Recombinant vector and recombinant strain for efficiently expressing lipase as well as construction method and application of recombinant vector and recombinant strain

Abstract

The invention discloses a recombinant vector and a recombinant strain for efficiently expressing lipase as well as a construction method and application of the recombinant vector and the recombinant strain, and relates to the technical field of molecular biology, and the recombinant vector comprises a nucleotide sequence for coding lipA, a nucleotide sequence for coding lipH and a BSFP0720 promoter. The BSFP0720 promoter sequence in the recombinant vector can promote the cells containing the recombinant vector to overexpress the lipase coded by the lipA gene under the condition of not adding an inducer; besides, co-expression of the lipA gene sequence and the lipH gene sequence promotes correct folding of the lipase coded by the lipA gene sequence after expression and promotes maturation and secretion of the lipase, so that the expression quantity of the lipase is improved. After the recombinant vector is expressed in the COO gene knockout strain, the lipase purification process is shortened, the lipase purity is improved, and when the recombinant vector is applied to an in-vitro detection kit, the detection accuracy is high.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a recombinant vector for efficiently expressing lipase, a recombinant strain, its construction method, and its applications. Background Technology

[0002] Lipases (EC 3.1.1.3) are hydrolases that catalyze the breakdown of triglycerides and are widely distributed in higher organisms and microorganisms. In the field of in vitro diagnostics, lipases play a crucial role in triglyceride (TG) assay kits. In the initial reaction, lipases hydrolyze triglycerides in serum samples into glycerol and fatty acids, providing substrates for subsequent reactions. Therefore, lipases, as a core raw material for diagnostic reagents, possess significant commercial value due to their efficient, high-quality, and low-cost industrial production.

[0003] Currently, the main methods for obtaining lipases include: (1) directly extracting lipases naturally from microorganisms, and (2) inducing microorganisms to express lipases using substrates such as olive oil. However, both of these methods have the disadvantages of low yield and high cost. Summary of the Invention

[0004] The main objective of this invention is to propose a recombinant vector, recombinant strain, construction method, and application for efficient expression of lipase, aiming to solve the problems of low yield and high cost in existing methods for obtaining lipase.

[0005] To achieve the above objectives, this invention proposes a recombinant vector for efficient expression of lipase, the recombinant vector comprising: a nucleotide sequence encoding lipA, a nucleotide sequence encoding lipH, and a BSFP_0720 promoter.

[0006] In one embodiment, the lipA and / or lipH genes are derived from Pseudomonas aeruginosa.

[0007] Preferably, the nucleotide sequence of the BSFP_0720 promoter is shown in SEQ ID NO.1;

[0008] And / or, the nucleotide sequence encoding lipA is shown in SEQ ID NO.2;

[0009] And / or, the nucleotide sequence encoding lipH is shown in SEQ ID NO.3.

[0010] In one embodiment, the recombinant vector is an integrative plasmid vector pBBR1MCS-2, a pUCP vector, or a pTAC vector;

[0011] Preferably, the recombinant vector is an integrative plasmid vector pBBR1MCS-2.

[0012] The present invention also provides a recombinant strain that efficiently expresses lipase, the recombinant strain comprising the aforementioned recombinant vector;

[0013] Preferably, the recombinant strain is derived from Pseudomonas aeruginosa.

[0014] In one embodiment, the recombinant strain cannot express cholesterol oxidase;

[0015] Preferably, the strain is *Pseudomonas aeruginosa* with the cholesterol oxidase gene knocked out.

[0016] This invention also provides a method for constructing a recombinant bacterial strain, comprising:

[0017] (1) Construct the aforementioned recombinant carrier;

[0018] (2) The recombinant vector was transformed into Pseudomonas aeruginosa;

[0019] Preferably, the *Pseudomonas aeruginosa* strain does not express cholesterol oxidase;

[0020] More preferably, the *Pseudomonas aeruginosa* strain is a strain with the cholesterol oxidase gene knocked out.

[0021] This invention also provides a method for high-efficiency expression of lipase, comprising:

[0022] (1) Cultivate the aforementioned recombinant strain under conditions conducive to lipase expression;

[0023] (2) Use the cultured recombinant strain to isolate and purify lipase.

[0024] The present invention also provides the application of the aforementioned recombinant vector or the aforementioned recombinant strain in the preparation of lipase.

[0025] The present invention also provides a lipase, which is prepared by the aforementioned method.

[0026] The present invention also provides an application of the aforementioned lipase in the preparation of in vitro diagnostic reagents;

[0027] Preferably, the in vitro diagnostic reagent includes a triglyceride detection reagent or a cholesterol detection reagent;

[0028] More preferably, the in vitro diagnostic reagents include reagents for detecting total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and small-dense low-density lipoprotein cholesterol.

