Feruloyl esterase mutant as well as engineering strain and application thereof

Abstract

The invention provides feruloyl esterase mutants FAEP171C-W176C, FAEP171C-W176C-M19W and FAEP171C-W176C-M19W-N12I as well as an application of the mutants in the preparation of ferulic acid, and particularly provides a feruloyl esterase mutant FAEP171C-W176C-M19W-N12I. According to the feruloyl esterase mutant, pichia pastoris serves as host bacteria, feruloyl esterase is introduced through a genetic engineering means, and the feruloyl esterase is subjected to molecular modification, so that the feruloyl esterase mutant with increased residual enzyme activity and high ferulic acid yield after heat treatment is obtained. According to the enzyme, oryzanol in rice bran oil residues is used as a substrate for a catalytic experiment, the yield of ferulic acid reaches 90.32%, compared with an enzyme without mutation through codon optimization, the yield is increased by 72.47%, and the enzyme has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a ferulic acid esterase mutant, an engineered strain containing the ferulic acid esterase mutant and a construction method thereof, and an application of the mutant in the production of ferulic acid. Background Art

[0002] Ferulic acid (FA), also known as 4-hydroxy-3-methoxycinnamic acid, exists in the form of high-concentration phenolic acid in the cell walls of many plants. Ferulic acid contains a phenol nucleus and a long side chain, and can quickly form a resonance-stabilized phenoxy free radical, thus having the effect of eliminating free radicals. This enables FA to protect DNA, proteins, and oils from damage by reactive oxygen species. Therefore, ferulic acid can be used to prevent or treat diseases related to oxidative stress, such as Alzheimer's disease, diabetes, hypertension, etc. In addition, ferulic acid also has antibacterial and ultraviolet absorption capabilities, and is widely used in fields such as food preservatives and sunscreens. The structural formula of ferulic acid is as follows:

[0003]

[0004] The main methods for synthesizing ferulic acid are chemical synthesis method, plant extraction method, and biological enzyme method. Among them, the chemical and plant extraction methods have high energy consumption, difficult separation of inhibitors, high cost, and low extraction rate. In contrast, the biological enzyme method shows significant advantages. Feruloyl esterase (FAE) can hydrolyze the ester bond in the plant cell wall to release ferulic acid. Using ferulic acid esterase to directionally hydrolyze the ferulic acid ester bond in raw materials such as rice bran and wheat bran has the advantages of mild reaction conditions, high product purity, and no pollution.

[0005] As a by-product of grain processing, rice bran oil foot has the advantages of low raw material cost and resource recycling. Rice bran oil foot contains rich oryzanol. Using ferulic acid esterase to catalyze the ester bond of oryzanol to release ferulic acid is a feasible path. However, the substrate of rice bran oil foot is complex and its structure is compact. It is difficult for enzyme molecules to penetrate, and the ester bond of oryzanol is difficult to expose, which has become the main bottleneck for the industrial application of producing ferulic acid by biological enzyme method using rice bran oil foot.

[0006]

[0007] Although ferulic acid esterase is widely used to hydrolyze ester bonds in plant cell walls to release ferulic acid, the disadvantage of low enzyme activity is still obvious. In addition, FAE derived from fungi has poor thermal stability. So far, there are few reports on the commercial production of FAE enzyme preparations. Therefore, it is of great significance to obtain ferulic acid esterase with high thermal stability and strong catalytic activity. In order to increase the contact between ferulic acid esterase and oryzanol ester bonds in rice bran oil foots, it is necessary to improve enzyme stability and enzyme solubility at the same time. Rice bran oil foots are lipophilic substances. To increase the solubility of ferulic acid esterase in the lipid phase, it is necessary to reduce the hydrophilicity of the enzyme. Therefore, the ratio of hydrophilic and hydrophobic amino acids on the surface of ferulic acid esterase can be adjusted to improve the lipid solubility of the enzyme in rice bran oil foots, enhance the contact between the enzyme and the substrate oryzanol, and promote the release of ferulic acid. Li Tongbiao et al. [Li Tongbiao, Zhou Chenyan, etc. Effect of introducing hydrophobic amino acids on the thermal stability of GH11 family xylanase [J]. Science and Technology of Food Industry, 2016, (01): 145-148.] introduced hydrophobic amino acids into xylanase, so that the optimal temperature of the mutant enzyme was increased from 40 °C to 48 °C. Therefore, adjusting the hydrophilic and hydrophobic amino acids of ferulic acid esterase can not only increase the lipid solubility of ferulic acid esterase in rice bran oil foots, but also increase the stability of ferulic acid esterase, which is a feasible transformation method. With the rapid development of synthetic biology, using genetic engineering technology to transform ferulic acid esterase to obtain engineering bacteria with highly active enzymes has become an important research direction. Summary of the Invention

[0008] The object of the present invention is to overcome the defects of the prior art and improve the ability of Pichia pastoris engineering strains to produce highly active ferulic acid esterase through genetic engineering technology.

[0009] The idea of the present invention is on the one hand to mutate and transform ferulic acid esterase FAE, the key enzyme for oryzanol hydrolysis, in order to improve the thermal stability of ferulic acid esterase, and on the other hand to attempt site-directed mutagenesis of the ferulic acid esterase gene in order to improve the activity and lipid solubility of ferulic acid esterase in rice bran oil, and to construct engineering strains using Pichia pastoris as the starting strain to verify its ability to produce ferulic acid.

