Application of lipoprotein lipase in promoting synthesis of rhamnolipid from pseudomonas aeruginosa

Abstract

The invention provides application of lipoprotein lipase in promoting synthesis of rhamnolipid from pseudomonas aeruginosa, and belongs to the technical field of microbial fermentation. The invention provides an application of lipoprotein lipase in promotion of synthesis of rhamnolipid from pseudomonas aeruginosa (Pseudomonas aeruginosa), and a preparation method of the lipoprotein lipase in the promotion of synthesis of rhamnolipid from the pseudomonas aeruginosa (Pseudomonas aeruginosa). The lipoprotein lipase is lipoprotein lipase from pseudomonas aeruginosa. The traditional wild pseudomonas aeruginosa strain PAO1 needs more than 120 hours to achieve higher yield, while the method disclosed by the invention can achieve higher yield within 48 hours, so that the fermentation period is shortened by at least 50%, and the production time cost is greatly reduced. The method is simple and convenient, only the lipoprotein lipase needs to be exogenously added, the production cost can be reduced, and industrial requirements are met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to application of a lipoprotein lipase in promoting synthesis of rhamnolipids by Pseudomonas aeruginosa. BACKGROUND

[0002] As a kind of biological surfactant, rhamnolipids have good emulsifying property, biodegradability and environmental compatibility, and have wide application prospects in the fields of oil exploitation, sewage treatment, medicine and the like. The production of rhamnolipids from waste oil by microbial fermentation can realize resource utilization of waste resources and reduce the production cost of biological surfactants, and is a hot research direction at present.

[0003] Pseudomonas aeruginosa (Pseudomonas aeruginosa) Pseudomonas aeruginosa ) is a kind of model strain capable of synthesizing rhamnolipids, but the utilization efficiency of waste oil and the yield of rhamnolipids of the wild type strain still need to be improved, and the culture cycle of the traditional wild type Pseudomonas aeruginosa is long. SUMMARY

[0004] Therefore, the application provides application of a lipoprotein lipase in promoting synthesis of rhamnolipids by Pseudomonas aeruginosa (Pseudomonas aeruginosa) Pseudomonas aeruginosa ), which can effectively improve the yield of rhamnolipids and shorten the culture cycle.

[0005] In order to achieve the above purpose, the application provides the following technical scheme. The application provides application of a lipoprotein lipase in promoting synthesis of rhamnolipids by Pseudomonas aeruginosa (Pseudomonas aeruginosa) Pseudomonas aeruginosa ); The lipoprotein lipase is a lipoprotein lipase derived from Pseudomonas aeruginosa.

[0006] Preferably, the Pseudomonas aeruginosa includes Pseudomonas aeruginosa PAO1 and / or recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL .

[0007] Preferably, the working concentration of the lipoprotein lipase is above 0.6 U / ml.

[0008] The application provides a method for promoting synthesis of rhamnolipids by Pseudomonas aeruginosa, which utilizes a lipoprotein lipase and Pseudomonas aeruginosa to culture in a fermentation medium containing oil, so as to obtain rhamnolipids. The lipoprotein lipase is a lipoprotein lipase derived from Pseudomonas aeruginosa.

[0009] Preferably, the ratio of the lipoprotein lipase to oil is 26-38 U:5 g.

[0010] Preferably, the fermentation medium comprises the following components: oil 100-150 g / L, NaNO3 13-17.0 g / L, MgSO4·7H2O 0.4-0.6 g / L, KCl 0.8-1.2 g / L, and K2HPO4 0.2-0.4 g / L.

[0011] Preferably, during the culture, trace element solution is added in batches. Preferably, the trace element solution comprises the following components: sodium citrate 1.8-2.2 g / L, FeCl3·6H2O 0.25-0.3 g / L, ZnSO4·7H2O 1.2-1.6 g / L, CoCl2·6H2O 1.0-1.4 g / L, CuSO4·5H2O 1.0-1.4 g / L, and MnSO4·H2O 0.6-1.0 g / L. When the trace element solution is added in batches, each addition accounts for 2-6 ‰ of the volume of the fermentation medium. The interval between adjacent two batches of addition is 15-25 h.

[0012] Preferably, the culture time is 20-120 h, and the culture temperature is 35-37 °C.

[0013] Preferably, the Pseudomonas aeruginosa comprises Pseudomonas aeruginosa PAO1 and / or recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL .

[0014] Preferably, the preparation method of the lipoprotein lipase is to separate the recombinant protein from the fermentation broth of the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL .

[0015] Compared with the prior art, the present application has the following advantages: The present application provides a use of a lipoprotein lipase in promoting Pseudomonas aeruginosa to synthesize rhamnolipid. Pseudomonas aeruginosa The lipoprotein lipase of the present application can effectively improve the yield of rhamnolipid. The present application compares the effects of lipoprotein lipases from different sources on the synthesis of rhamnolipid by Pseudomonas aeruginosa strains. The results show that the yield of rhamnolipid in the wild type control group is 9.37 g, while the yield of rhamnolipid in the wild type control group with added lipoprotein lipase is increased to 14.29 g / L. The lipoprotein lipase combined with the overexpression strain PAO1-pBBR1MCS-5- LPLThe rhamnolipid yield reaches the maximum of 14.51 g / L. This shows that the lipoprotein lipase can effectively promote the synthesis of rhamnolipid by the Pseudomonas aeruginosa.

[0016] The application provides a method for promoting the synthesis of rhamnolipid by Pseudomonas aeruginosa, which comprises culturing lipoprotein lipase and Pseudomonas aeruginosa in a fermentation medium containing oil to obtain rhamnolipid; the lipoprotein lipase is a lipoprotein lipase derived from Pseudomonas aeruginosa. The method can significantly improve the yield of rhamnolipid in 48 hours and shorten the culture period. The traditional wild-type Pseudomonas aeruginosa strain PAO1 needs more than 120 hours to reach a high yield, while the method can achieve high yield in 48 hours, shortens the fermentation period by at least 50%, and greatly reduces the production time cost. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a rhamnolipid standard curve diagram based on the anthrone method; Figure 2 It is a result diagram of rhamnolipid yield and oil consumption in 48h fermentation liquid; Figure 3 It is a recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL It is a result diagram of the number of colonies of Pseudomonas aeruginosa PAO1 fermented for 48h and the glycerol content in the fermentation liquid; Figure 4 It is a rhamnolipid spectrum diagram detected by HPLC; Figure 5 It is a rhamnolipid ESI-MS spectrum diagram; Figure 6 It is a rhamnolipid MS / MS diagram. DETAILED DESCRIPTION

[0018] The application provides an application of a lipoprotein lipase in promoting the synthesis of rhamnolipid by Pseudomonas aeruginosa (Pseudomonas aeruginosa). Pseudomonas aeruginosa The lipoprotein lipase is a lipoprotein lipase derived from Pseudomonas aeruginosa.

[0019] In the application, the Pseudomonas aeruginosa preferably comprises Pseudomonas aeruginosa PAO1 and / or a recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL The Pseudomonas aeruginosa PAO1 is purchased from the Biosynth Carlsbad Company. The strain PAO1-pBBR1MCS-5- LPL is prepared according to the scheme in Example 3 of a patent with the patent name of a recombinant vector expressing LPL, a recombinant bacteria and an LPL production method and the publication number CN120060316A.

[0020] In this invention, the lipoprotein lipase is preferably derived from the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL The expression was obtained. The preferred amino acid sequence of the lipoprotein lipase is as shown in SEQ ID NO: 1 (MKKKSLLPLGLAIGLASLAASPLIQASTYTQTKYPIALAHGALGFDNILGVDYWFGIPSALRRDGAQVYVTEVSQLDTSEVRGEQLLQQVEEIVALSGQPKVNLIGHSHGGPTIRYVAAVRPDLIASATSVGAPHKGSDTADFLRQIPPGSAEAAILSGLVNSLGALISFLSSGSTGTQNALGSLESLSSEGAARFNAKYPHGVPTSACGEGAYKVNGVSYYSWSGSSPLTNFLDPSDAFLGASSLTFKNGTANDGLVGTCSSHLGMVIRDNYKMNHLDEVNQVFGLTSLFEASPVSVYRQHANRLKNASL).