[0029] In the technical solution of this invention, the BSFP_0720 promoter sequence in the recombinant vector can induce overexpression of the lipA gene-encoded lipase in cells containing the recombinant vector without the addition of an inducer. Furthermore, the co-expression of the lipA and lipH gene sequences promotes the correct folding of the lipA gene-encoded lipase after expression and promotes lipase maturation and secretion, thereby increasing the expression level of the lipase. Expressing the recombinant vector of this invention in cholesterol oxidase (COO) gene knockout strains avoids the COO removal step in the lipase purification process, simplifies the purification process, and improves the purity of the lipase. Therefore, the lipase of this invention is suitable for use in in vitro detection kits with high detection accuracy. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram illustrating the detection principle of the TG reagent provided by the present invention.

[0032] Figure 2 This is a schematic diagram illustrating the detection principle of the CHOL reagent provided by the present invention.

[0033] Figure 3 Image (A) is a schematic diagram of the original composition of the pBBR1MCS-2 carrier in Example 1 provided by the present invention. Figure 3 (B) is a schematic diagram of the composition of the recombinant plasmid pBBR1-lipA in Example 1 provided by the present invention;

[0034] Figure 4 Image (A) shows the PCR identification results of the COO gene of the strains in Example 3 and Comparative Example 1 provided by this invention. Figure 4 (B) is a graph showing the COO enzyme activity analysis results of the fermentation broth in Example 3 and Comparative Example 1 provided by the present invention;

[0035] Figure 5 Image (A) shows the SDS-PAGE images of the supernatant after 6, 9, 12, and 15 hours of fed-batch fermentation in Example 3 of this invention. Figure 5 (B) is a graph showing the enzyme activity results of lipase in the supernatant during fed-batch fermentation at 6, 9, 12, and 15 hours in Example 3 of this invention. Figure 5Image C is an SDS-PAGE image of the purified lipase solution in Example 3 provided by the present invention. Figure 5 Image (D) is an SDS-PAGE image of the lyophilized lipase powder in Example 3 provided by the present invention. Figure 5 (E) is a graph showing the CE-SDS purity analysis results of the lipase lyophilized powder in Example 3 provided by the present invention;

[0036] Figure 6 Image (A) is an SDS-PAGE image of lipase lyophilized powder stored at different temperatures for different times in Example 3 of this invention. Figure 6 (B) is a graph showing the residual lipase activity of the lyophilized lipase powder in Example 3, which was stored at different temperatures for different times according to the present invention. Figure 6 (C) is a graph showing the effect of different temperatures on the lipase activity in Example 3 provided by the present invention. Figure 6 (D) is a graph showing the effect of different pH values ​​on lipase activity in Example 3 provided by the present invention;

[0037] Figure 7 The graph shows the correlation between the detected value and the theoretical value of the TG reagent in Example 4 provided by the present invention.

[0038] Figure 8 The graph shows the correlation between the detected value and the theoretical value of the CHOL reagent in Example 5 provided by the present invention.

[0039] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Lipases (EC 3.1.1.3) are hydrolases that catalyze the breakdown of triglycerides and are widely distributed in higher organisms and microorganisms. In the field of in vitro diagnostics, lipases play a crucial role in triglyceride (TG) detection kits. In the initial reaction, lipases hydrolyze triglycerides in serum samples into glycerol and fatty acids. Then, glycerol undergoes further reactions catalyzed by glycerol kinase and glycerol-3-phosphate oxidase, producing hydrogen peroxide. In the presence of chromogenic reagents such as peroxidase, 4-aminoantipyrine (4-AAP), and sodium N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS) or N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), hydrogen peroxide undergoes a Trinder reaction, forming a colored quinone imine, leading to an increase in absorbance. The increase in absorbance reflects the TG content in serum. Therefore, lipase, as a core raw material for diagnostic reagents, has enormous commercial value due to its efficient, high-quality, and low-cost industrial production.

[0042] Currently, the main methods for obtaining lipases include: (1) directly extracting lipases naturally from microorganisms, and (2) inducing microorganisms to express lipases using substrates such as olive oil. However, both of these methods have the disadvantages of low yield and high cost.

[0043] In view of this, the present invention provides a recombinant vector for efficient expression of lipase, the recombinant vector comprising: a nucleotide sequence encoding lipA, a nucleotide sequence encoding lipH, and a BSFP_0720 promoter.

[0044] In the technical solution of this invention, the BSFP_0720 promoter sequence in the recombinant vector can induce overexpression of the lipA gene-encoded lipase in cells containing the recombinant vector without the addition of an inducer. Furthermore, the co-expression of the lipA and lipH gene sequences promotes the correct folding of the lipA gene-encoded lipase after expression and promotes lipase maturation and secretion, thereby increasing the expression level of the lipase. Expressing the recombinant vector of this invention in COO gene knockout strains avoids the COO removal step in the lipase purification process, simplifies the purification process, and improves the purity of the lipase. Therefore, the lipase of this invention is suitable for use in in vitro detection kits with high detection accuracy.

[0045] It should be noted that the BSFP_0720 promoter sequence can be derived from the Burkholderia stabilis FERMP-21014 strain or can be synthesized artificially. The nucleotide sequence encoding the lipA gene can be a nucleotide fragment including the lipA gene or a nucleotide fragment of the lipA gene alone. The lipA gene can be derived from a natural strain or can be synthesized artificially.

