A thermophilic bacillus stearothermophilus lipase mutant and use thereof

Abstract

The application relates to a heat-resistant starch-eating bacillus cereus lipase mutant and application thereof, the amino acid sequence of the lipase mutant CTL-B1 is shown as SEQ ID NO. 3, the nucleotide sequence of the corresponding coding gene is shown as SEQ ID NO. 4, the heat stability of the lipase mutant is obviously better than that of a wild type, and the lipase mutant still has good hydrolysis activity to short chains, medium chains and long chains, is excellent in esterification linoleic acid and esterification linolenic acid reactions, increases the application range of the lipase with strong hydrolysis capacity, and has wide prospects.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to the construction, expression, and thermostability characterization of a thermophilic Bacillus amyloliquefaciens lipase (CTL). Background Technology

[0002] Triacylglycerol lipase (EC3.1.1.3), also known as triacylglycerol hydrolase, is a class of enzymes that catalyze the hydrolysis of glycerol esters into glycerol and free fatty acids. It can catalyze various reactions such as alcoholysis, acidolysis, ammonolysis, glycerololysis, esterification, and transesterification, making it a highly efficient biocatalyst. It possesses stereospecificity, regiospecificity, substrate selectivity, and site selectivity, as well as being characterized by mild catalytic processes, low energy consumption, few byproducts, and environmental friendliness. Therefore, it has wide applications in various fields such as food, pharmaceuticals, environment, papermaking, textiles, detergents, leather processing, biodiesel, biosensors, and fine chemicals.

[0003] For example, lipases can esterify linoleic acid and linolenic acid into monoglycerides, diglycerides, and triglycerides. Linoleic acid is an essential fatty acid in human and animal nutrition and is used in medicine to treat hyperlipidemia and arteriosclerosis. It also has many health benefits, such as regulating blood lipids, preventing thrombosis, and acting as a prostaglandin precursor. While linoleic acid has high bioavailability in its free fatty acid form, it is unstable and easily oxidized; whereas linoleic acid in its glyceride form has high bioavailability and good stability. The ability of lipases to synthesize linolenic acid and linoleic acid glycerides requires further investigation.

[0004] Generally, under high-temperature conditions, hydrolysis reactions involving lipases can reduce substrate viscosity, increase substrate solubility, and improve diffusion coefficients and reaction rates, resulting in more efficient reactions in industry. However, enzymes are prone to inactivation under extreme conditions. In recent years, studies have discovered new active lipases from thermophilic microorganisms. Although the wild-type lipases mentioned have improved heat resistance, their stability, activity, and yield remain inefficient. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a CTL mutant of lipase and its application. This CTL mutant of lipase has significantly better thermal stability than the wild type, while still maintaining good hydrolytic activity for short, medium and long chains.

[0006] A first aspect of the present invention is to provide a mutant CTL-B1 of a lipase CTL, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] Firstly, the lipase CTL was obtained through screening, and its amino acid sequence is shown in SEQ ID NO.1. The CTL enzyme gene sequence is shown in SEQ ID NO.2. The lipase CTL gene was obtained through gene synthesis and expressed in Escherichia coli Rosetta-gami B(DE3) as the host. The CTL pure enzyme was obtained by nickel column purification. The mutant CTL-B1 was obtained by replacing the amino acids in the loop region of the obtained lipase CTL (R269E / G270S / V271I / V272L).

[0008] In some embodiments, the digestive enzyme is DpnI enzyme.

[0009] A second aspect of the present invention is to provide the encoding gene of the above-mentioned mutant CTL-B1.

[0010] In some embodiments, the nucleotide sequence of the CTL-B1 is shown in SEQ ID NO.4.

[0011] In some embodiments, the nucleotide sequence of the CTL-B1 encodes an amino acid sequence as shown in SEQ ID NO. 3.

[0012] A third aspect of the present invention is to provide an expression vector into which the above-mentioned gene encoding lipase CTL or its mutant CTL-B1 gene is inserted, the form of which includes, but is not limited to, plasmids and other non-plasmid vectors, such as plasmids, expression cassettes, recombinant vectors or recombinant plasmids.

