Application of lipoprotein lipase tolerant to organic reagent in biodiesel preparation

Abstract

The invention provides application of lipoprotein lipase tolerant to an organic reagent in biodiesel preparation, and belongs to the technical field of enzyme engineering. The invention provides an application of lipoprotein lipase expressed by a recombinant pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL in the production of biodiesel by catalyzing oil and fat. The lipoprotein lipase has good tolerance to one or more than two conditions in organic reagents, high temperature and acidic and alkaline environments. According to the method, the lipoprotein lipase produced by the pseudomonas aeruginosa is used for catalyzing the waste edible oil and fat, so that the biodiesel can be efficiently synthesized. The lipoprotein lipase has wide application prospects in industrial diesel oil production in the future, the scheme is high in feasibility, and remarkable environmental benefits can be brought.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme engineering, and particularly relates to application of a lipoprotein lipase resistant to organic reagents in biodiesel production. BACKGROUND

[0002] Lipoprotein lipase (LPL) is an important industrial hydrolytic enzyme, widely distributed in various organisms from animals to bacteria, and participates in in vivo fat metabolism, can catalyze hydrolysis of various forms of long-chain triacylglycerol to form fatty acids and glycerol, and has a large demand in biological processes, and is one of important enzymes in global market. The lipase can catalyze hydrolysis of oil at an oil-water interface, and can also occur in complex catalytic reactions such as esterification, transesterification, lactone exchange, acidolysis, etc. in a microaqueous phase and a nonaqueous phase. The lipoprotein lipase can catalyze ester exchange reaction of oil and short-chain alcohol to synthesize biodiesel, and the enzyme method for synthesizing biodiesel has advantages such as mild reaction condition, less alcohol consumption, no pollution emission, and low requirement for raw materials, etc., and has become a trend of development of biodiesel today. At present, it has been reported that a variety of lipases can be used for catalytic synthesis of biodiesel, such as C. antarctica lipase and Rh. oryzae lipase, etc.

[0003] However, most enzymes are unstable under organic reagents, and the organic reagents can destroy the structure of the lipoprotein lipase in the process of synthesizing biodiesel, so that the lipoprotein lipase is inactivated, which seriously affects the efficiency of synthesizing biodiesel. SUMMARY

[0004] Based on this, the application provides application of a lipoprotein lipase in catalysis of oil for producing biodiesel, the lipoprotein lipase has high resistance to organic reagents, and can efficiently catalyze oil for synthesizing biodiesel.

[0005] In order to achieve the above purpose, the application provides the following technical scheme:

[0006] The application provides application of a lipoprotein lipase in catalysis of oil for producing biodiesel, the lipoprotein lipase is expressed by a recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL.

[0007] Preferably, the lipoprotein lipase comprises a lipoprotein lipase treated by an organic reagent.

[0008] Preferably, the volume fraction of the organic reagent is above 50%.

[0009] Preferably, the lipoprotein lipase comprises a lipoprotein lipase treated by high temperature of 30-70 DEG C and / or pH value of 5-9.

[0010] The oil and fat includes waste edible oil and fat.

[0011] The application provides application of recombinant Pseudomonas aeruginosa PAO1-pBBR1MCS-5-LPL in catalyzing oil and fat to produce biodiesel.

[0012] The application provides a method for catalyzing oil and fat to produce biodiesel by using lipoprotein lipase expressed by recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL.

[0013] Preferably, the preparation method of the lipoprotein lipase comprises the following steps: separating proteins in fermentation liquor of the strain PAO1-pBBR1MCS-5-LPL, and obtaining the lipoprotein lipase through purification.

[0014] Preferably, the oil and fat includes waste edible oil and fat.

[0015] The reaction system of the catalyzed oil and fat contains an organic reagent; the organic reagent includes short-chain alcohol.

[0016] Preferably, the temperature of the catalyzed oil and fat is 20-80 DEG C; the pH value of the catalyzed oil and fat is 4.5-11; and the time of the catalyzed oil and fat is no more than 48 h.

[0017] Preferably, the reaction system of the catalyzed oil and fat is as follows: waste edible oil and fat 40-60 g, distilled water 40-60 g, methanol 20 g and lipoprotein lipase 0.2 mL.

