A lipase, encoding gene and preparation method and application thereof

Abstract

The present invention discloses a lipase, a coding gene, a preparation method and an application thereof, and belongs to the field of protein engineering technology. The lipase provided by the present invention has high activity under medium temperature conditions, and has a wider pH application range. It is most stable in an environment of pH 5.0, and the relative enzyme activity reaches more than 140%. It shows excellent stability in organic solvents, surfactants, and metal ions. P-nitrophenyl palmitate shows high specificity and a maximum catalytic efficiency of 2.9×10 4 L·mol ‑1 ·s ‑1 .

Description

Technical Field

[0001] The present invention relates to the field of protein engineering technology, and in particular to a lipase, a coding gene, and a preparation method and application thereof. Background Art

[0002] Lipases (triacylglycerol acyl hydrolases) are a class of hydrolases that catalyze the hydrolysis of insoluble triacylglycerols into glycerol, glyceryl acyl groups, and free fatty acids. Lipases are a subclass of esterases and have the characteristics of long-chain triacyl-glycerols, extremely low water solubility, and catalysis at the lipid-water interface. Lipases are found in many plants, animals, insects, and microorganisms. Among them, lipases of microbial origin are more valuable than lipases from plants or animals due to their diverse catalytic activities, high yields, simple genetic manipulation, fast growth rates, good properties, functions under extreme conditions, stability in organic solvents, chemical selectivity and enantioselectivity, and the lack of any auxiliary factors. In industry, they have received more attention than plant and animal lipases. Due to their high versatility, lipases are considered to be the most important group of biocatalysts in the field of biotechnology and are widely used in many different industries such as food, detergents, pharmaceuticals, leather, textiles, cosmetics, biodiesel production, and papermaking.

[0003] Aspergillus niger lipase belongs to fungal lipase among microbial lipases and is an acidic lipase. Our research group has screened a strain of Aspergillus niger GZUF36 (Aspergillus niger CCTCC No. M2012538) that produces extracellular lipase with high 1,3-selectivity and resistance to organic solvents. Since the expression level of lipase is too low to be mass-produced, a suitable vector is generally selected for heterologous expression. Therefore, it is particularly important to design and construct a suitable heterologous expression vector. Summary of the invention

[0004] The purpose of the present invention is to provide a lipase, an encoding gene and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] In one aspect, the present invention provides a lipase, the amino acid sequence of the lipase is shown as SEQ ID NO.1.

[0007] The present invention also provides a DNA molecule encoding the lipase, and the nucleotide sequence of the DNA molecule is shown as SEQ ID NO.2.

[0008] The present invention also provides an expression vector, comprising the DNA molecule.

[0009] The present invention also provides a recombinant strain, comprising the expression vector.

[0010] The present invention also provides a method for preparing the lipase, which comprises utilizing the recombinant strain to express the lipase, and separating and purifying the lipase to obtain the lipase.

[0011] The invention also provides a fat hydrolysis method, which utilizes the lipase to catalyze and degrade the fat.

[0012] The invention also provides application of the lipase in preparing products for catalyzing fat hydrolysis.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The present invention uses MD simulation to obtain the B-factor value of the homology model, screens out high B-factor residue sites, uses FoldX to perform virtual saturation mutations on high B-factor residues, obtains ΔΔG, and combines the amino acid sequence alignment of thermophilic lipase to obtain the amino acid sequence of mutant B lipase. Compared with EXANL1, the highest enzyme activity of mutant B is increased by 3.66%, and the enzyme activity in the range of 35-45°C is about 3-13% higher than that of EXANL1, indicating that mutant B has higher activity under medium temperature conditions. In addition, the pH application range of mutant B lipase is wider, and it is most stable in an environment of pH 5.0, with a relative enzyme activity of more than 140%. It shows excellent stability in organic solvents, surfactants, and metal ions, and p-nitrophenyl palmitate shows high specificity and a maximum catalytic efficiency of 2.9×10 4 L·mol -1 ·s -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 Results of mutation design based on B-factor; (a) B-factor of the homology model calculated after MD simulation; (b) site distribution in the mutation scheme; (c) RMSD comparison between the B-factor mutant and the homology model; (d) RMSF comparison between the B-factor mutant and the homology model.

[0017] Figure 2This is the verification of mutant lipase heterologous expression in E. coli; (a). Double restriction enzyme digestion verification of mutant B recombinant plasmid; (b). SDS-PAGE electrophoresis analysis of mutant B; in (a): 1: plasmid digested with BamHI and NotI; 2: undigested plasmid; M: DNA ladder. In (b): M is a marker, and lanes 1-4 are mutant B whole cells, supernatant, inclusion body, and affinity chromatography purified fusion protein GST-B.

[0018] Figure 3 This is the SDS-PAGE analysis of mutant B purification. (a) Mutant B column affinity chromatography purification; (b) Mutant B anion exchange chromatography purification. M: marker; lane 1 in (a) is crude enzyme solution, lane 2 is flow-through, lane 3 is wash 1, lanes 4-6 are elutions 1-3; lanes 1-2 in (b) are gradient elutions, and lane 3 is after concentration.

[0019] Figure 4 This is the electrophoresis of thrombin cleavage and tag removal of mutant B. Among them, lane 1 is mutant B after enzyme cleavage, lane 2 is flow-through, lanes 3-5 are washes, lanes 6-7 are elutions, lane 8 is after flow-through concentration, and M is a marker.

