A marine-derived fatty acid decarboxylase, and preparation method and application thereof

Abstract

The application discloses a marine-derived fatty acid decarboxylase as well as a preparation method and application thereof, relates to the technical field of enzyme engineering, and the nucleotide sequence of the coding gene of the marine-derived fatty acid decarboxylase is shown as SEQ ID NO. 2, and the marine-derived fatty acid decarboxylase is derived from Salinicoccus roseus. The marine-derived fatty acid decarboxylase shows good decarboxylation activity on saturated straight-chain fatty acids with carbon atom numbers of 9-20 and C18:1 unsaturated fatty acids, wherein the decarboxylation efficiency on palmitic acid (C16:0) can reach 69.35% in 5 hours, and is significantly better than the reported OleT JE Meanwhile, the fatty acid decarboxylase has excellent salt tolerance, and still maintains more than 70% relative enzyme activity after being incubated in 0-5 M NaCl for 24 hours, can be used for preparing alpha-olefins, and has good industrial application prospect in the fields of biofuels and biological chemical industry.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a marine-derived fatty acid decarboxylase, its preparation method, and its application. Background Technology

[0002] With the overexploitation of fossil fuels leading to resource depletion and ecological degradation, accelerating the development and utilization of renewable energy has become a key measure to address the global energy crisis and climate issues. Alpha-olefins, as an ideal biofuel, possess advantages such as high energy density, low freezing point, and low hygroscopicity, showing broad application prospects in replacing fossil fuels.

[0003] Currently, the main chemical synthesis methods for α-olefins include ethylene polymerization and fatty acid decarbonylation, but these methods generally suffer from problems such as harsh reaction conditions, high energy consumption, and high costs. In contrast, biosynthesis methods, due to their milder reaction conditions and high product selectivity, align with the principles of green and sustainable development. Among them, the P450 fatty acid decarboxylase OleT (terminal olefin-forming fatty acid decarboxylase) is an enzyme that can catalyze the decarboxylation of free fatty acids to produce α-olefins in one step. It only requires H2O2 as a co-substrate and has outstanding advantages such as low production cost and a broad substrate spectrum (C4-C22), attracting much attention in the field of hydrocarbon fuel production. However, currently, only OleT has been reported and studied in depth. JE OleT MC OleT PRN OleTP CL More than ten types of enzymes, mainly derived from terrestrial microorganisms, exist, while research on marine-derived OleTs is relatively scarce, and these OleTs generally suffer from low catalytic efficiency. Furthermore, glycerides, as an economical raw material for the production of alkane (olefin) fuels, are widely derived from natural animal and vegetable oils, waste cooking oil, and non-edible waste oils. Although they can be converted into α-olefins through a cascade catalysis of lipases and OleTs, very few types of OleTs can currently be effectively applied to glyceride catalysis, especially decarboxylases suitable for high-salt conditions, which are rarely reported.

[0004] Therefore, there is an urgent need to develop a novel OleT with high catalytic activity, broad substrate spectrum, and good salt tolerance, and to establish a simple, mild, and applicable α-olefin biosynthesis process suitable for a variety of glycerides (including complex raw materials such as waste cooking oil) to achieve the green and efficient preparation of α-olefins. Summary of the Invention

[0005] In view of this, embodiments of the present invention provide a marine-derived fatty acid decarboxylase, its preparation method, and its application.

[0006] A first aspect of the present invention provides a marine-derived fatty acid decarboxylase, the encoding nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] In some embodiments, the amino acid sequence of the marine-derived fatty acid decarboxylase is shown in SEQ ID NO.1.

[0008] A second aspect of the present invention provides a recombinant expression vector comprising the encoding nucleotide sequence of a marine-derived fatty acid decarboxylase as described in the first aspect.

[0009] In some embodiments, the backbone vector of the recombinant expression vector is pET28a(+).

[0010] A third aspect of the present invention provides a recombinant engineered bacterial strain obtained by transfecting a host bacterium with the recombinant expression vector as described in the second aspect.

[0011] In some embodiments, the host bacterium is Escherichia coli BL21(DE3).

[0012] The fourth aspect of the present invention provides a method for preparing a marine-derived fatty acid decarboxylase, comprising: inoculating the recombinant engineered strain described in the third aspect into a culture medium containing vitamin B1, then adding 5-aminolevulinic acid, and after induction culture, collecting the bacterial cells, and then crushing and centrifuging them to obtain the product.

[0013] In some embodiments, the final concentration of vitamin B1 is 0.2~0.8mM.

[0014] In some embodiments, the culture medium comprises: 0.2-0.8% (w / v) yeast extract, 0.8-1.2% (w / v) peptone, 0.01-0.1% (w / v) glucose, 0.1-0.3% (w / v) lactose, 20-30 mM disodium hydrogen phosphate, 20-30 mM potassium dihydrogen phosphate, 40-60 mM ammonium chloride, 2-8 mM sodium sulfate, 1-3 mM magnesium sulfate, and 0.1-0.8 (w / v) glycerol.

[0015] In some embodiments, the final concentration of the 5-aminolevulinic acid is 0.1~1mM.

[0016] In some embodiments, the induction culture is carried out at a temperature of 16-20°C for a time of 18-36 hours.

[0017] In some embodiments, the centrifugation process further includes purification.

[0018] A fifth aspect of the present invention provides a method for catalytic decarboxylation of fatty acids, comprising: mixing the marine-derived fatty acid decarboxylase described in the first aspect, hydrogen peroxide, and fatty acids in a reaction system, and reacting therein.

