Lipase mutants and their use in the production of diglycerides

Abstract

This invention discloses a lipase mutant and its application in the preparation of diglycerides, belonging to the fields of enzyme engineering technology and enzyme immobilization technology. This invention utilizes a previously constructed lipase... Δ N39 Ocri-Lip100 V271L -LipB underwent semi-rational modification, using alanine to scan amino acid residues near the 5Å mark of the substrate pocket, and constructed a new type of mutant through saturation mutagenesis. ΔN39 Lip271L T62S The mutant exhibits an enzyme activity of 361.53 U / mg, representing a 364.81% increase compared to the unmutated form. This invention also utilizes this mutant to hydrolyze different edible oils to prepare diglycerides. It was found that hydrolysis of the enzyme with different edible oils at a 0.4:1 ratio for 2 hours yielded diglycerides with a content of 40.54%–64.07%. The lipase mutant of this invention possesses high enzyme activity and good catalytic activity, giving it advantages in diglyceride preparation and demonstrating significant potential for industrial application.

Description

Technical Field

[0001] This invention relates to lipase mutants and their application in the preparation of diglycerides, belonging to the fields of enzyme engineering technology and enzyme immobilization technology. Background Technology

[0002] Lipases, also known as triacylglycerol hydrolases, are widely distributed in nature. Most are secreted by microorganisms, with a smaller portion found in the organs and tissues of animals and plants. The significant interfacial activation properties of lipases result in their highest activity at the oil-water interface. When a hydrophobic substrate forms tiny droplets, the lipase adsorbs onto the surface and undergoes a conformational change, exposing its "cap" active site, thereby catalyzing the reaction. Lipases also participate in esterification and transesterification reactions.

[0003] Lipases hold an irreplaceable position in various industrial sectors due to their broad substrate adaptability and tolerance to organic solvents. In the food industry, lipases can be used in cheese production, chocolate processing, and oil modification, improving the flavor, texture, and stability of food products. In the detergent industry, lipases effectively degrade grease stains, enhancing detergent cleaning efficiency and reducing washing temperatures, thus achieving energy savings. In the pharmaceutical field, lipases not only participate in the synthesis of chiral drugs and the preparation of functional lipids but also show potential in treating certain metabolic diseases. In biodiesel production, lipases catalyze transesterification reactions of oils, converting them into biodiesel, contributing to sustainable energy development. In the field of bioremediation, lipases can also degrade environmental oil pollutants.

[0004] Although there are many types of lipases in nature, wild-type lipases have limitations in industrial applications. Existing lipases (such as wild-type) generally suffer from low enzyme activity and poor catalytic efficiency, making them difficult to meet industrial requirements in practical reactions. To improve performance, immobilization is often used, but immobilized lipases also face significant drawbacks: the immobilization process may lead to conformational changes and damage to the active site, while the substrate mass transfer restriction introduced by the carrier further reduces enzyme activity and catalytic efficiency; under harsh conditions, the activity loss of immobilized enzymes can be even more severe than that of free enzymes. In contrast, free enzymes do not require immobilization, do not suffer from conformational constraints and mass transfer resistance, can maintain higher intrinsic enzyme activity and catalytic efficiency, and are more flexible and controllable in operation. Therefore, there is an urgent need to screen for new lipases with high catalytic efficiency. Summary of the Invention

[0005] To address the problems of low enzyme activity, poor catalytic efficiency, and limited application of existing lipase technologies, this invention provides a method for modifying lipase mutants, constructing a high-throughput screening system, and synergistically improving stability and catalytic activity. The lipase is heterologously expressed in *E. coli*, and the resulting lipase solution is used for the preparation of diglycerides. The lipase obtained through this method can be scaled up, and both enzyme activity and catalytic efficiency are significantly improved.

[0006] The first objective of this invention is to provide a lipase mutant having mutations at one or more of the following sites relative to the parent: G60A, T62A, Y67A, H130A, G133A, P157A, S161A, F163A, L211A, L292A, L310A, V311A, and L331A.