[0010] Based on this, the present invention provides ferulic acid esterase mutants FAE V41C-V56C 、FAE G47C-N53C 、FAE P171C-W176C 。Among them, the amino acid sequence of the said FAE V41C-V56C is shown in SEQ ID NO.4; the amino acid sequence of FAE G47C-N53C is shown in SEQ ID NO.5; the amino acid sequence of FAE P171C-W176C is shown in SEQ ID NO.6.

[0011] Particularly, the present invention also provides ferulic acid esterase with improved enzyme activity, and the said ferulic acid esterase is ferulic acid esterase mutant FAE P171C-W176C-M19W 、FAEP171C-W176C-M19H , FAE P171C-W176C-M19C , FAE P171C-W176C-M19K and FAE P171C -W176C-T77A . Among them, the FAE P171C-W176C-M19W has the amino acid sequence shown in SEQ ID NO.7; FAE P171C-W176C-M19H has the amino acid sequence shown in SEQ ID NO.8; FAE P171C-W176C-M19C has the amino acid sequence shown in SEQ ID NO.9; FAE P171C -W176C-M19K has the amino acid sequence shown in SEQ ID NO.10; FAE P171C-W176C-T77A has the amino acid sequence shown in SEQ ID NO.11.

[0012] In addition, the present invention also provides ferulic acid esterase with improved fat solubility in rice bran oil foot, thereby increasing the contact between the enzyme and the substrate. The ferulic acid esterase mutant is FAE P171C-W176C-M19W-N12I , FAE P171C-W176C-M19W-E14L , FAE P171C -W176C-M19W-R18L , FAE P171C-W176C-T77A-R168L , FAE P171C-W176C-M19W-D213L and FAE P171C-W176C-M19W-E245L . Among them, the FAE P171C-W176C-M19W-N12I has the amino acid sequence shown in SEQ ID NO.12; the FAE P171C-W176C-M19W-E14L has the amino acid sequence shown in SEQ ID NO.13; the FAE P171C-W176C-M19W-R18L has the amino acid sequence shown in SEQ ID NO.14; the FAE P171C-W176C-T77A-R168L has the amino acid sequence shown in SEQ ID NO.15; the FAE P171C-W176C-M19W-D213L has the amino acid sequence shown in SEQ ID NO.16; FAE P171C-W176C-M19W-E245L has the amino acid sequence shown in SEQ ID NO.17.

[0013] After further screening, among them, the ferulic acid esterase mutant FAE P171C-W176C-M19W-N12I has the best effect. The mutant FAE P171C-W176C-M19W-N12I is obtained by mutating proline at position 171 of ferulic acid esterase to cysteine, tryptophan at position 176 to cysteine, methionine at position 19 to tryptophan, and asparagine at position 12 to isoleucine.

[0014] In the present invention, the ferulic acid esterase is derived from Hortaea werneckii, and the nucleic acid sequence of the ferulic acid esterase gene is shown in SEQ ID NO.1; the nucleic acid sequence of the ferulic acid esterase after removing introns and codon optimization is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.3.

[0015] Furthermore, the present invention also provides an expression vector containing the above-mentioned ferulic acid esterase mutants FAE V41C-V56C , FAE G47C-N53C , FAE P171C-W176C , FAE P171C-W176C-M19W , FAE P171C-W176C-M19H , FAE P171C-W176C-M19C , FAE P171C-W176C-M19K , FAE P171C -W176C-T77A , FAE P171C-W176C-M19W-N12I , FAE P171C-W176C-M19W-E14L , FAE P171C-W176C-M19W-R18L , FAE P171C -W176C-T77A-R168L , FAE P171C-W176C-M19W-D213L or FAE P171C-W176C-M19W-E245L , and a genetically engineered strain containing the expression vector.

[0016] According to a preferred embodiment, the host strain of the genetically engineered strain is Pichia pastoris.

[0017] The present invention also provides the application of the ferulic acid esterase mutant FAE P171C-W176C-M19W-N12I in the preparation of ferulic acid esters.

[0018] In the present invention, when constructing the above-mentioned mutants, the pPICZB plasmid carrying the ferulic acid esterase gene was linearized with PmeⅠ and transferred into Pichia pastoris X33. After resistance screening and colony PCR verification, Pichia pastoris containing the ferulic acid esterase gene or mutant gene was obtained; the verified transformant was transferred to 30 ml of BMGY medium and cultured at 30 °C and 230 rpm for 16 h to obtain a seed solution; the seed solution was transferred to a 50 ml centrifuge tube and centrifuged at 5000 rpm for 5 min at room temperature. The supernatant was discarded, and the cells were diluted with BMMY to OD 600 = 1.0. The diluted mixture was transferred to a 250 ml shake flask and continued to be cultured. Methanol and CuSO4 were added once every 24 h to make the final concentration of methanol 1% and the final concentration of CuSO4 0.1 mM. The culture was carried out at 30 °C and 230 rpm for 96 - 120 h, and the fermentation broth was purified by ProteinIsoRNi-IDAResin column to obtain ferulic acid esterase.

[0019] The present invention verified that the ferulic acid esterase mutant FAE P171C-W176CCatalytic oryzanol can achieve the conversion of ferulic acid, while ferulic acid esterase FAE P171C-W176C After heat treatment at 50 °C for 30 min, the enzyme activity was detected. The residual enzyme activity of the wild-type codon-optimized ferulic acid esterase was marked as 100%, and the ferulic acid esterase FAE P171C-W176C The residual enzyme activity was 160.61%, which was 60.61% higher than the residual activity of the control ferulic acid esterase FAE after heat treatment.