[0019] In some embodiments, the fatty acid is selected from at least one of C9-C20 saturated straight-chain fatty acids and C18 unsaturated fatty acids.

[0020] In some embodiments, the reaction temperature is 30~70°C.

[0021] In some embodiments, the pH of the reaction is 6 to 8.5.

[0022] In some embodiments, the buffer solution of the reaction system is a phosphate buffer solution.

[0023] In some embodiments, the NaCl concentration in the reaction system is 0.2~0.5M.

[0024] In some embodiments, the reaction system further comprises an organic solvent. Preferably, the organic solvent is DMSO.

[0025] A sixth aspect of the present invention provides a method for preparing α-olefins by cascade catalysis of glycerides, comprising mixing and reacting the marine-derived fatty acid decarboxylase, lipase, hydrogen peroxide and fatty acid glycerides described in the first aspect in a reaction system.

[0026] In some embodiments, the glyceride is selected from at least one of trilaurate, trimyristic acid, tripalmitate, trioleate, soybean oil, olive oil, coconut oil, peanut oil, and corn oil.

[0027] In some embodiments, the concentration of the glyceride is 0.5~2 mM. More preferably, it is 1 mM.

[0028] In some embodiments, the lipase is selected from at least one of lipase Novozyme 435, lipase TL IM, or lipase RMIM. More preferably, it is lipase TL IM.

[0029] In some embodiments, the amount of lipase added is 0.2-1.8% (w / v).

[0030] In some embodiments, the reaction temperature is 30-40°C, more preferably 35°C.

[0031] In some embodiments, the reaction system further comprises an organic solvent.

[0032] Preferably, the organic solvent is selected from at least one of ethanol, isopropanol, ethyl acetate, and DMSO. More preferably, it is DMSO.

[0033] The marine-derived fatty acid decarboxylase, its preparation method, and its application of the present invention have at least the following beneficial effects:

[0034] The marine-derived fatty acid decarboxylase of this invention exhibits excellent decarboxylation activity for both saturated straight-chain fatty acids with 9-20 carbon atoms and C18:1 unsaturated fatty acids. Specifically, the decarboxylation efficiency for palmitic acid (C16:0) reaches 69.35% after 5 hours, significantly superior to the previously reported OleT enzyme. JE Meanwhile, the fatty acid decarboxylase of the present invention has excellent salt tolerance, and retains more than 70% of its relative enzyme activity after incubation in 0-5 M NaCl for 24 hours.

[0035] Furthermore, this invention optimizes the enzyme-catalyzed decarboxylation of free fatty acids and the method for one-step preparation of α-olefins from glycerides via a cascade catalysis of the enzyme and lipase. The optimized cascade catalytic reaction system increases the yield of α-olefins from trimyristic acid glycerides to 1.27 mM, which is 2.01 times that before optimization. Moreover, this system is applicable to a variety of natural oils and waste cooking oils, and has the advantages of broad substrate spectrum, mild reaction conditions, simple operation, and green environmental protection. It has good industrial application prospects in the fields of biofuels and biochemicals. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0037] Figure 1 For the present invention OleT SR SDS-PAGE protein electrophoresis analysis diagram;

[0038] Figure 2 For the present invention OleT SR Results of verification of catalytic fatty acid decarboxylation activity;

[0039] Figure 3 For the present invention OleT SR With OleT PRN OleT JE Catalytic fatty acid substrate spectrum;

[0040] Figure 4 The reaction temperature of this invention affects OleT SR The effect of enzyme activity is shown in the figure.

[0041] Figure 5 The reaction pH of the present invention affects OleT SR The effect of enzyme activity is shown in the figure.

[0042] Figure 6 The present invention relates to NaCl for OleT SR The effect of enzyme activity is shown in the figure.

[0043] Figure 7 For the present invention OleT SR Figure showing the tolerance results to NaCl;

[0044] Figure 8 This is a graph showing the comparison of the hydrolytic abilities of different lipases on triglycerides of different fatty acids in this invention.

[0045] Figure 9 This is a graph showing the comparison of the hydrolytic capacity of different lipases on equimolar mixed fatty acid triglycerides according to the present invention.

[0046] Figure 10 The lipase and OleT of this invention SR Figure showing the effect of addition order on olefin yield;

[0047] Figure 11 This is a graph showing the effect of H2O2 addition concentration and frequency on olefin yield in this invention;

[0048] Figure 12 The graph shows the effect of different organic solvents on olefin yield according to this invention.

[0049] Figure 13 This is a graph showing the effect of reaction temperature on olefin yield in this invention;

[0050] Figure 14 The graph shows the effect of the addition of trimyristic glyceride on olefin yield according to the present invention.

[0051] Figure 15 This is a graph showing the effect of lipase dosage on olefin yield in this invention.

[0052] Figure 16 For the present invention OleT SR Figure showing the effect of crude enzyme solution dosage on olefin yield;

[0053] Figure 17 This is a graph showing the effect of the amount of ethyl acetate added on the olefin yield in this invention.

[0054] Figure 18 For the present invention OleT SR Figure showing the comparison of decarboxylation effects before and after optimization of the reaction with lipase cascade catalyzed trimyristic acid glyceride;

[0055] Figure 19 For the present invention OleT SR The decarboxylation effect of the lipase cascade reaction system on different glycerides is shown in the figure. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0057] In the embodiments described in this specification, the plasmid and pET28a(+)-OleT SR pET28a(+)-OleT PRN (GenBank accession number: WP_083091567.1) was synthesized by Sangon Biotech (Shanghai) Co., Ltd.; empty vector plasmids pET28a(+) and pET28a(+)-OleT JE (GenBank accession number: ADW41779.1) were preserved in our laboratory; E. coli BL21(DE3) competent cells were purchased from Weidi Biotechnology Co., Ltd.; immobilized lipases Novozyme435, TL IM, and RM IM were purchased from Novozymes China Ltd., Denmark; plasmid extraction kits were purchased from Sangon Biotech (Shanghai) Co., Ltd.; all chemicals were purchased from Sigma-Aldrich, TCI, Maclean's, or Aladdin, which had the highest purity and could be used without further purification.