[0021] The lipoprotein lipase is preferably derived from the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL The recombinant protein was isolated from the fermentation broth of the strain PAO1-pBBR1MCS-5-. The preferred method for preparing the lipoprotein lipase includes the following steps: [The text abruptly ends here, so the translation also ends here.] LPL The supernatant was separated from the fermentation broth to obtain the lipoprotein lipase. In this embodiment of the invention, the strain PAO1-pBBR1MCS-5- LPL The fermentation broth was prepared by inoculating strain PAO1-pBBR1MCS-5- at a 10% inoculum. LPL The inoculum was inoculated into LB medium for activation culture. The activated strain PAO1-pBBR1MCS-5- LPL Inoculate the seed culture medium at a rate of 5%–10% to obtain a seed culture solution; then inoculate the seed culture solution into a fermentation medium at a rate of 10% to obtain the strain PAO1-pBBR1MCS-5- LPLFermentation broth. The NaCl concentration in the LB medium is preferably 8-12 g / L, more preferably 9-11 g / L, and most preferably 10 g / L. The activation culture temperature is preferably 35-39 °C, more preferably 36-38 °C, and most preferably 37 °C. The activation culture time is preferably 10-14 h, more preferably 11-13 h, and most preferably 12 h. The activation culture rotation speed is preferably 200-240 rpm, more preferably 220 rpm. The seed culture medium is preferably the same as the activation culture medium. The seed culture time, temperature, and rotation speed are preferably the same as those of the activation culture. The fermentation medium preferably comprises the following components in the following amounts: oil 100-150 g / L, NaNO3 13-17.0 g / L, MgSO4·7H2O 0.4-0.6 g / L, KCl 0.8-1.2 g / L, and K2HPO4 0.2-0.4 g / L. More preferably, it comprises the following components in the following amounts: oil 125 g / L, NaNO3 15.0 g / L, MgSO4·7H2O 0.5 g / L, KCl 1.0 g / L, and K2HPO4 0.3 g / L. The pH of the fermentation medium is preferably 6.0-7.0, more preferably 6.0. During the fermentation process, a trace element solution is preferably added. When added in batches, the volume of each addition is preferably 2-6‰ of the volume of the fermentation medium, more preferably 4‰. The interval between two consecutive batch additions is preferably 15-25 h, more preferably 18-22 h, and most preferably 20 h. In this embodiment of the invention, the supplementation time is preferably the 0h, 20h and 40h of fermentation. The trace element solution preferably comprises the following components in the following amounts: sodium citrate 1.8~2.2 g / L, FeCl3·6H2O 0.25~0.3 g / L, ZnSO4·7H2O 1.2~1.6 g / L, CoCl2·6H2O 1.0~1.4 g / L, CuSO4·5H2O 1.0~1.4 g / L, and MnSO4·H2O 0.6~1.0 g / L; more preferably, it comprises the following components in the following amounts: sodium citrate 2.0 g / L, FeCl3·6H2O 0.28 g / L, ZnSO4·7H2O 1.4 g / L, CoCl2·6H2O 1.2 g / L, CuSO4·5H2O 1.2 g / L, and MnSO4·H2O 0.8 g / L. The nitrogen source in the fermentation medium includes sodium nitrate, and the inorganic salts include potassium dihydrogen phosphate and magnesium sulfate. The trace elements include iron, zinc, cobalt, copper, and manganese. Supplementing the fermentation medium with a trace element solution is beneficial for the growth of the strain and the production of rhamnolipids. The strain PAO1-pBBR1MCS-5- was obtained. LPLAfter fermentation, the proteins in the fermentation broth are separated to obtain the lipoprotein lipase. The separation method preferably includes centrifugation and collection of the supernatant. The centrifugation speed is preferably 11000 rpm, and the centrifugation time is preferably 10 min. In this invention, the working concentration of the lipoprotein lipase is preferably 0.6 U / ml or higher, more preferably 0.8~2 U / ml, and even more preferably 0.9 U / ml.

[0022] In this invention, lipoprotein lipase derived from *Pseudomonas aeruginosa* can effectively promote the synthesis of rhamnolipids by *P. aeruginosa*. Examples of this invention compare the effects of adding or removing lipoprotein lipase on *P. aeruginosa* strains PAO1 (wild-type strain) and PAO1-pBBR1MCS-5-. LPL The effect of lipoprotein lipase overexpression strains on rhamnolipid synthesis was investigated. Results showed that the lipolipid production was highest in the lipoprotein lipase overexpression strain (14.51 g / L), followed by the wild-type strain with lipoprotein lipase (14.29 g / L), the lipoprotein lipase-overexpression strain without lipoprotein lipase (12.81 g / L), and the wild-type strain without lipoprotein lipase (9.37 g / L). This indicates that lipoprotein lipase can effectively promote rhamnolipid synthesis in *Pseudomonas aeruginosa*. The lipoprotein lipase overexpression strain PAO1-pBBR1MCS-5-... LPL The lipoprotein lipase, when combined with exogenously added lipoprotein lipase, promotes the synthesis of rhamnolipids. Furthermore, the embodiments of this invention further compared the effects of lipoprotein lipases from different sources on rhamnolipid production, showing that lipoprotein lipase from *Pseudomonas aeruginosa* was the most effective. In the embodiments of this invention, the enzyme activity of lipoprotein lipase was determined using 4-nitrophenyl butyrate (PNPB) as a substrate.