[0007] In one embodiment, the mutation is based on the parental amino acid sequence shown in SEQ ID NO. 14, by truncating amino acids 2 to 40, and having any of the following mutations: Mutate glycine at position 60 to alanine; or Mutate the threonine at position 62 to either alanine or serine; or Mutate the tyrosine residue at position 67 to alanine; or Mutate histidine at position 130 to alanine; or Mutate glycine at position 133 to alanine; or Mutate proline at position 157 to alanine; or Mutate serine at position 161 to alanine; or Mutate the phenylalanine at position 163 to alanine; or Mutate leucine at position 211 to alanine; or Mutate leucoid at position 292 to alanine; or Mutate leucine at position 310 to alanine or serine; or Mutate valine at position 311 to alanine; or The leucine at position 331 was mutated to alanine.

[0008] In one embodiment, the lipase mutant is based on the parent shown in SEQ ID NO.14, with amino acids 2 to 40 truncated and threonine at position 62 mutated to serine.

[0009] In one embodiment, the lipase mutant is based on the parent shown in SEQ ID NO.14, with amino acids 2 to 40 truncated, threonine at position 62 mutated to serine, and phenylalanine at position 163 mutated to alanine.

[0010] In one embodiment, the lipase mutant is based on the parent shown in SEQ ID NO.14, with amino acids 2 to 40 truncated, and threonine at position 62 mutated to serine, and leucine at position 310 mutated to serine.

[0011] The present invention also provides a gene encoding the lipase mutant.

[0012] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13.

[0013] The present invention also provides a recombinant expression vector containing the said gene.

[0014] The present invention also provides microbial cells expressing the lipase mutant, or containing the gene, or containing the recombinant expression vector.

[0015] In one embodiment, the microbial cells include, but are not limited to, Escherichia coli.

[0016] In one embodiment, the *Escherichia coli* uses *Escherichia coli* BL21(DE3) as the host and pET-28a as the vector to express a lipase mutant with an outer membrane secretion protein (OmpA) linked to its N-terminus.

[0017] In one embodiment, the amino acid sequence of the outer membrane secretory protein OmpA is shown in SEQ ID NO.9.

[0018] In one embodiment, the *E. coli* has the membrane rivet protein Lpp and / or the outer membrane protein OmpA knocked out from the genome.

[0019] The present invention also provides a method for preparing the lipase mutant, wherein recombinant Escherichia coli expressing the lipase mutant is cultured in a culture medium at 35-37°C until OD200 reaches zero. 600 =1, add IPTG to a final concentration of 0.2±0.05 mmol / L, cool to 25±1℃ and incubate for at least 24 h, then collect the lipase mutant.

[0020] The present invention also provides the application of the lipase mutant in hydrolyzing rapeseed oil, perilla oil, peanut oil, walnut oil and flaxseed oil.

[0021] In one embodiment, the application includes, but is not limited to, the preparation of diglycerides from one or more of hydrolyzed rapeseed oil, perilla oil, peanut oil, walnut oil, or flaxseed oil.

[0022] In one embodiment, the application involves reacting the lipase mutant at a concentration of ≥361.53 U / g vegetable oil at 50-55°C for at least 2 hours.

[0023] In one embodiment, the reaction process is also stirred.

[0024] The present invention also provides the application of the lipase mutant in the field of oil processing.

[0025] Beneficial effects: (1) The present invention uses the previously modified lipase mutant ΔN39 Ocri-Lip100 V271L -LipB underwent semi-rational modification, employing a strategy of alanine scanning combined with site-directed and saturation mutagenesis of amino acid residues around the substrate pocket, successfully screening for mutants. ΔN39 Lip271L T62S The specific enzyme activity of this mutant reached 361.53 U / mg, which is 4.65 times higher than the specific enzyme activity before modification (77.78 U / mg), and 364.81% higher than the specific enzyme activity of the lipase mutant in the prior art (patent application publication number CN120866274A), demonstrating excellent catalytic performance.

