Highly active lipase mutant and application thereof in synthesis of functional oil

Abstract

The application discloses a high-activity lipase mutant and application thereof in synthesis of functional oil, and belongs to the technical field of enzyme engineering and the technical field of enzyme immobilization. B. cepacian The lipase from OCRI-Lip100 is truncated and mutated, and the soluble expression efficiency in E. coli is improved by adding a solubility tag at the C terminal and co-expressing with a foldase. The lipase mutant prepared by immobilization with DMONs nanomaterials has a prolonged half-life at 60 DEG C and improved catalytic activity relative to free enzyme, and has good industrial application potential.

Description

Technical Field

[0001] This invention relates to a highly active lipase mutant and its application in the synthesis of functional oils, belonging to the fields of enzyme engineering technology and enzyme immobilization technology. Background Technology

[0002] Lipases (EC 3.1.1.3) are a class of enzymes that catalyze the hydrolysis of fats and oils at the oil-water interface, releasing free fatty acids, diglycerides, monoglycerides, and glycerol. Furthermore, in organic media, lipases can catalyze esterification, alcoholysis, acidolysis, ammonolysis, transesterification, and transesterification of substrates, making them important industrial enzymes with diverse catalytic functions.

[0003] Lipases are widely found in nature, and their catalytic differences among species are significant. Discovering and screening high-lipase-producing strains from specific oil-rich environments, and then using microbial cells as a chassis for heterologous protein expression modification, has been widely applied in animal feed, food modification, biodiesel, environmental remediation, biopharmaceuticals, papermaking, and medical diagnostics. Patent application CN119776196A discloses a method for screening and applying highly active lipases, laying the foundation for optimizing heterologous lipase expression. However, because Burkholderia is an opportunistic pathogenic strain, its cultivation methods are complex, and scale-up production is difficult.

[0004] On the other hand, due to the poor environmental resistance of free lipases, their catalytic activity may decrease or even be completely inactivated during feed processing, storage, and animal digestion, thus failing to perform their biocatalytic function. Therefore, achieving the immobilization of highly active and thermally stable free lipases is an urgent problem to be solved in order to promote the industrial application of lipases. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a lipase mutant and a method for its recombinant expression and immobilization, wherein the previously screened Burkholderia ( Burkholderia cepacian Lipase derived from OCRI-Lip100 was modified and mutated for heterologous expression in E. coli, and the purified lipase solution was immobilized with DMONs nanomaterials using a nickel column. The lipase obtained through this method can be scaled up, and both enzyme activity and catalytic stability are significantly improved.

[0006] The first object of the present invention is to provide a lipase mutant having improvements of (a) and / or (b):

[0007] (a) A truncated amino acid sequence, such as the amino acid sequence from position 2 to 40 at the N-terminus of the lipase shown in SEQ ID NO.1;

[0008] (b) Based on (a), at least one of the following mutations was performed on the truncated lipase: Q39P, T258L, V271L.

[0009] In one embodiment, the truncation is performed by shortening the 2nd to 40th amino acids of the lipase shown in SEQ ID NO.1 to obtain the truncated lipase shown in SEQ ID NO.10.

[0010] In one embodiment, the mutation is based on a lipase truncated form with an amino acid sequence as shown in SEQ ID NO. 10, and has any of the following mutations:

[0011] Mutate glutamine at position 39 to proline; or

[0012] Mutate threonine at position 258 to leucine; or

[0013] Mutate valine at position 271 to leucine; or

[0014] Mutate valine at position 271 to leucine and glutamine at position 39 to proline; or

[0015] Mutate valine at position 271 to leucine and threonine at position 258 to leucine; or

[0016] Mutate valine at position 271 to leucine and threonine at position 274 to lysine; or

[0017] The valine at position 271 was mutated to leucine, the glutamine at position 39 to proline, and the threonine at position 258 to leucine.

[0018] A second objective of this invention is to provide a gene encoding the lipase mutant.

[0019] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.9.

[0020] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned genes.

[0021] In one embodiment, the expression vector includes, but is not limited to, pET series plasmids.

[0022] In one embodiment, the expression vector is pET-28a.