[0023] In one embodiment of the present invention, in order to screen lipoprotein lipases that promote rhamnolipid synthesis, the differences in rhamnolipid synthesis between lipoprotein lipases from different bacterial sources were compared. The results showed that the lipoprotein lipase from Pseudomonas aeruginosa was the most effective (14.29 g / L), while the lipoprotein lipases from thermophilic fungi and Mucor could not effectively increase the content of rhamnolipids.

[0024] This invention provides a method for promoting the synthesis of rhamnolipids by Pseudomonas aeruginosa, which involves culturing lipoprotein lipase and Pseudomonas aeruginosa in an oil-containing fermentation medium to obtain rhamnolipids. The lipoprotein lipase is preferably a lipoprotein lipase derived from Pseudomonas aeruginosa.

[0025] In this invention, the *Pseudomonas aeruginosa* and the lipoprotein lipase are preferably the same as those described above, and will not be repeated here. The oil preferably includes waste edible oil. The oil preferably contains at least one of the following components: triglycerides, free fatty acids, and glycerol. In an embodiment of this invention, the triglyceride content in the oil is approximately 90% by mass.

[0026] In this invention, the ratio of lipoprotein lipase to oil is preferably 26-38 U: 5g, more preferably 28-34 U: 5g, and most preferably 32 U: 5g.

[0027] In this invention, the fermentation medium is preferably the same as the fermentation medium described above, and will not be repeated here.

[0028] In this invention, during the cultivation period, a trace element solution is added in batches; the amount of trace element solution added in each batch is 2-6‰ of the volume of the fermentation medium, more preferably 4‰. The trace element solution and the timing of its addition are preferably the same as those for the trace element solution. The cultivation time is preferably 20-120 hours, more preferably 30-80 hours, further preferably 40-60 hours, and most preferably 48 hours. The cultivation temperature and rotation speed are preferably the same as those for the activation cultivation, and will not be repeated here.

[0029] The method described in this invention can effectively promote the synthesis of rhamnolipids by *Pseudomonas aeruginosa*, significantly increasing rhamnolipid yield and shortening the fermentation cycle. Traditional wild-type *Pseudomonas aeruginosa* PAO1 requires over 120 hours to achieve high yields, while the method described in this invention can achieve high yields within 48 hours, shortening the fermentation cycle by at least 50% and significantly reducing production time and costs. The method also efficiently improves the utilization efficiency of waste oils and promotes resource conversion. The oil degradation rates of the wild-type group with PAO1-lipase and the overexpression group with PAO1-lipase were significantly higher than those of the wild-type control group and the overexpression control group. Lipoprotein lipase, as a key lipase, can rapidly hydrolyze waste oils into substrates such as glycerol that are usable by microorganisms, reducing oil residue. This achieves efficient resource utilization of waste oils and provides sufficient carbon sources for rhamnolipid synthesis, reducing raw material waste. The method described in this invention is simple, requiring only the exogenous addition of lipoprotein lipase, which reduces production costs, adapts to industrial needs, and is suitable for simple production scenarios. The present invention allows for the on-demand control of the dosage of exogenous lipoprotein lipase (e.g., adding it only during the 0-48h period when the lipid concentration is high), avoiding the metabolic resource occupation caused by the continuous synthesis of enzyme protein by overexpressing strains, and ensuring more efficient energy allocation in rhamnolipid synthesis.