[0013] A fourth aspect of the present invention is to provide a recombinant genetically engineered bacterium containing the above-mentioned genes.

[0014] The fifth aspect of the present invention is to provide a method for preparing the above-mentioned recombinant genetically engineered bacteria, wherein the above-mentioned lipase CTL gene or its mutant CTL-B1 gene is cloned into the expression vector plasmid pET22b-CTL, and transformed into competent Escherichia coli cells to obtain recombinant genetically engineered bacteria.

[0015] In some embodiments, the expression vector is the plasmid pET22b-CTL.

[0016] In some embodiments, the Escherichia coli is Rosetta-gami B(DE3).

[0017] A sixth aspect of the present invention is to provide a method for preparing a mutant CTL-B1 of lipase CTL, wherein the above expression vector is mixed with competent Escherichia coli cells, transformed, and recombinant genetically engineered bacteria are obtained, and the recombinant genetically engineered bacteria are expressed and purified to obtain the thermophilic Bacillus lipase mutant.

[0018] A seventh aspect of the present invention is to provide the use of lipase CTL or its mutant CTL-B1.

[0019] The application of the lipase CTL or its mutant CTL-B1 in esterified linoleic acid and esterified linolenic acid.

[0020] The eighth aspect of the present invention is a method for providing esterified linoleic acid and esterified linolenic acid, wherein the enzyme used in the synthesis is the above-mentioned lipase CTL or its mutant CTL-B1.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention uses the amino acid sequence of lipase CTL obtained from sequence analysis in a gene library as a template, and rationally designs a series of mutation sites based on this. Based on research experience with enzyme mutants, the mutant is constructed, designed, and recombinantly expressed. Finally, a lipase mutant with moderate heat resistance, longer half-life, better enzyme activity, and better thermal stability is obtained through screening and modification.

[0023] This invention involves a series of mutations in a lipase CTL with a half-life of only 21.59 min at its optimum temperature of 50 °C. Mutant B1 replaces four amino acids in the loop region: R269E / G270S / V271I / V272L; mutant B2 replaces V271E / V272T; mutant C1 deletes four amino acids in the loop region: Q299 / S300 / P301 / A302; mutant C2 deletes eight amino acids in the loop region: T297 / Y298 / Q299 / S300 / P301 / A302 / P303 / V304. Recombinant expression of these mutants, through preliminary screening, yielded mutant B1 with improved thermostability. Its half-life was measured at 121.60 min, 5.63 times longer than the wild type; its Tm value was 1.5 °C higher than the wild type. This invention provides a mutant with good hydrolytic activity for short, medium and long chains, and significantly better thermal stability. It not only performs well in the esterification of linoleic acid and esterification of linolenic acid, but also expands the application range of lipases with strong hydrolytic ability. Attached Figure Description

[0024] Figure 1 This is an SDS-PAGE gel image of CTL.

[0025] Figure 2 This is an SDS-PAGE gel image of the CTL mutant.

[0026] Figure 3 This is the optimal hydrolysis temperature for CTL.

[0027] Figure 4This is the temperature tolerance curve for CTL.

[0028] Figure 5 The optimal pH for the hydrolysis reaction of CTL is given.

[0029] Figure 6 The relative hydrolytic activity of CTLs for different substrates.

[0030] Figure 7 This is a diagram of the product composition of the esterification reaction of lipase CTL.

[0031] Figure 8 The thermal stability of CTL wild-type WT and its mutant B1 at 50℃ was calculated by fitting the inactivation rate constant at 50℃.

[0032] Figure 9 The optimal temperature for CTL wild-type WT and its mutant B1.

[0033] Figure 10 The CD spectra of wild-type CTL WT and its mutant B1 at 28℃ and the results of CD spectrum analysis using CDNN software.

[0034] Figure 11 Full-wavelength scan of CTL wild-type WT and its mutant B1 at 30-96℃.