[0058] Unless otherwise specified, the PBS buffer of this invention contains 100 mM KH2PO4, 100 mM K2HPO4, 500 mM NaCl, 5% (w / v) glycerol, and pH 7.0.

[0059] Unless otherwise specified, all data in this invention were plotted and statistically analyzed using GraphPad Prism 9.3.1 software. For comparisons of three or more groups, one-way ANOVA was used. For pairwise comparisons between groups that did not show significant differences in one-way ANOVA and the control group, unpaired t-tests were used. Significance is indicated by *: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, and no significance is indicated.

[0060] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0061] Example 1: Marine-derived fatty acid decarboxylase OleT SR Preparation method

[0062] 1. Expressing OleT SR Construction and culture of recombinant strains

[0063] In this embodiment, the marine-derived fatty acid decarboxylase was screened from *Salinicoccus roseus* and named OleT. SR .

[0064] The fatty acid decarboxylase OleT SR The amino acid sequence information is as follows:

[0065] MSTIKKHKGLDNTLKVMKEGYLYTTNQRRRLGAENIFETRALGGKRVLVISGKAAELFYDNDKTERSGTLPKRLVNTLFGKGAIHTTTGKKHIDRKALFMSLMTEGNLKHLRELT RNHWYMNTHRMEQMDQINIYRESIILLTKVGTKWAGVQAPEEKIEEIATDMDIMIDSFKGLGTAFKGYKESVAARRRVEDWLEDQIIQTRKGKIFPPEGTALYEFAHWEDYKGNPMD SR LCAIDLMNTFRPLIAINRFVSFGLLAMHEYPISKEKINNDPDYAYMFSQEVRRFYPFVPFLPGKAKTDIQFEGHDIEKDTMLVLDIYGTMHQDDVFENADEFYPERFLDWDGSPFDLIPQGGGDYYTNHRCAGEWMTIIIMEETMKYFAGKITYDVPEQDLTVDLNSIPGYIKSGFIIENVAEKVDRK (SEQ ID NO.1).

[0066] The fatty acid decarboxylase OleT SR The encoding nucleotide sequence information is as follows:

[0067]

[0068] The encoding nucleotide sequence was ligated into pET28a(+), and then transfected into E. coli BL21(DE3) strain to obtain the plasmid pET28a(+)-OleT. SR The recombinant E. coli BL21(DE3) monoclonal strain was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and then incubated in a constant temperature shaker at 37°C and 200 rpm for 12-13 h. When the bacterial culture became significantly turbid, it was ready for inoculation with 100 mL of LB liquid medium at 5% (v / v). The culture was then incubated at 37°C and 220 rpm for approximately 2-3 h, when the OD... 600 When the OD value reaches 0.6-0.8, inoculate 500 mL of self-inducing liquid culture medium containing 0.5 mM vitamin B1 at 5% (v / v). Then incubate at 37℃ and 220 rpm for 2 h. 600 When the concentration reaches 0.8-1.0, add 250 μL of 1 M 5-aminolevulinic acid solution and induce culture at 18℃ and 220 rpm for 24 h to obtain fermentation broth for later use.

[0069] E. coli cells containing the empty plasmid pET28a(+) vector (denoted as emptyWC) were prepared using the same method and were prepared for later use.

[0070] The self-induction culture medium formula includes: 0.5% (w / v) yeast extract, 1% (w / v) peptone, 0.05% (w / v) glucose, 0.2% (w / v) lactose, 25 mM disodium hydrogen phosphate, 25 mM potassium dihydrogen phosphate, 50 mM ammonium chloride, 5 mM sodium sulfate, 2 mM magnesium sulfate, and 0.5 (w / v) glycerol.

[0071] 2. OleT SR Preparation of crude enzyme solution

[0072] After the induction expression was completed, the fermentation broth was centrifuged (6000 rpm, 4℃, 25 min), the supernatant was discarded, and the bacterial cells were collected and stored at -20℃ for later use. The bacterial cells were resuspended at a ratio of bacterial cells to Buffer A of 1:10 (w / v). Buffer A was prepared by adding 100 mM K₂HPO₄, 100 mM KH₂PO₄, 300 mM NaCl, 20 mM imidazole, and 5% (w / v) glycerol. The pH was adjusted to 8.0 during preparation. After thorough shaking, the suspension was filtered through a 0.22 μm aqueous filter and stored at 4℃ for later use. The resuspended bacterial solution was placed on ice and ultrasonically disrupted. The disrupted solution was then centrifuged at high speed (10000 rpm, 4℃, 35 min), and the supernatant was collected to obtain OleT. SRThe crude enzyme solution was stored at 4°C for later use. The crude enzyme solution for empty WC was prepared using the same method.