[0020] The present invention verified that using the ferulic acid esterase mutant FAE P171C-W176C-M19W to catalyze oryzanol, the yield of ferulic acid increased during the conversion by the mutant enzyme, and the yield of ferulic acid reached 84.44%.

[0021] The present invention also verified that the site-directed mutant enzyme FAE P171C-W176C-M19W-N12I catalyzes oryzanol in rice bran oil foot, and the yield of ferulic acid by the mutant enzyme increases. The site-directed mutation is obtained by mutating proline at position 171 of ferulic acid esterase to cysteine, tryptophan at position 176 to cysteine, methionine at position 19 to tryptophan, and asparagine at position 12 to isoleucine.

[0022] Among them, using the ferulic acid esterase mutant FAE P171C-W176C-M19W-N12I to catalyze oryzanol in rice bran oil, the yield of ferulic acid reached 90.32%.

[0023] Beneficial effects

[0024] (1) To obtain an expression strain producing ferulic acid esterase, a strain with excellent performance, stable and high yield needs to be selected as the host strain. Although Escherichia coli is easy to operate, it lacks a post-translational modification system for genes, and there is not enough time after translation for the target protein to fold into the correct structure, easily forming inactive inclusion bodies. Pichia pastoris not only has simple gene manipulation, but also can perform necessary post-translational modifications on exogenous ferulic acid esterase to ensure that ferulic acid esterase has biological activity. Therefore, choosing Pichia pastoris as the host strain and introducing ferulic acid esterase by genetic engineering means is beneficial to obtaining high-yield and highly active enzymes, providing sufficient catalysts for catalyzing oryzanol.

[0025] (2) Disulfide bonds can enhance the rigidity of the enzyme molecular structure, reduce the entropy value of the enzyme molecule, and are the most crucial factors for stabilizing the local structure of proteins and preventing enzyme inactivation and denaturation caused by high temperature. Existing research has shown that introducing disulfide bonds into enzyme molecules can significantly increase the stability of enzyme molecules. Therefore, using bioinformatics software to analyze the potential disulfide bond sites of ferulic acid esterase and performing molecular modification on ferulic acid esterase to obtain a ferulic acid esterase mutant FAE P171C-W176C .

[0026] (3) Rice bran oil foot is a complex mixture containing oryzanol and many other components. To improve the activity of ferulic acid esterase in catalyzing the release of ferulic acid from oryzanol in rice bran oil foot, the present invention uses bioinformatics software to analyze ferulic acid esterase, mutate the amino acids near the catalytic active center, and obtain FAE P171C-W176C-M19W , and the 19th amino acid of this enzyme is mutated from methionine to tryptophan on the basis of FAE P171C-W176C . The indole ring of tryptophan has stronger hydrophobicity and binds tightly to the benzene ring of ferulic acid through hydrophobic interaction, increasing the affinity between the substrate and the enzyme. The mutated catalytic active center is more accessible to the ester bond of oryzanol. This enzyme uses oryzanol in rice bran oil foot as a substrate for catalytic experiments, and the ferulic acid yield is 80.95%. While Ding JW et al. [Ding JW, Kua GKB, Ng KH, Yang KL. Immobilization of a Recombinant Cholesterol Esterase from Mustela putorius furo for the Hydrolysis of γ-Oryzanol. J Agric Food Chem. 2025 Mar 12;73(10):6018-6029. doi:10.1021 / acs.jafc.4c09820. Epub 2025 Mar 3. PMID:40026141.] used immobilized cholesterol esterase to catalyze the release of ferulic acid from oryzanol, and the yield was only 23.5%.

[0027] (4) Rice bran oil foot is a fat-soluble complex substance, and the solubility of ferulic acid esterase in the medium directly affects the catalytic activity of the enzyme. To further improve the catalytic activity of ferulic acid esterase towards oryzanol, the present invention uses bioinformatics software to analyze the amino acids on the surface of ferulic acid esterase, and increases the solubility of the enzyme in rice bran oil foot by mutating the hydrophilic amino acids on the enzyme surface to hydrophobic amino acids, and obtains FAE P171C-W176C-M19W-N12I , and the 12th amino acid of this enzyme is mutated from asparagine to isoleucine on the basis of FAE P171C-W176C-M19W . Asparagine is a polar amino acid, and when mutated to a hydrophobic amino acid, it enhances the compatibility of ferulic acid esterase in the non-polar environment of the oil foot and increases the solubility of the enzyme molecule in rice bran oil foot. This enzyme uses oryzanol in rice bran oil foot as a substrate for catalytic experiments, and the ferulic acid yield reaches 90.32%. Compared with the enzyme without codon optimization and mutation, the yield increases by 72.47%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 For the molecular docking results of FAE P171C-W176C protein and oryzanol compound;

[0029] Figure 2 Results of the effects of ferulic acid esterase M19 and T77 mutations on ferulic acid yield;

[0030] Figure 3 Results of the effects of ferulic acid esterase N12, E14, R18, R168, D213L, and E245L mutations on ferulic acid yield;

[0031] Figure 4 Results of the catalysis of oryzanol in rice bran oil by ferulic acid esterase and its mutants;

[0032] Figure 5 For pPICZB-FAE P171C-W176C-M19W-N12I Expression vector map. Detailed implementation mode

[0033] The following examples are used to explain the technical solutions of the present invention non-restrictively.

[0034] In the present invention, unless otherwise specified, "%" for indicating concentration is mass percentage.