[0018] The adding method of the methanol includes adding in batches during the catalytic reaction.

[0019] Compared with the prior art, the application has the following advantages:

[0020] The application provides application of lipoprotein lipase expressed by recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL in catalyzing oil and fat to produce biodiesel. The lipoprotein lipase has high tolerance to organic reagents. Through tolerance experiments of the lipoprotein lipase to organic solvents (methanol, ethanol, glycerol and dimethyl sulfoxide), it is known that the lipoprotein lipase has high tolerance to organic reagents. The lipoprotein lipase can be used to catalyze oil and fat to efficiently synthesize biodiesel; when the relative conversion rate of the lipoprotein lipase is 100%, the relative conversion rate of LPL produced by E. coli is only 32.4%. The lipoprotein lipase can be used for industrialized diesel production in the future.

[0021] In the present application, the lipoprotein lipase preferably comprises a lipoprotein lipase treated at a high temperature of 30-70 DEG C and / or a pH value of 5-9; and the oil and fat comprises waste edible oil and fat. The lipoprotein lipase in the present application has good tolerance to high temperature and alkaline environment. The experimental results of temperature tolerance and pH tolerance show that the relative activity of the lipoprotein lipase is above 83.65% after being treated at a high temperature of 30-70 DEG C for 30 min, and the relative activity is above 57.7% after being treated at a pH value of 5-9 for 30 min. The waste edible oil and fat has a huge production amount, and the main components are triglyceride, free fatty acid, glycerol, etc. The lipoprotein lipase has a wide application prospect in future industrialized diesel production and can bring significant environmental benefits. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 2 is a diagram of SDS-PAGE purification results of the lipoprotein lipase, wherein lane M: Protein Marker; lane 1: purification results of Ecoli-LPL; lane 2: purification results of the lipoprotein lipase expressed by the strain PAO1-pBBR1MCS-5-LPL. DETAILED DESCRIPTION

[0023] The present application provides a kind of lipoprotein lipase in the application of catalyzing oil and fat production biodiesel, the lipoprotein lipase is by the recombinant pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL expression obtained.

[0024] In the present application, the strain PAO1-pBBR1MCS-5-LPL is prepared according to the scheme in the patent with the publication number CN120060316A and the patent name A recombinant vector for expressing LPL, a recombinant bacterium and a method for producing LPL.

[0025] In the present application, the method for preparing the lipoprotein lipase preferably comprises the following steps: separating the supernatant of the fermentation broth of the strain PAOl-pBBRlMCS-5-LPL; and purifying the enzyme in the supernatant to obtain the lipoprotein lipase. In the present application, the method for preparing the fermentation broth of the strain PAOl-pBBRlMCS-5-LPL is as follows: inoculating the strain PAOl-pBBRlMCS-5-LPL into LB liquid medium with a final concentration of 40 μg / mL of gentamicin at an inoculation amount of 1%, and culturing at 37°C and 220 rpm for 12 h to obtain a seed liquid; and inoculating the seed liquid into 50 mL of fermentation medium at an inoculation amount of 1%, and culturing at 26°C for 48 h to obtain the fermentation broth of the strain PAOl-pBBRlMCS-5-LPL. The fermentation medium preferably contains the following components: beef extract powder 5 g / L, tryptone 15 g / L, glucose 10 g / L, potassium dihydrogen phosphate 2 g / L, anhydrous magnesium sulfate 0.25 g / L, and potassium chloride 0.5 g / L; and the pH value of the fermentation medium is preferably 5.0-7.0, more preferably 6.0. The fermentation medium is advantageous for increasing the expression amount of the lipoprotein lipase. After obtaining the fermentation broth of the strain PAOl-pBBRlMCS-5-LPL, the supernatant is separated, and the supernatant is a crude enzyme solution. The crude enzyme solution is purified to obtain the lipoprotein lipase. The method for purification preferably comprises ion exchange chromatography. The lipoprotein lipase prepared by the method of the present application can catalyze oil and fat to produce biodiesel, and can efficiently convert waste edible oil and fat into biodiesel, with a relative conversion rate of 100% in 24 h, while the relative conversion rate of the lipoprotein lipase Ecoli-LPL produced by E. coli BL21 (DE3) is only 32.4%.