[0035] Figure 12 The thermal melting curves of CTL wild-type WT and its mutant B1 at 28-96℃ are shown. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0037] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0038] Materials and reagents:

[0039] The plasmid pET22b-CTL was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0040] Escherichia coli TOP10 and Rosetta-gami B(DE3) competent cells, Weidi Biotechnology Co., Ltd.;

[0041] The SanPrep column-based plasmid DNA mini-extraction kit, the SanPrep column-based PCR product purification kit, and the DNA polymerase (2×PrimerStar Max) are all from Sangon Biotech (Shanghai) Co., Ltd.

[0042] All chemicals were purchased from Sigma-Aldrich or Aladdin.

[0043] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0044] To facilitate understanding of this technology, some terms and phrases are defined below.

[0045] During their research, the researchers of this invention accidentally discovered a moderately thermostable lipase CTL derived from *Caldibacillus thermoamylovorans*. They successfully expressed and purified it, and investigated its basic enzymatic properties, finding that it exhibits hydrolytic activity against short-chain, medium-chain, and long-chain triglycerides. While demonstrating good moderate thermostable performance, its poor tolerance to high temperatures limits its application. The researchers further investigated and designed a mutant that can enhance its enzyme activity under certain temperature conditions and prolong its half-life at the optimal temperature.

[0046] Enzyme activity definition: The hydrolytic activity of lipase is determined using the olive oil emulsion titration method. Under certain reaction conditions, the amount of enzyme required to release 1 μmol of free fatty acid per minute through hydrolysis is defined as 1 unit of enzyme activity (U).

[0047] The amino acid sequence (WP_034771740.1) of the wild-type lipase CTL is shown below: SEQ ID NO.1

[0048] ETSGNDYPIVLVHGLGGWGKGEFLGYRYWGGLKDIEFYLNQTGHRTYVATVGPVSSNWDRAVELYYYIKGGTVDYGAAHAKEHGHARFGRTYPGIYGQWDETNKIHLIGHSMGGQTSRMLVELLKSGSQKEQEYYSQHPEEGISPLFTGGKNWVHSVTSLATPHNGSTFADQEQIVSFIKDFIIHLASAAGQKQESLIYDFKLDQWGLKRQPGESFHAYMNRVMTSPIWQSNDISAYDLTTFGAQELNQWMKTYPDVYYLSYTGNASYRGVVTGNYYPIGTMHPLFTLISMQMGSYTRQSPAPVIDRSWLPNDGIVNVVSAKYPFGHPNSPYDGAIKQGVWNSFPVMEGWDHMDFINFIGSNTPGYFSIYGYYNDVANRVHSLPK

[0049] The nucleotide sequence of the wild-type lipase CTL is shown below: SEQ ID NO. 2 (1155 bp)

[0050]

[0051] The amino acid sequence of the mutant CTL-B1 is shown below: SEQ ID NO.3

[0052] ETSGNDYPIVLVHGLGGWGKGEFLGYRYWGGLKDIEFYLNQTGHRTYVATVGPVSSNWDRAVELYYYIKGGTVDYGAAHAKEHGHARFGRTYPGIYGQWDETNKIHLIGHSMGGQTSRMLVELLKSGSQKEQEYYSQHPEEGISPLFTGGKNWVHSVTSLATPHNGSTFADQEQIVSFIKDFIIHLASAAGQ KQESLIYDFKLDQWGLKRQPGESFHAYMNRVMTSPIWQSNDISAYDLTTFGAQELNQWMKTYPDVYYLSYTGNASYESTLTGNYYPIGTMHPLFTLISMQMGSYTRQSPAPVIDRSWLPNDGIVNVVSAKYPFGHPNSPYDGAIKQGVWNSFPVMEGWDHMDFINFIGSNTPGYFSIYGYYNDVANRVHSLPK

[0053] The nucleotide sequence of the mutant CTL-B1 is shown below: SEQ ID NO.4 (1155bp)

[0054]