[0020] Figure 5 The optimal reaction temperature (a) and temperature stability (b) of EXANL1 and mutants; Note: The highest enzyme activity of EXANL1 (15.174 U / mg) was taken as 100% relative enzyme activity.

[0021] Figure 6 The optimal reaction pH (a) and pH stability (b) of EXANL1 and mutants.

[0022] Figure 7 Comparison of the tolerance of EXANL1 and mutants to organic solvents; Note: Control in EXANL1 is 100% relative enzyme activity (7.229 U / mg).

[0023] Figure 8 The effect of surfactant on EXANL1 and mutants; Note: Control in EXANL1 is 100% relative enzyme activity (7.229 U / mg).

[0024] Fig. 9 The effects of metal ions on EXANL1 and mutants.

[0025] Fig.10 The selectivity of EXANL1 and its mutants for p-nitrophenol ester substrates of different chain lengths.

[0026] Fig.11The kinetics of EXANL1 and mutant B towards different substrates.

[0027] Fig.12 Molecular docking and interaction prediction of EXANL1 with p-NPC8 (a), C12 (b), and C16 (c).

[0028] Fig.13 Molecular docking and interaction prediction between mutant B and p-NPC8 (a), C12 (b), and C16 (c). DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0035] An embodiment of the present invention provides a lipase, the amino acid sequence of the lipase is shown as SEQ ID NO.1.

[0036] The embodiment of the present invention provides a DNA molecule encoding the lipase, and the nucleotide sequence of the DNA molecule is shown as SEQ ID NO.2.

[0037] An embodiment of the present invention provides an expression vector, comprising the DNA molecule.

[0038] The embodiment of the present invention provides a recombinant strain, including the expression vector.

[0039] The embodiment of the present invention provides a method for preparing the lipase, which comprises using the recombinant strain to express the lipase, and separating and purifying the lipase to obtain the lipase.

[0040] In some specific embodiments, the mutant lipase is purified by affinity chromatography, and the eluate is dialyzed with a dialysis buffer solution at a volume ratio of 1:100 at 4°C for 24 hours to remove glutathione and salt therein; the dialyzed enzyme solution is further purified using an AKTA Purifier protein purification system and a Q 6FF anion column.

[0041] In some specific embodiments, after separation and purification, the step of enzymatic cleavage with thrombin to remove the GST tag is further included.

[0042] The embodiment of the present invention provides a method for hydrolyzing fat, using the lipase to catalyze the degradation of fat.

[0043] The embodiment of the present invention provides the use of the lipase in preparing a product that catalyzes fat hydrolysis.

[0044] Example 1

[0045] 1. Semi-rational design of lipase EXANL1

[0046] Mutant design based on B-factor calculation

[0047] The amino acid sequence of EXANL1 (GenBank ID: MK592411.1) was uploaded to the Swiss-model website, and a homology model was constructed using the 6UNV crystal structure with a homology of 54.58% as a template. The B-factor of the constructed lipase model structure was calculated by MD simulation, and the amino acid sites with large B-factor values ​​in this model structure were screened out. The screened mutation sites were scanned by PositionScan using FoldX software, and the free energy changes after each amino acid mutated to other amino acids were counted, and sites with ΔΔG < -0.5kcal / mol were retained. The amino acid sequence of the thermophilic lipase with a single crystal structure was then compared using the multiple sequence alignment method to observe the amino acid type corresponding to the thermophilic lipase at the high B-factor site in the homology model structure, and different amino acid sites were used as potential mutation sites. The screened pre-mutation sites were further screened to eliminate those that were far from the active center. The remaining amino acids were used as mutation sites.

[0048] The homology modeling model was evaluated using SAVES. The pull-through diagram showed that 86.6% of the amino acid residues were in the most reasonable area, 13.4% of the amino acid residues were in the area where they could be allowed to exist, and no residues were in the unallowed position, indicating that the structure of the homology modeling conformed to the stereochemical conformational rules and was reasonable. The Verify 3D score showed that more than 80% of the residues had a 3D / 1D value greater than 0.2, and the model quality was qualified. The part below 0.2 needed further correction. The score of the homology model structure was 82.96%, indicating that a good prediction model was obtained. ERRAT is used to evaluate the overall properties of non-bonded interactions in protein molecules. Generally speaking, the larger the ERRAT value, the higher the quality of the model. The homology model ERRAT score was 93.130, indicating that the model was a high-quality model. In summary, the homology model constructed using Swiss-model has a reasonable and reliable structure and can be used for further calculations and mutation design.