[0023] A fourth object of the present invention is to provide microbial cells that express the lipase mutant, or contain the gene, or contain the recombinant expression vector.

[0024] The fifth objective of this invention is to provide a recombinant engineered bacterium expressing lipase, using Escherichia coli as the starting strain, expressing the lipase mutant with a solubilizing tag added to the N-terminus.

[0025] In one embodiment, the solubilizing tag is a maltose-binding protein (MBP) tag or a histidine tag.

[0026] In one embodiment, the recombinant engineered bacteria co-express a lipase mutant containing a maltose-binding protein or histidine lysing tag and a folding enzyme LipB.

[0027] In one embodiment, the nucleotide sequence encoding the maltose-binding protein MBP is shown in SEQ ID NO.4.

[0028] In one embodiment, the nucleotide sequence encoding the folding enzyme LipB is shown in SEQ ID NO.8.

[0029] A seventh objective of this invention is to provide an immobilized lipase, wherein the immobilized lipase is carried by DMONs nanomaterials.

[0030] In one embodiment, the method for preparing the DMONs nanomaterial includes the following steps:

[0031] Step S1: The catalyst triethanolamine is slowly dissolved in ultrapure water, and the structure directing agent hexadecylmethylammonium bromide and sodium salicylate are added. The mixture is stirred and dissolved to obtain a silicon source reaction mixed solution.

[0032] Step S2: Add tetraethyl orthosilicate and 1,2-bis(triethoxysilyl)ethane to the silicon source reaction mixture, shake and stir to obtain a crude sample of DMONs nanomaterials;

[0033] Step S3: Reflux the crude DMONs nanomaterial sample in hydrochloric acid solution, wash with ethanol, and vacuum dry to obtain clean and purified DMONs nanomaterials.

[0034] In one embodiment, the method involves adding DMONs nanomaterials to a lipase preparation solution and obtaining an immobilized lipase with DMONs nanomaterials as a carrier through adsorption.

[0035] This invention also provides the application of the lipase or immobilized lipase in the field of oil processing technology.

[0036] Beneficial effects:

[0037] (1) The present invention uses Burkholderia bacteria screened in the early stage B. cepacianLipase derived from OCRI-Lip100 was mutated at certain sites and a solubilization tag was added, shortening the N-terminus by 2-40 amino acids. The resulting truncated mutant was heterologously expressed in *E. coli*. Lipase was produced by adding recombinant bacteria to a substrate-containing system using induced fermentation, followed by purification of the product using a nickel column. This method enables rapid lipase production with simple culture conditions and high expression levels.

[0038] (2) The present invention also prepared lipase immobilized with DMONs nanomaterials, which can extend the half-life of the immobilized lipase to 34 minutes at the optimal temperature of 60℃; the immobilized lipase was stored at 25℃ for 28 days and the lipase activity was significantly higher than that of the free enzyme. It has the characteristics of stable enzymatic properties, greatly improved enzyme activity, and easy recycling. Attached Figure Description

[0039] Figure 1 This is a map of the recombinant plasmid expressing lipase in Escherichia coli in Example 1 of the present invention;

[0040] Figure 2 The growth curve and enzyme activity detection diagram of the recombinant strain constructed in Example 1 of this invention;

[0041] Figure 3 This is an image of the recombinant lipase truncated form and the fusion lysosomal tag enzyme activity diagram from Example 4 of the present invention;

[0042] Figure 4 This is a diagram illustrating the lipase expression host, folding enzyme screening, and protein purification in Example 5 of the present invention.

[0043] Figure 5 This is a diagram showing the optimized culture conditions for recombinant Escherichia coli expressing lipase in Example 6 of the present invention;

[0044] Figure 6 This is an enzyme activity diagram of the recombinant Escherichia coli expressing a lipase mutant in Example 7 of the present invention;

[0045] Figure 7 This is a comparison diagram of the enzyme activity of immobilized and free lipase in Example 8 of the present invention. Detailed Implementation

[0046] I. Reagents and Materials:

[0047] PrimeSTAR Max DNA polymerase was purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0048] 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.