[0073] 3. OleT SR Purification

[0074] OleT SR The purification instruments and chromatography columns used were an AKTA Purifier, a Ni-NTA affinity chromatography column, and a HiPrep™ 26 / 10 gel filtration chromatography column. The column was first equilibrated with loading buffer (containing 100 mM K₂HPO₄, 100 mM KH₂PO₄, 300 mM NaCl, 20 mM imidazole, 5% (w / v) glycerol, pH 8.0). After equilibration, the crude enzyme solution was pumped in. After loading, the nickel column was washed with loading buffer until equilibration was achieved at a flow rate of 4 mL / min. Then, impurity removal buffer (containing 100 mM K₂HPO₄, 100 mM KH₂PO₄, 300 mM NaCl, 100 mM imidazole, 5% (w / v) glycerol, pH 8.0) was used to remove impurities, and the nickel column was washed until equilibration was achieved at a flow rate of 4 mL / min. Then, elute with elution buffer (containing 100 mM K₂HPO₄, 100 mM KH₂PO₄, 300 mM NaCl, 500 mM imidazole, 5% (w / v) glycerol, pH 8.0) at a flow rate of 3 mL / min. Collect the eluted sample and detect it by SDS-PAGE electrophoresis. For samples containing the peak corresponding to the target protein, replace the salt in a salt-replaced column. Add the protein to the salt-replaced column equilibrated with salt-replaced buffer (100 mM K₂HPO₄, 100 mM KH₂PO₄, 500 mM NaCl, 5% (w / v) glycerol, pH 7.5), continue eluting with the salt-replaced buffer, collect the eluted protein, concentrate and aliquot, and store at 4°C for later use.

[0075] Experimental results are as follows Figure 1 As shown, OleT is displayed. SR After purification by nickel column and elution with 500 mM imidazole, good expression and purification results were obtained.

[0076] Example 2 OleT SR Verification of the decarboxylation activity of catalytic fatty acids

[0077] This embodiment uses the OleT obtained above. SR The crude enzyme solution served as the experimental group, while the crude enzyme solution in empty WC served as the control group. The reaction buffer served as the blank control. The decarboxylation effect on fatty acids was tested. The specific experiments are as follows:

[0078] 1. Experimental Methods

[0079] First, add 500 μL of OleT to a 4 mL brown reaction flask. SR The reaction mixture consisted of crude enzyme solution, 390 μL reaction buffer (100 mM K₂HPO₄, 100 mM KH₂PO₄, 500 mM NaCl, 5% (v / v) glycerol, pH 7.5), 80 μL DMSO, 10 μL H₂O₂ solution (100 mM stock solution), and 20 μL DMSO stock solutions of different fatty acids (100 mM stock solution), with a total reaction volume of 1 mL. The fatty acids were selected from C6 to C20 saturated straight-chain fatty acids.

[0080] The reaction was then carried out at 30℃ and 500 rpm for 5 h. After the reaction started, 10 μL of H2O2 was added every half hour. The reaction was terminated with 40 μL of 6N HCl. An equal volume of ethyl acetate was added to the reaction mixture, along with an appropriate amount of anhydrous magnesium sulfate powder to remove water from the organic phase. The mixture was stirred at 500 rpm for 25 min, followed by centrifugation at 13500 rpm and 4℃ for 15 min. The upper organic phase was then aspirated using a 1 mL syringe, filtered through a 0.22 μm organic phase filter membrane, and transferred to a 2 mL chromatographic vial for gas chromatography analysis.

[0081] In the experiment, an Agilent 8860 gas chromatography system equipped with a flame ionization detector (FID) was used for the detection, identification, and quantitative analysis of fatty acids and alkenes. An HP-5 column (30m × 0.32mm × 0.25μm) was used for detection. Specific parameter settings were as follows: injection volume 1 μL, injector temperature 170℃, detector temperature 300℃, no split ratio set. The temperature program was: 40℃ held for 5 min, then increased to 300℃ at a rate of 20℃ per minute, held for 1 min, for a total time of 12.5 min.

[0082] The control group will use the above-mentioned OleT SR The crude enzyme solution was replaced with empty WC crude enzyme solution, while other conditions remained unchanged, to catalyze the decarboxylation of fatty acids. The blank control group was prepared with OleT... SR The crude enzyme solution and H2O2 were replaced with reaction buffer, while other conditions remained unchanged. The experiment was set up in triplicate. A bar chart of fatty acid substrate-olefin yield was plotted with time on the x-axis and olefin yield on the y-axis.

[0083] 2. Experimental Results

[0084] Experimental results are as follows Figure 2 As shown, the empty WC crude enzyme solution has no decarboxylation effect on fatty acids, while OleT... SRThe crude enzyme solution exhibited decarboxylation activity against C10-C20 saturated straight-chain fatty acids, but no decarboxylation activity was detected against C6-C8 saturated straight-chain fatty acids, indicating that OleT... SR It possesses the activity of catalyzing the decarboxylation of C10-C20 saturated straight-chain fatty acids.

[0085] Example 3 OleT SR Enzymatic properties characterization

[0086] This embodiment addresses the aforementioned OleT. SR The substrate preference, optimal reaction temperature, optimal reaction pH, and optimal salt concentration were investigated. Specific experiments are as follows:

[0087] 1. Substrate preference detection

[0088] By selecting saturated straight-chain fatty acids (C9-C20) and unsaturated fatty acids (C18:1, C18:2) of different chain lengths as reaction substrates, the study investigated OleT... SR Substrate range. The final concentration of the pure enzyme solution was set at 50 μM in a 1 mL reaction system, as follows:

[0089] Table 1:

[0090] Substrate solution (100 mM) 20 μL Reaction Buffer 390 μL <![CDATA[H2O2 solution (100 mM)]]> 10 μL <![CDATA[Purified OleT SR > 50 μM DMSO 80 μL Total volume 1mL

[0091] The reaction buffer contains 100 mM K2HPO4, 100 mM KH2PO4, 500 mM NaCl and 5% (v / v) glycerol, with a pH of 7.5.