[0026] (2) This invention screens for various secretion signal peptides (such as OmpA) and uses CRISPR / Cpf1 technology to knock out host bacteria. E. coli The endogenous outer membrane protein gene OmpA and the anchoring protein gene Lpp of BL21(DE3) significantly improved the secretion efficiency of lipase mutants, increasing it by 25.6% compared to the non-knockout strains. Simultaneously, this invention designed a chromogenic screening medium combining tributyrate, neutral red, and rhodamine B, establishing a high-throughput plate screening method based on clear zones and color changes. This method can rapidly and intuitively screen highly active mutants from a library of over 600 mutant strains, significantly improving screening efficiency.

[0027] (3) This invention also provides a method for efficiently preparing diglycerides using a lipase mutant. By optimizing the lipase ratio, after reacting at 55°C for 2 hours, the diglyceride content in the hydrolyzed rapeseed oil system can reach 64.07%, which is much higher than the 18.58% under unoptimized conditions. This mutant can be universally used to hydrolyze edible oils such as rapeseed oil, walnut oil, peanut oil, and perilla oil, while efficiently preparing diglycerides. The theoretical value of diglycerides can reach 40.5%~64.1%, showing broad substrate adaptability and good industrial application prospects. Attached Figure Description

[0028] Figure 1 The lipase mutant and substrate in Example 1 of this invention p-NPP molecular docking diagram and display of amino acids in the substrate pocket within 3-5 Å range.

[0029] Figure 2 The images show the activity of the lipase mutant in hydrolyzing substrates and its protein expression effect; where a represents the relative enzyme activity of the mutant; and b represents a gel image of the purified protein from mutant T62A.

[0030] Figure 3 The diagram shows the establishment of the high-throughput screening method in Example 3 of the present invention; wherein, a is a schematic diagram of screening extracellular secretory signal peptides of lipase; b is the effect of screening different secretory signal peptides and endogenous gene knockout on extracellular enzyme activity and secretion; c is the formation of a clear zone of extracellular secretory lipase on a plate.

[0031] Figure 4 These are the high-throughput screening results and fully saturated mutation results of this invention.

[0032] Figure 5 This is an enzyme activity diagram of the recombinant Escherichia coli expressing lipase mutant constructed in this invention.

[0033] Figure 6 The graph shows the diglyceride content of edible oils hydrolyzed by lipase mutants; where a is a gas chromatogram of the hydrolyzed edible oil products; and b is a graph showing the diglyceride and triglyceride content of different edible oils hydrolyzed by lipase. Detailed Implementation

[0034] I. Reagents and Materials: PrimeSTAR Max DNA polymerase was purchased from Baori Biotechnology (Beijing) Co., Ltd. The 5000 bp DNA Marker, 2000 bp DNA Marker, 10000 bp DNA Marker and Gold MixVer2 polymerase were purchased from Beijing Qingke Biotechnology Co., Ltd. Phanta UniFi Master Mix polymerase was purchased from Nanjing Novizan Biotechnology Co., Ltd. Gel recovery kit, plasmid extraction kit and protein standard marker were purchased from Thermo Fisher Scientific. p-Nitrophenol (p-NP), p-nitrophenyl palmitate (p-NPP), etc. were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All analytical grade reagents were purchased from Sinopharm Group; Primer synthesis and sequencing were performed at Beijing Qingke Biotechnology Co., Ltd.

[0035] II. Culture medium: LB solid medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, agar powder 15.

[0036] LB liquid medium (g / L): peptone 10, yeast extract 5, sodium chloride 10.

[0037] TB shake flask fermentation medium (g / L): peptone 20-24, yeast extract 10-12, dipotassium hydrogen phosphate 9-10, potassium dihydrogen phosphate 2-3, glycerol 4-5.