[0035] The strains and plasmids involved in the examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0036] Table 1 Strains and plasmids involved in the present invention

[0037]

[0038]

[0039]

[0040] Table 2 Primers involved in the present invention

[0041]

[0042]

[0043] Note: Lowercase letters are mutation sites.

[0044] The present invention relates to the following media:

[0045] LLB: 1% peptone, 0.5% yeast extract, 0.5% NaCl; 2% agar powder is added to the solid medium.

[0046] YPD liquid medium: 2% glucose, 1% yeast powder, 2% peptone; 2% agar powder is added to the solid medium.

[0047] BMGY medium: 1% glycerol, 2% tryptone, 1% yeast powder, 1.34% YNB, 4×10 -5% biotin, 10% phosphate buffer at pH 6.0; 2% agar powder is added to the solid medium.

[0048] BMMY medium: 1% yeast powder, 2% tryptone, 1.34% YNB, 4×10 -5 % biotin, 10% phosphate buffer at pH 6.0, 1.5% methanol; 2% agar powder is added to the solid medium.

[0049] In the present invention, unless otherwise specified, the fermentation method of each recombinant Pichia pastoris is as follows:

[0050] The recombinant Pichia pastoris is cultured in 30 ml of BMGY (containing bleomycin resistance) liquid medium at 230 rpm and 30 °C for 16 h to obtain a seed solution. The seed solution is transferred to 50 ml of BMMY medium to make the cell OD 600 reach 1.0, and cultured at 230 rpm and 30 °C. Methanol and CuSO4 are added once every 24 h until the final concentration of methanol is 1% and the final concentration of CuSO4 is 0.1 mM, and cultured for 96 - 120 h.

[0051] In the present invention, unless otherwise specified, the detection method for the enzyme activity of each ferulic acid esterase and its mutant is as follows:

[0052] Dissolve 1 mM oryzanol in 1 ml of reaction buffer (containing 0.1 M sodium phosphate at pH = 7.5, 0.15 M sodium chloride, 0.18 mM sodium taurocholate, and 6% (v / v) acetone), keep it warm in a 35 °C constant temperature water bath for 5 min, immediately add 100 μl of appropriately diluted ferulic acid esterase purified by ProteinIsoRNi-IDA Resin, react the reactants at 30 °C for 30 min, take out the reaction solution and immediately boil to terminate the reaction, centrifuge at 12,000 rpm for 5 min, and analyze the ferulic acid concentration in the supernatant by HPLC after filtering through a 0.22 μm filter membrane. The detection conditions are as follows:

[0053] Aglient 1200 high performance liquid chromatograph, ultraviolet detector, C18 chromatographic column, mobile phase A is 100% methanol (v / v), mobile phase B is 0.1% formic acid, and the gradient elution program is that mobile phase A increases from 10% to 100% in 0 - 15 min, mobile phase A remains at 100% in 15 - 20 min, and mobile phase A decreases from 100% to 10% in 20 - 30 min. The injection volume is 10 μl, the flow rate is 1 ml / min, and the detection wavelength is 320 nm.

[0054] Under this reaction condition, the amount of ferulic acid esterase required to catalyze the formation of 1 μmol of ferulic acid from oryzanol per minute is defined as 1 enzyme activity unit U.

[0055] The method for extracting oryzanol at the start of the reaction is as follows:

[0056] Add 1 ml of ethyl acetate to 1 ml of the reaction starting mixture, vortex for 5 min, centrifuge at 12,000 rpm for 10 min, aspirate the upper organic layer, add 1 ml of ethyl acetate to the lower reaction system for extraction twice more, combine the extracts, dry them with a nitrogen blower, add 1 ml of ethyl acetate and vortex, filter through a 0.22 μm filter membrane, and analyze the oryzanol concentration by HPLC. The detection conditions are as follows:

[0057] Aglient 1200 high-performance liquid chromatograph, ultraviolet detector, C18 chromatographic column, mobile phase C is 0.1% formic acid and 0.05% trifluoroacetic acid (v / v), mobile phase D is 50% acetonitrile and 50% methanol (v / v), the gradient elution program is that mobile phase D remains at 10% from 0 to 5 min, mobile phase D increases from 70% to 100% from 5 to 20 min, the mobile phase remains at 100% from 20 to 55 min, and mobile phase D decreases from 100% to 10% from 55 to 65 min. The injection volume is 10 μl, the flow rate is 1 ml / min, and the detection wavelength is 320 nm.

[0058] Under these reaction conditions, the calculation method of ferulic acid yield is as follows:

[0059]

[0060] C FA —— Ferulic acid concentration at the end of the reaction

[0061] C Ory —— Oryzanol concentration at the start of the reaction

[0062] In the present invention, the rice bran oil foot is from Zhejiang Delakang Food Co., Ltd.

[0063] Construction of a ferulic acid esterase thermostability-increasing mutant in Example 1

[0064] (1) Construction of the mutant vector

[0065] Remove the intron from the original gene of ferulic acid esterase (GenBank: OTA25173.1) derived from Hortaea werneckii and perform codon optimization. The optimized nucleic acid sequence is SEQ ID NO.2, which is synthesized by GenScript Biotech Corporation and ligated into the pPICZB vector digested with EcoRⅠ and SalⅠ to form the vector pPICZB-FAE.