[0046] The nucleotide sequence encoding the lipH gene can be a nucleotide fragment including the lipH gene or a nucleotide fragment of the lipH gene alone. The lipH gene can be derived from natural strains or synthesized artificially.

[0047] In some embodiments of the present invention, the lipA and / or lipH genes are derived from *Pseudomonas aeruginosa*. Preferably, the BSFP_0720 promoter sequence is shown in SEQ ID No. 1; and / or, the nucleotide sequence encoding lipA is shown in SEQ ID No. 2; and / or, the nucleotide sequence encoding lipH is shown in SEQ ID No. 3.

[0048] In some embodiments of the present invention, the recombinant vector includes the integrative plasmid vector pBBR1MCS-2, the pUCP vector, or the pTAC vector; preferably, the recombinant vector is the integrative plasmid vector pBBR1MCS-2. That is, the original vector in the recombinant vector can be of various types, such as the pBBR1MCS-2 vector, the pUCP vector, or the pTAC vector. The recombinant vector can be stably expressed in host cells. Preferably, the recombinant vector is the integrative plasmid vector pBBR1MCS-2, which is particularly suitable for expressing exogenous genes in Gram-negative bacteria.

[0049] This invention also provides a recombinant strain for efficiently expressing lipase, the recombinant strain comprising the aforementioned recombinant vector; preferably, the recombinant strain is derived from *Pseudomonas aeruginosa*. The natural host, *Pseudomonas aeruginosa*, possesses a complete lipase folding and secretion mechanism, providing the necessary microenvironment for lipase maturation. For example, the endogenous periplasmic molecular chaperone Skp of *Pseudomonas aeruginosa* effectively prevents lipase misfolding, which is crucial for lipase maturation and secretion pathways. Therefore, compared with other bacteria, *Pseudomonas aeruginosa* is the optimal expression host for producing fully active lipase. More preferably, the recombinant strain is derived from *Pseudomonas aeruginosa* M18. Furthermore, the co-expression of the lipA and lipH gene sequences in the aforementioned recombinant vector, combined with the advantage of *Pseudomonas aeruginosa* in secreting lipase, not only enables efficient lipase expression but also eliminates the need for cell lysis during lipase purification, reducing initial impurities in the enzyme solution and simplifying the operation process. Simultaneously, the lipase expressed by the recombinant *Pseudomonas aeruginosa* strain not only exhibits high lipase activity but also high cholesterol esterase (COE) activity.

[0050] When the recombinant strain originates from a strain that expresses the COO gene, such as *Pseudomonas aeruginosa*, although the lipase yield is high, the presence of the COO gene in the strain leads to the endogenous expression of a constant level of COO, resulting in COO doping in the final lipase. The lipase of this invention, due to its COE activity, can be used simultaneously to prepare reagents for detecting triglycerides (TG) and cholesterol markers (e.g., CHOL). However, the COO doping in the lipase causes cross-reactions, increasing hydrogen peroxide production and interfering with the signal, leading to inaccurate TG detection results. While existing purification techniques can reduce COO to undetectable levels, complete removal is difficult, and even trace amounts of COO in the lipase can result in poor detection accuracy. In some embodiments of this invention, the recombinant strain does not express cholesterol oxidase; preferably, the strain is *Pseudomonas aeruginosa* with the cholesterol oxidase gene knocked out.

[0051] In the technical solution of this invention, the recombinant strain cannot express cholesterol oxidase; that is, during the lipase expression process of the recombinant strain, cholesterol oxidase is not expressed, thus ensuring that the final lipase does not contain COO incorporation, omitting the step of removing COO during lipase purification. When the recombinant strain is a Pseudomonas aeruginosa strain with the COO gene knocked out, its lipase expression level is high and there is no COO doping, resulting in high lipase purity.

[0052] This invention also provides a method for constructing a recombinant bacterial strain, comprising:

[0053] (1) Construct the aforementioned recombinant carrier;

[0054] (2) The recombinant vector was transformed into Pseudomonas aeruginosa;

[0055] Preferably, the *Pseudomonas aeruginosa* strain does not express cholesterol oxidase;

[0056] More preferably, the *Pseudomonas aeruginosa* strain is a strain with the cholesterol oxidase gene knocked out.

[0057] Specifically, the method for constructing the recombinant strain includes: transforming the aforementioned recombinant vector into host bacteria to obtain genetically engineered recombinant bacteria. Transformation refers to the process of introducing the recombinant vector into host bacteria, and the main steps include: preparing competent cells, transformation, recovery culture, screening positive clones, and verification. Through transformation, recombinant plasmids can be rapidly introduced into host bacteria to obtain genetically engineered recombinant bacteria that can be stably passaged.

[0058] This invention also provides the aforementioned recombinant vector, or the aforementioned recombinant strain, for the preparation of lipase. Lipase can be expressed stably and efficiently using the aforementioned recombinant vector or recombinant strain, and the resulting lipase has high yield and purity.

[0059] This invention also provides a lipase prepared by the aforementioned method. The lipase obtained by the above method exhibits good enzyme activity and high yield. Its enzyme activity includes lipase activity and COE activity.