[0038] High-throughput screening medium (g / L): glyceryl tartrate 8-10, Tween-80 8-10, peptone 16-18, yeast extract 8-10, sodium chloride 5-6, dipotassium hydrogen phosphate 9.5-10.5, potassium dihydrogen phosphate 1.5-2, agar powder 18-20, rhodamine B 0.05-0.10, neutral red solution 0.015-0.018.

[0039] Phosphate-buffered saline (PBS) (g / L): disodium hydrogen phosphate 1-2, potassium dihydrogen phosphate 0.2-0.5, sodium chloride 8-10, potassium chloride 0.1-0.3, pH adjusted to 8.0 with hydrochloric acid.

[0040] Protein purification buffer A: Sodium chloride 155 mM, Tris-HCl 55 mM, pH=7.5.

[0041] Protein purification buffer B: Sodium chloride 155 mM, Tris-HCl 55 mM, imidazole 550 mM, pH=7.5.

[0042] III. Enzyme Purification Methods After the shake-flask culture was completed, the culture medium was centrifuged at 5000×g, 4℃ for 30 min. The precipitate was collected and resuspended with an equal volume of 20 mM PBS (pH=8.0) buffer. The cells were washed twice, and the cells were sonicated under ice-water bath conditions. After centrifugation at 10000×g for 30 min at 4℃, the protein mixture after cell disruption was obtained. The protein mixture was filtered through a 0.45 μM sterile filter membrane to obtain the cell disruption supernatant, and then subjected to nickel-column affinity chromatography in an AKTA protein purification system. The protein purification procedure first equilibrated the Ni-His Trap FF column with 10-15 mL of protein purification solution A. The filtered protein mixture was then loaded onto the column, followed by gradient elution with 4% and 8% protein purification solutions B. The target protein was eluted with 40% protein purification solution B.

[0043] IV. Detection Methods: High-throughput screening method: Using glyceryl tartrate, neutral red, and rhodamine B as colorimetric indicators, the recombinant mutant vector was transformed into Escherichia coli BL21(DE3)ΔLPPΔOmpA competent cells, and then cultured to the recombinant cell concentration OD100 using chemical transformation. 600 When the ratio is 1-1.5, centrifuge at 5000 rpm for 2 minutes, spread on a high-throughput screening plate and incubate upside down at 37℃ for 18-28 hours. Based on the color development and clear zone size of the recombinant strain, screen for recombinant mutants with high lipase activity and strong secretion efficiency.

[0044] Methods for determining lipase activity: p -NPP was used as the substrate at a concentration of 10 g / L, dissolved in a mixture of gum arabic and tricolaton. p -NPP substrate. The reaction system for enzyme activity assay consisted of 1.5 mL of 150 μL enzyme solution and 1.35 mL substrate. p -NPP substrate mixture solution, reacted in a 40℃ water bath for 5-10 min, centrifuged at 12000×g for 5 min, and then 200 μL of supernatant was taken to measure the absorbance at 410 nm. Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of substrate p-nitrophenol per minute as 1 U.

[0045] Preparation method of diglycerides: Centrifuge 10 mL of crude enzyme solution at 12000 rpm for 5 min, discard the supernatant, and freeze-dry under vacuum at -65℃. Homogenize rapeseed oil and other ingredients at an oil-water ratio of 5:3 to form a stable emulsion, add 0.1 g of freeze-dried crude enzyme to the system, and react at 45℃ and 220 rpm for 1 h. After the reaction, take 20 μL of the reaction solution, add 1 mL of n-hexane, shake and mix well, and use gas chromatography to identify the content of diglycerides and triglycerides in the reaction system.