[0026] In the present application, the lipoprotein lipase preferably comprises a lipoprotein lipase treated with an organic reagent. The volume fraction of the organic reagent is preferably above 50%. The lipoprotein lipase of the present application still has good biological activity in the presence of the organic reagent, and can be used for producing biodiesel. The organic reagent preferably comprises at least one of methanol, ethanol, dimethyl sulfoxide (DMSO), and glycerol. In the present application, the lipoprotein lipase is mixed with methanol, ethanol, dimethyl sulfoxide (DMSO), and glycerol respectively at a volume ratio of 1:1, and incubated at 30°C for 3 h, and then the activity of the lipoprotein lipase is determined. The results show that the relative activity of the lipoprotein lipase is above 86.93%, while the relative activity of the lipoprotein lipase Ecoli-LPL produced by E. coli BL21 (DE3) is only above 20.3%, indicating that the lipoprotein lipase has high stability to the organic reagent. In the present application, the enzyme activity of the lipoprotein lipase is determined by using 4-nitrophenyl butyrate (PNPB) as a substrate.

[0027] In the present application, the lipoprotein lipase preferably comprises a lipoprotein lipase treated at a high temperature of 30-70 DEG C and / or a pH value of 5-9; and the oil and fat preferably comprises waste edible oil and fat. The lipoprotein lipase has good tolerance to high temperature, acid and alkaline environment. The temperature tolerance and pH tolerance experiment results show that the relative activity of the lipoprotein lipase is above 83.65% after being treated at a high temperature of 30-70 DEG C for 30 min, and the relative activity of the lipoprotein lipase is above 57.7% after being treated at a pH value of 5-9 for 30 min.

[0028] In the present application, the oil and fat preferably comprises waste edible oil and fat. The waste edible oil and fat preferably contains at least one of the following components: triglyceride, free fatty acid and glycerol. In addition, the waste edible oil and fat also contains residual salt and polar compounds such as aldehyde, ketone, hydroxy acid and polymer generated by the oxidation of oil and fat caused by high-temperature frying. In the present application, the oil and fat is waste edible oil and fat, and the mass percentage of triglyceride is about 90%. The results of the present application show that the lipoprotein lipase can efficiently convert waste edible oil and fat into biodiesel. In the present application, the lipoprotein lipase can catalyze the reaction of triglyceride and methanol to generate fatty acid methyl ester and glycerol, so the biodiesel contains fatty acid methyl ester and glycerol.

[0029] The present application provides a recombinant Pseudomonas aeruginosa PAO1-pBBR1MCS-5-LPL for catalyzing the production of biodiesel from oil and fat.

[0030] In the present application, the recombinant Pseudomonas aeruginosa PAO1-pBBR1MCS-5-LPL can express

[0031] The lipoprotein lipase can be used for catalyzing the production of biodiesel from oil and fat. The lipoprotein lipase for catalyzing the production of biodiesel from oil and fat is preferably the same as described above, and will not be repeated here.

[0032] The present application provides a method for catalyzing the production of biodiesel from oil and fat by enzyme method, using the lipoprotein lipase expressed by the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL to catalyze the production of biodiesel from oil and fat.

[0033] The present application provides a method for catalyzing the production of biodiesel from oil and fat by enzyme method, using the lipoprotein lipase expressed by the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL to catalyze the production of biodiesel from oil and fat.

[0034] In the present application, the preparation method of the lipoprotein lipase comprises the following steps: separating the protein in the fermentation broth of the strain PAO1-pBBR1MCS-5-LPL, and purifying to obtain the lipoprotein lipase.

[0035] In the present application, the oil and fat preferably comprises waste edible oil and fat; the waste edible oil and fat is preferably the same as described above, and will not be repeated here.

[0036] The reaction system of the catalytic oil and fat contains an organic reagent; the organic reagent is preferably the same as described above, and will not be repeated here. The organic reagent more preferably comprises a short-chain alcohol. The short-chain alcohol preferably comprises methanol and / or ethanol.