[0066] The 3D structure of FAE (Uniprot accession number A0A1Z5SWH4) was predicted using the Disulfide by Design 2.0 software, and potential disulfide bonds with bond energies (χ 3 angle) between +97 (±30) were screened. A total of 20 pairs were predicted. The 3D structure of feruloyl esterase was observed using Pymol software. The disulfide bonds inside the molecule were discarded, and the potential disulfide bond sites within the range of the catalytic triad (S86-H230-D155) and the ligand were discarded. If the same amino acid formed disulfide bonds with different amino acids, the pair of amino acids with the largest B-factor value of the formed disulfide bond was selected as the research object. Since the disulfide bonds near the edge of the enzyme would not have a great impact on enzyme activity and might greatly improve the thermal stability of the enzyme, the potential disulfide bonds on the surface of the FAE protein were selected. After screening, 10 pairs remained. The Damietta protein design toolkit online software was used for prediction. According to the predicted total energy ΔG total (ΔG total <0 indicating a decrease in total energy and the mutation making the conformation more stable, otherwise it would reduce protein stability), the 3 groups of mutants with the lowest mutation energy were screened out, namely V41C-V56C, P171C-W176C, and G47C-N53C. For each disulfide bond mutant, a combination of two mutation sites was designed, that is, two amino acid residues were mutated to cysteine Cys, and the two Cys might form a disulfide bond. The screened potential disulfide bonds were experimentally verified.

[0067] The above mutation sites were experimentally verified: The full-gold Fast Mutagenesis System kit was used to construct mutation vectors. Using the pPICZB-FAE plasmid as a template, PCR amplification was carried out using the primers V41C-FOR / REV, P171C-FOR / REV, and G47C-FOR / REV in Table 1 respectively to obtain FAE V41C 、FAE P171C and FAE G47CThe single point mutation product was amplified by PCR at 94°C for 3 min; 94°C for 20 s, 55°C for 20 s, 72°C for 2 min, 25 cycles; 72°C for 10 min. The reaction system was as follows: 0.5 μl template, 0.5 μl 10 μM primers, 2×TransStart FastPfu Fly PCR SuperMix 12.5 μl, 11 μl ddH2O. The amplified products were added with 0.5 μl DMT enzyme and incubated at 37°C for 1 h to digest the template plasmid. The digested products were detected by agarose gel electrophoresis. 2 μl of the correct product was transferred into the DMT competent cells provided by the kit using the heat shock method, and then spread on LLB (bleomycin concentration was 100 μg / ml) solid plates and cultured overnight at 37°C in the dark. Single colony transformants were selected for plasmid sequencing. The correct plasmid was named pPICZB-FAE V41C 、pPICZB-FAE G47C and pPICZB-FAE P171C .

[0068] pPICZB-FAE V41C 、pPICZB-FAE G47C and pPICZB-FAE P171C The double-point mutation plasmid pPICZB-FAE was constructed using the full-stranded gold Fast Mutagenesis System kit and amplified with V56C-FOR / REV, N53C-FOR / REV, and W176C-FOR / REV primers. V41C-V56C 、pPICZB-FAE G47C-N53C and pPICZB-FAE P171C-W176C .

[0069] FAE confirmed by sequencing V41C-V56C The amino acid sequence of FAE is shown in SEQ ID NO.4. G47C-N53C The amino acid sequence of FAE is shown in SEQ ID NO.5. P171C-W176C The amino acid sequence is shown in SEQ ID NO.6.

[0070] (2) Construction of mutant strains and protein expression verification

[0071] The correctly sequenced transformants were cultured overnight in LLB (bleomycin concentration was 100 μg / ml), plasmids were extracted, and plasmids pPICZB-FAE and pPICZB-FAE were digested with PmeⅠ. V41C-V56C 、pPICZB-FAE G47C-N53C and pPICZB-FAE P171C -W176CCoagulant recovery: According to the instruction manual of Shanghai Weidi Biotechnology X33 Pichia pastoris competent cells, 1 μg of linearized product was electrotransformed into X33. After 4 h of resuscitation, it was spread on a YPD (bleomycin concentration: 100 μg / ml) resistant plate for screening. After culturing for 72 h, transformants were selected and colony PCR was verified using pPICZB-FOR / REV. The verified strains were named X33-1, X33-2, X33-3, and X33-4, namely Pichia pastoris X33 into which the intron-removed and codon-optimized FAE was transferred, and Pichia pastoris X33 strains into which FAE V41C-V56C , FAE G47C-N53C , and FAE P171C-W176C were transferred respectively.

[0072] Meanwhile, transformants were picked and transferred to 30 ml of BMGY medium, cultured at 30 °C and 230 rpm for 16 h to obtain seed liquid. The seed liquid was transferred to a 50 ml centrifuge tube, centrifuged at 5000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were diluted with BMMY to OD 600 = 1.0. The diluted mixture was transferred to a 250 ml flask and continued to be cultured. Methanol and CuSO4 were added once every 24 h to make the final concentration of methanol 1% and the final concentration of CuSO4 0.1 mM, and cultured at 30 °C and 230 rpm for 96 - 120 h.

[0073] The fermentation broth was centrifuged at 7000 rpm for 4 min at 4 °C, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane and then the protein was purified using a ProteinIsoRNi-IDAResin column. During the purification process, the miscellaneous proteins were eluted with a phosphate buffer containing 20 mM imidazole, and the target protein was eluted with a phosphate buffer containing 250 mM imidazole. The purified protein was detected by SDS-PAGE electrophoresis for the target protein band. The verification results were correct, indicating that FAE, FAE V41C-V56C , FAE G47C-N53C , and FAE P171C-W176C purified enzymes were obtained.