[0055] The amino acid sequence of the mutant CTL-B2 is shown below: SEQ ID NO.5

[0056] ETSGNDYPIVLVHGLGGWGKGEFLGYRYWGGLKDIEFYLNQTGHRTYVATVGPVSSNWDRAVELYYYIKGGTVDYGAAHAKEHGHARFGRTYPGIYGQWDETNKIHLIGHSMGGQTSRMLVELLKSGSQKEQEYYSQHPEEGISPLFTGGKNWVHSVTSLATPHNGSTFADQEQIVSFIKDFIIHLASAAGQ KQESLIYDFKLDQWGLKRQPGESFHAYMNRVMTSPIWQSNDISAYDLTTFGAQELNQWMKTYPDVYYLSYTGNASYRGETTGNYYPIGTMHPLFTLISMQMGSYTRQSPAPVIDRSWLPNDGIVNVVSAKYPFGHPNSPYDGAIKQGVWNSFPVMEGWDHMDFINFIGSNTPGYFSIYGYYNDVANRVHSLPK

[0057] The nucleotide sequence of the mutant CTL-B2 is shown below: SEQ ID NO.6 (1155bp)

[0058]

[0059] The amino acid sequence of the mutant CTL-C1 is shown below: SEQ ID NO.7

[0060] ETSGNDYPIVLVHGLGGWGKGEFLGYRYWGGLKDIEFYLNQTGHRTYVATVGPVSSNWDRAVELYYYIKGGTVDYGAAHAKEHGHARFGRTYPGIYGQWDETNKIHLIGHSMGGQTSRMLVELLKSGSQKEQEYYSQHPEEGISPLFTGGKNWVHSVTSLATPHNGSTFADQEQIVSFIKDFIIHLASAA GQKQESLIYDFKLDQWGLKRQPGESFHAYMNRVMTSPIWQSNDISAYDLTTFGAQELNQWMKTYPDVYYLSYTGNASYRGVVTGNYYPIGTMHPL FTLISMQMGSYTRPVIDRSWLPNDGIVNVVSAKYPFGHPNSPYDGAIKQGVWNSFPVMEGWDHMDFINFIGSNTPGYFSIYGYYNDVANRVHSLPK

[0061] The nucleotide sequence of the mutant CTL-C1 is shown below: SEQ ID NO.8 (1143bp)

[0062]

[0063] The amino acid sequence of the mutant CTL-C2 is shown below: SEQ ID NO.9

[0064] ETSGNDYPIVLVHGLGGWGKGEFLGYRYWGGLKDIEFYLNQTGHRTYVATVGPVSSNWDRAVELYYYIKGGTVDYGAAHAKEHGHARFGRTYPGIYGQWDETNKIHLIGHSMGGQTSRMLVELLKSGSQKEQEYYSQHPEEGISPLFTGGKNWVHSVTSLATPHNGSTFADQEQIVSFIKDFIIHLAS AAGQKQESLIYDFKLDQWGLKP

[0065] The nucleotide sequence of the mutant CTL-C2 is shown below: SEQ ID NO.10 (1131bp)

[0066]

[0067] The present invention will be further described in detail below with reference to specific embodiments.

[0068] Example 1: Design and construction of CTL and its mutant CTL-B1

[0069] (1) Using the target gene sequence as a template, select mutation sites for primer design. The primer list is shown in Table 1 below:

[0070] Table 1. Primer Design for Mutants

[0071]

[0072] (2) Using plasmid pET22b-CTL as an amplification template, PCR amplification was performed using the primers described above.

[0073] Synthesize the primers in Table 1, and amplify the target gene and vector according to the corresponding system and procedure in Table 2 below using a PCR instrument to obtain the corresponding target gene and vector.

[0074] Table 2. PCR Reaction System and Procedure

[0075]

[0076] The amplified target gene and vector were recovered according to the SanPrep column PCR product gel recovery operation manual.

[0077] The recovered target gene and vector were stored in a storage tube at -20°C for later use.

[0078] (3) Digestion, purification and transformation of PCR products.

[0079] The PCR products are digested with DpnI enzyme to remove the original template contained in the PCR products.

[0080] The digestion system consisted of: 1 μL of DpnI enzyme, 1 μL of 10× buffer, 8 μL of PCR product, and incubation at 37℃ for 1 h.