[0092] The reaction was carried out at 30°C and 500 rpm for 5 hours, and subsequent steps were the same as in Example 2 above. A control group was also added, with OleT... SR Replace with OleT JE And OleT PRN All other conditions remained unchanged. The experiment was conducted in triplicate, and a bar chart of fatty acid substrate-olefin conversion was plotted.

[0093] Experimental results are as follows Figure 3 As shown, purified OleT SR It can catalyze the decarboxylation of C9-C20 saturated straight-chain fatty acids and oleic acid (C18:1), with a preference for medium and long-chain fatty acids, and the best effect is observed for C14:0 and C16:0 fatty acids; under the same conditions and after 5 h of reaction, the decarboxylation activity of this enzyme is generally superior to that of OleT. JE The decarboxylation efficiency for palmitic acid was 69.35%, which is higher than that of OleT. JE 1.3 times higher; this enzyme has a higher decarboxylation efficiency than OleT for saturated straight-chain fatty acids with fewer than C12. PRN .

[0094] 2. Optimal reaction temperature detection

[0095] Within a temperature range of 4-100℃ (set to 4, 15, 30, 35, 40, 45, 50, 60, 80, 100℃), the effect of different reaction temperatures on OleT was measured. SR The influence of enzyme activity, including methods for enzyme activity determination:

[0096] In a 4 mL brown reaction flask, add 5 mM myristic acid DMSO stock solution (C14, 100 mM stock solution) and 50 μM purified OleT. SR Enzyme, reaction buffer (100 mM K2HPO4, 100 mM KH2PO4, 500 mM NaCl, 5% (w / v) glycerol, pH 7.0), and finally 2 mM H2O2 were added, with a total reaction volume of 1 mL.

[0097] The reaction was carried out at different temperatures and 500 rpm for 15 min. The reaction was terminated by adding 40 μL of 6N HCl. Extraction was performed in the reaction flask by adding 1 mL of ethyl acetate and an appropriate amount of anhydrous magnesium sulfate (to remove moisture from the organic phase), centrifuging for 15 min, collecting the upper organic phase, filtering through a membrane, and performing GC analysis. The experiment was conducted in triplicate.

[0098] The highest measured enzyme activity was defined as 100%. Relative enzyme activities at different temperatures were calculated, and a line graph of temperature versus relative enzyme activity was plotted. OleT SR Enzyme activity is defined as the amount of enzyme required to release 1 nmol of 1-tetracene per minute at 30°C and 500 rpm, which is defined as 1 U.

[0099] Experimental results are as follows Figure 4 As shown, OleT SR The optimal reaction temperature is 30℃. Within the range of 30-60℃, its relative enzyme activity is higher than 75%. At 80℃, it can still maintain about 40% of the relative enzyme activity, which shows that it has a certain degree of high temperature resistance.

[0100] 3. Optimal reaction pH detection:

[0101] Within a temperature range of pH 5.0–10.0, the effect of different reaction pH values ​​on OleT was determined using the reaction system at the above-mentioned optimal reaction temperature. SRThe effect of pH on enzyme activity was investigated. Different pH buffers were used: citrate (pH 5.0, 6.0), phosphate (pH 6.0, 7.0, 7.5, 8.0), Tris-HCl (pH 8.0, 9.0), and glycine-NaOH (pH 9.0, 10.0). The experiment was conducted in triplicate. The highest measured enzyme activity was defined as 100%. Relative enzyme activities at different pH values ​​were calculated, and a line graph of pH versus relative enzyme activity was plotted.

[0102] Experimental results are as follows Figure 5 As shown, OleT SR The optimal reaction buffer is phosphate buffer, with the best reaction effect at pH 7.

[0103] 4. Detection of optimal NaCl concentration for reaction:

[0104] Using the above-mentioned optimal reaction temperature and optimal reaction pH, the effects of NaCl on OleT at concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1, 2, 4, and 5 M were determined. SR The effect of enzyme activity.

[0105] The experiment was conducted in triplicate. The highest enzyme activity measured was defined as 100%. The relative enzyme activity at different NaCl concentrations was calculated, and a line graph of NaCl concentration versus relative enzyme activity was plotted.

[0106] Experimental results are as follows Figure 6 As shown, OleT SR The optimal NaCl concentration is 0.3 M, and it exhibits good tolerance in salt concentrations of 0-5 M.

[0107] Example 4 OleT SR Salt tolerance test

[0108] This embodiment describes the purified OleT as described above. SR The salt tolerance was tested. The specific experimental method is as follows:

[0109] At 4℃, OleT SR The pure enzyme solution was incubated for 24 h in NaCl solutions of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1, 2, 4, and 5 M, respectively. Myristic acid substrate was then added, and OleT was measured using the reaction system at the optimal reaction temperature described in Example 3. SR The enzyme activity was calculated. Three parallel groups were set up for the experiment. Using the enzyme activity of the unincubated pure enzyme solution as 100%, the residual enzyme activity after incubation in different NaCl solutions for 24 h was calculated, and a bar chart of NaCl concentration versus residual enzyme activity was plotted.

[0110] Experimental results are as follows Figure 7 As shown, OleT SR It can adapt well to 0-5 M salt concentration environments. After incubation in 0-1 M NaCl solution for 24 h, the enzyme still retains more than 70% of its residual enzyme activity.

[0111] Example 5: Application in the catalytic preparation of α-olefins from glycerol esters

[0112] This embodiment provides an OleT solution by optimizing the preparation raw materials and reaction conditions. SR A method for the efficient catalytic preparation of α-olefins from glycerides via a lipase cascade includes the following experiments.