[0046] V. Sequence Description The numbering of the mutation sites described in this application is based solely on the full-length amino acid sequence of the wild-type Lip-12c protein shown in SEQ ID NO.14; the sequence is numbered sequentially starting from the first amino acid residue at the N-terminus of the protein, with the first residue numbered 1 and the sequence increasing sequentially. ΔN39 Lip271L T62A For example, ΔN39 Lip271L T62A This indicates a mutant formed by truncating amino acids 2 to 40 and mutating threonine at position 62 to alanine, based on the amino acid sequence shown in SEQ ID NO.14.

[0047] The primer sequences involved in the embodiments are shown in Table 1.

[0048] Table 1 Primer Sequences

[0049] Example 1: Design of lipase 310L mutant and construction of expression strain Based on the lipase Lip-12c with the amino acid sequence shown in SEQ ID NO.14, amino acids 2 to 40 were truncated to obtain the lipase shown in SEQ ID NO.1. ΔN39 The Lip271L sequence. Based on the lipase shown in SEQ ID NO.1. Δ N39 The Lip271L protein structure was visualized, and solvent molecules and other substances besides amino acids were removed from the protein structure file. The active sites 131S, 308D, and 330H, as well as the binding sites 61L, 132Q, 286D, 332D, 336Q, and 340V, were selected, and the amino acids within a 3-5 Å range surrounding these sites were removed. Figure 1 The other amino acids were mutated to alanine. The gene sequence shown in SEQ ID NO.2 was synthesized, and the resulting gene was ligated into the pET-28a plasmid using homologous recombination to obtain the recombinant plasmid pET-28a- ΔN39 Lip271L, recombinant plasmid pET-28a- ΔN39 Lip271L was converted to [the desired state] via chemical transformation. Escherichia coli Recombinant Escherichia coli strains were obtained from BL21(DE3) competent cells.

[0050] Example 2: Semi-rational modification and expression of lipase mutants With pET-28a- ΔN39 BL21(DE3) of Lip271L was used as the control strain. The recombinant *E. coli* constructed in Example 1 and the control strain were inoculated into LB liquid medium and cultured at 37°C for 6-8 h. They were then transferred to 250 mL flat-bottomed shake flasks containing TB medium and cultured at 37°C for 1.5-2.0 h. 0.06 mM IPTG was added to induce recombinant protein expression, and the culture temperature was adjusted to 25°C and cultured for 18 h. The recombinant cells in the shake flasks were centrifuged at 8000 rpm at 4°C for 30 min, the supernatant was discarded, and the cells were reselected 2-3 times with pH 8.0 PBS buffer. The cells were then disrupted using an ultrasonic homogenizer and centrifuged at 12000 rpm at 4°C for 15-20 min to obtain the crude enzyme solution for intracellular expression.

[0051] The crude enzyme solution obtained above was mixed with p-nitrophenol palmitate at a concentration of 10 g / L. p-NPP) was mixed at a volume ratio of 1:9 and reacted at 40℃ for 5 min. After terminating the reaction, the mixture was centrifuged at 12000×g for 5 min, and 200 μL of the supernatant was taken to measure the absorbance at a wavelength of 410 nm. Finally, the activities of the lipase mutant series of hydrolyzed substrates were calculated. The results are as follows: Figure 2 As shown, mutant ΔN39 Lip271L T62A mutant ΔN39 Lip271L F163A and mutants ΔN39 Lip L310A Enzyme activity was significantly increased compared to the control ( ΔN39 The enzyme activity of Lip271L was increased by 12.08%, 79.35%, and 57.43% compared to that of 77.78 U / mg.