[0060] This invention also provides an application of lipase in the preparation of in vitro diagnostic reagents. Because the lipase of this invention has high purity, lipase activity, and COE enzyme activity, it is suitable for large-scale industrial production and has a low cost. Therefore, it can replace existing commercial lipases or COE enzymes in the preparation of related in vitro diagnostic products.

[0061] Preferably, the in vitro diagnostic reagent includes a triglyceride detection reagent, comprising the aforementioned lipase. Existing triglyceride detection reagents may include reagent R1 and reagent R2. Reagent R1 includes a buffer solution and lipase; reagent R2 includes a buffer solution, peroxidase, lipase, glycerol-3-phosphate oxidase, glycerol kinase, adenosine triphosphate disodium salt (ATP), and 4-aminoantipyrine (4-AAP). The lipase of the present invention has high lipase activity and can replace the lipase in reagents R1 and R2 of existing triglyceride detection reagents. Furthermore, due to the high yield of the lipase of the present invention, the reagent production cost is reduced.

[0062] Specifically, the reaction principle of the triglyceride detection reagent is as follows: Figure 1 As shown.

[0063] Preferably, the in vitro diagnostic reagent includes a cholesterol detection reagent. More preferably, the in vitro detection reagent includes reagents for total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and small-dense low-density lipoprotein cholesterol. The lipase of the present invention also exhibits high COE activity, making it suitable for clinical detection of cholesterol-related biomarkers such as total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and small-dense low-density lipoprotein cholesterol. The detection of the above-mentioned cholesterol ester-related biomarkers uses the same detection principle, that is, cholesterol esters in the test sample are hydrolyzed into cholesterol and fatty acids by COE, and then COO uses cholesterol as a substrate to produce cholesterol-4-en-3-one and hydrogen peroxide. Hydrogen peroxide undergoes the same Trinder reaction as in TG detection, thereby reflecting the cholesterol ester content in the sample.

[0064] Taking total cholesterol (CHOL) as an example, CHOL reagents can include reagent R1 and reagent R2. R1 includes buffer, TOOS and COE; R2 includes buffer, peroxidase, COE, COO and 4-AAP.

[0065] Specifically, the detection principle of CHOL reagent is as follows: Figure 2 As shown.

[0066] It should be noted that the TG detection reagent and cholesterol detection reagent of the lipase of this invention have high detection accuracy.

[0067] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0068] Experimental materials

[0069] LB agar medium: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar.

[0070] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.

[0071] LB agar medium containing 50 μg / mL gentamicin: After sterilizing the above LB agar medium, cool it to 45-50℃, add gentamicin to a final concentration of 50 μg / mL, mix well, pour into a petri dish and let it solidify naturally.

[0072] SOC medium: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5mM KCl, 10mM MgCl2, 20mM glucose.

[0073] LB agar medium containing 50 μg / mL kanamycin: After sterilizing the above LB agar medium, cool it to 45-50℃, add kanamycin to a final concentration of 50 μg / mL, mix well, pour into a petri dish and let it solidify naturally.

[0074] Fermentation medium: 0.1% MgSO4·7H2O, 0.4% KH2PO4, 1% Na2HPO4·12H2O, 0.4% (NH4)2SO4, 0.01% CaCl2, 0.6% glucose and 50 μg / mL kanamycin.

[0075] Feeding medium: 30% glucose and 50 μg / mL kanamycin.

[0076] Example 1 Construction of recombinant plasmid

[0077] The sequences of the promoter BSFP_0720 from Burkholderia stabilis FERMP-21014, the lipase gene lipA (Uniprot ID: P26876) from Pseudomonas aeruginosa M18 (purchased from Beina Biotechnology, BNCC337099), and the secretory chaperone gene lipH (Uniprot ID: Q01725) from Pseudomonas aeruginosa M18 were sequentially tandem to obtain a new DNA molecule. BstBI and AgeI restriction endonuclease sites were added to the 5' and 3' ends of the new DNA molecule, respectively. Then, it was ligated into the vector pBBR1MCS-2 (purchased from Addgene, catalog number #85168). The T7 promoter and lac operon in the vector pBBR1MCS-2 were removed to obtain the recombinant plasmid, which was designated pBBR1-lipA. The original composition of the carrier pBBR1MCS-2 is as follows: Figure 3 As shown in (A), the composition of the recombinant plasmid pBBR1-lipA is as follows: Figure 3 As shown in (B). Among them, Pseudomonas aeruginosa was purchased from Beina Biotechnology, catalog number BNCC337099.