[0074] (3) Determination of mutant protein stability

[0075] The FAE, FAE V41C-V56C , FAE G47C-N53C , and FAE P171C-W176C purified enzymes were taken out after heating at 50 °C for 20 min, ice-bathed for 20 min, and equilibrated at room temperature for 5 min. The content of ferulic acid released from oryzanol catalyzed by the purified enzyme and the 50 °C heat-treated enzyme was detected respectively. Taking the residual activity of FAE as 100%, the residual activities of the purified enzymes of each mutant were compared with the FAE enzyme, and the results are shown in Table 3.

[0076] Table 3 Ferulic acid yield and residual enzyme activity of ferulic acid esterase and mutants after heat treatment at 50 °C

[0077]

[0078]

[0079] As can be seen from Table 3, the ferulic acid yield of the unmutated ferulic acid esterase FAE after heat treatment was only 35.67% ± 2.81%, and the ferulic acid yields of the three different mutants after heat treatment were also only 27.45% - 54.75%; the highest residual enzyme activity after treatment at 50°C was that of FAE P171C-W176C , and the maximum increase in its residual enzyme activity reached 160.61% ± 3.98%. The residual enzyme activity of FAE V41C-V56C slightly increased, reaching 113.4% ± 2.64%. The residual enzyme activity of FAE G47C-N53C decreased to 89% ± 5.66%. While FAE P171C-W176C has the characteristic of increasing residual enzyme activity, its ferulic acid yield is also 53.5% higher than that of FAE. According to the principle of selecting mutant enzymes with improved thermal stability while basically ensuring the ferulic acid yield, FAE P171C-W176C was selected for subsequent experiments.

[0080] Example 2 Construction of Mutants with Increased Substrate Affinity of Ferulic Acid Esterase

[0081] (1) Construction of Mutant Vectors

[0082] The amino acid sequence of FAE P171C-W176C (SEQ ID NO.6) was input into AlphaFold for modeling. The substrate oryzanol molecule (PubChem CID: 5282164) was downloaded from PubChem. Using the FAE P171C-W176C protein model as the receptor and the oryzanol molecule as the ligand, the receptor and ligand were hydrogenated and balanced using Autoduck Vina 1.2.5, and the molecular docking was completed using the docking algorithm. After determining the grid box range according to the docking results, the docking was performed again. The results showed that the receptor molecule and the ligand binding pocket had good spatial steric complementarity characteristics, as shown in Figure 1 .

[0083] Centering on the ligand, the ligand molecule and the catalytic triad (S86 - H230 - D155) Amino acids within the range, excluding N25 and D76 that have formed hydrogen bonds with the substrate, leaving a total of 14 amino acids remaining (M19, N21, L24, P26, G28, I29, V75, T77, G78, R79, F81, L148, T221, L249 respectively). Subject the 14 candidate mutant enzymes to alanine scanning mutagenesis using the Damiettaprotein design toolkit. The results show that mutating M19 and T77 to alanine results in ΔG total with the lowest energy. Again, use the Damietta protein design toolkit to perform saturation mutagenesis on M19 and T77. Select mutations with increased affinity and a mutation energy less than -9 as mutations with a positive effect. A total of 5 mutation sites are selected, namely M19W, M19H, M19C, M19K, and T77A, and conduct experimental verification on the selected mutants.

[0084] Perform site-directed mutagenesis on each of the above mutation sites, using pPICZB-FAE P171C-W176C plasmid as a template, and using M19W-FOR / REV, M19H-FOR / REV, M19C-FOR / REV, M19K-FOR / REV, and T77A-FOR / REV in Table 1 as primers, perform PCR reactions using the TransGen Biotech mutagenesis kit. The specific experimental procedure is the same as the process of constructing the mutant plasmid in Example 1, and sequence verification is performed on the constructed plasmid.

[0085] The correct sequenced FAE P171C-W176C-M19W has an amino acid sequence as shown in SEQ ID NO.7, FAE P171C-W176C-M19H has an amino acid sequence as shown in SEQ ID NO.8, FAE P171C-W176C-M19C has an amino acid sequence as shown in SEQ ID NO.9, FAE P171C-W176C-M19K has an amino acid sequence as shown in SEQ ID NO.10, FAE P171C-W176C-T77A has an amino acid sequence as shown in SEQID NO.11.

[0086] (2) Construction of mutant strains and verification of protein expression

[0087] Transform the correctly sequenced pPICZB-FAE P171C-W176C-M19W , pPICZB-FAE P171C-W176C-M19H , pPICZB-FAE P171C -W176C-M19C , pPICZB-FAE P171C-W176C-M19K , pPICZB-FAE P171C-W176C-T77A, PmeⅠ was used for enzyme digestion and gel recovery, and the mutant plasmid strain construction method in Example 1 was used for construction. The correct strains verified by colony PCR were named X33-5, X33-6, X33-7, X33-8 and X33-9. Transformants were selected and cultured, purified and tested by SDS-PAGE electrophoresis according to the protein expression verification method in Example 1. The verification results were correct, indicating that FAE was obtained. P171C-W176C-M19W 、FAE P171C -W176C-M19H 、FAE P171C-W176C-M19C 、FAE P171C-W176C-M19K and FAE P171C-W176C-T77A Purify the enzyme.

[0088] (3) Determination of catalytic activity of mutant proteins

[0089] FAE, FAE P171C-W176C 、FAE P171C-W176C-M19W 、FAE P171C-W176C-M19H 、FAE P171C-W176C-M19C 、FAE P171C -W176C-M19K 、FAE P171C-W176C-T77A The mixture was equilibrated at room temperature for 5 min. The contents of ferulic acid released by oryzanol catalyzed by the mutant enzyme were detected at 35° C. according to the experimental steps in Example 1. The ferulic acid yield was calculated. Figure 2 .