[0049] Phanta UniFi Master Mix polymerase was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0050] Gel recovery kit, plasmid extraction kit and protein standard marker were purchased from Thermo Fisher Scientific.

[0051] p-Nitrophenol (p-NP), p-nitrophenyl palmitate (p-NPP), etc. were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0052] All analytical grade reagents were purchased from Sinopharm Group;

[0053] Primer synthesis and sequencing were performed at Beijing Qingke Biotechnology Co., Ltd.

[0054] II. Culture medium:

[0055] LB solid medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, agar powder 15.

[0056] LB liquid medium (g / L): peptone 10, yeast extract 5, sodium chloride 10.

[0057] TB shake flask fermentation medium (g / L): peptone 20-24, yeast extract 10-12, potassium dihydrogen phosphate 9-10, potassium dihydrogen phosphate 2-3, glycerol 4-5.

[0058] 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.

[0059] Protein purification buffer A: Sodium chloride 155 mM, Tris-HCl 55 mM, pH=7.5.

[0060] Protein purification buffer B: Sodium chloride 155 mM, Tris-HCl 55 mM, imidazole 550 mM, pH=7.5.

[0061] III. Detection Methods:

[0062] Lipase activity assay: p-NPP was used as the substrate at a concentration of 10 g / L, dissolved in a mixture of gum arabic and triton. The reaction system for the enzyme activity assay consisted of 1.5 mL containing 150 μL of enzyme solution and 1.35 mL of the p-NPP substrate mixture. The reaction was carried out in a water bath at 40°C for 5–10 min, followed by centrifugation at 12000 × g for 5 min. 200 μL of the supernatant was then collected, and the absorbance was measured at 410 nm. The unit of enzyme activity is defined as 1 U, which is the amount of enzyme required to convert 1 μmol of p-nitrophenol per minute.

[0063] Lipase purification method: A 5 mL nickel column was connected to an AKTA instrument and equilibrated with 10 column volumes of 50 mmol / L phosphate buffer (pH=7.4). 20 mL of the crude enzyme solution obtained above was loaded onto the nickel column on ice. Imidazole gradient elution was performed, with imidazole prepared in phosphate buffer (50 mmol / L, pH=7.4) at concentrations of 10 mmol / L, 30 mmol / L, and 200 mmol / L. The eluted target protein was then desalted and deimidazoleized.

[0064] IV. The primer sequences involved in the embodiments are shown in Table 1.

[0065] Table 1 Primer Sequences

[0066]

[0067] Example 1: Construction of recombinant Escherichia coli expressing lipase Lip-12c

[0068] The nucleotide sequence encoding the lipase Ocri-Lip100, as shown in SEQ ID NO.2, was synthesized. The obtained gene was ligated into the pET-28a plasmid using homologous recombination to obtain the recombinant plasmid (…). Figure 1 The recombinant plasmid, named pET-Ocri-Lip100, was transformed into pET-Ocri-Lip100 via chemical transformation. Escherichia coli Recombinant Escherichia coli strains were obtained from BL21(DE3) competent cells.

[0069] Example 2: Expression of lipase Ocri-Lip100

[0070] BL21(DE3) containing pET-28a was used as the control strain. The recombinant *E. coli* strain constructed in Example 2 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. 0.2 mM IPTG was added to induce recombinant protein expression, and the culture temperature was adjusted to 30°C and cultured for 36 h. The recombinant cells in the shake flasks were centrifuged at 5500 rpm at 4°C for 8-10 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 disruptor and centrifuged at 12000 rpm at 4°C for 15-20 min to obtain the crude enzyme solution for intracellular expression.

[0071] The intracellular expression crude enzyme solution obtained from the above-mentioned cell disruption was mixed with 10 g / L p-nitrophenol palmitate (p-NPP) 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 aspirated to measure the absorbance at a wavelength of 410 nm. Finally, the activity of the lipase Ocri-Lip100 in hydrolyzing the substrate was calculated. The results are as follows: Figure 2 As shown, the enzyme activity of strain 1 is 0.001 U / mL.

