Lipoxygenase mutants and uses thereof

Abstract

The application discloses a lipoxygenase mutant and application thereof, and belongs to the technical field of molecular biology and enzyme engineering. The lipoxygenase mutant disclosed by the application has the following mutations on the basis of the amino acid sequence shown in SEQ ID NO. 1: H58E / I149K / A323G / R356S / S391G, D82I, D82V, S136V, S136A, I149R, I149K, A323G, R356S, A369G, N372R, V380W, S391G, S391A, S445G, S445A, D82I / I149K, D82I / S136V, H58E or H58D. The lipoxygenase mutant provided by the application has obviously improved enzyme activity and stability in catalyzing reactions, and improves the feasibility of industrial application.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and enzyme engineering, and more specifically to lipoxygenase mutants and their applications. Background Technology

[0002] Lipoxygenases (LOXs) are iron- or manganese-containing oxidases found in plants, animals, bacteria, and fungi. They catalyze the oxidation of polyunsaturated fatty acids into corresponding highly reactive hydroperoxides. The hydroperoxides produced by lipoxygenases can be used in various applications, such as bleaching colored components, modifying lipids from different raw materials, producing lipid-derived chemicals, and producing aromatic compounds.

[0003] Many applied studies have used soybean-derived lipoxygenases, such as US Patent 5464761, which uses soybean-derived lipoxygenases to produce fatty alcohols or aldehydes; and patent CN1816282, which uses soybean or other legume-derived lipoxygenases for bleaching dairy products. Besides soybean-derived lipoxygenases, other plant-derived lipoxygenases have also been reported, such as potato-derived lipoxygenases, tomato-derived lipoxygenases, apple-derived lipoxygenases, tea-derived lipoxygenases, and rice-derived lipoxygenases reported in CN118086378A.

[0004] There are also numerous reports on lipoxygenases from microbial sources, such as CN1452656B, which discloses a source from *Tetranychus gracilis* (…). Gaeumannomyces graminis Lipooxygenase from Pseudomonas aeruginosa (CN114277005B) is disclosed. Pseudomonas aeruginosa Lipoxygenase, CN104293805A discloses algae ( Porphyra thallus Lipooxygenase from algae, CN117844770A discloses... Porphyra haitanensis Sources of lipoxygenase for the production of fatty alcohols include CN119709889A, which discloses the production of decacarbonaldehyde from lipoxygenases derived from algae and Fusarium, and CN104630164A, which reports the production of low-temperature lipoxygenases from marine microorganisms.

[0005] Naturally derived lipoxygenases generally suffer from poor thermal stability and low specific activity, thus limiting their industrial application. For example, US patent 5464761 reports that when preparing fatty hydroperoxides using soybean-derived lipoxygenases, the soybean flour needs to be freshly prepared, and the reaction needs to be carried out at a low temperature of 10°C. Burkholderia thailandensis The activity of the bacterial lipoxygenase was reduced to less than 35% after incubation at 40 degrees Celsius for 30 minutes.

[0006] Therefore, providing lipoxygenase mutants and their applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides lipoxygenase mutants and their applications.

[0008] This invention provides microorganisms Brunnivagina elsteri Some variants of lipoxygenase have shown significant improvements in specific enzyme activity or thermostability, laying the foundation for industrial applications.

[0009] This invention provides a polypeptide with lipoxygenase activity, its encoding gene sequence, a recombinant preparation method for the polypeptide, its enzymatic properties, and its applications. The polypeptide provided by this invention possesses lipoxygenase activity, exhibiting significantly improved specific activity or superior thermal stability. Therefore, this polypeptide undoubtedly has broad application prospects in multiple fields such as the food and pharmaceutical industries.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] Lipoxygenase mutant, which originated from Brunnivagina elsteri (Genebank ID: WP_095722942) is predicted to be a mutant of lipoxygenase (amino acid sequence as shown in SEQ ID NO.1) formed by mutation at least one of the following sites: H58, D82, S136, I149, A323, R356, A369, N372, V380, S391, S445. This lipoxygenase mutant can catalyze the production of hydroperoxides from polyunsaturated fatty acids such as linoleic acid or linolenic acid, and exhibits higher enzyme activity.