[0078] Example 2: Construction of COO gene knockout strain

[0079] The COO gene knockout method from the whole genome of *Pseudomonas aeruginosa* M18 (GenBank: AB920752.1) was as follows: The upstream and downstream homologous recombination arms of the COO gene were amplified from the genome of *Pseudomonas aeruginosa* M18 strain using ultrafidelity DNA polymerase. The upstream and downstream homologous recombination arms were ligated using fusion PCR to obtain the targeting fragment ΔCOO, which was then cloned into the suicide plasmid pCVD442GS (containing a gentamicin resistance gene) to obtain the targeting plasmid pCVD442GS-ΔCOO. pCVD442GS-ΔCOO was then transformed into *E. coli* β2155 using electroporation to obtain the donor strain β2155 / pCVD442GS-ΔCOO. The donor bacterium β2155 / pCVD442GS-ΔCOO was conjugated with the recipient bacterium *Pseudomonas aeruginosa* M18. Gentamicin-resistant *P. aeruginosa* clones with integrated targeting plasmids were screened on gentamicin-containing medium and named PA / pCVD442GS-ΔCOO. Several PA / pCVD442GS-ΔCOO clones were cultured on LB agar containing 10% sucrose until single colonies formed. Strains (i.e., single clones) with successfully knocked-out COO genes were screened and identified using upstream primer COO-inF, downstream primer COO-inR, and PCR. The sequence of upstream primer COO-inF is shown in SEQ ID No. 4, and the sequence of downstream primer COO-inR is shown in SEQ ID No. 5. Furthermore, the enzymatic activity of COO in the fermentation broth was detected to confirm successful knockout; strains with successfully knocked-out COO were designated PA / ΔCOO.

[0080] SEQ ID No. 4: GGTACTGCAGGTAGAACTTTGATCAC.

[0081] SEQ ID No. 5: CCGTTCAACTACCCCTTCCCGAC.

[0082] Example 3: Construction of engineered strains, expression and purification of lipase

[0083] 1. Construction of engineered strains

[0084] The COO gene knockout strain PA / ΔCOO from Example 2 was activated on LB agar medium, and a single colony was selected and inoculated into 2 mL of LB medium. The culture was then incubated overnight at 300 rpm and 37°C to obtain the first culture. 0.5 mL of the first culture was inoculated into 50 mL of LB medium and incubated for 2–3 hours until OD (Organic Demand) was reached. 600The pH reached 0.8, yielding a second culture. The second culture was cooled on ice and centrifuged at 4°C and 5000 rpm for 10 min to collect the precipitate, i.e., the target cells. The collected target cells were resuspended in 10 mL of pre-chilled electroporation buffer (pH 7.5), and the centrifugation and washing steps were repeated twice. The final cell pellet was resuspended in 800 μL of pre-chilled electroporation buffer to obtain competent cells. The electroporation buffer contained 1 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 1 mM MgCl2, and 300 mM sucrose.

[0085] 50 ng of the recombinant plasmid pBBR1-lipA from Example 1 was added to 100 μL of competent cells, mixed, and placed on ice for 10 min. The mixture was then transferred to a 0.2 cm electroporation tube and electroporated using a Bio-Rad Gene PulserXcell electroporation system with the following parameters: C = 25 μF, PC = 200 Ω, V = 2.5 kV. Immediately after electroporation, the mixture was added to 1 mL of SOC medium and incubated at 37 °C and 300 rpm for 2 h. After incubation, 100 μL of the incubation solution was spread onto LB agar medium containing 50 μg / mL kanamycin and cultured overnight to obtain the engineered strain containing the pBBR1-lipA plasmid.

[0086] 2. Lipase expression

[0087] A single-clone engineered strain containing the pBBR1-lipA plasmid was inoculated into 1.5 L of LB medium and cultured at 30 °C and 250 rpm for 15 hours to obtain a seed culture. All seed cultures were transferred to a 200 L fermenter, which was pre-filled with 80 L of fermentation medium. After fermentation at 30 °C for 15 hours, feeding was initiated, and the feeding period lasted for a total of 15 hours. The resulting fermentation broth was then harvested, yielding approximately 110 L.

[0088] 3. Purification of lipase

[0089] The fermentation broth was centrifuged to remove the cell precipitate, yielding a supernatant. Ammonium sulfate (final mass concentration of ammonium sulfate in the system was 20%) was added to the supernatant, mixed, centrifuged, and the precipitate was collected. The precipitate was then washed twice with 10 mM phosphate buffer (PB) containing 20% ​​ammonium sulfate (pH 7.5). The washed precipitate was resuspended in 20 L of PB and centrifuged again to remove insoluble particles, yielding the salted-out supernatant.

[0090] The supernatant after salting out was first passed through a 2L octyl hydrophobic packing material (purchased from Bailinke) to collect the flow-through liquid. Then, the flow-through liquid was passed through a 10L NanoGel packing material.TM -50Q packing material (purchased from Suzhou Nanomicro) was used for elution, and the eluent was PB containing 200mM ammonium sulfate. After elution, a purified lipase solution was obtained.

[0091] The purified lipase solution was concentrated to 16-18 mg / mL by ultrafiltration. After concentration, it was dialyzed three times at low temperature with 10 mM PB (pH 7.5), each time for 6-8 hours. After dialysis, 0.1% NP-40, 1% bovine serum albumin (BSA), and 2% trehalose were added to the lipase solution as excipients. The solution was then freeze-dried to obtain lyophilized lipase powder, which was then dried and stored.