[0030] In this invention, the amount of glycerol produced after hydrolysis of the oil can be used to reflect lipase activity. The extraction of rhamnolipin involves centrifuging the fermentation broth, adding acid to the supernatant to adjust the pH to 2-3 to precipitate the rhamnolipin, then extracting with a mixed ethyl acetate solution, and concentrating the extract to obtain crude rhamnolipin. The content of rhamnolipin is determined using the anthrone method and high-performance liquid chromatography (HPLC). The qualitative method for rhamnolipin includes HPLC qualitative analysis: using a C18 column, with acetonitrile-water (60:40, v / v) as the mobile phase, and detecting rhamnolipin homologues at a wavelength of 210 nm. Before HPLC analysis, the sample needs to be derivatized: triethylamine and 4-bromobenzoyl bromide are added, reacted at 60°C for 30 min, and then used for mass spectrometry identification. The mass spectrometry identification employed electrospray ionization (ESI) negative ion mode, and the structures of monorhamnolipids and dirhamnolipids were confirmed by detecting characteristic ion peaks at m / z 505 and m / z 685.

[0031] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] 1. Materials and Sources Fermentation medium: 125 g / L waste oil, 15.0 g / L NaNO3, 0.5 g / L MgSO4·7H2O, 1.0 g / L KCl, 0.3 g / L K2HPO4, adjusted to pH 6.5 with 0.1 M pH 6.5 sodium phosphate buffer.

[0033] Fermentation medium preparation method: Add 150 mL of distilled water to a 250 mL Erlenmeyer flask, and dissolve 3.0 g of NaNO3, 0.1 g of MgSO4·7H2O, 0.2 g of KCl, and 0.06 g of K2HPO4 in sequence. Adjust the pH to 6.5 with 0.1 M sodium phosphate buffer with a pH of 6.5. Add waste oil: Add 6.25 g of waste oil (pretreated to remove impurities) to 50 mL of the above aqueous system to make the waste oil concentration 125 g / L, thus obtaining the fermentation medium.

[0034] Trace element solution: sodium citrate 2.0 g / L, FeCl3・6H2O 0.28 g / L, ZnSO4・7H2O 1.4 g / L, CoCl2・6H2O 1.2 g / L, CuSO4・5H2O 1.2 g / L, MnSO4・H2O 0.8 g / L, sterilized at 121℃ for 20 min, and filtered through a 0.22 μm filter membrane.

[0035] Example 1 1. Seed liquid preparation: Pseudomonas aeruginosa strain PAO1 was inoculated into LB medium (containing 10 g / L NaCl) and activated by culture at 37℃ and 220 rpm for 12 h; the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- was then removed from -80℃. LPL Glycerol tubes were inoculated into 5 mL LB medium (containing 10 g / L NaCl) and activated at 37 °C and 220 rpm for 12 h to obtain strain PAO1-pBBR1MCS-5- LPL Activation solution.

[0036] Take 5 mL of the activation solution of Pseudomonas aeruginosa strain PAO1 and the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- respectively. LPL The activation solution was transferred to 50 mL of LB seed culture medium and cultured at 37℃ and 120 rpm for 12 h to obtain seed culture of Pseudomonas aeruginosa strain PAO1 and recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5-. LPL Seed liquid.

[0037] 2. Sources of lipoprotein lipase: thermophilic fungi ( Thermomyces lanuginosus ) Lipoprotein lipase source: purchased from Blue Ocean Biotechnology Co., Ltd., and prepared into an enzyme solution with water; Mucor ( Mucor megnus ) Lipoprotein lipase source: purchased from Blue Ocean Biotechnology Co., Ltd., and prepared into an enzyme solution with water; PAO1-pBBR1MCS-5- LPL Lipoprotein lipase source: PAO1-pBBR1MCS-5- LPL The seed culture was transferred to 50 mL of fermentation medium at a 10% inoculation rate and placed in a 250 mL shake flask. The flask was incubated at 37 °C and 200–220 rpm for 48 h with shaking. At 0, 20, and 40 h of shaking incubation, 0.2 mL of trace element solution (added at a ratio of 4 mL / L) was added each time, and the mixture was incubated with shaking for another 48 h. The supernatant of the fermentation broth was then separated to obtain PAO1-pBBR1MCS-5- LPL The source of the lipoprotein lipase solution.

[0038] 3. Fermentation: The two seed cultures were transferred to 50 mL of fermentation medium at a 10% inoculation rate (0.5 mL seed culture) and placed in 250 mL shake flasks. The mixtures were then shaken and cultured at 37 °C and 200-220 rpm. At the 0, 20 and 40 h of shaking culture, 0.2 mL of trace element solution (added at a ratio of 4 mL / L) was added. The mixtures were shaken and cultured for 48 h to obtain the fermentation broth.