[0113] 1. Screening of lipases

[0114] In this experiment, three immobilized lipases, Novozyme435, TL IM, and RM IM, were used to catalyze the hydrolysis of trilaurate (C12), trimyristate (C14), tripalmitate (C16), and trioleate (C18:1), respectively, to evaluate the hydrolytic activity of different lipases on various fatty acid triglycerides.

[0115] (1) Evaluation of hydrolytic capacity of single fatty acid triglycerides

[0116] The specific experimental methods for evaluating the hydrolytic ability of different lipases on triglycerides of different single fatty acids are as follows:

[0117] In a 4 mL brown reaction flask, add 5 mg of lipase (Novozyme435, TL IM, or RM IM), 200 μL of 20 mM fatty acid triglyceride emulsion, and bring the reaction volume to 1.2 mL with PBS buffer. The fatty acid triglyceride emulsion is prepared by weighing 4 g of polyvinyl alcohol (PVA) into 100 mL of pure water, stirring at 90°C until the PVA particles are completely dissolved, then adding different fatty acid triglycerides (trilaurate, trimyristate, tripalmitate, or trioleate), homogenizing, and obtaining the final product.

[0118] The reaction was carried out at 30℃ and 500 rpm for 6 h. After the reaction was completed, 900 μL of the reaction mixture was transferred to a 2 mL centrifuge tube, and 900 μL of ethyl acetate and an appropriate amount of anhydrous magnesium sulfate were added for extraction. After vortexing and centrifugation, the upper organic phase was collected, and the fatty acid content was determined. The experiment was conducted in triplicate.

[0119] The results of the detection of the hydrolytic capacity of different lipases for triglycerides of different fatty acids are as follows: Figure 8 As shown, lipase TL IM has the best hydrolytic effect on triglycerides of different fatty acids, and its optimal substrate is trimyristic acid glyceride.

[0120] (2) Evaluation of the hydrolytic capacity of mixed fatty acid triglycerides

[0121] The specific experimental methods for evaluating the hydrolytic ability of different lipases on mixed fatty acid triglycerides are as follows:

[0122] In a 10 mL brown reaction flask, add 20 mg of lipase (Novozyme435, TL IM, or RM IM), 800 μL of mixed fatty acid glyceride emulsion (200 μL of each fatty acid glyceride emulsion, with a final concentration of 4 mM), and make up the reaction volume to 1.2 mL with PBS buffer.

[0123] The reaction was carried out at 30℃ and 500 rpm for 10 h. The experiment was set up in triplicate.

[0124] Results of the hydrolytic capacity of different lipases on equimolar mixed fatty acid triglycerides are as follows: Figure 9 As shown, the total free fatty acid content generated by lipase TL IM was 6.20 mM, which was 2.22 times and 1.50 times that of lipases Novozym 435 and RM IM, respectively. Myristic acid accounted for the largest proportion of the total free fatty acid content, further verifying the above results. Therefore, lipase TL IM was selected as the preferred enzyme for the cascade reaction in subsequent experiments.

[0125] 2. Regarding lipase and OleT... SR Optimize the order of addition

[0126] This experiment compared lipases TLIM and OleT. SR The effect of the order of addition of crude enzyme solution on the reaction. The specific experimental method is as follows:

[0127] In a 4 mL brown reaction flask, add 20 mg lipase TL IM, 300 μL 20 mM trimyristic acid glyceride emulsion, and 500 μL OleT SR The crude enzyme solution was then replenished with PBS buffer to make up the reaction volume to 1 mL.

[0128] The experiment was set up with four groups, as follows:

[0129] (A) Simultaneous addition of lipase and OleT SR crude enzyme solution;

[0130] (B) First add lipase and react for 10 h, then add OleT. SR crude enzyme solution;

[0131] (C) Add only lipase;

[0132] (D) Do not add lipase, use PBS buffer instead of OleT SR Crude enzyme solution.

[0133] The reaction was carried out at 30℃ and 500 rpm for 6 h. After the reaction started, 10 μL of H2O2 (100 mM mother liquor) was added every hour. The reaction was terminated by adding 40 μL of 6 N HCl, and olefin detection was performed.

[0134] Experimental results are as follows Figure 10 As shown, lipase and OleT were added simultaneously. SR The total amount of olefins produced by catalyzing glycerides is higher than that produced by adding lipase first and then OleT. SR The corresponding total amount of olefins generated. The above results indicate the simultaneous addition of lipase and OleT. SR One-pot reaction is more conducive to the hydrolysis and decarboxylation of fatty acid triglyceride substrates.

[0135] 3. Optimize the amount and frequency of H2O2 addition.

[0136] This experiment selected two addition methods (adding once every half hour or every hour) and two addition amounts (10 μL or 20 μL each time), with a H2O2 stock solution concentration of 100 mM. The specific experimental methods are as follows:

[0137] In a 4 mL brown reaction flask, add 20 mg of optimized immobilized lipase, 300 μL of 20 mM myristate triglyceride emulsion, and 500 μL of OleT. SR The crude enzyme solution was then replenished with PBS buffer to make up the reaction volume to 1 mL.

[0138] The experiment was set up in four groups, as follows:

[0139] (A) Add 10 μL every half hour;

[0140] (B) Add 10 μL per hour;

[0141] (C) Add 20 μL every half hour;

[0142] (D) Add 20 μL per hour.

[0143] The reaction was carried out at 30℃ and 500 rpm for 2 h. The reaction was terminated by adding 40 μL of 6 N HCl, and the olefins were then detected.