[0049] After MD simulation of the homology modeling structure, the B-factor of each residue in the homology model was calculated using the rms command in GROMACS. The obtained structure was displayed using PyMOL, and the amino acid residues with the largest B-factor were selected. The results are shown in Table 1. Figure 1In the non-blue region of a, the largest B-factor is Arg 8 located on the N-terminal loop, reaching 290.65, and the smallest is Asp 38, with a B-factor of 43.55. FoldX was used to perform saturation mutations on these 15 amino acid residues, and their unfolding free energy ΔΔG was calculated. A total of 56 mutation results that met the requirements were obtained (see Table 2). The ΔΔG after saturation mutations of Lys 57, Leu 111 and Leu 275 were basically > 0. In NCBI, Blast was used to search the sequence in the PDB database, and the thermophilic lipase with crystal structure (PDB: 6OR3) was selected. Its sequence homology with EXANL1 was 50.18%. Mega 7.0 was used for sequence alignment, and ESPrit3 was used to draw the amino acid residues in the thermophilic lipase corresponding to the amino acid residues with high B-factor values ​​in the EXANL1 sequence (Table 3). Combined with the ΔΔG results, the amino acid sequence of thermophilic lipase was mainly screened, and the following mutation sites were finally screened out: D37P, D38A, E53K, T65S, ​​N66G, F68V, D102N, L111I, K123S, D170N, D218E, E275T. This was used as a library to construct a multi-point mutant named mutant B. The protein sequence of mutant B is shown in SEQ ID NO.1.

[0050] SEQ ID NO.1:

[0051] APTPLDVRSVSTSTLDELQLFAQWSAAAYCSNNIDS PA SNVTCTADACPSVE K ASTKMLLEFDL SG N V GGTAGFLAADNTNKRLVVAFRGSSTVKNWIADL N FILQDNDD I CTGCKVHTGFW S AWEAAADNLTSKIKSAMSTYSGYTLYFTGHSLGLATLGATVLRN N GYSVELYTYGCPRVGNYALAEHITSQGSGANFRVTHLNDIVPRLPPM E FGFSQPSPEYWITSGTGASVTASDIELIEGINSTAGNAGEATVDVLAHLWYFFAIS T CLL.

[0052] Note: The underlined position is the mutation site.

[0053] Table 1 High B-factor sites in homology modeling models

[0054]

[0055] Table 2 Results of FoldX virtual saturation mutation (ΔΔG<-0.5 kcal / mol) of high B-factor amino acid residues

[0056]

[0057]

[0058] Table 3 Alignment results of EXANL1 high B-factor sites with thermophilic lipase sequences

[0059]

[0060] 2. Construction of recombinant strains and mutants

[0061] The coding gene of mutant B was inserted into the pGEX-4T-1 vector through the two restriction sites of BamHI and NotI to obtain a recombinant plasmid. The coding sequence of mutant B is shown in SEQ ID NO.2.

[0062] SEQ ID NO.2:

[0063] GCGCCGACCCCGCTGGATGTTCGTAGCGTTAGCACCAGCACCCTGGATGAACTGCAGCTGTTCGCGCAGTGGAGCGCGGCGGCGTACTGCAGCAACAACATCGATTCTCCGGCGAGCAACGTTACCTGCACCGCGGATGCGTGCCCGAGCGTTGAAAAAGCGAGCACCAAAATGCTGCTGGAATTCGATCTGTCTGGTAACGTGGGCGGCACCGCGGGTTTCCTGGCGGCGGATAACACTAACAAACGTCTGGTTGTTGCGTTCCGTGGTAGCTCTACCGTTAAAAACTGGATCGCGGATCTGAACTTCATCCTGCAGGATAACGATGATATCTGCACCGGTTGCAAAGTTCACACCGGTTTCTGGAGCGCGTGGGAAGCAGCGGCGGATAACCTGACCAGCAAAATCAAATCCGCGATGTCTACCTACAGCGGTTACACCCTGTACTTCACCGGTCACAGCCTGGGTGGTGCGCTGGCGACCCTGGGCGCAACCGTTCTGCGTAACAACGGTTACAGCGTTGAACTGTACACCTACGGTTGCCCGCGCGTTGGTAACTACGCGCTGGCTGAACACATCACCAGCCAGGGTAGCGGCGCTAACTTCCGTGTTACCCACCTGAACGATGGTGTTCCGCGTCTGCCGCCGATGGAATTCGGTTTCAGCCAGCCGTCCCCAGAATATTGGATTACTTCTGGCACCGGTGCTAGCGTTACCGCTTCTGATATTGAACTTATTGAAGGTATTAACAGCACCGCAGGTAACGCGGGTGAAGCAACCGTTGATGTTCTGGCGCACCTGTGGTATTTCTTCGCGATTAGCACCTGCCTGCTGtga。

[0064] Preparation of mutant lipase

[0065] From the mutant glycerol tube prepared by Sangon Biotech (Shanghai) Co., Ltd., 50 μL of cloned bacterial solution was added to 50 mL of LB liquid medium (containing a final concentration of 50 μg / mL ampicillin sodium Amp), and cultured at 37°C, 200 r / min for 12 hours as seed solution. Subsequently, 2 mL (2%) of seed solution was taken in a clean bench and added to 100 mL of LB liquid medium containing a final concentration of 100 μg / mL Amp, and cultured at 37°C, 200 r / min for 3 hours. The absorbance value was measured at 600 nm using an ultraviolet spectrophotometer. When the absorbance value at 600 nm was greater than 0.6, 100 μL of IPTG (100 mM) solution was added to the fermentation broth, and the expression of the target protein was induced at low temperature at 16°C, 200 r / min. The low temperature induction expression time was 20 hours. After induction, centrifuge at 6000g for 10 minutes, collect the precipitate, wash the bacteria three times with pre-cooled saline to remove the culture medium, and weigh the wet weight of the bacteria after centrifugation. Add the bacterial disruption solution at a solid-liquid ratio of 1:20, and disrupt the bacteria in an ultrasonic cell disruptor under ice bath conditions to release the target protein. The disruption conditions are: 250W, disruption time 5s, interval time 8s, and disruption time 30min. After the disruption, centrifuge the disruption solution at 10000g for 20 minutes, collect the supernatant, which is the crude enzyme solution of the mutant, and store the crude enzyme solution in a 4°C refrigerator for later use.