[0081] After the digestion process was completed, the product was purified by PCR. Following the operating procedure of the SanPrep column PCR product purification kit, 10 μL of ddH2O was added for elution to obtain the purified PCR product.

[0082] 10 μL of the PCR product of the above mutation was mixed with 50 μL of E. coli TOP10 competent cells, incubated on ice for 30 min, then heat-shocked in a 42°C water bath for 90 s, and then rapidly cooled on ice for 3 min.

[0083] Add 750 μL of low-salt LB liquid medium and incubate at 37°C in a shaker for 45 min.

[0084] After centrifugation at 10000 rpm for 1 min, discard 650 μL of supernatant, resuspend the bacterial cells, and spread them onto a plate containing a final concentration of 50 ng / mL. -1 LB solid medium resistant to ampicillin was incubated at 37°C for 10-12 hours, and single colonies were picked and sent for sequencing.

[0085] After confirming correct sequencing, single clones were inoculated into 5 mL of low-salt LB liquid medium and cultured at 37°C and 250 rpm for 12–16 h. Plasmids were extracted using the SanPrep column-based plasmid DNA mini-extraction kit, and the extracted plasmids were heat-shocked and transformed into E. coli Rosetta-gami B(DE3) competent cells.

[0086] Figure 1 This is an SDS-PAGE gel image of CTL eluted with 300 mM imidazole after purification by a nickel column. The image shows that CTL was well expressed and purified. M: Standard protein marker; Lane 1: Total cell lysis buffer; Lane 2: Cell lysis supernatant; Lane 3: Cell lysis precipitate; Lane 4: Nickel column purification buffer; Lane 5: 300 mM imidazole elution buffer.

[0087] Example 2: Expression and purification of CTL and mutant B1

[0088] Recombinant E. coli Rosetta-gami B(DE3) strain containing plasmid pET22b-CTL and mutants pET22b-CTL-B1, pET22b-CTL-B2, pET22b-CTL-C1, and pET22b-CTL-C2 was incubated at 37°C with 50 μg·mL⁻¹ of plasmid. -1 Ampicillin and 34 μg·mL -1 The culture was carried out in Terrific broth (TB medium) containing chloramphenicol and penicillin. When the OD600 reached 5.0, 0.5 mM isopropyl β-D-1-thiogalactoside (IPTG) was added, and the culture was carried out at 18 °C for 20 h. After centrifugation at 6000 rpm / min for 20 min at 4 °C, the culture was stored at -80 °C for later use.

[0089] The instruments, chromatography columns, and purification chromatography columns used for CTL and mutant purification, as well as for sample loading and collection, were: His Prep™ FF16 / 10 and HiPrep™ 26 / 10 salt-changing columns.

[0090] Prepare a equilibration chromatography column containing 50 mM PBS, 300 mM NaCl, 20 mM imidazole, 10% (v / v) glycerol, and pH 8.0. After equilibration, pump in the crude enzyme solution, and equilibrate with the loading buffer after sample loading. Prepare an elution buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, 300 mM imidazole, 10% (v / v) glycerol, and pH 8.0 for elution. Collect the eluted sample and detect it by SDS-PAGE electrophoresis. Replace the salt in the sample containing the peak corresponding to the target protein using a salt-replacement column. Prepare a salt-replacement buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, and pH 8.0. Add the protein to the salt-replacement column equilibrated with the salt buffer, and continue eluting with the salt-replacement buffer. Collect the eluted protein, aliquot it, pre-freeze in liquid nitrogen, and store at -80°C for later use.

[0091] Figure 2 This is an SDS-PAGE gel image of CTL mutants purified by nickel column elution with 300 mM imidazole. The image shows that the CTL mutants achieved good expression and purification results. M: Standard protein marker; Lane 1: CTL-B1 purified enzyme; Lane 2: CTL-B2 purified enzyme; Lane 3: CTL-C1 purified enzyme; Lane 4: CTL-C2 purified enzyme.