[0090] Depend on Figure 2 It can be seen that the original enzyme and FAE P171C-W176C The ferulic acid yields were 70.95% and 74.79%, respectively, which were comparable to those of the original enzyme and FAE. P171C-W176C In comparison, FAE P171C-W176C-M19W The ferulic acid yield of the purified mutant enzyme increased to 84.44% ± 1.25%, while FAE P171C-W176C-M19C 、FAE P171C-W176C-M19K and FAE P171C-W176C-T77A The ferulic acid yields of the purified mutant enzymes decreased to 65.46%±2.98%, 67.42%±2.71% and 57.03%±3.81%, respectively. P171C-W176C-M19H Ferulic acid yield and FAE P171C-W176C The yield did not change much, reaching 74.61% ± 2.53%, so FAE was selected P171C-W176C-M19W Conduct follow-up experiments.

[0091] Example 3 Construction of hydrophobic amino acid mutants of ferulic acid esterase

[0092] (1) Construction of mutant vector

[0093] FAE P171C-W176C-M19WThe amino acid sequence (SEQ ID NO.7) was input into AlphaFold for modeling. The 3D modeling structure of the mutant enzyme was downloaded, and Pymol was used to observe the FAE P171C-W176C-M19W The hydrophilic and hydrophobic amino acid distribution was observed. With the ligand as the center, the ligand molecule was removed and the amino acids within the catalytic triad (S86-H230-D155) were removed. A total of 83 hydrophilic amino acids remained. Site-directed mutagenesis was performed on the 83 candidate amino acids following the principle of mainly changing the hydrophilicity and hydrophobicity of amino acids while minimizing the change of other physical and chemical properties. The mutant enzyme was predicted using the Damietta protein design toolkit. The results showed that the mutant enzymes with N12I, E14L, R18L, R168L, D213L, and E245L mutations had the lowest ΔG total energy. The selected mutants were experimentally verified.

[0094] Site-directed mutagenesis was performed on each of the above mutation sites. Using the pPICZB-FAE P171C-W176C-M19W plasmid as a template and the N12I-FOR / REV, E14L-FOR / REV, R18L-FOR / REV, R168L-FOR / REV, D213L-FOR / REV, and E245L-FOR / REV in Table 1 as primers, PCR reactions were carried out using the TransGen mutagenesis kit. The specific experimental process was the same as that of the mutant plasmid construction in Example 1. The constructed plasmids were verified by sequencing.

[0095] The amino acid sequence of the correctly sequenced FAE P171C-W176C-M19W-N12I is shown in SEQ ID NO.12, the amino acid sequence of FAE P171C -W176C-M19W-E14L is shown in SEQ ID NO.13, the amino acid sequence of FAE P171C-W176C-M19W-R18L is shown in SEQID NO.14, the amino acid sequence of FAE P171C-W176C-M19W-R168L is shown in SEQ ID NO.15, the amino acid sequence of FAE P171C -W176C-M19W-D213L is shown in SEQ ID NO.16, the amino acid sequence of FAE P171C-W176C-M19W-E245L is shown in SEQID NO.17.

[0096] (2) Construction of mutant strains and verification of protein expression

[0097] The correctly sequenced pPICZB-FAE P171C-W176C-M19W-N12I , pPICZB-FAE P171C-W176C-M19W-E14L , pPICZB-FAE P171C-W176C-M19W-R18L , pPICZB-FAE P171C-W176C-M19W-R168L, pPICZB-FAE P171C-W176C-M19W-D213L and pPICZB-FAE P171C-W176C-M19W-E245L Use PmeⅠ to digest and recover by gel electrophoresis respectively, and construct them using the method for constructing mutant plasmid strains in Example 1. The strains verified correctly by colony PCR were named X33-10, X33-11, X33-12, X33-13, X33-14, and X33-15 respectively. Pick the transformants respectively and carry out culture, purification, and SDS-PAGE electrophoresis detection according to the protein expression verification method in Example 1. The verification results are correct, indicating that FAE P171C-W176C-M19W-N12I , FAE P171C-W176C-M19W-E14L , FAE P171C -W176C-M19W-R18L , FAE P171C-W176C-M19W-R168L , FAE P171C-W176C-M19W-D213L and FAE P171C-W176C-M19W-E245L purified enzymes.

[0098] (3) Determination of catalytic activity of mutant proteins

[0099] Equilibrate FAE, FAE P171C-W176C-M19W , FAE P171C-W176C-M19W-N12I , FAE P171C-W176C-M19W-E14L , FAE P171C -W176C-M19W-R18L , FAE P171C-W176C-M19W-R168L , FAE P171C-W176C-M19W-D213L and FAE P171C-W176C-M19W-E245L at room temperature for 5 min, and detect the content of ferulic acid released from oryzanol by the mutant enzymes respectively under the experimental conditions in Example 1 at 35℃, and calculate the ferulic acid yield. The results are shown in Figure 3 .