[0012] The term "mutation" mentioned above includes, but is not limited to, the substitution, deletion, or addition of amino acids.

[0013] Preferably, the above-mentioned lipoxygenase mutant is a mutant formed by mutation of one or more of the following sites in the amino acid sequence of lipoxygenase SEQ ID NO.1: H58, D82, S136, I149, A323, R356, A369, N372, V380, S391, and S445. Preferably, sites D82, S136, A323, A369, S391, and S445 are mutated to aliphatic amino acids (G, A, V, L, and I), respectively; H58 is mutated to acidic amino acids E or D; R356 is mutated to neutral polar amino acids S or T; I149 and N372 are mutated to basic amino acids R or K; and V380 is mutated to aromatic amino acids W, Y, or F.

[0014] Preferably, the above-mentioned lipoxygenase mutant is a mutant formed by one or more mutations selected from the following groups in the amino acid sequence shown in SEQ ID NO.1 of lipoxygenase: H58E, D82V, S136V, I149K, A323G, R356S, A369G, N372R, V380W, S391G, S445G.

[0015] More preferably, the above-mentioned lipoxygenase mutant is the H58E, I149K, A323G, R356S, S391G mutant (H58E / I149K / A323G / R356S / S391G) shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.3.

[0016] A second aspect of the invention provides a nucleotide sequence encoding the above-described lipoxygenase mutant.

[0017] Preferably, the nucleotide sequence encoding the lipoxygenase mutant SEQ ID NO.3 is SEQ ID NO.4.

[0018] A third aspect of the invention provides a plasmid containing the aforementioned gene. The plasmid includes a vector for expressing the aforementioned gene, preferably a PET series vector, such as pET22b, pET24a, pET28a, etc., but not limited thereto.

[0019] A fourth aspect of the present invention provides a microorganism for expressing the above-described lipoxygenase mutant. For example, the microorganism is a transformant transformed with the above-described plasmid.

[0020] The aforementioned microorganisms can be selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0021] The fifth aspect of the invention provides the use of the above-mentioned lipoxygenase mutant or the above-mentioned microorganism in the production of fatty hydroperoxides.

[0022] Specifically, in the production of fatty hydroperoxides, linoleic acid, linolenic acid, eicosapentaenoic acid, eicosapentaenoic acid, etc. can be used as substrate raw materials; preferably, linoleic acid and linolenic acid are used as substrates, and the above-mentioned lipoxygenase mutants or microorganisms are used as catalysts to catalyze the reaction.

[0023] As an alternative implementation, the aforementioned microorganisms can be in the form of bacterial cells or their cell fragments, serving as catalysts for the oxidation reaction.

[0024] Compared to wild-type lipoxygenase SEQ ID NO.1, the lipoxygenase mutant (H58E / I149K / A323G / R356S / S391G) SEQ ID NO.3 provided by this invention exhibits significantly improved enzyme activity and stability in the catalytic reaction, thereby enhancing the feasibility of industrial application.

[0025] The application of lipoxygenase mutants, or the coding gene of lipoxygenase mutants, or expression vectors containing the coding gene of lipoxygenase mutants, or recombinant cells containing the coding gene of lipoxygenase mutants, in improving the conversion rate of linoleic acid, linolenic acid, eicosapentaenoic acid, or eicosapentaenoic acid, wherein the lipoxygenase mutant has the following mutations based on the amino acid sequence shown in SEQ ID NO.1: H58E / I149K / A323G / R356S / S391G. Specifically, using linoleic acid, linolenic acid, eicosapentaenoic acid, or eicosapentaenoic acid as substrates, and using the coding gene of lipoxygenase mutants, or the coding gene of lipoxygenase mutants, or expression vectors containing the coding gene of lipoxygenase mutants, or recombinant cells containing the coding gene of lipoxygenase mutants as catalysts to catalyze the reaction.