[0072] Example 3: Construction of lipase truncated form

[0073] To improve the soluble expression of lipase in *E. coli*, the secondary structure of the lipase Ocri-Lip100 protein was predicted using online software (https: / / www.novopro.cn / tools / signalp) and primer pairs were used. ΔN39 (Ocri-Lip100)-F / R was amplified using Ocri-Lip100 as a template to truncate the N-terminus of amino acids 2-40 (39 amino acids in total). The PCR product was transformed into competent E. coli JM109 cells, incubated on ice for 25-35 min, heat-shocked at 42℃ for 90 s, and then 600 μL of fresh LB broth was added. The cells were incubated at 37℃ for 35-40 min, centrifuged at 5000 rpm for 2 min, and the supernatant was discarded, retaining 200 μL. After thorough mixing, the mixture was spread onto solid LB broth containing kanamycin and incubated upside down at 37℃ for 12-16 h. Single colonies were selected for sequencing verification, and the correct transformant was named pET- ΔN39 Ocri-Lip100.

[0074] Example 4: Lipase truncated form ΔN39 Ocri-Lip100 soluble expression

[0075] In lipase ΔN39 Ocri-Lip100 was fused with one or more C-terminal fusion-promoting tags, such as MBP (SEQ ID NO.3), SUMO (SEQ ID NO.4), and GST (SEQ ID NO.5), as shown in Table 1. Amplification was performed using PCR, and the amplified products were ligated using homologous recombination to obtain positive clones and recombinant plasmids fused with fusion-promoting tags. All were transformed and cultured according to the method in Example 1. Enzyme activity in the crude enzyme solution was measured, and the results are as follows: Figure 3 As shown, the enzyme activities of the truncated lipase and the lipase fused with the solubilizing tag were 0.13 U / mL, 0.29 U / mL, 0.20 U / mL and 0.16 U / mL, respectively, with the truncated lipase fused with the MBP tag showing the most significant increase in enzyme activity.

[0076] Example 5: Lipase truncated form ΔN39 Optimization of Ocri-Lip100 soluble expression

[0077] (1) Host optimization

[0078] To further improve the expression level and catalytic efficiency of lipase, the recombinant plasmid pET-MBP- ΔN39 Ocri-Lip100 was transformed into one or more starting strains of *Escherichia coli* Rosetta (DE3), *Escherichia coli* Origami B (DE3), and *Escherichia coli* Origami B pGpro7 (DE3) according to the method in Example 1. After shake-flask culture, the recombinant strains were broken up and crude lipase solution was obtained. The enzyme activity was compared with that of the recombinant strain *Escherichia coli* BL21 (DE3) / pET-MBP- ΔN39 The results were compared with Ocri-Lip100. The enzyme activity increased from 0.29 U / mL to 1.23 U / mL.

[0079] (2) The truncated lipase variant ΔN39Ocri-Lip100 was co-expressed with the folding enzyme.

[0080] In plasmid pET- MBP- ΔN39 A folding enzyme was expressed based on Ocri-Lip100, and the folding enzyme was constructed into the recombinant plasmid pET-MBP- using homologous recombination. ΔN39On the Ocri-Lip100, expression cassettes co-expressing TrxA folding enzyme (SEQ ID NO. 6), TrxB folding enzyme (SEQ ID NO. 7), or LipB folding enzyme (SEQ ID NO. 8) were ligated using RBS1 (AAGGAGCG). Primers are shown in Table 1. PCR amplification was performed, and the amplified products were ligated using homologous recombination. The transformation culture method and crude enzyme solution acquisition method are as described in Examples 1-2. Figure 4 As shown, the lipase activities fused with folding enzymes LipB, TrxA, and TrxB were 32.50 U / mL, 1.34 U / mL, and 2.02 U / mL, respectively. Co-expression of LipB folding enzyme showed the most significant increase in activity, compared to strain BL21(DE3) / pET-MBP- ΔN39 Ocri-Lip100 enzyme activity increased by 26.4 times.

[0081] Example 6: Optimization of Culture Conditions for Recombinant Escherichia coli

[0082] Recombinant strain BL21(DE3) / pET-MBP- was induced with different final concentrations of IPTG. ΔN39 Ocri-Lip100-lipB expresses lipase.