[0066] The protein concentration was determined by the Bradford method, and the lipase activity was determined by the p-nitrophenol colorimetric method (p-NPP).

[0067] The mutants were constructed and the recombinant plasmids were double-digested for verification. The results are shown in Figure 2 (a) The recombinant plasmid is transformed into competent E. coli cells, positive recombinants are selected for sequencing, and then fermented in LB liquid medium. Inducers are added at low temperature to induce the expression of the target protein. The supernatant is collected by centrifugation and SDS-PAGE is used to verify whether the target protein is successfully expressed. Figure 2 (b) is the affinity chromatography purification electrophoresis of mutant B. In lane 1, there is an obvious thick band in the middle of the molecular weight of 48-63kDa, and its size is about 58kDa. Lanes 2 and 3 are the eluted target proteins, and their molecular weights are consistent with the size in lane 1. The enzyme activity was determined using the p-NPP method and the results showed that the enzyme was active, indicating that mutant B was successfully expressed.

[0068] 4. Purification of mutant lipase

[0069] The mutant lipase was purified by affinity chromatography, and the eluate was dialyzed with a dialysis buffer solution at a volume ratio of 1:100 at 4°C for 24 hours to remove glutathione and salt. The dialyzed enzyme solution was further purified using an AKTA Purifier protein purification system and a Q 6FF anion column.

[0070] 5. SDS-PAGE electrophoresis

[0071] The mutant proteins at each purification stage were analyzed by electrophoresis using 12% separation gel. The equipment used was a DYY-6C Beijing Liuyi electrophoresis instrument, and the electrophoresis conditions were: 80V, 30min to run the concentrated gel, and then adjust the voltage value to 120V to run the separation gel. After the end, the protein was stained with Coomassie R250 staining solution, and the staining was carried out in a shaker at 30°C, 100r / min for 2h in the dark, and then decolorized with a decolorizing solution.

[0072] The mutant was ultrasonically disrupted to release the target protein from the Escherichia coli cells, and the supernatant was obtained by centrifugation. The supernatant was filtered using a 0.45 μm water filter to remove insoluble precipitates. The supernatant was first purified using a glutathione affinity chromatography column (GST column), then dialyzed to remove glutathione and salts, and then further purified using an anion exchange chromatography column.

[0073] 1). GST column affinity chromatography purification

[0074] The affinity chromatography results of mutant B are shown in Figure 3 As shown in (a), lanes 3-5 are elutions 1, 2, and 3, respectively. It can be seen that there is a band in the three eluates between the molecular weight of 48-63kDa, and there are some impurities. The enzyme activity assay shows that it has hydrolytic activity against p-NPP, indicating that the band is the fusion protein GST-B. The eluate is collected and dialyzed for further purification. After the mutant is purified by affinity chromatography, there are still a lot of impurities in the eluate, which has not reached electrophoretic purity, so further purification is required to remove the impurities that cannot be separated by affinity chromatography.

[0075] 2).Q column-ion exchange chromatography purification

[0076] The isoelectric point (pI) of EXANL1 and mutants was predicted using Protparam in the Expasy online server. The results showed that EXANL1 pI: 4.41 and mutant B pI: 4.73. Therefore, a strong anion column-Q column was selected to purify the mutants by ion exchange chromatography. After anion exchange chromatography purification, the purity of the lipase protein expressed by mutant B was further improved ( Figure 3 (b)), but a small amount of impurity protein was still eluted. The target protein of this purity was used for subsequent enzymatic property studies.

[0077] 6. Removal of GST label

[0078] The purified fusion protein was cleaved with thrombin to remove the GST tag.

[0079] The protein concentration of mutant B after purification by anion chromatography was determined, and an appropriate amount of thrombin was added for enzyme digestion. After enzyme digestion, the GST tag and the target protein were separated by GST affinity chromatography column, and the flow-through, washing liquid and eluate were collected for SDS-PAGE verification. The electrophoresis results are as follows: Figure 4 Lane 1 shows that the fusion protein of mutant B is divided into two parts after enzyme cleavage: 32kDa target protein and 26kDa tag protein. The flow-through after passing through the column (lane 2) shows 32kDa target protein and a small amount of tag protein. No protein is washed out in the washing solution (lanes 3-5), and GST tag protein is clearly visible in the eluate. The above results show that thrombin can fully cut the GST tag from the fusion protein, but the GST affinity chromatography column cannot fully adsorb the cut GST tag on the column and separate it from the target protein. Therefore, further treatment is required to obtain a target protein with high uniformity.

[0080] In summary, this example uses MD simulation to obtain the B-factor value of the homology model, screens out high B-factor residue sites, uses FoldX to perform virtual saturation mutations on high B-factor residues, obtains ΔΔG, and combines the amino acid sequence alignment of thermophilic lipase to design the mutation scheme of mutant B: D37P, D38A, E53K, T65S, ​​N66G, F68V, D102N, L111I, K123S, D170N, D218E, E275T.

