes, Candida antarctica lipase, Aspergillus oryzae lipase, Rhizopus oryzae lipase, Yarrowia lipolytica lipase, Mucor miehe) lipase, Serratia marcescens lipase, Pseudomonas sp. lipase or Bacillus sp. lipase;

The P450 fatty acid decarboxylase is OleT _JE derived from Jeotgalicoccus sp.) or P450 _BSβ derived from Bacillus subtilis.

The catalyst is obtained as follows:

1) By constructing a recombinant plasmid containing the target lipase gene, transforming the recombinant plasmid into Escherichia coli competent cells, obtaining a recombinant Escherichia coli genetically engineered strain expressing lipase and P450 fatty acid decarboxylase; the obtained Escherichia coli genetically engineered strain is used as the starting strain through two-stage fermentation;

2) The above-mentioned fermented liquid is the growth state cell culture liquid as a catalyst;

The above fermentation broth is centrifuged to collect recombinant Escherichia coli cells overexpressing lipase and P450 fatty acid decarboxylase, that is, resting whole cells are used as catalysts;

The above fermentation liquid is ultrasonically disrupted to obtain a cell-free crude extract of lipase and P450 fatty acid decarboxylase as a catalyst;

Alternatively, the above fermented broth uses the pure enzyme obtained by affinity chromatography of the recombinant enzyme His-tag and Ni-NTA as a catalyst.

The above-mentioned recombinant plasmids were respectively constructed by using inducible plasmids and lipase or P450 fatty acid decarboxylase by enzyme digestion and enzyme ligation, and then the respectively obtained recombinant plasmids were transformed into Escherichia coli competent cells to obtain the constructed high-efficiency lipase Or P450 fatty acid decarboxylase genetically engineered bacteria;

The genetically engineered bacteria obtained above are accumulated by the biomass of the bacteria before induction until the density of the bacteria reaches OD ₆₀₀ =0.6-0.8, and then subjected to two-stage fermentation of enzyme expression induced by IPTG, and the further growth of the bacteria is maintained.

The inducible plasmid is a pET plasmid series controlled by T7 promoter; such as pRSFDuet1 or pET28b or pET22b, and the Escherichia coli is Escherichia coli BL21 (DE3).

The engineered bacteria obtained above were respectively cultured in LB medium at 37°C under shaking at a speed of 200-250rpm until overnight, and continued at a temperature of 37°C after overnight, and the culture solution was mixed with 1-3 (v/v)% The inoculum amount was cultured in LB medium under shaking at 200-250rpm until the cell density reached OD ₆₀₀ =0.6-0.8; then 0.1-1mM IPTG was added to the medium at 18-20°C, and the induction time was 18 -20h, that is, to obtain the fermentation broth of different engineering bacteria respectively;

The engineering bacterium is the antibiotic required for the stable plasmid of 25-100 μg/ml added to the LB medium of the lipase gene engineering bacterium fermentation culture;

The engineering bacterium is P450 fatty acid decarboxylase genetically engineered bacterium fermented and cultured by adding 25-100 μg/ml of antibiotics required for stable plasmids and 1 mM vitamin B12.

The antibiotics required for the stable plasmid are ampicillin, kanamycin, chloramphenicol or streptomycin according to different plasmids.

The present invention has advantages:

The present invention uses cheap and easy-to-obtain glycerides as raw materials to catalyze the synthesis of aliphatic olefins through the coupling of lipase hydrolysis and P450 decarboxylase. Compared with the traditional hydrogenation chemical process and de novo biosynthesis pathway of aliphatic hydrocarbons, only two enzyme steps are required The catalyst-promoting step, the reaction conditions and the ratio of enzymes are easy to adjust, the conversion rate of the substrate is high, the energy consumption is low, and there is no pollution.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1

The method for the preparation of fatty olefins catalyzed by the coupling of lipase and P450 fatty acid decarboxylase in this embodiment is:

(1) Construction of genetically engineered bacteria: the pRSFDuet1 plasmid was constructed with the lipase Tl l gene of Thermomyces lanuginosus or the OleT _JE gene derived from Jeotgalicoccus sp. Recombinant plasmids, the resulting recombinant plasmids were transformed into Escherichia coli competent cells Escherichia coliBL21 (DE3), positive transformants were screened using LB resistance plates containing 50 μg/ml kanamycin, and high-expression lipase or P450 fatty acid decarboxylase were obtained respectively. Recombinant Escherichia coli genetically engineered strains.

(2) Preparation of lipase and P450 fatty acid decarboxylase pure enzyme: inoculate the single bacterium colony of the recombinant genetically engineered strain of above-mentioned screening into the 250ml shaking flask that 20ml LB medium is housed, in shaker speed 250rpm, cultivate overnight at 37°C, According to the inoculum amount of 1% (v/v), inoculate the overnight seed solution into a 2L shaker flask with 500ml LB, and cultivate it on a shaking table at 37°C and 250rpm. When the cell density reaches OD ₆₀₀ =0.6-0.8, add 0.2 mM IPTG, start the enzyme-induced expression at 20°C, and keep the further growth of the bacteria until 20h.