[0092] Example 3: Enzymatic characterization of lipase CTL (1) Determination of lipase CTL activity by olive oil emulsion titration method:

[0093] Weigh 4g of olive oil emulsion into a 50mL Erlenmeyer flask with a stopper, add 5mL of 20mM pH 8.0 phosphate buffer, and add 15mL of industrial alcohol to the blank control. Preheat in a water bath at 150rpm and 50℃ for 5min. After preheating, add 1mL of enzyme solution diluted appropriately, and then react for 10min. After the reaction, immediately add 15mL of industrial alcohol and shake at 150rpm and 50℃ for 1min to completely inactivate the protein. Each sample needs to have one blank and three parallel tests. Then remove the Erlenmeyer flask and let it stand until it cools to room temperature. Add 3 drops of phenolphthalein as an indicator to the above Erlenmeyer flask and titrate with 0.05M NaOH until the solution changes from milky white to slightly red. The formula for calculating the hydrolytic enzyme activity (1) is as follows:

[0094]

[0095] Where A represents lipase activity, U·mL -1 ;

[0096] c represents the concentration (M) of NaOH used in the titration;

[0097] N represents the dilution factor;

[0098] V1 represents the volume of sodium hydroxide titrated in the experimental group, in mL;

[0099] V2 represents the volume of sodium hydroxide titrated in the blank group, in mL;

[0100] T represents the reaction time, in minutes.

[0101] (2) Optimal reaction temperature and temperature tolerance of wild-type and mutant lipase CTLs:

[0102] The hydrolytic activity of lipase CTL was determined using the olive oil emulsion titration method at pH 8.0 and different temperatures (20, 30, 40, 45, 50, 55, 60, 70, 80, and 90 °C). The highest enzyme activity measured at each temperature was defined as 100%, and the enzyme activities measured at other temperatures were expressed as relative activities. Temperature-relative enzyme activity curves for wild-type lipase CTL were obtained. The results show that the optimal temperature for CTL is 50 °C. (See details...) Figure 3 .

[0103] The enzyme was incubated at 40, 50, 60, and 70°C for two hours each, with hydrolytic enzyme activity measured every 30 minutes. The enzyme activity before heat treatment (0 h) was defined as 100%, and the enzyme activities measured at other time points were expressed as relative activities. Time-residual enzyme activity curves were plotted. The results showed that CTL had a half-life greater than 30 minutes at 40°C. However, incubation at temperatures above 40°C resulted in a rapid decrease in CTL hydrolytic enzyme activity and destruction of the protein structure. (See details...) Figure 4 .

[0104] (3) Optimal reaction pH for lipase CTL:

[0105] At the optimal reaction temperature, the hydrolytic activity of lipase CTL was determined by titration using olive oil emulsion at pH values ​​of 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. The highest hydrolytic activity measured at different pH values ​​was defined as 100%, and the enzyme activities measured at other pH values ​​were expressed as relative activities. pH-relative enzyme activity curves for CTL wild-type and its mutants were obtained. The results showed that CTL exhibited the highest relative hydrolytic activity at pH 7.5 and pH 8.0, with the optimal pH being 8.0. (See details...) Figure 5 .

[0106] (4) The ability of CTL lipases to hydrolyze different substrates:

[0107] The hydrolytic activity of the substrates, olive oil, was determined by replacing them with glyceryl tartrate (C4:0), coconut oil (C12:0, C14:0), and camellia oil (C18:1).

[0108] (5) Initial screening of esterification ability of linoleic acid / linolenic acid:

[0109] Weigh 5g of reaction substrate (glycerol to linoleic acid / linolenic acid molar ratio of 1:3) into a 10mL reaction flask, add 1mg of lyophilized enzyme powder, and add 2% (w / w) of water. Then place it in a 40℃ constant temperature oil bath magnetic stirrer at 200rpm. Extract oil samples at different reaction times (1, 3, 6, 9, 12, 24h). Detect the content of glycerides and fatty acids in the extracted samples using high performance liquid chromatography equipped with a Waters differential detector.

[0110] The specific steps are as follows:

[0111] a) Sample preparation: Take 50 μL of sample each time, add it to 1 mL of mobile phase, vortex and extract thoroughly, add anhydrous sodium sulfate to remove water from the sample, centrifuge at 12000 rpm for 3 min, and take the supernatant to add to the chromatographic bottle for detection and analysis.