[0092] Example 4: Preparation and Detection Method of TG Reagent

[0093] This embodiment provides a TG reagent, which includes reagent R1 and reagent R2. R1 contains 200 mM 3-morpholinopropanesulfonic acid (MOPS), 2.5 kU / L lipase from Example 3, and 10 mM TOPS; R2 contains 200 mM MOPS, 5 kU / L peroxidase, 10 kU / L lipase from Example 3, 7.5 kU / L glycerol-3-phosphate oxidase, 5 kU / mL glycerol kinase, 2 mM adenosine triphosphate disodium salt (ATP), and 1.5 mM 4-AAP.

[0094] The lipases used in this embodiment are all lipases prepared in Example 3, and their lipase activity is 30 U / mg.

[0095] Example 5: Preparation and detection method of CHOL reagent

[0096] This embodiment provides a CHOL reagent, which includes reagent R1 and reagent R2. R1 contains 50 mM HEPES, 2 mM TOOS and 3 kU / L of the lipase from Example 3; R2 contains 50 mM HEPES, 10 kU / L of peroxidase, 3 kU / L of the lipase from Example 3, 1.5 kU / L of COO and 1.5 mM 4-AAP.

[0097] The lipases used in this embodiment are all the lipases prepared in Example 3, but the COE enzyme activity is used for feeding. The COE enzyme activity of the lipase in Example 3 is 93 U / mg.

[0098] Comparative Example 1

[0099] The difference between Comparative Example 1 and Example 3 is that:

[0100] Replace the monoclonal engineered strain in step 2 with the wild-type Pseudomonas aeruginosa strain M18, which does not contain the pBBR1-lipA plasmid.

[0101] Performance testing

[0102] Enzyme quantity, enzyme activity, purity, and enzymatic properties testing:

[0103] 1. Determination of enzyme quantity and enzyme activity

[0104] (1) COO activity

[0105] COO activity refers to the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute (equivalent to 0.5 μmol of quinone imine dye) at 37°C using cholesterol as a substrate, and is measured spectrophotometrically at 546 nm to detect the quinone imine dye. The COO activity of the supernatant from the fermentation broth after fermentation in Example 3 and Comparative Example 1 was determined, and the results are as follows: Figure 4 As shown.

[0106] Figure 4 In (A), "Marker" represents the standard molecular weight of the pre-stained protein, "Knockout" represents the PCR identification result of the engineered strain containing the pBBR1-lipA plasmid in Example 3 (i.e., the recombinant strain with the COO gene knocked out), and "Control" represents the PCR identification result of the wild-type Pseudomonas aeruginosa M18 strain in Comparative Example 1 (i.e., the strain without the COO gene knocked out). Figure 4 As shown in (A), the PA / ΔCOO strain has successfully knocked out the endogenous COO gene.

[0107] Figure 4 In (B), FS represents the COO enzyme activity in the supernatant of the fermentation broth of wild-type Pseudomonas aeruginosa strain M18 in Comparative Example 1; PL represents the COO enzyme activity in the purified lipase from the supernatant of the fermentation broth of wild-type Pseudomonas aeruginosa strain M18 in Comparative Example 1; and GK represents the COO enzyme activity in the supernatant of the fermentation broth of the engineered strain containing the pBBR1-lipA plasmid in Example 3. Figure 4 As shown in Figure (B), the supernatant of the fermentation broth produced by the engineered strain showed no COO enzyme activity, further confirming the knockout of the COO gene.

[0108] (2) Lipase expression level, lipase activity and COE activity of lipase

[0109] The expression level of lipase in the supernatant of the fermentation broth at 6, 9, 12, and 15 hours of fed-batch fermentation in Example 3 was detected by SDS-PAGE. The results are as follows: Figure 5 As shown in (A).

[0110] Figure 5 In (A), M represents the molecular weight of the pre-stained protein standard, and 6h, 9h, 12h, and 15h represent the supernatant samples taken at 6, 9, 12, and 15 hours respectively during fed-batch fermentation. Figure 5 As shown in (A), when glucose is used as the sole carbon source, lipase is effectively secreted in engineered strains containing the pBBR1-lipA plasmid, and the expression level of lipase gradually increases over time.

[0111] Lipase activity refers to the amount of enzyme that produces 1 μmol of glycerol per minute (equivalent to 0.5 μmol of quinone imine dye) at 37°C using olive oil as a substrate. The COE activity of lipase refers to the amount of enzyme that produces 1 μmol of cholesterol per minute (equivalent to 0.5 μmol of quinone imine dye) at 37°C using cholesterol ester as a substrate. Quinone imine dye was detected at 546 nm using spectrophotometry. The results are as follows... Figure 5 As shown in (B).