[0039] Based on the type of seed culture and the type of lipoprotein lipase added during shaking culture, the fermentation system was divided into the following groups: Wild-type control group: Inoculated with seed culture of wild-type strain PAO1, and no lipoprotein lipase was added during the shaking culture; Overexpression control group: Inoculated with strain PAO1-pBBR1MCS-5- LPL No lipoprotein lipase was added during the seed culture with shaking. Wild-type plus PAO1-lipase group: Inoculated with wild-type strain PAO1 seed culture, and after shaking culture for 0 h, 40UPAO1-pBBR1MCS-5- was added. LPL Lipoprotein lipase solution of the source; Wild-type plus Tl-lipase group: Inoculated with wild-type strain PAO1 seed culture, 40U of thermophilic fungus was added at 0h of shaking culture. Thermomyces lanuginosus ) lipoprotein lipase solution; Wild-type plus Mm-lipase group: Inoculated with wild-type strain PAO1 seed culture, 40U of Mucor was added at 0h of shaking culture. Mucor megnus ) lipoprotein lipase solution; Overexpression plus PAO1-lipase group: inoculated with strain PAO1-pBBR1MCS-5- LPL Seed culture, shake culture, add 40U PAO1-pBBR1MCS-5- at hour 0. LPL The source of the lipoprotein lipase supernatant.

[0040] Repeat 3 times per group.

[0041] Example 2 Determination method of rhamnolipids in fermentation broth (anthrone method) The rhamnolipid content in the fermentation broth of the wild-type control group, overexpression control group, wild-type group with PAO1-lipase, overexpression group with PAO1-lipase, wild-type group with Tl-lipase, and wild-type group with Mm-lipase in Example 1 was detected by the following methods.

[0042] a. Reagent preparation: Anthrone reagent (0.2g anthrone dissolved in 100mL concentrated sulfuric acid, prepared on the same day); rhamnolipid standard solution (1mg / mL stock solution, diluted to 0.1~0.5mg / mL series standard solutions).

[0043] b. Standard curve preparation: Take 1.0 mL of rhamnose standard solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively, add 4.0 mL of anthrone reagent, heat in a boiling water bath for 10 min, cool in ice water for 10 min, and measure the absorbance at 620 nm. Plot a standard curve with rhamnose concentration on the x-axis and absorbance on the y-axis. Figure 1 The regression equation is: y = 1.467x + 0.01833 (R² > 0.99).

[0044] c. Sample Determination: For different fermentation times, take 2 mL of fermentation broth, centrifuge at 4℃ and 8000 rpm for 10 min, and collect the supernatant; adjust the pH to 2.0 with 1M hydrochloric acid, add 4 mL of ethyl acetate, shake and extract for 10 min, centrifuge at 12000 rpm for 5 min, and collect the organic phase; repeat the extraction with ethyl acetate once, combine the organic phases, evaporate to dryness under nitrogen, and reconstitute with 1 mL of distilled water as the sample to be tested. Take 1.0 mL of the sample to be tested, operate according to the standard curve method, and calculate the rhamnolipid concentration based on the absorbance. The results are shown in [Figure number missing]. Figure 2 .

[0045] The results showed that at 48 h of fermentation, the rhamnolipid yield was highest in the PAO1-lipase overexpression group (14.51 g / L), followed by the wild-type PAO1-lipase group (14.29 g / L), the overexpression control group (12.81 g / L), and the wild-type control group (9.37 g / L). The strain PAO1-pBBR1MCS-5- overexpressing lipoprotein lipase... LPL The synergistic effect of exogenous lipoprotein lipase (overexpression plus PAO1-lipase group) significantly increased rhamnolipid production in the early stage of fermentation, demonstrating the synergistic effect of "overexpression + exogenous addition". Furthermore, the wild-type group with Mm-lipase had a yield of 11.2 g / L, and the wild-type group with Tl-lipase had a yield of 10.5 g / L, indicating that the exogenous addition of P. aeruginosa strain PAO1-derived lipoprotein lipase was significantly more effective than lipoprotein lipases from other species.

[0046] Example 3 Determination of lipid degradation rate and determination of glycerol content 1. The degradation rate in the fermentation broth of the wild-type control group, the overexpression control group, the wild-type group with PAO1-lipase and the overexpression group with PAO1-lipase in Example 1 was detected by the following method.

[0047] Method for detecting oil content: Take 50 mL of sample at different fermentation times, add petroleum ether at a ratio of 1:1 (v / v), shake to mix, centrifuge at 4618 g and 4℃ for 30 min, collect the organic phase, evaporate to dryness and weigh to obtain the oil content.