[0026] The application of lipoxygenase mutants or the coding gene of lipoxygenase mutants or expression vectors containing the coding gene of lipoxygenase mutants or recombinant cells containing the coding gene of lipoxygenase mutants in improving the specific activity of lipoxygenase, wherein the lipoxygenase mutant has the following mutations based on the amino acid sequence shown in SEQ ID NO.1: D82I, D82V, S136V, S136A, I149R, I149K, A323G, R356S, A369G, N372R, V380W, S391G, S391A, S445G, S445A, D82I / I149K or D82I / S136V.

[0027] The application of lipoxygenase mutants or the coding gene of lipoxygenase mutants or expression vectors containing the coding gene of lipoxygenase mutants or recombinant cells containing the coding gene of lipoxygenase mutants in improving the thermostability of lipoxygenase, wherein the lipoxygenase mutant has the following mutations based on the amino acid sequence shown in SEQ ID NO.1: H58E, H58D, I149R, I149K, R356S, A323G, N372R, A369G, V380W or S391G.

[0028] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses lipoxygenase mutants and their applications, which have the following beneficial effects:

[0029] This invention provides a lipoxygenase mutant with high specific enzyme activity. The activity of the lipoxygenase mutant provided in this invention is significantly improved, reaching 282% of that of the wild-type lipoxygenase. Therefore, the use of this enzyme can significantly improve its industrial application.

[0030] This invention provides a lipoxygenase mutant with significantly improved stability. The relative residual activity after incubation at 50°C for 1 hour is also significantly improved compared to the wild type, reaching 215% of the wild type. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 This is a schematic diagram of a vector for lipoxygenase expression.

[0033] Figure 2 SDS-PAGE images of wild-type lipoxygenase cell fermentation; M - protein marker; 1 - whole-cell sample of uninduced sample by ultrasonic disruption; 2 - supernatant of uninduced sample by ultrasonic disruption and centrifugation; 3 - whole-cell sample of induced sample by ultrasonic disruption and centrifugation; 4 - supernatant of induced sample by ultrasonic disruption and centrifugation.

[0034] Figure 3 The reactivity of wild-type lipoxygenase and lipoxygenase mutants at different temperatures was measured.

[0035] Figure 4 The reactivity of wild-type lipoxygenase and lipoxygenase mutants under different pH conditions was measured.

[0036] Figure 5 The graph shows the residual activity of wild-type lipoxygenase and lipoxygenase mutants after incubation at different temperatures.

[0037] Figure 6 The reaction curves for wild-type lipoxygenase and lipoxygenase mutants catalyzing linoleic acid / linolenic acid substrates are shown. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The initial lipoxygenase SEQ ID NO.1, which is the object of modification in this invention, is a wild-type enzyme derived from... Brunnivagina elsteri (Genebank ID: WP_095722942). This enzyme is predicted to be a lipoxygenase in the gene bank, consistent with reports from [source missing]. Rivularia sp The homology of PCC 7116 with lipoxygenase (Qi Y., et al. ACS SustainableChem. Eng. 2020, 8, 5558.) is 65%.

[0040] To improve the performance of wild-type lipoxygenase (amino acid sequence as shown in SEQ ID NO.1), gene mutation was performed using error-prone PCR, followed by performance evaluation using 96-well plate culture medium. Through extensive screening, some mutants with improved performance were finally obtained. Subsequently, some mutation sites were integrated to obtain the variant enzyme of SEQ ID NO.3.

[0041] For ease of expression, wild-type lipoxygenase and its mutants will be collectively referred to as "lipoxygenase" in this article.

[0042] Amino acid mutations include substitution, deletion, or addition. Among these, amino acid substitutions include conserved substitutions and non-conserved substitutions.