[0081] For the amino acid sequence of the mutation scheme, primers were designed and mutant plasmids were constructed, which were transformed and expressed in E. coli. Double enzyme digestion and SDS-PAGE verification were performed to prove that the mutant was successfully constructed and the mutant successfully expressed the target protein. The mutant was purified by affinity chromatography and ion exchange chromatography to obtain a relatively pure mutant lipase.

[0082] Example 2

[0083] 1. Optimal reaction temperature and temperature stability of mutant lipase

[0084] 1.1 Taking p-nitrophenyl palmitate (p-NPC16) as substrate and the reaction buffer solution as disodium hydrogen phosphate-citric acid (50mM, pH 4.0), the enzyme solution was added to the buffer solution containing the substrate after incubation at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃, and the hydrolysis activity of lipase on p-NPC16 at different temperatures was determined. The effect of different temperatures on the enzyme activity of mutant lipase was expressed as relative enzyme activity, and the group with the highest absolute enzyme activity was defined as 100% relative enzyme activity, and the corresponding temperature was the optimal reaction temperature.

[0085] Depend on Figure 5 As shown in (a), the lipase EXANL1 reaches the maximum enzyme activity at a temperature of 45°C, while the optimal reaction temperature of mutant B is at 40°C. In the range of 35-45°C, the enzyme activities of EXANL1 and mutant B are both above 68%, indicating that EXANL1 and mutant B are both mesophilic lipases. Compared with EXANL1, the maximum enzyme activity of mutant B increased by 3.66%, and the enzyme activity in the range of 35-45°C was about 3-13% higher than that of EXANL1, indicating that mutant B has higher activity under mesophilic conditions. When the temperature is greater than 45°C, the enzyme activity of mutant B begins to drop sharply. When the temperature reaches 60°C, mutant B is inactivated.

[0086] 1.2 Determination of temperature stability: The mutant lipase was placed in a water bath set at 45°C, 50°C, 55°C, and 60°C for incubation for 60 minutes. After the incubation, the mutant was taken out and allowed to return to room temperature. The residual enzyme activity was measured at the optimal reaction temperature. The relative enzyme activity was used to characterize the thermal stability of the mutant lipase. The untreated enzyme activity was defined as 100% relative enzyme activity.

[0087] Figure 5 (b) reflects the changes in enzyme activity of EXANL1 and mutant B after incubation at 40℃, 45℃, 50℃, 55℃, and 60℃ for 1h. After incubation at 40℃ for 1h, mutant B retained 85.21% of the relative enzyme activity. After incubation at 45℃ for 1h, the relative enzyme activity of mutant B was measured at 66.66%. After incubation in a water bath at 50℃ for 1h, the relative enzyme activity of mutant B was measured at 1.72%. After incubation at 55℃ for 1h, the recovery rate of mutant B was 0.21%. After incubation at 60℃ for 1h, mutant B lost its activity.

[0088] 2. Optimal reaction pH and pH stability of mutant lipase

[0089] 2.1 Determination of the optimal reaction pH: At the optimal reaction temperature, the appropriately diluted lipase was added to 50 mM buffer solutions of different pH values ​​(disodium hydrogen phosphate-citric acid: pH 2.0-8.0, sodium carbonate-sodium bicarbonate: pH 9.0-10.0) to determine the enzyme activity. The effect of different pH values ​​on the activity of the mutant lipase was expressed as relative enzyme activity. The highest absolute enzyme activity was defined as 100% relative enzyme activity, and the corresponding pH was the optimal reaction pH.

[0090] Figure 6 (a) is the optimum pH of EXANL1 and mutant lipase. It can be seen that both EXANL1 and mutant B are active mainly under acidic conditions, with the optimum pH of 4.0. The relative enzyme activity of mutant B at pH 4.0 is higher than that of EXANL1. When the pH is greater than 4, the relative enzyme activity drops sharply, indicating that they are suitable for acidic environments and are acidic lipases. At pH 6.0 and above, EXANL1 retains weak activity or inactivates, while mutant B maintains activity (3%-103%) at pH 2-9, indicating that the mutation broadens the pH application range of mutant B. The reason for this change may be the change of multiple acidic amino acids (D37P, D102N, D170N, E275T) involved in mutant B to neutral amino acids, causing changes in the electrostatic interaction of the protein and ultimately changing the microenvironment of the catalytic pocket.

[0091] 2.2 Determination of pH stability: To investigate the stability of the mutant lipase at different pH levels, the enzyme solution was mixed with pH 2.0-8.0 (50 mM disodium hydrogen phosphate-citric acid buffer solution) and pH 9.0-10.0 (50 mM carbonate buffer solution) at a volume ratio of 1:3 and placed at 4°C for 48 h. After the treatment, the residual enzyme activity after different pH treatments was measured under the optimal conditions, and the untreated enzyme activity was taken as 100% relative enzyme activity.