The engineering bacterium is added 50 μg/ml kanamycin to the LB medium fermented by lipase genetically engineered bacteria;

50 μg/ml kanamycin and 1 mM vitamin B12 were added to the LB medium for the fermentation culture of the genetic engineering bacteria of P450 fatty acid decarboxylase as the engineering bacteria.

Collect the recombinant Escherichia coli cells expressing lipase or P450 fatty acid decarboxylase in the cells by centrifugation, and then ultrasonically disrupt the cells to obtain cell-free crude extracts of lipase or P450 fatty acid decarboxylase, respectively. The cell-free crude extracts of the recombinant enzyme His-tag and Ni-NTA affinity chromatography were used to prepare the respective pure enzymes as catalysts.

(3) Coupling of lipase and P450 fatty acid decarboxylase to catalyze the preparation of aliphatic olefins: 0.5mM soybean oil added by exogenous sources is used as substrate, 0.3% hydrogen peroxide (v/v) of 1mM supplied by exogenous sources is used as cofactor, with the above The obtained pure enzyme is used as a catalyst, wherein the purified lipase in the pure enzyme is 2 μM, the purified P450 fatty acid decarboxylase is 4 μM, and the reaction medium is a phosphate buffer solution with a pH of 7.8, reacted for 6 hours at 30° C. in a water bath, and passed through the lipase-mediated Guided hydrolysis coupled with P450 fatty acid decarboxylase-mediated decarboxylation to produce aliphatic olefins. After the coupling reaction is finished, add ethyl acetate containing the internal standard of seventeen-carbon fatty acid in the same volume as the reaction system for extraction and analysis, and calculate the conversion rate of soybean oil into fatty olefins (pentadecene and heptadecene) by using the internal standard method was 31%.

Example 2

The method for the preparation of fatty olefins catalyzed by the coupling of lipase and P450 fatty acid decarboxylase in this embodiment is:

(1) Construction of genetically engineered bacteria: construct a recombinant plasmid by combining pRSFDuet1 plasmid with Thermomyces lanuginosus lipase T1 l gene or OleT _JE gene derived from Jeotgalicoccus sp., and transform the recombinant Transfer the plasmid to E. coli competent cells Escherichia coli BL21(DE3), use LB resistance plates containing 50 μg/ml kanamycin to screen positive transformants, and obtain recombinant E. coli genetic engineering that highly expresses lipase and P450 fatty acid decarboxylase respectively strain.

(2) Preparation of lipase and P450 fatty acid decarboxylase cell-free crude extract: Inoculate a single colony of the recombinant genetically engineered strains screened above into a 250ml shaker flask with 20ml LB medium, and shake at a rotating speed of 250rpm at 37°C Cultivate overnight, inoculate the overnight seed liquid into a 2L shaker flask filled with 500ml LB according to the inoculation amount of 1% (v/v), and culture on a shaker at 37°C and 250rpm until the cell density reaches OD ₆₀₀ =0.6-0.8 , adding 0.2mM IPTG, starting the enzyme-induced expression at 20°C, and maintaining the further growth of the bacteria until 20h.

The engineering bacterium is added 50 μg/ml kanamycin to the LB medium fermented by lipase genetically engineered bacteria;

50 μg/ml kanamycin and 1 mM vitamin B12 were added to the LB medium for the fermentation culture of the genetic engineering bacteria of P450 fatty acid decarboxylase as the engineering bacteria.

Recombinant Escherichia coli cells expressing lipase or P450 fatty acid decarboxylase in the cells were collected by centrifuging the fermented broth obtained above, that is, the resting whole cells of the two enzymes were respectively obtained as catalysts.

(3) Coupling of lipase and P450 fatty acid decarboxylase to catalyze the preparation of aliphatic olefins: microalgae oil (0.5 mM) added by exogenous sources as substrate, 0.3% (v/v) hydrogen peroxide (1.5 mM) supplied by exogenous sources As a cofactor, resting state whole cell lipase (20mg) and resting state whole cell P450 fatty acid decarboxylase (30mg) are used as mixed catalysts, the reaction medium is phosphate buffer solution with a pH of 7.8, and the reaction is carried out in a water bath at 30°C 12h, aliphatic olefins were prepared by coupling lipase-mediated hydrolysis to P450 fatty acid decarboxylase-mediated decarboxylation. After the coupling reaction is finished, an equal volume of ethyl acetate containing an internal standard of seventeen carbon fatty acids is added for extraction analysis, and the internal standard method is used to calculate that the conversion rate of microalgae oil into aliphatic olefins (pentadecene and heptadecene) is 20%.

Example 3

(1) Construction of genetically engineered bacteria: construct pRSFDuet-tll recombinant plasmid by connecting pRSFDuet1 plasmid with Thermomyces lanuginosus lipase T1 l gene fragment, and connect pACYCDuet1 plasmid with source The recombinant plasmid pACYCDuet-oleT _JE was constructed from the OleT _JE gene fragment of Jeotgalicoccus sp., and the two recombinant plasmids were co-transformed into E. coli competent cells Escherichia coli BL21 (DE3). Positive transformants were screened on the LB resistance plate of prime, and a recombinant Escherichia coli genetically engineered strain that efficiently co-expressed lipase and P450 fatty acid decarboxylase was obtained.