[0112] Figure 5 (B) shows the lipase activity and COE activity results of the supernatant of the fermentation broth in Example 3 and Comparative Example 1 at 6, 9, 12 and 15 h of fed-batch fermentation. Figure 5 In section (B), the red line (dots) represents the COE activity produced by the monoclonal engineered strain containing the pBBR1-lipA plasmid in Example 3, and the blue line (squares) represents its corresponding lipase activity; the blue line (upright triangle) represents the lipase activity produced by wild-type Pseudomonas aeruginosa M18 in Comparative Example 1, and the green line (inverted triangle) represents its corresponding COE enzyme activity. Figure 5 As shown in Figure (B), after all fermentation was completed (15 hours of initial feeding + 15 hours of subsequent feeding), the lipase activity of the fermentation broth in Example 3 reached 68 kU / L, while the corresponding COE activity was 214 kU / L, with the COE activity being approximately 3.1 times that of the lipase activity. In contrast, the natural Pseudomonas aeruginosa in Comparative Example 1, under the same culture conditions, only produced 0.6 kU / L of COE activity or 0.19 kU / L of lipase activity after fermentation (the two lines almost overlap). That is, the engineered bacteria in Example 3 produced 356 times more COE or lipase activity than the natural bacteria in Example 1, meaning that the lipase yield of the engineered bacteria in Example 3 was 356 times higher than that of the natural bacteria in the Comparative Example.

[0113] 2. Purity of lipase

[0114] The purity of the lipase in Example 3 was assessed by SDS-PAGE and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). The results are as follows: Figure 5 As shown in (C), (D) and (E).

[0115] Figure 5 In (C), M represents the standard molecular weight of the prestained protein, and P represents the purified lipase solution; Figure 5 In the (D) designation, M represents the standard molecular weight of the pre-stained protein, and L represents lyophilized lipase powder. Figure 5 As shown in (C), (D) and (E), the lipase has excellent purity, and the purity of the lipase in the finished product is 94.32% (excluding BSA).

[0116] 3. Water content of lipase

[0117] The lyophilized lipase powder was a light brown powder. The moisture content of the lyophilized lipase powder in Example 3 was measured using a Karl Fischer moisture analyzer (V10S, Mettler Toledo). Its moisture content was less than 5%.

[0118] 4. Accelerated stability of lipase at different temperatures

[0119] The lyophilized lipase powder from Example 3 was accelerated at 37°C for 7, 14, and 21 days, and at 45°C for 7 days, with a sample stored at -20°C for 21 days used as a control. The degree of protein degradation or aggregation was detected by SDS-PAGE, and the results are as follows: Figure 6 As shown in (A), the residual lipase activity was also detected, and the results are as follows. Figure 6 As shown in (B).

[0120] Figure 6 In the diagram (A), M represents the standard molecular weight of the pre-stained protein; 1 indicates lipase lyophilized powder stored at -20℃ for 21 days; 2 indicates lipase lyophilized powder accelerated at 37℃ for 7 days; 3 indicates lipase lyophilized powder accelerated at 37℃ for 14 days; 4 indicates lipase lyophilized powder accelerated at 37℃ for 21 days; and 5 indicates lipase lyophilized powder accelerated at 45℃ for 7 days. Figure 6 As shown in (A) and (B), after the lipase lyophilized powder in Example 3 was accelerated at 37°C for 21 days and at 45°C for 7 days, the residual lipase activity was greater than 96% compared with the lipase lyophilized powder stored at -20°C for 21 days (i.e., the relative enzyme activity was calculated based on the lipase activity of the lipase lyophilized powder stored at -20°C for 21 days). This indicates that the lipase prepared in Example 3 has good stability and is suitable for long-term storage.

[0121] 5. Enzymatic properties of lipases

[0122] (1) The effect of chemical substances on lipase activity

[0123] Various chemicals were added to a 10 KU / L reconstituted solution of the lyophilized lipase powder prepared in Example 3 (reconstituted with water), and the mixture was incubated at 25°C for 1 h. The residual lipase activity was then tested, and the results are shown in Table 1.

[0124] Table 1. Characteristics of lipases against chemical interference

[0125] chemical substances concentration Residual enzyme activity <![CDATA[AgNO3]]> 2mM 101％ <![CDATA[CoCl2]]> 2mM 99％ <![CDATA[NiSO4]]> 2mM 100％ <![CDATA[FeSO4]]> 2mM 98％ <![CDATA[CuSO4]]> 2mM 95％ <![CDATA[ZnCl2]]> 2mM 93％ <![CDATA[CaCl2]]> 2mM 95％ <![CDATA[MnSO4]]> 2mM 102％ <![CDATA[BaCl2]]> 2mM 102％ EDTA 2mM 59％ 2,2'-Bipyridine 2mM 94％ 1,10-Phenanthroline 2mM 94％ maleimide 2mM 99％ Dimethyl sulfoxide 2mM 101％ Sodium cholate 20mM 99％ boric acid 20mM 97％ <![CDATA[NaN3]]> 20mM 100％ SDS 0.1％ 83％ Tween20 0.1％ 105％ Tween80 0.3％ 93％ Brij35 1％ 101％ Triton X-100 1％ 91％

[0126] As shown in Table 1, the lipase lyophilized powder prepared in Example 3 exhibits good tolerance to the most common chemicals, enabling it to adapt to complex clinical samples and different reagent formulations. 5 mM EDTA and 3.5 mM SDS have a significant impact on lipase activity; after treatment at 25°C for 1 h, the residual lipase activities were 59% and 83%, respectively.

[0127] (2) Effect of temperature on lipase activity

[0128] The lyophilized lipase powder prepared in Example 3 was reconstituted to 10 KU / L and incubated at different temperatures for 10 min. The residual lipase activity was then measured, and the results are as follows: Figure 6 As shown in (C).