[0037] In the present application, the temperature of the catalytic oil and fat is 20℃-80℃, more preferably 30℃-70℃, more preferably 35℃-60℃, and most preferably 40℃; the pH value of the catalytic oil and fat is 4.5-11, more preferably 5-10, and most preferably 9; the time of the catalytic oil and fat is preferably not more than 48h, and more preferably not more than 24h.

[0038] In the present application, the reaction system of the catalytic oil and fat is: waste edible oil and fat 40-60g, distilled water 40-60g, methanol 20g, and lipoprotein lipase 0.2mL; wherein the method of adding methanol comprises adding it in batches during the catalytic reaction.

[0039] In the present application, the waste edible oil and fat and distilled water are mixed, and then the pH value is adjusted to 4.5-5, and then the lipoprotein lipase and methanol are added. Adjusting the pH value to 4.5-5 can maintain the activity and stability of the lipoprotein lipase, inhibit the saponification reaction, and adapt to the characteristics of the waste edible oil and fat (the waste edible oil and fat itself has a high acid value).

[0040] In order to further illustrate the present application, the schemes provided by the present application are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0041] 1. Material source

[0042] Strain PAO1-pBBR1MCS-5-LPL and strain Ecoli.BL21(DE3)-pBBR1MCS-5-LPL are prepared according to the scheme in the patent with publication number CN120060316A and patent name A recombinant vector for expressing LPL, a recombinant bacteria and a method for producing LPL.

[0043] 2. LPL activity detection method

[0044] (1) Detection principle:

[0045] 4-nitrophenyl butyrate (PNPB) is a widely used substrate in lipase hydrolytic activity assays. LPL hydrolyzes it to produce p-nitrophenol (PNP), which is yellow and has a high sensitivity with a maximum absorbance at 400 nm. The released p-nitrophenol is continuously determined by monitoring the increase in absorbance at 400 nm.

[0046] (2) Definition of enzyme activity unit:

[0047] 1.0 micromole (10 -6 micromoles) of p-nitrophenol per minute at 37°C and pH 7.2.

[0048] (3) Main reagents:

[0049] Reagent 1: 100 mmol / L NaH2PO4, 150 mmol / L NaCl, 0.5% Triton X-100, pH 7.2.

[0050] Reagent 2: 50 mmol / L PNPB (4-nitrophenyl butyrate) dissolved in 1 mL of acetonitrile.

[0051] Reagent 3: LPL enzyme solution.

[0052] (4) Sample determination:

[0053] 0.9 mL of reagent 1 and 0.1 mL of reagent 3 are mixed and preheated at 37°C for 2 min, then 0.01 mL of reagent 2 is added. After mixing, the absorbance change at 400 nm is immediately determined for 5 min to obtain △A 400 nm / min. Water is used instead of enzyme solution as a blank group, and the same method is used for determination.

[0054] 5. Enzymatic activity calculation formula of lipoprotein lipase:

[0055]

[0056] 1.01 = determination volume (mL); df = dilution factor; 14.8 = microliter extinction coefficient of p-nitrophenol at 400 nm; 0.1 = volume of enzyme used (mL). The operator between the parentheses is multiplication.

[0057] Example 1

[0058] Collection and purification of LPL

[0059] 1. Recombinant expression of strain PAO1-pBBR1MCS-5-LPL

[0060] The strain PAOl-pBBRlMCS-5-LPL was inoculated into LB liquid medium with a final concentration of 40 μg / mL of gentamicin at an inoculation amount of 1%, and cultured at 37°C, 220 rpm for 12 h, and the bacterial liquid was used as a seed liquid. Then the seed liquid was inoculated into 50 mL of fermentation medium at an inoculation amount of 1%, and fermented at 26°C for 48 h to obtain the fermentation liquid of the strain PAOl-pBBRlMCS-5-LPL.