[0052] Example 3: Construction of a high-throughput screening system (1) Screening of secretory signal peptides To improve the efficiency of lipase mutant screening, the recombinant strains were modified to express lipases and become secretory strains capable of secreting into the culture medium. The following proteins were screened: DsbA (SEQ ID NO.3), a disulfide bond forming protein; PelB (SEQ ID NO.5), a pectic acid lyase secretory protein; PhoA (SEQ ID NO.7), an alkaline phosphatase secretory protein; and OmpA (SEQ ID NO.9), an outer membrane protein. The transmembrane sequences (nucleotide sequences SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.10, respectively) of these proteins were amplified using primer pairs DsbA-F / R, PelB-F / R, PhoA-F / R, and OmpA-F / R and ligated into the vector pET-28a- ΔN39 Lip271L F163A Simultaneously, the above inducible plasmids were constructed into constitutive plasmids, named pET-28a-DsbA. ΔN39 Lip271L F163A pET-28a-PelB ΔN39 Lip271L F163A pET-28a-PhoA ΔN39 Lip271L F163A and pET-28a-OmpA ΔN39 Lip271L F163ATo improve the efficiency of high-throughput screening, CRISPR / Cpf1 gene editing was used to knock out the endogenous outer membrane secretion protein gene OmpA (Gene ID: WP_193328575.1) and the anchoring protein Lpp (Gene ID: WP_001487254.1) in the BL21(DE3) genome. The resulting strain was named... E. coli BL21(DE3)Δ lpp Δ ompA .

[0053] pET-28a-DsbA constructed in Example 3 ΔN39 Lip271L F163A pET-28a-PelB ΔN39 Lip271L F163A pET-28a-PhoA ΔN39 Lip271L F163A and pET-28a-OmpA ΔN39 Lip271L F163A Transformed into strains E. coli BL21(DE3)Δ lpp Δ ompA In the middle, the medium was cultured in TB liquid medium at 30℃ for 16 h, and the results were as follows: Figure 3 As shown, the mutant F163A expresses OmpA in its recombinant plasmid at the N-terminus. E. coli BL21(DE3)Δ lpp Δ ompA / pET-28a-OmpA Δ N39 Lip271L F163A Lipase secretion was most effective compared to recombinant plasmids expressing OmpA alone. E. coli BL21(DE3) / pET-28a-OmpA ΔN39 Lip271L F163A The secretion efficiency is high at 25.6%.

[0054] (2) Design and optimization of high-throughput screening media In a screening medium containing 10–15 g / L tributyrate, 1.6%–1.6% neutral red, and 0.05–0.1 g / L rhodamine B as a colorimetric indicator, emulsification was performed using a 10 nm stainless steel averaging head at 10,000–20,000 rpm for 3–5 min until the solution was uniformly milky white. Recombinant *E. coli* strains expressing a mutant lipase secretion signal peptide were then used. E. coli BL21(DE3)Δ lpp Δ ompA / pET-28a-OmpA ΔN39 Lip271LF163A The sample was spread on a solid culture medium plate containing a colorimetric indicator. Lipase secreted from the sample reacted with the substrate to form a clear zone and a colorimetric reaction. Mutant strains with higher lipase activity were screened based on the size of the clear zone and the color change. Figure 3 c) High-throughput culture was then scaled up using a plate shaker. Results showed clear changes in the size of the transparent zone on plates containing the three screening indicators, indicating the feasibility of the high-throughput screening method established using these indicators.

[0055] Example 4: Fully saturated mutagenesis of lipase mutants Based on the screening of substrate pockets 3-5 Å containing F163A, L310A, and T62A in Example 1, degenerate primers A163N-F / R, A310N-F / R, and A163N-F / R were designed. These primers were then used with pET-28a-OmpA constructed in Example 3. ΔN39 Lip271L F163A pET-28a-OmpA ΔN39 Lip271L L310A and pET-28a-OmpA ΔN39 Lip271L T62A Use the template as a base and perform a full saturation mutation. Transform the recombinant plasmid carrying the mutated gene into E. coli BL21(DE3)Δ lpp Δ ompA The obtained mutants were plated on a selection medium containing an indicator, and a mutant library of >600 strains was constructed. The lipase secretion behavior was as follows: Figure 4 As shown. Sequencing results of the recombinant strain show that the mutant OmpA ΔN39 Lip271L T62S OmpA ΔN39 Lip271L F163A and OmpA ΔN39 Lip271L L310S It has the highest relative enzyme activity.