[0043] "Conservative substitution" refers to the interchangeability of residues with similar side chains, and therefore generally includes replacing amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. For example, but not limited to, an amino acid with an aliphatic side chain can be replaced with another aliphatic amino acid such as alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain can be replaced with another amino acid with a hydroxyl side chain such as serine and threonine; an amino acid with an aromatic side chain can be replaced with another amino acid with an aromatic side chain such as phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain can be replaced with another amino acid with a basic side chain such as lysine and arginine; an amino acid with an acidic side chain can be replaced with another amino acid with an acidic side chain such as aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid can be replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0044] "Non-conservative substitution" refers to the substitution of amino acids in a polypeptide with amino acids that have significantly different side-chain properties. Non-conservative substitution can utilize amino acids between rather than within groups and affects: (a) the structure of the peptide backbone in the substitution region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side-chain volume. For example, but not limited to, exemplary non-conservative substitutions could be the substitution of acidic amino acids with basic or aliphatic amino acids; the substitution of aromatic amino acids with small amino acids; and the substitution of hydrophilic amino acids with hydrophobic amino acids.

[0045] Mutations at some sites in the amino acid sequence of wild-type lipoxygenase are not single mutations. For example, the mutation at position 149 can be either I149K or I149R, in which case it can be described as I149KR. The I149K mutation refers to the mutation in which the isoleucine residue (I or Ile) at position 149 of the amino acid sequence shown in SEQ ID NO.1 is replaced by a lysine residue (K or Lys), and so on.

[0046] For ease of expression, the abbreviation of amino acids for proteins can be either three letters or one letter, as is well known to those skilled in the art.

[0047] To express lipoxygenase in Escherichia coli, the coding gene for the codon-optimized wild-type lipoxygenase SEQ ID NO.1 is SEQ ID NO.2; the coding gene for the lipoxygenase mutant SEQ ID NO.3 is SEQ ID NO.4.

[0048] The transformant host can be any microorganism suitable for expressing lipoxygenase, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0049] When used as a biocatalyst for the production of fatty acid hydroperoxides, the lipoxygenase of the present invention can be in the form of an enzyme or in the form of a bacterial cell. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, and enzymes immobilized on a carrier; the bacterial cell form includes live cells, dead cells, and immobilized cells.

[0050] As another alternative implementation, microbial cells expressing the aforementioned lipoxygenase can be used as biocatalysts for enzyme-catalyzed reactions. The microorganisms can be in the form of cells or their cell fragments. Cellular forms include both live and dead cells. When microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli cease fermentation and proliferation and are instead used for enzyme-catalyzed reactions, they are themselves naturally immobilized enzymes. Furthermore, they do not require disruption or even extraction and purification; they can be used as enzyme preparations for catalytic reactions.

[0051] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content refers to the mass percentage.

[0052] Example 1: Synthesis and Cloning of Lipoxygenase Sequence

[0053] The amino acid sequence of wild-type lipoxygenase is shown in SEQ ID NO.1.

[0054] MIASSKGNSTGSPDSQSLEVIRQKYQYNYTHIPPLAMLDTLPEGEQFSRDWLFLLAQ H LRVVFVNTIITNRGNRGSLSVRD D IKYFILEAVMKGAIPIQVSVIGRLLQIIPQILLKGLSIDFRELDNLFFSALKE S GLSIFKDSLDKV I ELMYEGQPTGHVKNLREYEKLLPQMELPAIANGFGQDDVFAYMQVAGYNPLMIQRVTSLGENFPVTDEHYQGMMGEDDSLVAAGREGRLYLADYRILDGAVNGTYPKTQKYLYAPIALFAVPRGKEQNRMLRAIAIQCGQNPRENPIVTPKSGKYAWMFAKTVVHIADANYHE A VSHLGRTHLFVGAFVIATHRQLPANHPLSLL R PHFVGTLAINEE A QR N LIAAGGG V DSLLGATIDN S RVFAVRGLQSYGFNTAMLPKRLELRGVDDTNLLPVYPYRDDGLLIWNAIHEWV SSEQ ID NO.1.