[0092] The residual enzyme activity of EXANL1 and mutant lipase was measured after standing in different pH buffer solutions for 48 h. Figure 6 As shown in (b), EXANL1 had the highest relative enzyme activity at pH 4.0, was relatively stable in the pH 2.0-8.0 environment, retaining more than 70% of the enzyme activity, retained only 24.64% of the enzyme activity at pH 9.0, and retained only 4.33% of the enzyme activity at pH 10.0, indicating that EXANL1 was acid- and alkali-resistant; mutant B was most stable in the pH 5.0 environment, with a relative enzyme activity of more than 140%, was relatively stable in the pH 3.0-7.0 environment, retaining 66% of the enzyme activity, and its stability decreased significantly in the pH 2.0 environment, and was inactivated in the pH 10.0 environment.

[0093] 3. Tolerance of mutant lipase to organic solvents

[0094] The lipase solution was mixed with different types of organic solvents in a volume ratio of 1:1, and the lipase solution without organic solvent was used as a comparison. After incubation at 4°C for 2 hours, the residual enzyme activity of the lipase was determined. If there is an experimental group with stratification, only the aqueous phase is taken for enzyme activity determination. The types of organic solvents used in the experiment include acetone, acetonitrile, formic acid, isooctane, n-octanoic acid, n-heptane, dichloromethane, dimethyl sulfoxide, methanol, xylene, and vinyl acetate. The enzyme activity of the untreated lipase was taken as 100% relative enzyme activity, and the enzyme activity of the remaining lipases treated in different organic solvents was expressed as relative enzyme activity to explore the tolerance of the mutant lipase to organic solvents.

[0095] Eleven organic solvents with different oil-water partition coefficients were selected to investigate their effects on the enzymatic activity of lipase EXANL1 and mutant lipases. Figure 7 ). The results showed that both EXANL1 and mutant B lipases had high stability in hydrophobic organic solvents with large Log P values: isooctane (Log P 3.8), n-heptane (Log P 4.66), and xylene (Log P 3.1). After incubation in the above three organic solvents at a concentration of 50% for 2 hours, the enzyme activity was significantly increased and all were above 120%. EXANL1 and mutant B both had a certain tolerance to vinyl acetate (Log P 0.73), but mutant B had poor tolerance and the relative enzyme activity after incubation was only about 12% of that of EXANL1. Mutant B had a certain tolerance in four polar solvents: acetone (Log P-0.24), acetonitrile (Log P-0.34), dimethyl sulfoxide (LogP-1.35), and methanol (Log P-0.77), while EXANL1 lost its enzyme activity after treatment. The reason may be that polar solvents form interactions with enzyme surface residues that are not conducive to stability through hydrogen bonds and hydrophobic interactions.

[0096] 4. Tolerance of mutant lipase to surfactants

[0097] A series of surfactant solutions with a concentration of 0.1% were prepared, and the lipase solution and the surfactant were mixed at a volume ratio of 1:1, so that the final concentration of the surfactant was 0.05%. The surfactants included: hexadecyltrimethylammonium bromide (CTAB), sodium N-lauroyl creatine, sodium dodecyl sulfate (SDS), Tween 20, Tween 40, Tween 60, Tween 80, and Triton X-100. After the mixed solution was placed at 4°C for 2 hours, the residual enzyme activity of the mutant lipase after treatment with different surfactants was determined, and the enzyme activity of the lipase without adding surfactant but incubated was taken as 100% relative enzyme activity.

[0098] Eight surfactants, including sodium N-lauroyl sarcosinate, SDS, CTAB, Triton X-100, Tween 20, Tween 40, Tween 60, and Tween 80, were selected and their effects on the enzymatic activity of lipase were investigated at a final concentration of 0.05%. Figure 8 The changes in enzyme activity of EXANL1 and mutant B after incubation in a surfactant with a final concentration of 0.05% for 2 hours are shown. EXANL1 is tolerant to all selected surfactants, and is most stable to Triton X-100. After treatment, the enzyme activity is significantly increased to 123.7%. It has poor tolerance to SDS and CTAB. Except for Tween 20, it has good tolerance to the other Tween series surfactants, and still retains more than 75% of the relative enzyme activity after treatment. Mutant B is tolerant to all surfactants except CTAB, but its tolerance to Tween series surfactants is significantly reduced compared to EXANL1, but its tolerance to Triton is better than EXANL1 (relative enzyme activity increased by about 35%).

[0099] 5. Tolerance of mutant lipase to metal ions

[0100] The purified mutant lipase was mixed with 10mM KCl, CaCl2, NaCl, MgCl2, FeCl3, FeCl2, CuCl2, MnCl2, and ZnCl2 solutions at a volume ratio of 1:1, so that the final concentration of the metal ion solution was 5mM. The mixed solution was placed at 4°C for 2 hours, and the residual enzyme activity of the mutant lipase treated with different metal ions was measured. The enzyme activity of the untreated mutant lipase was 100%.

[0101] Fig. 9 It is shown that at a final concentration of 5 mM K + EXANL1 was activated (103%), Fe 2+ EXANL1 was completely inactivated at a final concentration of 5 mM Ca 2+ 、Na + Mg 2+ , Mn 2+ , Cu 2+ After 2 h of treatment, more than 50% of the activity was retained. + 、Na + Mg 2+ , Mn 2+ 、Zn 2+ The mutant B was activated, and the enzyme activities were increased to 126.79%, 109.06%, 125.56%, 221.64%, and 119.53%, respectively. 2+The activation effect of Fe was the most significant, increasing by more than 121% compared with EXANL1. 3+ , Fe 2+ , Cu 2+ It has a significant inhibitory effect on both EXANL1 and mutant B.