(2) Preparation of lipase and P450 fatty acid decarboxylase cell-free crude extract: Inoculate a single colony of the recombinant genetically engineered strains screened above into a 250ml shaker flask with 20ml LB medium, and shake at a rotating speed of 250rpm at 37°C Cultivate overnight, inoculate the overnight seed solution into a 2L shaker flask filled with 500ml LB according to 1% (v/v) inoculation amount, and culture on a shaker at 37°C and 250rpm until the cell density reaches OD ₆₀₀ =0.6-0.8 , adding 0.2mM IPTG, starting the enzyme-induced expression at 20°C, and maintaining the further growth of the bacteria until 20h.

The engineering bacterium is lipase genetically engineered bacteria fermented in LB medium, adding 50 μg/ml kanamycin and 25 μg/ml chloramphenicol;

50 μg/ml kanamycin, 25 μg/ml chloramphenicol and 1 mM vitamin B12 were added to the LB medium for fermentation and culture of genetically engineered bacteria with P450 fatty acid decarboxylase as the engineering bacteria.

Recombinant Escherichia coli cells expressing lipase or P450 fatty acid decarboxylase in the cells were collected by centrifuging the fermented broth obtained above, that is, the resting whole cells of the two enzymes were respectively obtained as catalysts.

(3) Coupling of lipase and P450 fatty acid decarboxylase to catalyze the preparation of aliphatic olefins: microalgae oil (0.5 mM) added by exogenous sources as substrate, 0.3% (v/v) hydrogen peroxide (1.5 mM) supplied by exogenous sources As a cofactor, the resting state whole cells (50mg) co-expressed by the two enzymes are used as the catalyst, the reaction medium is phosphate buffer solution with a pH of 7.8, and the reaction is carried out at 30°C for 12 hours in a water bath, and the hydrolysis reaction mediated by lipase Coupling of P450 Fatty Acid Decarboxylase-Mediated Decarboxylation to Produce Aliphatic Alkenes. After the coupling reaction is finished, an equal volume of ethyl acetate containing an internal standard of seventeen carbon fatty acids is added for extraction analysis, and the internal standard method is used to calculate that the conversion rate of microalgae oil into aliphatic olefins (pentadecene and heptadecene) is 30%.

Example 4

(1) Construction of genetically engineered bacteria: pET22b-tll recombinant plasmid was constructed by enzyme-digesting enzyme connecting pET22b plasmid and Thermomyces lanuginosus lipase T1 l gene fragment, and enzyme-digesting enzyme connecting pACYCDuet1 plasmid and source The recombinant plasmid pACYCDuet-oleT _JE was constructed from the OleT _JE gene fragment of Jeotgalicoccus sp., and the two recombinant plasmids were co-transformed into E. coli competent cells Escherichia coli BL21 (DE3). Positive transformants were screened on the LB resistance plate, and recombinant Escherichia coli genetically engineered strains that efficiently co-expressed lipase and P450 fatty acid decarboxylase were obtained.

(2) Preparation of lipase and P450 fatty acid decarboxylase cell-free crude extract: Inoculate a single colony of the recombinant genetically engineered strains screened above into a 250ml shaker flask with 20ml LB medium, and shake at a rotating speed of 250rpm at 37°C Cultivate overnight, inoculate the overnight seed solution into a 2L shaker flask filled with 500ml LB according to 1% (v/v) inoculation amount, and culture on a shaker at 37°C and 250rpm until the cell density reaches OD ₆₀₀ =0.6-0.8 , adding 0.2mM IPTG, starting the enzyme-induced expression at 20°C, and maintaining the further growth of the bacteria until 12h.

100 μg/ml ampicillin and 25 μg/ml chloramphenicol were added to the LB medium for fermentation culture of lipase genetically engineered bacteria as described engineering bacteria;

100 μg/ml ampicillin, 25 μg/ml chloramphenicol and 1 mM vitamin B12 were added to the LB medium for fermentation and culture of genetically engineered bacteria with P450 fatty acid decarboxylase as the engineering bacteria.

The fermentation liquid of the co-expressed growing cell culture liquid obtained above is used as an in-situ catalyst.

(3) Coupling of lipase and P450 fatty acid decarboxylase to catalyze the preparation of aliphatic olefins: olive oil (0.5 mM) added from an external source was used as a substrate, and 0.3% (v/v) hydrogen peroxide (1.5 mM) supplied from an external source was used as The cofactor, using the above-mentioned in situ growth state cell culture medium as a catalyst, reacted in a water bath at 28°C for 36 hours, and prepared aliphatic olefins by coupling the hydrolysis reaction mediated by lipase to the decarboxylation reaction mediated by P450 fatty acid decarboxylase. After the coupling reaction is finished, an equal volume of ethyl acetate containing seventeen-carbon fatty acid internal standard is added for extraction analysis, and the internal standard method is used to calculate that the conversion rate of olive oil into aliphatic olefins (pentadecene and heptadecene) is 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 subtilis_BSβ。

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.