[0083] (1) Optimization of IPTG concentration

[0084] The recombinant Escherichia coli BL21(DE3) / pET-MBP- constructed in Example 5 was used. ΔN39 Ocri-Lip100-lipB was inoculated into LB liquid medium and cultured at 37°C for 6–8 h. The inoculum was then transferred to 250 mL flat-bottomed shake flasks containing TB medium and cultured at 37°C for 1.5–2.0 h. IPTG was then added at final concentrations of 0.02 mM, 0.04 mM, 0.06 mM, 0.10 mM, 0.20 mM, 0.50 mM, and 1.00 mM, respectively. The mixture was then cooled to 30°C and cultured for a total of 36 h. The enzyme activity of the crude enzyme solution was measured according to the method in Example 2. The results showed that the lipase activity induced with a final concentration of 0.06 mM reached 35.04 U / mL.

[0085] (2) Optimization of induction timing

[0086] The culture method was the same as in Part (1) of this embodiment, except that after culturing at 37°C for 2 h, 3 h, 3.5 h and 4 h respectively, the lipase was induced with IPTG at a final concentration of 0.06 mM and then cultured for 32 h. The results showed that the lipase activity induced after 4 h of culture reached 37.56 U / mL.

[0087] (3) Optimization of induction time

[0088] The culture method is the same as in Part (1) of this embodiment. The difference is that after culturing at 37°C for 4 h, IPTG with a final concentration of 0.06 mM was used to induce for 10-24 h. The results showed that the recombinant mean lipase expression efficiency was the highest when the inducing agent was added and cultured for another 12 h. The enzyme activity of the crude enzyme solution was 39.89 ± 0.30 U / mL.

[0089] Example 7: Preparation of mutants with improved lipase stability

[0090] The patent application document with publication number CN119776196A evaluated the wild type. Burkholderia cepacian The enzymatic properties of lipases produced by OCRI-Lip100 were analyzed, and it was found that lipase Lip-12c exhibited the highest catalytic activity at 60°C, but its stability was poor. To further improve the thermostability of the lipase, this embodiment used AI-assisted design and the online software FireProt (https: / / loschmidt.chemi.muni.cz / fireprot / ) to evaluate the thermostability of the lipase and select mutation sites. Site-directed mutagenesis was performed at Q39, V89, S141, T155, T258, V271, and K274 sites. The recombinant plasmid pET-MBP- carrying the truncated lipase variant constructed in Example 5 was used. ΔN39 Using Ocri-Lip100-lipB as a template, PCR amplification was performed, and the primers used are shown in Table 1. The PCR-amplified product containing the homologous recombination sequence was introduced into E. coli JM109 competent cells via chemical transformation. Single colonies were selected for PCR to obtain positive transformants with successful sequencing. The transformants were cultured according to the method in Example 2, and the crude enzyme solution was collected and purified. The purified mutant enzyme was placed in PBS buffer (pH=8.0) and incubated at 60°C for 5 min. After terminating the reaction, it was centrifuged at 12000×g for 5 min, and 200 μL of the supernatant was aspirated to measure the absorbance at 410 nm. ΔN39 The enzyme activity of Ocri-Lip100 was 100% after incubation at 60℃ for 5 min, and the enzyme activity and stability of the mutant V217L screened out were significantly higher than those of the control. Figure 6 The enzyme activity was 57.55 U / mL, which was 137.52% higher than the control.

[0091] Example 8: Preparation of Nanomaterials and Immobilized Enzymes

[0092] 1. Preparation of nanomaterials DMONs

[0093] The catalyst triethanolamine was dissolved in ultrapure water, and the structure-introducing agents hexadecylmethylammonium bromide and sodium salicylate were added and stirred to obtain a silicon source mixture. Tetraethyl orthosilicate and 1,2-bis(triethoxysilyl)ethane were further added to the silicon source mixture, and after shaking and mixing, a crude DMONs nanoparticle sample was obtained. The crude DMONs nanoparticle sample was refluxed in hydrochloric acid solution, washed with ethanol, and vacuum dried to obtain clean and purified DMONs nanomaterials.