[0048] Calculate the oil consumption using Formula I, and calculate the oil degradation rate using Formula II.

[0049] Oil consumption = Initial oil quantity - Remaining oil quantity (Formula I) Oil degradation rate (%) = (Initial oil amount - Remaining oil amount) / Initial oil amount × 100% Formula II In Formula I and Formula II, the initial amount of oil is 6.25g.

[0050] The results of oil consumption and oil degradation rate after 48 hours of fermentation for each group are shown in Table 1 and 2. Figure 2 .

[0051] Table 1. Oil consumption and oil degradation rate after 48 hours of fermentation.

[0052] The results showed that after 48 hours of fermentation, the lipid degradation rate of the wild-type group with PAO1-lipase and the overexpression group with PAO1-lipase was significantly higher than that of the wild-type control group and the overexpression control group. This indicates that the exogenous addition of lipoprotein lipase can rapidly improve the lipid degradation efficiency in the early stage of fermentation, and this effect is not affected by whether the strain overexpresses lipoprotein lipase.

[0053] 2. The glycerol content in the fermentation broth of the wild-type control group, the overexpression control group, the wild-type group with PAO1-lipase, and the overexpression group with PAO1-lipase in Example 1 was detected by the following method.

[0054] The fermentation supernatant was centrifuged at 12,000 rpm for 2 min. Subsequently, the glycerol content was determined according to the manufacturer's instructions for the AmplexRed Glycerol Assay Kit (catalog number S0223S) from Beyotime Biotech.

[0055] Test results are shown Figure 3 The results showed that, compared with the wild-type control group and the overexpression control group, the glycerol content was increased in the wild-type plus PAO1-lipase group and the overexpression plus PAO1-lipase group.

[0056] Example 4 Methods for determining fermentation cell density The cell density in the fermentation broth of the wild-type control group, the overexpression control group, the wild-type group with PAO1-lipase, and the overexpression group with PAO1-lipase in Example 1 was detected by the following method.

[0057] Take the fermentation broth and perform serial dilutions (10⁻⁶) with ddH₂O. - ¹ to 10 -7), from the appropriate dilution (10 -4 Up to 10 -7 Take 0.1 mL of bacterial suspension from each of the two culture media and add it dropwise onto solid culture medium plates. Spread the plates evenly with a sterile spreader, invert the plates, and count the colonies on plates with a count between 30 and 300. Finally, calculate the bacterial density (CFU / mL) in the fermentation broth based on the colony count and dilution factor.

[0058] Test results are shown Figure 3 The results showed that, compared with the wild-type control group and the wild-type plus PAO1-lipase group, the colony counts of the overexpression control group and the overexpression plus PAO1-lipase group were reduced.

[0059] Example 5 HPLC and mass spectrometry identification of rhamnolipids The rhamnolipin in the fermentation broth of the wild-type control group, the overexpression control group, the wild-type group with PAO1-lipase, and the overexpression group with PAO1-lipase in Example 1 was detected by the following method.

[0060] 1. HPLC quantitative analysis Sample pretreatment: Take 10 mL of fermentation broth, centrifuge at 8000 rpm for 10 min at 4 °C, adjust the pH of the supernatant to 2.0 with 1 M hydrochloric acid, add 12.5 mL of ethyl acetate (v / v 1:1.25), shake and extract for 10 min, centrifuge at 12000 rpm for 5 min, and collect the organic phase; repeat the extraction once, combine the organic phases, evaporate to dryness under nitrogen, redissolve in 1 mL of methanol, and filter through a 0.22 μm filter membrane for later use.

[0061] Chromatographic conditions: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (60:40 v / v), containing 0.1% trifluoroacetic acid; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 10 μL; detection wavelength: 210 nm.

[0062] Quantitative method: Standard curves were plotted using dirhamnolipin and monorhamnolipin standards (concentration 0.1-1.0 mg / mL), and the rhamnolipin content in the samples was calculated using the external standard method.

[0063] 2. Mass spectrometry structure identification Derivatization: Take 500 μL of the above HPLC test solution, add 50 μL of triethylamine and 100 μL of 4-bromobenzoyl bromide (10 mg / mL acetonitrile solution), react at 60 °C in the dark for 30 min, add 100 μL of methanol to terminate the reaction, filter through a 0.22 μm filter membrane and use for mass spectrometry analysis.