[0061] 2. Collection of PAOl-LPL crude enzyme liquid

[0062] The fermentation liquid of the strain PAOl-pBBRlMCS-5-LPL was centrifuged at 12000 rpm for 5 min, and the LPL expressed in PAOl was collected from the supernatant of the fermentation liquid, i.e. the LPL crude enzyme liquid;

[0063] 3. Purification of PAOl-LPL

[0064] (1) A DEAE Sepharose ion exchange chromatography gravity column was loaded, 5 mL of the gravity column was taken, the lower gasket was loaded, sterile water was added to rinse the column tube and the gasket, 1 mL of DEAE filler was added to the gravity column, and the protection solution was dried;

[0065] (2) The filler was washed, and 10 times the column volume of sterile water was added to wash the filler;

[0066] (3) The filler was equilibrated, and 10 times the column volume of 20 mM PB buffer (pH 6.8) was added;

[0067] (4) A small amount of crude enzyme sample (pH 6.8) was added to the gravity column, and the filler was sucked out, and 50 mL of crude enzyme was incubated with the filler at 4°C for 2 h;

[0068] (5) After the incubation was completed, the crude enzyme and the filler were added to the gravity column, and the flow-out was collected;

[0069] (6) 10 times the column volume of 20 mM PB buffer (pH 6.8) was used to wash the impurities, and the impurity sample was collected;

[0070] (8) Elution was performed on the DEAE Sepharose using 0.1 M, 0.15 M, 0.35 M, and 0.45 M NaCl eluent, and the eluted sample was collected, which was the purified PAOl-LPL;

[0071] (9) The collected purified sample was run on SDS-PAGE, and the results are shown in Figure 1 .

[0072] Comparative Example 1

[0073] Collection and purification of Ecoli-LPL

[0074] 1. The recombinant expression of strain Ecoli.BL21(DE3)-pBBR1MCS-5-LPL is the same as that of strain PAOl-pBBR1MCS-5-LPL in Example 1.

[0075] 2. Collection of Ecoli-LPL crude enzyme solution

[0076] The bacteria in the fermentation broth of strain Ecoli.BL21(DE3)-pBBR1MCS-5-LPL are collected, resuspended with lysis buffer, and then subjected to ultrahigh pressure disruption to obtain the soluble protein Ecoli-LPL crude enzyme solution.

[0077] 3. The purification method of Ecoli-LPL is the same as that of PAOl-LPL in Example 1, and the eluted sample is purified Ecoli-LPL.

[0078] Example 2

[0079] Organic reagent tolerance experiment of LPL

[0080] The purified PAOl-LPL of Example 1 and the purified Ecoli-LPL of Example 2 are mixed with methanol, ethanol (analytical grade), DMSO and glycerol at a volume ratio of 1:1, respectively, and the mixture is incubated at 30°C for 3h. Then, the lipoprotein lipase activity is measured under standard conditions, and the same enzyme activity detection experiment is carried out with water as a control. The results of the organic reagent stability of the purified PAOl-LPL of Example 1 are shown in Table 1, and the results of the organic reagent stability of the purified Ecoli-LPL of Example 2 are shown in Table 2.

[0081] The enzyme activity of LPL produced by PAOl wild-type bacteria is only 1 U / mL.

[0082] Table 1 Organic reagent stability of PAOl-LPL

[0083] Organic reagent Enzyme activity (U / mL) Relative activity (%) Water (control) 336.7 100％ Methanol 292.7 86.93％ Ethanol 295.8 87.86％ DMSO 299.58 88.98％ Glycerol 391.3 116.24％

[0084] Table 2 Organic reagent stability of Ecoli-LPL

[0085] Organic reagent Enzyme activity (U / mL) Relative activity (%) Water (control) 80.6 100％ Methanol 16.3 20.3％ Ethanol 28.7 35.7％ DMSO 24.5 30.4％ Glycerol 40.7 50.6％

[0086] As can be seen from the data in Tables 1 and 2, the purified LPL of Example 1 has high stability in any one of methanol, ethanol, DMSO and glycerol.

[0087] Example 3

[0088] Optimum temperature and temperature tolerance experiment of PAOl-LPL

[0089] The purified PAOl-LPL of Example 1 was subjected to enzyme activity assay at 25°C, 30°C, 37°C, 40°C, 45°C, 50°C, 55°C and 60°C, respectively, to obtain the optimum temperature, with the relative enzyme activity at the optimum temperature being 100%. The detection results are shown in Table 3. The results show that the optimum temperature of the purified LPL of Example 1 is 40°C, and the enzyme activity does not change significantly under different temperature conditions.