[0055] The optimized wild-type lipoxygenase nucleotide sequence, synthesized by Sangon Biotech (Shanghai) Co., Ltd., is shown in SEQ ID NO.2.

[0056] ATGATCGCATC GTCTATCAACAT CTAA ;SEQ ID NO.2.

[0057] The synthesized wild-type lipoxygenase nucleotide sequence (as shown in SEQ ID NO.2) was cloned into plasmid pRSF-Duet-1 and stored in *E. coli* DH5α. The specific steps were as follows: plasmid pRSF-Duet-1 was digested with Fermentas' EcoRI and HindIII restriction endonucleases, and then purified using an Axygen gel extraction kit. Using Fermentas' T4 DNA ligase, the wild-type lipoxygenase sequence fragment and the plasmid fragment were ligated according to the product instructions. The ligation product was then heat-shocked and transformed into *E. coli* DH5α, and cultured overnight on LB agar plates containing 50 µg / ml kanamycin. The next day, single colonies were picked and cultured in LB liquid medium. The plasmid was extracted using an Axygen plasmid extraction kit and sent to Shanghai Sangon Biotech for sequencing. The constructed vector containing the wild-type lipoxygenase sequence (pRSF-Duet-1-Lipoxygenase) is shown in the attached image. Figure 1 .

[0058] The correctly sequenced recombinant expression vector was retransformed into E. coli BL21(DE3) competent cells using the heat shock method, then plated onto a plate containing 50 µg / ml kanamycin resistance, and incubated overnight at 37 °C. Positive transformants were then picked.

[0059] Example 2: Fermentation verification of lipoxygenase-producing strains

[0060] Single clones were picked from the transformation plate of Example 1 and inoculated into 5 ml of LB liquid medium (1% tryptone, 0.5% yeast extract, and 1% sodium chloride). After overnight incubation, 250 µl of each clone was inoculated into two 250 ml shake flasks containing 50 ml of TB medium (1.2% tryptone, 2% yeast extract, 1.25% dipotassium hydrogen phosphate, 0.2% potassium dihydrogen phosphate, and 0.5% glycerol). The flasks were incubated at 37°C and 180 rpm until the OD600 value reached 0.7. Induction was performed in one shake flask with 500 µl of 10 mM IPTG inducer solution, while the other shake flask contained the same volume of sterile water. The incubation temperature was adjusted to 20°C, and induction was performed for 20 hours.

[0061] After fermentation, 1 ml of fermentation broth was taken from each of the two shake flasks, centrifuged, and the supernatant was removed. Then, 1 ml of 0.05 M phosphate buffer (pH 8.0, containing 0.2 M sodium chloride) was added to the tubes to fully resuspend the cells, and the mixture was incubated on ice for 30 min. Following this, the cells were sonicated, and 40 µl of the sonicated fragments were collected for SDS-PAGE analysis. These samples were from both the uninduced and induced whole-cell sonicated samples. The remaining sonicated fragments were centrifuged, and 40 µl of the supernatant was collected for SDS-PAGE analysis. These samples were from both the uninduced and induced whole-cell sonicated samples. The SDS-PAGE results are shown in the attached image. Figure 2 . Figure 2 The results showed that wild-type lipoxygenase with a theoretical molecular weight of around 71 kDa could achieve good soluble expression in Escherichia coli.

[0062] Example 3: Detection of lipoxygenase activity

[0063] Weigh 0.07g of linoleic acid, dissolve it in 2ml of anhydrous ethanol, then add 1ml of 5% Tween 20 solution, and finally add water to bring the volume to 100ml. After stirring thoroughly, the substrate solution is obtained.