[0144] Experimental results are as follows Figure 11 As shown, adding 10 μL of H2O2 per hour resulted in the highest amount of olefins generated (0.48 mM). Therefore, H2O2 was subsequently supplemented by adding 10 μL per hour.

[0145] 4. Optimize the types of organic solvents and reaction times in the reaction system.

[0146] This experiment selected four polar organic solvents (ethanol, isopropanol, ethyl acetate, and DMSO) and eight reaction times (10, 20, 30, 60, 120, 360, 480, and 600 min). The specific experimental methods are as follows:

[0147] In a 4 mL brown reaction flask, add 5 mg of optimized lipase, 50 μL of 20 mM trimyristic acid glyceride emulsion (final concentration 1 mM), and 500 μL of OleT. SR Crude enzyme, 20 μL of different organic solvents (final ratio 2% (v / v)), and finally made up to 1 mL reaction volume with PBS buffer. Reacted at 30℃ and 500 rpm for different times, with H2O2 added according to the optimized amount and frequency. The reaction was terminated by adding 40 μL of 6N HCl, and olefin detection was performed.

[0148] Experimental results are as follows Figure 12 As shown, the olefin yields after adding four polar organic solvents were all higher than those in the blank group without any organic solvents. Ethyl acetate had the greatest effect on promoting decarboxylation efficiency, with an olefin yield of 0.73 mM after 6 h of reaction, which was 1.76 times that of the blank group. After adding ethyl acetate, the olefin concentration increased with increasing reaction time in the first 6 h of reaction. Therefore, ethyl acetate will be added to the cascade reaction in the subsequent reaction, and the reaction time will be determined to be 6 h.

[0149] 5. Optimize the reaction temperature

[0150] This experiment selected five reaction temperatures (30, 35, 40, 45, and 50℃) to study the effects of OleT... SR The process of preparing α-olefins from glycerides by lipase cascade catalysis was optimized, and the steps were as shown in Example 3, with other conditions and treatments remaining unchanged.

[0151] Experimental results are as follows Figure 13 As shown, the olefin yield of the cascade reaction is highest at a reaction temperature of 35°C. The olefin yield decreases when the reaction temperature is below or above 35°C.

[0152] 6. Optimize the concentration of the reaction substrate.

[0153] This experiment selected five substrate concentrations of trimyristic acid glyceride (0.5, 1, 2, 4, 8 mM) to study the effects of OleT... SR The process of preparing α-olefins from glycerides by lipase cascade catalysis was optimized, and the steps were as shown in Example 3, with other conditions and treatments remaining unchanged.

[0154] Experimental results are as follows Figure 14 As shown, the concentration of olefins generated in the cascade reaction is highest when the concentration of trimyristic acid glyceride is 1 mM. When the concentration of glyceride is below or above 1 mM, the olefin yield decreases.

[0155] 7. Optimize the dosage of lipase in the reaction.

[0156] This experiment set up five lipase TL IM concentrations (0.2, 0.6, 1, 1.4, or 1.8% w / v) to study the effects of OleT... SR The process of preparing α-olefins from glycerides by lipase cascade catalysis was optimized, and the steps were as shown in Example 3, with other conditions and treatments remaining unchanged.

[0157] Experimental results are as follows Figure 15 As shown, the olefin production rate increased slowly when the lipase dosage increased from 0.2% (w / v) to 1% (w / v). The olefin production reached its maximum when the lipase dosage was 1% (w / v). Further increases in lipase dosage led to a decrease in olefin production.

[0158] 8. Regarding the reaction OleT SR Optimization of crude enzyme solution dosage

[0159] This experiment sets up 5 types of OleT SR The effects of crude enzyme solution concentrations (0.3, 0.4, 0.5, 0.6, 0.7 v / v) on OleT SR The process of preparing α-olefins from glycerides by lipase cascade catalysis was optimized, and the steps were as shown in Example 3, with other conditions and treatments remaining unchanged.

[0160] Experimental results are as follows Figure 16 As shown, OleT SR The highest amount of olefins was generated when the crude enzyme solution was added at a concentration of 0.5. With the increase of OleT... SR With continued increase in the amount of crude enzyme solution added, the amount of olefins produced showed a decreasing trend.

[0161] 9. Optimize the amount of organic solvent added to the reaction system.

[0162] This experiment set up different organic solvent addition amounts (0.5%, 1%, 2%, 4%, 6% v / v) to affect OleT SR The process of preparing α-olefins from glycerides by lipase cascade catalysis was optimized, and the steps were as shown in Example 3, with other conditions and treatments remaining unchanged.

[0163] Experimental results are as follows Figure 17 As shown, the olefin yield is highest when the ethyl acetate addition is 2% (v / v); the olefin yield decreases when the ethyl acetate addition is lower or higher than 2% (v / v).

[0164] 10. Preparation of α-olefins under optimal conditions

[0165] This experiment tested OleT under the above-mentioned optimal conditions. SR The yield of α-olefins prepared from glycerides by lipase cascade catalysis was compared with that before optimization. The specific reaction parameters were set as follows:

[0166] Optimized group: reaction temperature 35℃, trimyristic acid glyceride dosage 1 mM, lipase dosage 1% (w / v), OleT SR The crude enzyme solution was added at a rate of 0.5% (v / v), and the ethyl acetate was added at a rate of 0.5% (v / v).

[0167] Unoptimized group: reaction temperature 30℃, trimyristic acid glyceride dosage 1 mM, lipase dosage 1% (w / v), OleT SR The crude enzyme solution was added at a rate of 0.5% (v / v), and the ethyl acetate was added at a rate of 2% (v / v). The steps were the same as in Example 3, with other conditions and treatment methods remaining unchanged.