[0090] Table 3 Optimum temperature of PAOl-LPL

[0091] Temperature Enzyme activity (U / mL) Relative activity (%) 25℃ 1494 56.6％ 30℃ 1665 63.3％ 37℃ 1760 66.9％ 40℃ 2630 100％ 45℃ 2450 93.1％ 50℃ 2410 91.6％ 55℃ 2330 88.5％ 60℃ 1960 74.5％

[0092] The purified LPL of Example 1 was incubated at 20°C, 30°C, 40°C, 50°C, 60°C and 70°C for 30 min, respectively, and then the lipoprotein lipase activity was determined under standard conditions. The enzyme activity was the highest at 40°C, and the enzyme activity at 40°C was taken as 100%.

[0093] The detection results are shown in Table 4. The results show that the purified LPL of Example 1 has strong high-temperature tolerance, and the relative enzyme activity at 50-70°C is higher than that of the non-incubated treatment, indicating that it has good tolerance to high temperature.

[0094] Table 4 Temperature tolerance of PAOl-LPL

[0095] Temperature Enzyme activity (U / mL) Relative activity (%) 20℃ 960 92.31％ 30℃ 1010 97.12％ 40℃ 1040 100％ 50℃ 970 93.27％ 60℃ 950 91.35％ 70℃ 870 83.65％

[0096] Example 4

[0097] Optimum pH and pH tolerance experiment of PAOl-LPL

[0098] The purified PAOl-LPL of Example 1 was subjected to enzyme activity assay at pH values of 5, 6, 7, 8, 9, 10 and 11, respectively, to obtain the optimum pH, with the relative enzyme activity at the optimum pH being 100%. The detection results are shown in Table 5. The results show that the optimum pH of the purified PAOl-LPL of Example 1 is 9.

[0099] Table 5 Optimum pH of PAOl-LPL

[0100] pH Enzyme activity (U / mL) Relative activity (%) 5 1090 31.5％ 6 1730 50％ 7 2020 58.3％ 8 3410 98.55％ 9 3460 100％ 10 2520 72.8％ 11 2010 58％

[0101] The purified PAOl-LPL was incubated at pH values of 5, 6, 7, 8, 9, 10 and 11 for 30 min, respectively, and then the lipoprotein lipase activity was determined under standard conditions. The relative enzyme activity at the pH with the highest enzyme activity was taken as 100%. The detection results are shown in Table 6. The results show that the purified PAOl-LPL of Example 1 has high tolerance to alkaline environment.

[0102] Table 6 pH tolerance of PAOl-LPL

[0103] pH Enzyme activity (U / mL) Relative activity (%) 5 1430 57.7％ 6 1460 58％ 7 2280 90.1％ 8 2530 100％ 9 2470 98％ 10 460 18.1％ 11 9.5 4％

[0104] Example 5

[0105] Application of LPL in biodiesel production

[0106] Take 15 g of canteen waste oil, add 70 ℃ hot distilled water 15 g, shake for 5 min, and then separate the layers. Measure the pH of the lower layer water sample. If the pH is <4.5, add an appropriate amount of 16% NaOH solution to the oil sample, and stir magnetically for 15 min. Then measure the pH again until the pH is adjusted to 4.5-5.0.

[0107] Take 100 g of waste oil sample with adjusted pH in a four-hole flask, and add 0.2 mL of PAO1-LPL purified in Example 1 and Ecoli-LPL purified in Example 2, respectively. Add methanol in the manner shown in Table 7, a total of 20 g. Place the four-hole flask in a 40 ℃ water bath, and start stirring. React for 24 h, and then take samples to measure the change in acid value according to the third method in GB5009.229-2025, and calculate the conversion rate.

[0108] The acid value and conversion rate results are shown in Table 8.