[0064] Take 0.5 ml of the above substrate solution, add 0.5 ml of 0.2 M pH 7.8 phosphate buffer, then add 5 µl of enzyme solution of a certain dilution, and incubate in a 25°C water bath for 10 min. Immediately after incubation, add 950 µl of 0.2 M pH 9.5 glycine-sodium hydroxide buffer to terminate the reaction. Take the terminated reaction solution, dilute it appropriately with the corresponding termination buffer, and then measure the absorbance at 234 nm. For the control sample, first add the termination buffer, then add the enzyme solution, dilute, and then measure the absorbance.

[0065] The enzyme activity unit is defined as the amount of enzyme required to produce 1 micromole of product per minute, where 1 unit is the amount of enzyme required. The molar extinction coefficient of the product is... ε =25000 M -1 cm -1 .

[0066] Example 4: Random PCR Mutation and Mutant Screening of Lipoxygenase

[0067] Use primer Lip-1 (AAAGAATTC) ATGATCGCATC ; SEQ ID NO.5) and Lip-2 (CAAGCTT TTAGATGTTGATAGAC(SEQ ID NO. 6) The lipoxygenase moiety in the pRSF-Duet-1-Lipoxygenase plasmid was amplified. PCR was performed using Taq polymerase from TaKaRa. The reaction mixture consisted of: 5 μl 10× buffer, 4 μl dNTP mixture (2.5 mM each), 1 μl primers, 0.5 μl plasmid template, 0.5 μl Taq polymerase, and an additional 0.3 mM MnCl2, followed by double-distilled water to a final volume of 50 μl. The PCR program was 98℃ for 5 min; 94℃ for 20 s, 56℃ for 40 s, 72℃ for 2 min, for 30 cycles; 72℃ for 10 min. The PCR product was purified using a PCR purification kit from Axygen. The purified PCR product was digested with NdeI and XhoI restriction enzymes and cloned into the corresponding restriction sites of the pRSF-Duet-1 vector. The resulting vector was then transformed into competent Escherichia coli BL21(DE3) strain. The samples were then plated onto screening plates containing kanamycin resistance and incubated overnight at 37°C.

[0068] Positive single clones were then selected and cultured in 96-well deep-well plates containing TB medium at 37°C for approximately 5 hours. Induction was then initiated by adding 0.1 mM IPTG to each well, while simultaneously adjusting the culture temperature to 20°C. After overnight induction, Triton X-100 was added to each well to a final concentration of 0.2%. The plates were then incubated at 20°C with shaking for approximately 30 minutes. 5 μL of the supernatant was collected for enzyme activity testing. Based on the activity levels, shake-flask rescreening was performed for verification, and mutation sites were analyzed using sequencing.

[0069] To screen for highly stable lipoxygenase mutants, after deep-well plate culture, a portion of the cells from each well was picked, and a certain amount of Triton X-100 was added for cell disruption. The plates were then incubated at 50°C for approximately one hour. Next, a reaction solution containing 50 μM methylene blue and 100 μM linoleic acid in 0.1 M pH 7.5 phosphate buffer was added to the plates for a colorimetric reaction. A lighter color indicated higher lipoxygenase activity, suggesting potentially higher thermostability, thus completing the screening for highly thermostable enzymes. Positive mutant strains were then subjected to shake-flask verification and sequence analysis. Table 1 lists the increased activity of the lipoxygenase mutants, and Table 2 lists the improved thermostability.

[0070] Table 1. Activity changes of lipoxygenase mutants

[0071]

[0072] Table 2. Thermal stability of lipoxygenase mutants

[0073]

[0074] Example 5 Integration and Validation of Mutation Sites

[0075] Some of the mutation sites in Tables 1 and 2 of Example 4 were integrated, and the gene was directly synthesized, ligated into the pRSF-Duet-1 vector, and then verified by shake flask. Finally, a lipoxygenase mutant (H58E / I149K / A323G / R356S / S391G) with significantly improved activity and stability was obtained. The amino acid sequence is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.4.