[0102] 6. Determination of substrate specificity of mutant lipase

[0103] p-Nitrophenyl acetate (p-NPC2), p-nitrophenyl butyrate (p-NPC4), p-nitrophenyl hexanoate (p-NPC6), p-nitrophenyl octanoate (p-NPC8), p-nitrophenyl laurate (p-NPC12), p-nitrophenyl myristate (p-NPC14) and p-nitrophenyl palmitate (p-NPC16) were used as substrates to detect the specificity of lipase to the substrate chain length. The hydrolysis reaction was carried out under the optimal reaction conditions of the mutant lipase, and the enzyme activity was determined at 405nm on a UV spectrophotometer. The enzyme activity that hydrolyzes the p-NPC16 substrate was set as 100% relative enzyme activity.

[0104] The substrate specificity of EXANL1 and mutants Fig.10 As shown, it can be seen that there are obvious differences in the relative enzyme activity changes of EXANL1 and mutant lipases to p-nitrophenyl ester substrates of different chain lengths. The substrate specificity of EXANL1 and mutant B is p-NPC8>p-NPC16>p-NPC12>p-NPC6>p-NPC4>p-NPC2. Both EXANL1 and mutant B lipases have the highest enzyme activity to p-NPC8, and the specificity is ranked as mutant B>EXANL1, and the relative enzyme activities are 134.48% and 100%, respectively. EXANL1 and mutant B have little difference in specificity to medium- and long-chain p-nitrophenol ester substrates, but have a large difference in short-chain p-nitrophenol ester substrates (p-NPC2, p-NPC4). For p-NPC2, the specificity is mutant B>EXANL1, and for p-NPC4, the specificity is mutant B>EXANL1.

[0105] 7. Determination of kinetic parameters of mutant lipase

[0106] The kinetic parameters of mutant lipases were determined using p-nitrophenol ester substrates (p-NPC8, p-NPC12 and p-NPC16). The concentrations of the substrates were 0.1-1.0 mmol·L -1 The hydrolase activity of lipase against different substrate concentrations was determined under the optimal reaction conditions using p-nitrophenol ester p-NPC8, p-nitrophenol ester p-NPC12 and p-NPC16 substrate solutions. max , K m Value, kcat The values ​​were calculated using the Lineweaver-Burkplot method and the Michaelis-Menten equation.

[0107] Using the Michaelis equation and double reciprocal plotting, see Fig.11 , the Michaelis constant K is obtained by calculation m , the maximum reaction rate V max , catalytic constant k cat , specificity constant k cat / K m The results are shown in Table 4. The kinetic parameters of lipase K m The value is negatively correlated with the affinity between the enzyme molecule and the substrate. EXANL1 and mutant B have the highest affinity for p-NPC12 substrate and the affinity for p-NPC8 is basically the same, indicating that the mutation site does not affect the active pocket suitable for medium-chain substrates. EXANL1 and mutant B have a K value of 0.04 for p-NPC16. m The values ​​were 9.20 and 0.83 mmol·L -1 , indicating that the affinity of mutant B to p-NPC16 is basically the same and significantly better than that of EXANL1. cat The maximum reaction rate V of EXANL1 and mutant B to C16 is max 333.33 and 48.31 mmol·min respectively -1 mg -1 , for V of p-NPC8 max Both at 70mmol·min -1 mg -1 Above. cat / K m The value is often used to measure the catalytic efficiency of enzymes. The results in the table show that the catalytic efficiency of EXANL1 for C12 is as high as 3.8×10 4 L·mol -1 ·s -1 Mutant B has a maximum catalytic efficiency of 2.9×10 4 L·mol -1 ·s -1 .

[0108] Table 4 Kinetic parameters of EXANL1 and mutant B for different substrates

[0109]

[0110] 8. Molecular Docking

[0111] The amino acid sequence of the mutant was subjected to homology modeling using the crystal structure model with the lid opened using Swiss-model. The homology model was energy minimized using GROAMCS2022.4 under the Amber99sb-ILDN force field. The ligand molecule was structurally optimized using Gaussian 09. Autodock vina (Trott et al., 2009) was used for molecular docking of protein and substrate. The model with the lowest docking energy was taken as the optimal configuration for interaction analysis, and the interaction was analyzed using DiscoveryStudio 4.5.

[0112] like Fig.12 As shown in Figure 2, the molecular docking and interaction prediction results of EXANL1 and p-NPC8 (a), C12 (b), and C16 (c) show that the interactions between EXANL1 and p-NPC8, C12, and C16 are mainly van der Waals forces, hydrogen bonds, and hydrophobic interactions. EXANL1 forms three hydrogen bonds with all three substrates, although the residues that form hydrogen bonds with p-NPC8 and C12 are However, the positions of interaction with the substrate are different: p-NPC8 is the oxygen atom on the nitro group, while p-NPC12 is the oxygen atom on the carbonyl group; the residue that forms a hydrogen bond with p-NPC16 is The binding free energies of EXANL1 to three p-nitrophenol ester substrates with different chain lengths are p-NPC8 (-6.6 kcal / mol), p-NPC12 (-6.3 kcal / mol), and p-NPC16 (-6.4 kcal / mol), respectively. Although the binding free energy of EXANL1 to p-NPC16 is higher than that to p-NPC8, p-NPC16 forms a hydrogen bond with Ser 154 in the catalytic triad.