[0100] It can be seen from Figure 3 that the ferulic acid yields of the original enzyme and FAE P171C-W176C-M19W reach 72.83% and 85.27% respectively. Compared with the original enzyme and FAE P171C-W176C-M19W , the ferulic acid yields of the purified enzymes of FAE P171C-W176C-M19W-N12I and FAE P171C-W176C-M19W-R168L mutants change little, reaching 86.70%±1.25% and 86.04%±3.64% respectively, while the ferulic acid yields of the purified enzymes of FAE P171C-W176C-M19W-E14L , FAE P171C-W176C-M19W-R18L , FAE P171C-W176C-M19W-D213L and FAE P171C-W176C-M19W-E245L mutants decrease to 68.99%±2.97%, 66.11%±2.54%, 58.18±4.14%, and 65.06%±2.47% respectively. The decrease in yield may be due to the change in surface charge distribution affecting substrate binding. Therefore, FAE P171C-W176C-M19W-N12I and FAE P171C -W176C-M19W-R168L were selected for subsequent experiments.

[0101] Example 4 Catalysis of Oryzanol in Rice Bran Oil Foot by Ferulic Acid Esterase Mutant

[0102] Weigh 10 g of rice bran oil foot into a beaker, add 0.3 g of sodium taurocholate, place it on a magnetic stirrer with a rotation speed of 200 rpm, preheat it at 35 °C for 10 min. After the preheating is completed, add 100 μl of purified ferulic acid esterase FAE, FAE P171C -W176C-M19W , FAE P171C-W176C-M19W-N12I and FAE P171C-W176C-M19W-R168L respectively, and react on a magnetic stirrer at 35 °C for 1 h. Detect the content of ferulic acid released by the mutant enzyme catalyzing oryzanol according to the detection method in Example 1, and calculate the ferulic acid yield. The results are shown in Figure 4 .

[0103] As can be seen from Figure 4 the results, the ferulic acid yield of the unmutated ferulic acid esterase FAE is 52.37% ± 2.06%. After introducing a disulfide bond and mutating the amino acids near the active center in site-directed mutant FAE P171C-W176C-M19W , the ferulic acid yield increased to 80.95% ± 1.24%. The ferulic acid yields of FAE P171C-W176C-M19W-N12I and FAE P171C-W176C-M19W-R168L increased to 90.32% ± 2.84% and 87.12% ± 5.79% respectively. Thus, it can be seen that the mutants FAE P171C-W176C-M19W-N12I and FAE P171C-W176C-M19W-R168L significantly improved the yield of ferulic acid catalytically released from oryzanol in rice bran oil foot.

[0104] The ferulic acid esterase mutants FAE P171C-W176C-M19W , FAE P171C-W176C-M19W-N12I and FAE P171C -W176C-M19W-R168L constructed in the present invention effectively improved the yield of ferulic acid catalytically released using oryzanol in rice bran oil foot as a substrate. Among them, FAE P171C-W176C-M19W-N12I had the best effect on catalyzing oryzanol to produce ferulic acid and had good application prospects.

[0105] The present invention verified through experiments that not any mutation can improve the thermal stability of ferulic acid esterase after introducing disulfide bond mutations. Among them, the residual enzyme activity of the mutant enzyme FAE P171C-W176C was improved compared with the wild-type enzyme FAE after heat treatment at 50 °C.

[0106] In order to further improve the yield of ferulic acid esterase catalyzing oryzanol in rice bran oil foot, it is necessary to improve the affinity and solubility of ferulic acid esterase with the substrate oryzanol in rice bran oil foot. Therefore, site-directed mutations were carried out on the amino acids near the catalytic active center and the hydrophilicity and hydrophobicity of the amino acids on the surface of ferulic acid esterase. Especially, the ferulic acid esterase mutant FAE P171C-W176C-M19W, FAE P171C-W176C-M19W-N12I and FAE P171C-W176C-M19W-R168L , The experimental results show that this method can effectively improve the yield of ferulic acid catalyzed with oryzanol as the substrate.

Claims

1. Ferulic acid esterase mutant FAE P171C-W176C , the mutant is obtained by mutating the 171st amino acid of ferulic acid esterase from proline to cysteine and the 176th amino acid from tryptophan to cysteine, and the amino acid sequence of the mutant is shown in SEQ ID NO.

6.

2. Use of the ferulic acid esterase mutant FAE according to claim 1 P171C-W176C in the preparation of ferulic acid.

3. An expression vector or engineered strain containing the ferulic acid esterase mutant FAE as described in claim 1 P171C-W176C .

4. Ferulic acid esterase mutant FAE P171C-W176C-M19W , the mutant is obtained by mutating the 171st amino acid of ferulic acid esterase from proline to cysteine, the 176th amino acid from tryptophan to cysteine, and the 19th amino acid from methionine to tryptophan. The amino acid sequence of the mutant is shown in SEQ ID NO.

7.

5. Containing the ferulic acid esterase mutant FAE as described in claim 4 P171C-W176C-M19W Expression vector or engineered strain.

6. Use of the ferulic acid esterase mutant FAE according to claim 4 P171C-W176C-M19W in the preparation of ferulic acid.

7. Ferulic acid esterase mutant FAE P171C-W176C-M19W-N12I , the mutant is obtained by mutating the 171st amino acid of ferulic acid esterase from proline to cysteine, the 176th amino acid from tryptophan to cysteine, the 19th amino acid from methionine to tryptophan, and the 12th amino acid from asparagine to isoleucine. The amino acid sequence of the mutant is shown in SEQ ID NO.

12.

8. An expression vector or engineered strain containing the ferulic acid esterase mutant FAE as described in claim 7. P171C-W176C-M19W-N12I ​ 9. Use of the ferulic acid esterase mutant FAE according to claim 7 P171C-W176C-M19W-N12I in the preparation of ferulic acid.

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