[0094] Optionally, the following steps can be performed: In a 2 L spherical reaction flask containing 500 mL of deionized water, 1.36 g of TEA was added as a catalyst, and the mixture was slowly stirred at 80 °C for 30 min until completely dissolved. Then, 7.60 g of CTAB and 2.52 g of NaSal were added as structure-directing agents, and stirring was continued for 1 h until completely dissolved. After that, a mixture of 40 mL of TEOS and 32 mL of BTEE was added as a silicon source, and the mixture was stirred vigorously for 12 h to synthesize DMONs. After the reaction was completed, the product was collected by high-speed centrifugation (20,000 rpm, 5 min) and washed three times with anhydrous ethanol. To remove residual structure-directing agents from the DMONs, the product was extracted by reflux with 2 M hydrochloric acid-ethanol solution at 60 °C for 6 h, repeated three times until the structure-directing agents were completely removed. The product was then thoroughly washed with ethanol and vacuum dried at room temperature.

[0095] 2. Combination and application of recombinant lipase mutants with DMON nanomaterials

[0096] The nanomaterial DMONs was added to the solution of purified lipase mutants to obtain DMONs nanomaterial-immobilized lipase through adsorption. The specific steps were as follows: 50 mg of DMONs was added to 1 mL of phosphate-buffered saline (PBS, 50 mM, pH 8.0), followed by 4 mL of purified and concentrated lipase V271L solution. The mixture was sonicated in an ice-water bath for 10 min to ensure complete dispersion of the carrier in the enzyme solution. Vacuum was applied for 10 min to remove air from the carrier, and the mixture was immobilized by self-assembly in a constant-temperature shaker (30℃, 200 rpm) for 30 min. After immobilization, the supernatant and precipitate were collected separately by high-speed centrifugation (12000 rpm, 5 min). The precipitate was lyophilized under vacuum at -65℃ to obtain immobilized enzyme V271L@DMONs, which was then stored at 4℃.

[0097] Immobilized enzyme V271L@DMONs was catalyzed using olive oil as a substrate at 60℃. The results showed that the immobilized lipase activity was still significantly higher than that of the free enzyme after 28 days of storage at 25℃. Figure 7 ).

[0098] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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, The amino acids from position 2 to 40 of the lipase shown in SEQ ID NO.1 were truncated, and the valine at position 271 of the truncated amino acid sequence was mutated to leucine.

2. A biomaterial, characterized in that, Including any one of (a) to (c): (a) The gene encoding the lipase mutant of claim 1; (b) A recombinant expression vector containing the gene described in (a); (c) Microbial cells expressing the lipase mutant of claim 1, or containing the gene of (a), or containing the recombinant expression vector of (b).

3. Recombinant Escherichia coli, characterized in that, Using Escherichia coli as the starting strain, the lipase mutant of claim 1 with a solubilization tag added to the C-terminus is expressed; the solubilization tag is a maltose-binding protein tag or a histidine tag.

4. The recombinant Escherichia coli according to claim 3, characterized in that, A lipase mutant containing an MBP or histidine lysosome tag and a folding enzyme are co-expressed; the folding enzyme is TrxA folding enzyme, TrxB folding enzyme, or LipB folding enzyme.

5. The recombinant Escherichia coli according to claim 3 or 4, characterized in that, The starting strains were Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), Escherichia coli Origami B (DE3), or Escherichia coli Origami B pGpro7 (DE3).

6. An enzyme preparation containing the lipase mutant of claim 1.

7. A method for preparing immobilized lipase, characterized in that, Add DMONs nanomaterials to the solution of the lipase described in claim 1, and use the DMONs nanomaterials as a carrier to self-assemble and obtain an immobilized lipase.

8. An immobilized lipase, characterized in that, Using DMONs nanomaterials as a carrier, it contains the lipase mutant of claim 1 with lipase catalytic activity.

9. The application of the lipase mutant of claim 1, or the recombinant Escherichia coli of any one of claims 3 to 5, or the enzyme preparation of claim 6, or the immobilized lipase of claim 8 in the fields of biology and medicine; the application is not for the purpose of disease diagnosis / treatment.

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