[0112] b) High-performance liquid chromatography (HPLC) detection conditions: A Phenomenex Luna 5u Silica(2) 100A (250mm × 4.60mm) column was installed, and the column temperature was set to 30℃, with a mobile phase flow rate of 1 mL / min. -1 The injection volume is 10 μL.

[0113] c) Analysis of Detection Results: Analysis of the standards for each substance revealed that the elution order in the chromatogram was as follows: triglycerides (TAG), fatty acids (FFA), 1,3-diglycerides (1,3-DAG), 1,2-diglycerides (1,2-DAG), and monoglycerides (MAG). The relative content of each substance could be calculated using the area normalization method.

[0114] like Figure 6 As shown, the relative hydrolytic activity of lipase CTL varies for different substrates. The hydrolytic activity for C4 to C18 triglycerides is 60% of the highest hydrolytic activity, indicating that lipase CTL has good hydrolytic activity for short-chain, medium-chain and long-chain lipases.

[0115] After 24 hours of reaction, the composition of the esterification product of lipase CTL was determined, and the following results were obtained. Figure 7 It can be seen that lipase CTL can esterify linoleic acid and linolenic acid into monoglycerides, diglycerides and triglycerides. This property indicates the ability of CTL to esterify linoleic acid and linolenic acid and its future application prospects.

[0116] The study on CTL wild-type and its mutants revealed that, when the enzyme activity of the mutants was measured at 50°C, mutants C1 and C2 were inactivated, with hydrolytic activities only 1.24±0.04% and 0.24±0.04% of the CTL wild-type, respectively. While mutant B2 still possessed 91.90±0.02% of the hydrolytic activity relative to the CTL wild-type, preliminary measurements showed that its residual enzyme activity after 1 hour of incubation was only 18.78±0.64%, lower than the CTL wild-type's 23.23±0.65%. Only mutant B1 exhibited good activity and thermostability. Therefore, subsequent thermostability experiments were conducted only on the effective mutant B1. Please see Example 4.

[0117] Example 4: Thermal stability characterization of CTL wild type and its mutants

[0118] (1) Half-life (t) 1 / 2 The determination of )

[0119] The purified enzyme solutions of CTL wild-type and mutant were diluted to 0.3 mg / mL. -1 The lipase was incubated in a 50℃ water bath for a certain period of time, and samples were taken at regular intervals. The samples were immediately placed in an ice-water bath for 30 minutes to equilibrate. The residual activity of CTL and its mutant B1 was determined at 50℃ using the olive oil emulsion titration method. A time-v / ln (residual activity) graph was plotted, and the slope was calculated using linear fitting. This slope represents the first-order inactivation constant (kT) of the lipase at that temperature. 1 / 2 Calculated using Formula 3.

[0120]

[0121]

[0122] Figure 8 The thermal stability plots of CTL wild-type and mutant B1 at 50℃ were calculated and fitted with the inactivation rate constant at 50℃. Figure 8 It can be seen that the inactivation rate after incubation at 50℃ is significantly slower after mutation. After fitting calculation, it was found that the half-life of wild-type WT at 50℃ is 21.59 min, while that of mutant B1 at 50℃ is 121.60 min, which is 5.63 times higher than that of WT. The enzyme activity was reduced to 96.16±0.07% of the original value as measured by the method in Example 3(1) (data not shown). (2) Determination of the optimal reaction temperature:

[0123] The purified enzyme solution was titrated at 40, 45, 50, 55, and 60 °C using the olive oil emulsion titration method to determine the hydrolytic activity of the effective mutant. The reaction temperature with the highest enzyme activity was defined as the optimum reaction temperature (Topt). Furthermore, at 30, 40, and 45 °C, the residual enzyme activity of mutant B1 was higher than that of the wild type. Below the optimum temperature, the enzyme activity was less affected by temperature. See details. Figure 9 .