[0113] like Fig.13 As shown in the figure, the molecular docking and interaction prediction results of mutant B with p-NPC8, C12, and C16 show that the interaction types between mutant B and the three substrates are consistent with EXANL1, including hydrogen bonds, van der Waals forces, and Pi-alkyl interactions in hydrophobic interactions. The binding free energy of docking shows that mutant B has the lowest binding free energy for p-NPC8, which is -6.9 kcal / mol, indicating that p-NPC8 is the preferred substrate of mutant B. Mutant B forms two hydrogen bonds with p-NPC8, C12, and C16, and one of the hydrogen bonds is formed with Ser154 in the catalytic triad. The hydrogen bonds formed by mutant B with p-NPC8 and C16 are: Ser 154 interacts with the oxygen atom on the nitro group in the substrate, His 118 forms a hydrogen bond with the carbonyl oxygen atom in the substrate, and in p-NPC12, Leu155 replaces His 188 and forms a hydrogen bond with another nitro oxygen atom.

[0114] EXANL1 and mutant B lipases have the highest affinity for the p-NPC8 substrate, but for the p-NPC12 substrate, the molecular docking results show that mutant B lipase has the highest affinity for it, but the results obtained by the kinetic parameter model show that EXANL1 lipase has the highest affinity for p-NPC12.

[0115] 9. Data Analysis and Statistics

[0116] In this embodiment, three parallel groups were set for all experimental results. Data displayed are mean ± standard deviation (SD). SPSS26 was used for difference significance analysis, and Origin 20 was used for graph drawing. Different letters above each graph and each curve indicate that the effects on the enzyme activity of lipase at different levels are significantly different (p < 0.05).

[0117] In summary, this example verifies that the mutant B lipase is a mesophilic lipase. Compared with EXANL1, the maximum enzyme activity of mutant B is increased by 3.66%, and the enzyme activity in the range of 35-45°C is about 3-13% higher than that of EXANL1, indicating that mutant B has higher activity under mesophilic conditions.

[0118] Mutant B lipase is an acidic lipase and maintains activity (3%-103%) at pH 2-9, indicating that the mutation broadens the pH application range of mutant B. It is most stable in an environment of pH 5.0, with a relative enzyme activity of more than 140%. It is relatively stable in an environment of pH 3.0-7.0, retaining 66% of the enzyme activity.

[0119] Mutant B lipase has high stability in hydrophobic organic solvents with large Log P: isooctane (Log P 3.8), n-heptane (LogP 4.66), and xylene (Log P 3.1). After incubation in the above three organic solvents at a concentration of 50% for 2 hours, the enzyme activity was significantly improved and was all above 120%; Mutant B has a certain tolerance in four polar solvents: acetone (Log P-0.24), acetonitrile (Log P-0.34), dimethyl sulfoxide (Log P-1.35), and methanol (Log P-0.77), while the enzyme activity was lost after treatment with EXANL1.

[0120] Mutant B was tolerant to all surfactants except CTAB, and its tolerance to Triton was better than that of EXANL1 (relative enzyme activity increased by about 35%).

[0121] Compared with EXANL1K + 、Na + Mg 2+ , Mn 2+ 、Zn2+ The mutant B was activated, and the enzyme activities were increased to 126.79%, 109.06%, 125.56%, 221.64%, and 119.53%, respectively. 2+ The activation effect was most significant, increasing by more than 121% compared to EXANL1.

[0122] The substrate specificity of mutant B was p-NPC8>p-NPC16>p-NPC12>p-NPC6>p-NPC4>p-NPC2. The relative enzyme activity of mutant B to p-NPC8 was 134.48%, and the specificity to p-NPC8, p-NPC2, and p-NPC4 was higher than that to EXANL1. The maximum catalytic efficiency of mutant B to p-NPC8 was 2.9×10 4 L·mol -1 ·s -1 The docking results of the p-NPC12 substrate molecule showed that mutant B lipase had the highest affinity to it.

[0123] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A lipase, characterized in that The amino acid sequence of the lipase is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the lipase according to claim 1, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.

2.

3. An expression vector, characterized in that: Comprising the DNA molecule according to claim 2.

4. A recombinant strain, characterized in that: Comprising the expression vector according to claim 3.

5. The method for preparing the lipase according to claim 1, characterized in that: The lipase is expressed by the recombinant strain according to claim 4, and the lipase is separated and purified to obtain the lipase.

6. The preparation method according to claim 5, characterized in that: Affinity chromatography was used for purification, and the eluate was dialyzed with dialysis buffer at a volume ratio of 1:100 at 4°C for 24 hours; the dialyzed enzyme solution was further purified using an AKTA Purifier protein purification system and a Q 6FF anion column.

7. The preparation method according to claim 6, characterized in that: After separation and purification, the method further includes an enzymatic cleavage step using thrombin.

8. A method for hydrolyzing fat, characterized in that: The lipase according to claim 1 is used to catalyze the degradation of fat.

9. Use of the lipase according to claim 1 in preparing products catalyzing fat hydrolysis.

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