[0109] Table 7 Methanol addition method

[0110] Time (h) Amount added (g) 0 4 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2

[0111] Table 8 Acid value and relative conversion rate

[0112] Sample Initial acid value (mgKOH / g) 24h acid value (mgKOH / g) Relative conversion (%) PAOl-LPL 50 20.4 100％ Ecoli-LPL 50 40.4 32.4％

[0113] Take PAO1-LPL as the relative conversion rate of 100%, and the conversion rate of Ecoli-LPL is: (Ecoli-LPL initial acid value - Ecoli-LPL 24h acid value) / (PAO1-LPL initial acid value - PAO1-LPL 24h acid value) x 100%, that is, (50-40.4) / (50-20.4) x 100%. The results in Table 8 show that PAO1-LPL can efficiently catalyze the synthesis of biodiesel from canteen waste oil.

[0114] Example 6

[0115] Principle of LPL stability

[0116] The polysaccharide precipitates with some specific compounds (such as metal ions) to form a complex or a combination. To explore whether the principle of LPL stability is that the recombinant P. aeruginosa secreted lipoprotein lipase forms an enzyme-lipopolysaccharide complex to enhance the stability, the precipitation and redissolution experiments of the lipoprotein lipase were carried out. The magnesium sulfate powder was added to the supernatant of the fermentation broth of the strain PAOl-pBBRlMCS-5-LPL in Example 1, 10 g of magnesium sulfate was added per 100 mL of the supernatant of the fermentation broth, and it was precipitated at 4°C overnight. The supernatant and the precipitate were separated by high-speed centrifugation at 10,000 rpm for 10 min. The precipitate was redissolved with 50 mM Tris-HCl buffer (pH 8.0) in an equal volume to obtain the redissolved material after precipitation.

[0117] The enzyme activities of the supernatant of the fermentation broth (before precipitation), the supernatant after precipitation, and the redissolved material after precipitation were detected, respectively, and the detection results are shown in Table 9.

[0118] Table 9: Enzyme activity detection results

[0119] Sample Enzyme activity U / mL Total enzyme activity Relative activity % Fermentation broth supernatant (before precipitation) (20 mL) 80 1600 100％ Supernatant after precipitation (20 mL) 40 800 50％ Resuspension after precipitation (20 mL) 53 1060 66.25％

[0120] The results show that the relative activity of the redissolved material after precipitation is 66.25%, indicating that the LPL produced by PAOl-pBBRlMCS-5-LPL carries lipopolysaccharide, which can enhance the stability, while the LPL produced by E. coli does not produce lipopolysaccharide precipitation, and the stability is relatively low.

[0121] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. The application of a lipoprotein lipase in the catalytic production of biodiesel from oils, wherein the lipoprotein lipase is expressed by a recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL.

2. The application according to claim 1, characterized in that, The lipoprotein lipase includes lipoprotein lipase treated with organic reagents.

3. The application according to claim 2, characterized in that, The volume fraction of the organic reagent is above 50%.

4. The application according to claim 1, characterized in that, The lipoprotein lipase includes lipoprotein lipase treated at high temperatures of 30°C to 70°C and / or at pH values ​​of 5 to 9. The oils include waste cooking oils.

5. Application of a recombinant Pseudomonas aeruginosa PAO1-pBBR1MCS-5-LPL in the catalytic production of biodiesel from oils.

6. A method for producing biodiesel from oils using enzymatic catalysis, characterized in that, The production of biodiesel was catalyzed by lipoprotein lipase expressed by recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-LPL.

7. The method according to claim 6, characterized in that, The method for preparing the lipoprotein lipase includes the following steps: separating the protein from the fermentation broth of the strain PAO1-pBBR1MCS-5-LPL, and purifying it to obtain the lipoprotein lipase.

8. The method according to claim 6, characterized in that, The oils include waste cooking oils; The reaction system of the catalytic oil contains organic reagents; the organic reagents include short-chain alcohols.

9. The method according to claim 6, characterized in that, The temperature of the catalytic oil is 20℃~80℃; the pH value of the catalytic oil is 4.5~11; and the catalytic time is no more than 48h.

10. The method according to any one of claims 6 to 9, characterized in that, The reaction system for catalyzing oils is as follows: 40-60g of waste edible oils, 40-60g of distilled water, 20g of methanol, and 0.2mL of lipoprotein lipase; The method of adding methanol includes adding it in batches during the catalytic reaction.

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