[0064] Mass spectrometry conditions: electrospray ionization (ESI) source, positive ion mode; scan range m / z 100-1000; capillary voltage 3.0 kV; cone voltage 50 V; ion source temperature 150 °C; desolvation gas temperature 350 °C; flow rate 700 L / h.

[0065] Structural confirmation: Detected through [M+H] + Positive ion peaks (m / z approximately 651 for dirhamnolipin and m / z approximately 505 for monorhamnolipin) and characteristic fragment ions, combined with comparison with standard quality spectra, confirmed the structure of rhamnolipin homologues.

[0066] See results Figure 4 , Figure 5 and Figure 6 The results showed that, based on the above HPLC quantitative analysis and mass spectrometry structural identification, rhamnolipids were clearly detected in the target sample, mainly in the form of monorhamnolipids and dirhamnolipids: (from the HPLC detection results...) Figure 4 From the mass spectrometry results, the retention time of the target component in the sample perfectly matches that of the monorhamnolipin and dirhamnolipin standards; Figure 5 and Figure 6 In positive ion mode, the characteristic precursor ion peaks of [monorhamnetin + H]⁺ at approximately m / z 505 and [dirhamnetin + H]⁺ at approximately m / z 651 were clearly observed. Further fragmentation analysis of the characteristic precursor ions revealed that the resulting fragment ion spectra completely matched the characteristic fragments of the standard quality spectra of the mono- and dirhamnetin lipolipids, without any interference from extraneous peaks or unknown components. In summary, this confirms that the fermentation system described in this invention can effectively synthesize and accumulate rhamnetin, and its structure meets the identification criteria for the target substance, providing a reliable material basis for the subsequent functional applications or further purification of rhamnetin.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A lipoprotein lipase in promoting the growth of Pseudomonas aeruginosa ( Pseudomonas aeruginosa Applications in the synthesis of rhamnolipids; The lipoprotein lipase is a lipoprotein lipase derived from Pseudomonas aeruginosa.

2. The application according to claim 1, characterized in that, The *Pseudomonas aeruginosa* strain includes *Pseudomonas aeruginosa* PAO1 and / or recombinant *Pseudomonas aeruginosa* strain PAO1-pBBR1MCS-5- LPL .

3. The application according to claim 1 or 2, characterized in that, The working concentration of the lipoprotein lipase is above 0.6 U / ml.

4. A method for promoting the synthesis of rhamnolipids by *Pseudomonas aeruginosa*, characterized in that, Rhamnolipids were obtained by culturing lipoprotein lipase and Pseudomonas aeruginosa in an oil-containing fermentation medium. The lipoprotein lipase is a lipoprotein lipase derived from Pseudomonas aeruginosa.

5. The method according to claim 4, characterized in that, The ratio of lipoprotein lipase to oil is 26~38U:5g.

6. The method according to claim 4, characterized in that, The fermentation medium comprises the following components in the following amounts: oil 100~150g / L, NaNO3 13~17.0g / L, MgSO4・7H2O 0.4~0.6g / L, KCl 0.8~1.2g / L and K2HPO4 0.2~0.4g / L.

7. The method according to claim 4, characterized in that, During the culture period, trace element solution was added in batches; The trace element solution comprises the following components in the following amounts: sodium citrate 1.8~2.2 g / L, FeCl3·6H2O 0.25~0.3 g / L, ZnSO4·7H2O 1.2~1.6 g / L, CoCl2·6H2O 1.0~1.4 g / L, CuSO4·5H2O 1.0~1.4 g / L, and MnSO4·H2O 0.6~1.0 g / L; When the trace element solution is added in batches, the amount added each time is 2-6‰ of the volume of the fermentation medium; The interval between two consecutive batches of additions is 15 to 25 hours.

8. The method according to claim 4, characterized in that, The culture time is 20-120 hours; the culture temperature is 35-37°C.

9. The method according to any one of claims 4 to 8, characterized in that, The *Pseudomonas aeruginosa* strain includes *Pseudomonas aeruginosa* PAO1 and / or recombinant *Pseudomonas aeruginosa* strain PAO1-pBBR1MCS-5- LPL .

10. The method according to claim 9, wherein the lipoprotein lipase is prepared from the recombinant Pseudomonas aeruginosa strain PAO1-pBBR1MCS-5- LPL The recombinant protein was isolated from the fermentation broth.

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