[0129] Depend on Figure 6 As shown in (C), the lipase prepared in Example 3 can withstand extreme high temperatures for a short time. After being treated at 60°C for 10 minutes, the residual lipase activity is greater than 90%.

[0130] (3) Effect of pH on lipase activity

[0131] The lipase lyophilized powder prepared in Example 3 was reconstituted to 10 KU / L using buffer solutions of different pH values ​​and incubated at 25°C for 24 h. The residual lipase activity was then measured, and the results are as follows: Figure 6 As shown in (D).

[0132] Depend on Figure 6 As shown in (D), the lipase prepared in Example 3 maintains high activity in buffer solutions with pH values ​​between 7 and 9. These data provide valuable reference points for the application conditions of lipase.

[0133] The detection accuracy of the TG reagent in Example 4 was analyzed. The specific detection method was as follows: 3 μL of calibrator (target value 2.86 mM) or serum sample was added to 240 μL of R1, thoroughly mixed, and incubated at 37°C for 5 minutes to measure absorbance A1. Then, 60 μL of R2 was added, thoroughly mixed, and absorbance A2 was measured after 5 minutes. The absorbance change (ΔA) was calculated as ΔA = A2 - A1. A working curve was plotted using the calibrator, and the TG concentration in the sample was determined based on the absorbance change on the curve. The entire process was performed on a fully automated biochemical analyzer (HITACHI 7180), with the main wavelength set to 546 nm and the secondary wavelength set to 700 nm. The detection results are as follows: Figure 7 As shown.

[0134] Depend on Figure 7 As can be seen, within the linear range of 0.5-10.0 mM, the TG reagent in Example 4 exhibited good linearity, and the R-squared value of the fitted curve was [value missing]. 2A value ≥ 0.999 indicates a strong correlation between the theoretical and measured values. This result demonstrates that the lipase of this invention can effectively exert its lipase activity, meeting the requirements for clinical TG detection.

[0135] The detection accuracy of the CHOL reagent in Example 5 was analyzed. The specific detection method was the same as that of the TG reagent, and its calibrator target value was 7.51 mM. The results are as follows: Figure 8 As shown.

[0136] Depend on Figure 8 As can be seen from the above, the CHOL reagent prepared based on the COE activity of lipase in Example 5 has a linear range of 1.0-18.0 mM, R 2 ≥0.999. This result indicates that the lipase of the present invention can effectively exert the COE effect, meeting the requirements for clinical detection of CHOL.

[0137] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A recombinant vector for high-efficiency expression of lipase, characterized in that, The recombinant vector includes: a nucleotide sequence encoding lipA, a nucleotide sequence encoding lipH, and the BSFP_0720 promoter.

2. The recombinant vector as described in claim 1, characterized in that, The lipA and / or lipH genes are derived from Pseudomonas aeruginosa; Preferably, the nucleotide sequence of the BSFP_0720 promoter is shown in SEQ ID NO.1; And / or, the nucleotide sequence encoding lipA is shown in SEQ ID NO.2; And / or, the nucleotide sequence encoding lipH is shown in SEQ ID NO.

3.

3. The recombinant vector as described in claim 1 or 2, characterized in that, The recombinant vector is an integrative plasmid vector pBBR1MCS-2, a pUCP vector, or a pTAC vector. Preferably, the recombinant vector is an integrative plasmid vector pBBR1MCS-2.

4. A recombinant strain that efficiently expresses lipase, characterized in that, The recombinant strain includes the recombinant vector as described in any one of claims 1 to 3; Preferably, the recombinant strain is derived from the genus *Pseudomonas aeruginosa*.

5. The recombinant strain according to claim 4, characterized in that, The recombinant strain cannot express cholesterol oxidase; Preferably, the strain is *Pseudomonas aeruginosa* with the cholesterol oxidase gene knocked out.

6. A method for constructing a recombinant bacterial strain, characterized in that, include: (1) Constructing the recombinant vector as described in any one of claims 1 to 3; (2) The recombinant vector was transformed into Pseudomonas aeruginosa; Preferably, the *Pseudomonas aeruginosa* strain does not express cholesterol oxidase; More preferably, the *Pseudomonas aeruginosa* strain is a strain with the cholesterol oxidase gene knocked out.

7. A method for efficient expression of lipase, characterized in that, include: (1) Cultivate the recombinant strain as described in claim 5 under conditions conducive to lipase expression; (2) Use the cultured recombinant strain to isolate and purify lipase.

8. The use of a recombinant vector as described in any one of claims 1 to 3, or the use of a recombinant strain as described in claim 4 or 5, in the preparation of lipase.

9. A lipase, characterized in that, The lipase is prepared by the method described in claim 7.

10. The use of the lipase as described in claim 9 in the preparation of in vitro diagnostic reagents; Preferably, the in vitro diagnostic reagent includes a triglyceride detection reagent or a cholesterol detection reagent; More preferably, the in vitro diagnostic reagents include reagents for detecting total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and small-dense low-density lipoprotein cholesterol.

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