[0056] To further verify the feasibility of the high-throughput mutant library saturation mutation screening results under scale-up conditions, the above mutant plasmid was transformed into *E. coli* BL21(DE3), yielding recombinant strains BL21(DE3) / pET-28a- ΔN39 Lip271L T62S BL21(DE3) / pET-28a- ΔN39 Lip271L F163A and BL21(DE3) / pET-28a- Δ N39 Lip271L L310S.

[0057] The recombinant strains were cultured in TB medium at 25°C for 16 h, and the enzyme activity in the fermentation broth was detected. The results are as follows: Figure 5 As shown, the A62 mutant library results indicate that lipase... ΔN39 The Lip271L serine enzyme with a threonine mutation at position 62 has the highest activity, at 134.8 ± 3.4 U / mL. ΔN39 Lip271L T62A The mutant was 2.2 times stronger; the A310 mutant library results showed that it would increase the lipase content. ΔN39 The leucine mutation at position 310 in Lip271L, which converts it to serine, results in an enzyme activity of 75.6 ± 6.5 U / mL. Δ N39 Lip271L L310A The activity was 1.14 times that of the A163 mutant library; however, no mutants with significantly increased enzyme activity were found in the A163 mutant library.

[0058] Further optimization of lipase mutants, in mutant ΔN39 Lip271L T62S Based on this, the phenylalanine at position 163 was mutated to alanine, and the leucine at position 310 was mutated to serine, resulting in mutants. ΔN39 Lip271L T62SF163A and ΔN39 Lip271L T62SL310S The results showed that a lipase mutant was found. ΔN39 Lip271L T62S It exhibits the highest specific enzyme activity, with a 364.81% increase in specific enzyme activity compared to the lipase mutant in the patent application document with publication number CN120866274A.

[0059] Example 5: Lipase mutant ΔN39 Lip271L T62S Hydrolyzed rapeseed oil 1. Lipase mutant ΔN39 Lip271L T62S Preparation of crude enzyme solution Expressing lipase mutants ΔN39 Lip271L T62S The plasmid was transformed into Escherichia coli BL21(DE3) by chemical transformation to obtain the recombinant strain BL21(DE3) / ΔN39 Lip271L T62S Spread the culture onto solid LB agar plates and incubate at 37°C for 12-16 h. Finally, select single colonies and inoculate them into 50 mL shake tubes containing 5 mL of liquid LB medium. Incubate at 37°C for 8-10 h, then transfer to 2.5 L shake flasks containing 900 mL of liquid TB medium and incubate until OD (outlet count) reaches 100%.600 At a concentration of 1, 0.2 mmol / L IPTG was added to induce protein expression, and the temperature was adjusted to 25℃ and cultured for 24 h. The recombinant cells cultured in shake flasks were centrifuged at 8000 rpm and 4℃ for 30-40 min, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (pH 8.0) 2-3 times. The cells were then sonicated at 30% power for 2 s with a 3 s pause for 25 min. Finally, the cells were centrifuged at 8000 rpm and 4℃ for 30-40 min to obtain the lipase-containing mutant. ΔN39 Lip271L T62S The crude enzyme solution (enzyme activity 134.8 ± 3.4 U / mL) was purified to obtain enzyme protein. The enzyme protein's specific activity was determined to be 361.53 U / mg, which was lower than the control. ΔN39 Lip271L is 4.65 times higher.

[0060] 2. Lipase mutant ΔN39 Lip271L T62S Optimization of conditions for hydrolyzing rapeseed oil Weigh 100 g of rapeseed oil into a 500 mL glass beaker, and add 100 mg of the lipase mutant prepared according to the aforementioned method. ΔN39 Lip271L T62S The enzyme protein was homogenized at 8000 rpm for 30 s at room temperature using a T10IKA homogenizer, followed by stirring at 55°C for 2 h. Samples were taken every 0.5 h during the reaction, and the composition of the hydrolysis products was analyzed by gas chromatography. The results are as follows: Figure 6 As shown in a, this lipase mutant can hydrolyze rapeseed oil and obtain 18.58% diglyceride oil.