[0168] like Figure 18 As shown, compared to unoptimized organic solvent addition, reaction temperature, lipase, and OleT... SR The system with increased crude enzyme solution yielded a maximum 1-tetracene yield of 1.27 mM after optimization of the cascade reaction system, which is 2.01 times that before optimization (0.63 mM).

[0169] Example 6: Application in the catalytic preparation of α-olefins from different substrates

[0170] This embodiment tested the optimized OleT. SR The decarboxylation effect of the lipase cascade catalytic reaction system on various other glycerides, including soybean oil, olive oil, coconut oil, and waste cooking oil, was investigated. Specific reaction parameters were set as follows:

[0171] The final concentrations of trilaurate, trimyristicin, tripalmitoylglycerol, and triolein were 1 mM. The final proportions of soybean oil, olive oil, coconut oil, and waste cooking oil were 1.25% (v / v). The amount of lipase added was 5 mg for each sample, and the amount of crude enzyme solution added was 500 μL. The reaction volume was made up to 1 mL with PBS buffer. The reaction was carried out at 35°C for 6 h. The steps were the same as in Example 3, with other conditions and treatment methods remaining unchanged.

[0172] The preparation method of the 25% (v / v) oil substrate includes: taking 30 mL of 4% (v / v) PVA solution, adding 10 mL of soybean oil, olive oil, coconut oil or waste cooking oil, homogenizing, and storing at 4℃.

[0173] Experimental results are as follows Figure 19As shown, the olefin yield of single oils is higher than that of complex oils, with tripalmitin having the highest olefin yield at 0.65 mM. Among the four oils with more complex compositions, coconut oil has the highest total olefin yield, while waste cooking oil has a slightly lower total olefin yield.

[0174] In summary, this invention discloses a marine-derived fatty acid decarboxylase, its preparation method, and its applications. This marine-derived fatty acid decarboxylase exhibits good decarboxylation activity for both saturated straight-chain fatty acids with 9-20 carbon atoms and C18:1 unsaturated fatty acids. Specifically, the decarboxylation efficiency for palmitic acid (C16:0) reaches 69.35% after 5 hours, significantly superior to the previously reported OleT enzyme. JE Meanwhile, the fatty acid decarboxylase of this invention exhibits excellent salt tolerance, retaining over 70% of its relative enzyme activity after 24 hours of incubation in 0-5 M NaCl. Furthermore, this invention optimizes the enzyme's catalytic decarboxylation of free fatty acids and the method for one-step preparation of α-olefins from glycerides via a cascade reaction of this enzyme and lipase. The optimized cascade catalytic reaction system increases the α-olefin yield of trimyristic acid glyceride to 1.27 mM, 2.01 times that before optimization. This system is applicable to various natural oils and waste cooking oils, possessing advantages such as a broad substrate spectrum, mild reaction conditions, simple operation, and environmental friendliness, demonstrating promising industrial application prospects in the fields of biofuels and biochemicals.

[0175] The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of the present invention.

Claims

1. A marine-derived fatty acid decarboxylase, characterized in that, The encoding nucleotide sequence is shown in SEQ ID NO.

2.

2. A recombinant expression vector, characterized in that, It contains the encoding nucleotide sequence of the marine-derived fatty acid decarboxylase as described in claim 1.

3. A recombinant engineered bacterial strain, characterized in that, It was obtained by transfecting the recombinant expression vector as described in claim 2 into a host bacterium.

4. A method for preparing a marine-derived fatty acid decarboxylase, characterized in that, include: The recombinant engineered strain described in claim 3 was inoculated into a culture medium containing vitamin B1 and then 5-aminolevulinic acid was added. After induction culture, the bacterial cells were collected, crushed, and centrifuged to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The final concentration of vitamin B1 is 0.2~0.8 mM; And / or, the final concentration of the 5-aminolevulinic acid is 0.1~1mM; And / or, the induction culture temperature is 16~20℃ and the time is 18~36h.

6. The preparation method according to claim 4 or 5, characterized in that, The centrifugation process also includes purification.

7. A method for catalytic decarboxylation of fatty acids, characterized in that, include: In the reaction system, the marine-derived fatty acid decarboxylase, hydrogen peroxide, and fatty acids described in claim 1 are mixed and reacted.

8. The method according to claim 7, characterized in that, The fatty acid is selected from at least one of C9-C20 saturated straight-chain fatty acids and C18 unsaturated fatty acids; And / or, the temperature of the reaction is 30~70°C; And / or, the pH of the reaction is 6 to 8.5; And / or, the buffer solution of the reaction system is a phosphate buffer solution; And / or, the NaCl concentration in the reaction system is 0.2~0.5M.

9. A method for preparing α-olefins via a cascade catalytic process of glycerides, characterized in that, The reaction system includes mixing the marine-derived fatty acid decarboxylase, lipase, hydrogen peroxide, and fatty acid glycerides as described in claim 1, and reacting them.

10. The method according to claim 9, characterized in that, The glyceride is selected from at least one of trilaurate, trimyristic acid, tripalmitate, trioleate, soybean oil, olive oil, coconut oil, peanut oil, and corn oil. And / or, the concentration of the glyceride is 0.5~2mM; And / or, the lipase is selected from at least one of lipase Novozyme435, lipase TL IM, or lipase RM IM; And / or, the amount of lipase added is 0.2~1.8% (w / v). And / or, the temperature of the reaction is 30~40℃; And / or, the reaction system may also contain an organic solvent.

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