[0124] (3) Determination of melting temperature:

[0125] The thermal melting temperature (Tm) refers to the temperature at which the light absorption reaches half of its maximum absorption (complete denaturation) during thermal denaturation (the double helix structure loses half). The secondary structure and Tm of proteins are characterized by measuring the circular dichroism (CD) spectrum of the sample using a circular dichroism chromatograph. The desalted pure enzyme solution was diluted to a final concentration of 0.2 mg / mL with PBS (pH 8.0) buffer. -1 A 300 μL sample was taken and a Hellma circular dichroic cuvette with a 1 mm optical path length was used. The full wavelength scanning range was 190-260 nm, the temperature was 28 °C, the step size was 1.0 nm, and the sampling time was 0.25 s. In the thermal distortion experiment, the sample was heated from 30 °C to 96 °C at a heating rate of 2 °C·min⁻¹, and the change in ellipticity at 190-250 nm was measured. The obtained curves were used to calculate the secondary structure of the protein using Pro-Data Viewer and CDNN software, and the Tm value of the protein was calculated using Global Analysis T-ramp software.

[0126] Figure 10 The CD spectra of CTL wild-type WT and its mutant B1 at 28℃, and the results of CD spectrum analysis using CDNN software. Figure 10 It can be seen that the peak value of mutant B1 at 210nm is lower than that of WT. Further analysis using CDNN software revealed that B1 has less α-Helix than WT, but more β-sheets and β-turns. This indicates that the multi-point mutation in the loop region alters the secondary structure of the protein, making it more stable.

[0127] Full-wavelength scanning of CTL wild-type WT and its mutant B1 was performed at 30-96℃. Figure 11 As can be seen, the secondary structure is continuously destroyed as the temperature increases, and the WT structure changes more rapidly. At 84℃, the structure has completely depolymerized. This indicates that B1 has improved stability compared to WT.

[0128] Figure 12 The graph shows the thermal melting curves of CTL wild-type WT and its mutant B1 at 28-96℃. The temperature Tm (℃) corresponding to the folding fraction of 0.5 is shown in the figure. It can be seen from the figure that the Tm of CTL is 58.7℃ and the Tm of mutant B1 is 60.2℃, which is 1.5℃ higher.

[0129] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A thermophilic Bacillus amyloliquefaciens lipase mutant, characterized in that, The sequence of the mutant is shown in SEQ ID NO.

3.

2. The encoding gene of the thermophilic amyloliquefaciens lipase mutant according to claim 1.

3. The encoding gene according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.4; or a sequence that is different from SEQ ID NO.4 due to the degeneracy of the genetic code, the amino acid sequence of which is shown in SEQ ID NO.

3.

4. An expression vector containing the encoding gene according to any one of claims 2-3, characterized in that, The encoding gene described in claim 2 or 3 is cloned into an expression vector.

5. A recombinant genetically engineered bacterium containing the coding gene of any one of claims 2-3 or the expression vector of claim 4.

6. A method for preparing the thermophilic Bacillus amyloliquefaciens lipase mutant according to claim 1, characterized in that, The expression vector described in claim 4 was mixed with competent Escherichia coli cells and transformed to obtain recombinant genetically engineered bacteria. The thermophilic Bacillus lipase mutant was expressed, purified, and obtained.

7. The preparation method according to claim 6, characterized in that, The expression vector is plasmid pET22b-CTL; and / or the Escherichia coli is Rosetta-gami B (DE3).

8. The preparation method according to claim 6 or 7, characterized in that, The expression vector was prepared by using the sequence shown in SEQ ID NO.2 as a template and the sequences shown in SEQ ID NO.2 as primers for amplification. The purified PCR product was then transferred into competent Escherichia coli cells and cultured to obtain a positive expression vector.

9. The use of the thermophilic amyloliquefaciens lipase mutant of claim 1 and / or the encoding gene of claim 2 in esterified linoleic acid and esterified linolenic acid.

10. A method for esterifying linoleic acid and esterifying linolenic acid, characterized in that, The lipase used in the synthesis is the thermophilic amyloliquefaciens lipase mutant as described in claim 1.

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