[0061] Further, a low-temperature molecular distillation method was used to remove the lighter oil phase at an evaporation temperature of 170°C, a condensation temperature of 20°C, a scraper speed of 300 rpm, and a vacuum pressure of 100 Pa. Subsequently, purified diglyceride oil was obtained by separation at 200°C, a scraper speed of 300 rpm, and a vacuum pressure of 100 Pa. The results showed that the conversion value of diglyceride could reach 34.21%.

[0062] Example 6: Lipase mutant ΔN39 Lip271L T62S Optimization of conditions for hydrolyzed rapeseed oil Based on Example 5, rapeseed oil and mutants were optimized. ΔN39 Lip271L T62S The ratio of lipase to rapeseed oil (g) to lipase (mg) in the reaction system was controlled to be 1:0.6 and 1:0.4, respectively. The results are shown in Table 2 and Figure 6 As shown in b, when the ratio of rapeseed oil to lipase is 1:0.4 (i.e., 0.4 mg of lipase is added to each gram of rapeseed oil), 64.07% diglyceride oil can be obtained in 2 hours.

[0063] Table 2 Optimization of Lipase and Rapeseed Oil Catalytic Ratio

[0064] Example 7: Lipase mutant ΔN39 Lip271L T62S Verification of the wide applicability of hydrolyzed oils Using lipase mutants ΔN39 Lip271L T62S Following the conditions of Example 5, peanut oil, corn oil, rice bran oil, sunflower oil, and pecan oil were hydrolyzed at 55°C at a ratio of 0.4 mg enzyme protein / g vegetable oil. After 2 hours of reaction, the content of diglycerides obtained from the hydrolysis of different edible oils by this mutant ranged from 40.54% to 47.01%. Except for hydrolyzed rapeseed oil, hydrolyzed sunflower seed oil had the highest diglyceride content, reaching 47.94%, followed by rice bran oil at 47.01%, peanut oil at 45.71%, while pecan oil and corn oil had relatively lower contents at 40.5%. These results indicate that this lipase mutant can hydrolyze different oils to prepare diglycerides, demonstrating good versatility.

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A lipase mutant, characterized in that, Based on the parental amino acid sequence shown in SEQ ID NO.14, the threonine at position 62 was mutated to serine, and amino acids 2 to 40 were deleted.

2. The gene encoding the lipase mutant of claim 1.

3. A recombinant expression vector containing the gene of claim 2.

4. A microorganism expressing the lipase mutant of claim 1 or containing the gene of claim 2.

5. Recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET-28a as the vector, the lipase mutant of claim 1, which has the outer membrane secretion protein OmpA signal peptide shown in SEQ ID NO.9 linked at the N-terminus, is expressed.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The membrane anchoring protein Lpp and / or the outer membrane protein OmpA located in the genome were knocked out.

7. The method for preparing the lipase mutant of claim 1, characterized in that, The recombinant Escherichia coli according to claim 5 or 6 is cultured in a culture medium at 35-37°C until OD reaches [value missing]. 600 =1, add IPTG to a final concentration of 0.15~0.25 mmol / L, cool to 24~26℃ and incubate for at least 24 h, then collect the lipase mutant.

8. The application of the lipase mutant of claim 1 in hydrolyzed rapeseed oil, perilla oil, peanut oil, walnut oil and flaxseed oil.

9. A method for preparing diglycerides, characterized in that, The lipase mutant of claim 1 was contacted with vegetable oil at a concentration of ≥361.53 U / g and reacted at 50-55°C for at least 2 h.

10. The application of the lipase mutant of claim 1 in the field of oil processing.

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