[0076] MIASSKGNSTGSPDSQSLEVIRQKYQYNYTHIPPLAMLDTLPEGEQFSRDWLFLLAQ E LRVVFVNTIITNRGNRGSLSVRDDIKYFILEAVMKGAIPIQVSVIGRLLQIIPQILLKGLSIDFRELDNLFFSALKESGLSIFKDSLDKV K ELMYEGQPTGHVKNLREYEKLLPQMELPAIANGFGQDDVFAYMQVAGYNPLMIQRVTSLGENFPVTDEHYQGMMGEDDSLVAAGREGRLYLADYRILDGAVNGTYPKTQKYLYAPIALFAVPRGKEQNRMLRAIAIQCGQNPRENPIVTPKSGKYAWMFAKTVVHIADANYHE G VSHLGRTHLFVGAFVIATHRQLPANHPLSLL S PHFVGTLAINEEAQRNLIAAGGGVDSLLGATIDN G RVFAVRGLQSYGFNTAMLPKRLELRGVDDTNLLPVYPYRDDGLLIWNAIHEWVSDYLTIYYKTDADVQNDINLQAWAAEVQAFEGGRIPDFGEENGGIQTREYLIDAIALIIFTASAQHAVV SEQ ID NO.3.

[0077]

[0078] The lipoxygenase mutant and wild-type lipoxygenase, with amino acid sequences as shown in SEQ ID NO.3, were subjected to activity testing using the method described in Example 3. The activity of the lipoxygenase mutant was 282% of that of the wild-type lipoxygenase. After incubating the lipoxygenase mutant and wild-type lipoxygenase at 50°C for 1 hour, the residual activity was tested using the activity testing method described in Example 3. The results showed that the residual activity of the lipoxygenase mutant was 215% of that of the wild-type lipoxygenase.

[0079] The enzymatic properties of the wild-type lipoxygenase (amino acid sequence as shown in SEQ ID NO.1) and the lipoxygenase mutant (amino acid sequence as shown in SEQ ID NO.3) of the present invention were tested.

[0080] To investigate the effect of different temperatures on the reactions of the two enzymes, enzyme solutions (ultrasonically disrupted and centrifuged supernatants) of wild-type lipoxygenase and lipoxygenase mutants were first prepared using the fermentation culture and ultrasonic disruption method described in Example 2. Then, the enzyme activity was detected at different temperatures using the lipoxygenase activity detection method described in Example 3. The reaction temperatures were 0℃, 8℃, 20℃, 25℃, 29℃, 30℃, 31.3℃, 34.2℃, 40℃, 45℃, 50.8℃, 53.8℃, and 55℃. The maximum enzyme activity value obtained at each temperature for each enzyme was taken as 100%, and the relative activity values ​​at the other temperatures were calculated to obtain the enzyme activity. Figure 3 The result. Figure 3 The results showed that the optimal reaction temperature for wild-type lipoxygenase was 31.3℃, while the optimal reaction temperature for lipoxygenase mutants was in the range of 31.3℃-34.2℃.

[0081] To investigate the effect of different pH values ​​on the reactions of the two enzymes, enzyme solutions of wild-type lipoxygenase and lipoxygenase mutants were first prepared using the fermentation and ultrasonic disruption method described in Example 2. Then, the enzyme activity was detected under different pH conditions using the lipoxygenase activity detection method described in Example 3. The reaction pH values ​​were 6.2, 6.6, 7.0, 7.4, 7.6, 7.8, 8.0, 8.5, 9.0, and 9.5. pH values ​​from 6.2 to 8.0 were obtained using 0.2 M phosphate buffer, while pH values ​​at 8.5, 9.0, and 9.5 were obtained using 0.2 M glycine-sodium hydroxide buffer. The maximum enzyme activity value obtained under each pH condition was taken as 100%, and the relative activity values ​​under the remaining pH conditions were calculated to obtain the enzyme activity. Figure 4 The result. Figure 4 The results showed that the optimal reaction pH for both wild-type lipoxygenase and lipoxygenase mutants was 7.8.

[0082] To detect the residual activity of the two enzymes after incubation at different temperatures for 1 hour, enzyme solutions of the two enzymes were prepared according to the method in Example 2. The enzyme solutions were then aliquoted into several PCR tubes at a rate of 50 μl / tube. These aliquoted PCR tubes were placed on a PCR instrument and incubated for 1 hour at different temperature gradients. The temperatures investigated were 0℃, 25℃, 35℃, 36.2℃, 39.2℃, 45℃, 50℃, 55.8℃, 58.8℃, and 60℃. After 1 hour of incubation, the enzymes were immediately placed in an ice bath, and enzyme activity was detected according to the method in Example 3. The activity detection value of the wild-type lipoxygenase or lipoxygenase mutant sample at 0℃ was taken as 100%, and the relative enzyme activity values ​​at the other temperatures were calculated. The results are shown in […]. Figure 5 . Figure 5 The results showed that the temperature stability of the lipoxygenase mutant was significantly better than that of the wild-type lipoxygenase. For example, after incubation at 50°C for 1 hour, the residual activity of the lipoxygenase mutant was 215% of that of the wild-type lipoxygenase.

[0083] Example 6: Reaction of wild-type lipoxygenase and lipoxygenase mutant catalyzing linoleic acid substrate

[0084] Wild-type lipoxygenase and lipoxygenase mutant E. coli cells (OD600 value 9.5) cultured overnight in 250 ml shake flasks were collected, with a volume of 50 ml per flask. 30 ml of 50 mM phosphate buffer was added, followed by sonication and centrifugation to collect the supernatant.

[0085] Weigh 10g of linoleic acid or linolenic acid, add 2ml of anhydrous ethanol and 2ml of 5% Tween 80, and add 50ml of 0.2M pH 8.0 phosphate buffer. Then, place in a 25℃ water bath and stir thoroughly for 5 minutes. Add 25ml of the supernatant from the sonication of wild-type lipoxygenase or a lipoxygenase mutant, purge with pure oxygen, and stir at 800rpm. Take samples every 30 minutes, measure the absorbance at 234nm, and calculate the conversion rate. The reaction progress curve is shown below. Figure 6 . Figure 6 The results showed that, using linoleic acid as a substrate, the lipoxygenase mutant could almost completely convert the substrate to hydroperoxide within 1.5 hours, while the maximum conversion rate of the wild-type lipoxygenase was around 70%. Using linolenic acid as a substrate, the lipoxygenase mutant could complete the linolenic acid-catalyzed hydroperoxide reaction within 1 hour, while the maximum conversion rate of the wild-type lipoxygenase was around 80%. These results indicate that the application performance of the lipoxygenase mutant is significantly superior to that of the wild-type lipoxygenase.

[0086] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A lipoxygenase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the following mutations are performed: H58E / I149K / A323G / R356S / S391G, H58E, or H58D.

2. The encoding gene of the lipoxygenase mutant according to claim 1.

3. An expression vector containing the encoding gene of the lipoxygenase mutant as described in claim 2.

4. Recombinant cells containing the encoding gene of the lipoxygenase mutant as described in claim 2.

5. The application of lipoxygenase mutants, or the coding gene of lipoxygenase mutants, or expression vectors containing the coding gene of lipoxygenase mutants, or recombinant cells containing the coding gene of lipoxygenase mutants, in improving the conversion rate of linoleic acid or linolenic acid, characterized in that, The lipoxygenase mutant is based on the amino acid sequence shown in SEQ ID NO.1 and undergoes the following mutations: H58E / I149K / A323G / R356S / S391G.

6. The application according to claim 5, characterized in that, Using linoleic acid or linolenic acid as a substrate, the reaction is catalyzed by a lipoxygenase mutant or the coding gene of the lipoxygenase mutant or an expression vector containing the coding gene of the lipoxygenase mutant or a recombinant cell containing the coding gene of the lipoxygenase mutant.

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