High-temperature-resistant feruloyl esterase and application thereof

Abstract

The invention provides a high temperature resistant feruloyl esterase and an application thereof. The feruloyl esterase wild type has a remarkable high-temperature-resistant characteristic and still shows relatively high thermal stability under the condition of 95 DEG C, and the thermal inactivation half-life period of the feruloyl esterase wild type can reach 5h. On the basis, after the feruloyl esterase mutant obtained through structural rationality modification continuously reacts for 5 h at the temperature of 75 DEG C, floccule precipitation is not observed, and stable catalytic activity is kept. In the application, the feruloyl esterase mutant disclosed by the invention and commercial xylanase have a synergistic effect, so that the release of bound ferulic acid in starch-removed wheat bran can be effectively promoted. The invention provides a novel enzymology tool for efficiently and quickly releasing the plant-derived ferulic acid under a high-temperature condition.

Description

Technical Field

[0001] This invention relates to a heat-resistant ferulic acid esterase and its application, belonging to the field of ferulic acid esterase preparation technology. Background Technology

[0002] Ferulic acid is a class of phenolic acid compounds widely found in plant cell walls. It possesses antioxidant, anti-inflammatory, and antibacterial properties, as well as potential health and pharmaceutical applications, and has been widely used in food, pharmaceuticals, cosmetics, and functional materials. However, in natural plant raw materials, ferulic acid mostly exists in the form of ester bonds bound to polysaccharides such as arabinoxylan and hemicellulose, making it difficult to release efficiently through physical or simple chemical methods.

[0003] Currently, the main industrial methods for releasing ferulic acid include alkaline hydrolysis, acid hydrolysis, and enzymatic hydrolysis. Chemical hydrolysis methods suffer from drawbacks such as harsh reaction conditions, severe equipment corrosion, numerous byproducts, and a heavy environmental burden. In contrast, enzymatic hydrolysis, due to its mild conditions, high specificity, and environmental friendliness, is considered the ideal route for releasing ferulic acid. Feruloylesterase (FAE) is a class of hydrolases that specifically hydrolyze the ester bonds between ferulic acid and polysaccharides. However, most reported feruloylesterases are derived from fungi or mesophilic microorganisms, with their optimal temperatures typically between 40–60°C. They exhibit poor thermal stability and are easily inactivated under high-temperature conditions, making them unsuitable for the high-temperature operating environments (such as above 80°C) commonly encountered in industrial processes.

[0004] Furthermore, the complex structure of plant cell walls often makes it difficult for a single enzyme system to fully disrupt the polysaccharide backbone. Previous studies have shown that hemicellulose-degrading enzymes such as xylanase can break down the polysaccharide backbone, providing more action sites for ferulic acid esterase. However, mature technical solutions for achieving multi-enzyme synergistic and efficient release of ferulic acid under high-temperature conditions are still lacking. Therefore, developing a ferulic acid esterase with excellent heat resistance, stable catalytic activity under high-temperature conditions, and the ability to synergistically act with xylanase to promote the release of ferulic acid from plant materials, along with its application methods, is of great significance. Summary of the Invention

[0005] This invention provides a heat-resistant ferulic acid esterase and its application, aiming to solve the technical problems of low activity, poor stability, and limited ferulic acid release efficiency of existing ferulic acid esterases under high-temperature industrial conditions and complex substrates.

[0006] The present invention adopts the following technical solution: This invention provides a thermoresistant ferulic acid esterase, wherein the ferulic acid esterase is selected from any of the following: (a) A protein with the amino acid sequence shown in SEQ ID NO:2; (b) A derivative protein of SEQ ID NO:2 having a mutant with one or more amino acid residues substituted, deleted or added, and having ferulic acid esterase activity, and having a heat-inactivating half-life of not less than 10 hours at 75°C.

[0007] In some embodiments, the thermoresistant ferulic acid esterase has a thermal inactivation half-life of not less than 20 hours at 75°C.

[0008] In some embodiments, the thermoresistant ferulic acid esterase has a thermal inactivation half-life of not less than 40 hours at 75°C.

[0009] In some embodiments, the thermoresistant ferulic acid esterase has a relative enzyme activity of not less than 30% after incubation at 99°C for 180 min.

[0010] In some embodiments, the thermostable ferulic acid esterase retains no less than 50% of its initial enzyme activity after continuous reaction at 75°C for 5 hours, and no visible protein flocculent precipitate is formed.

[0011] In some embodiments, the mutant is an F56Y mutant or an R164Q mutant.

[0012] A method for promoting the release of ferulic acid from plant materials, using the above-mentioned thermostable ferulic acid esterase for catalytic reaction.

[0013] The beneficial effects of this invention are as follows: The described ferulic acid esterase exhibits significantly superior heat resistance compared to conventional ferulic acid esterases, enabling it to adapt to high-temperature industrial reaction conditions. It can maintain catalytic activity for extended periods under high-temperature conditions and, when synergistically combined with xylanase, can effectively promote the release of bound ferulic acid from destarched wheat bran. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0015] Figure 1 : Gel image of ferulic acid esterase purification. Wherein, Proteinsupernatant: represents the protein supernatant after lysis and centrifugation; 300IM: represents the protein eluted with elution buffer containing 300IM.

[0016] Figure 2Figure 1: Thermostatity assay of ferulic acid esterase; where A is the optimal reaction temperature of wild-type ferulic acid esterase, B is the thermal stability of wild-type ferulic acid esterase under different temperature conditions, C is the relative enzyme activity of purified F56Y mutant after incubation at 75℃ for different times, and D is the SDS-PAGE analysis of commercial heat-resistant bovine serum albumin (BSA) and purified F56Y mutant after incubation at 75℃ for different times.

[0017] Figure 3 Selective precipitation of host protein in the crude enzyme system of mutant R164Q during high-temperature catalysis. In the figure, A is the SDS-PAGE analysis of the supernatant and precipitate of the crude enzyme preparation of mutant R164Q collected at different time points, and B is the enzyme activity of mutant R164Q in the supernatant, which was determined using p-nitrophenyl ferulic acid ester (pNPF) as substrate.

[0018] Figure 4 Graph showing the catalytic activity of ferulic acid esterase on different substrates. (The graph includes information on:) p NPC2 (p-nitrophenol acetate). p NPC4 (p-nitrophenol butyrate) p NPC8 (p-nitrophenol octanoate), p NPC12 (p-nitrophenol lauryl ester).

[0019] Figure 5 Figure: Effects of common metal ions and organic solvents on ferulic acid esterase activity; where A and C represent the effects on wild-type ferulic acid esters, and B and D represent the effects on ferulic acid ester R164Q.

[0020] Figure 6 Effect of pH on wild-type ferulic acid esterase activity.

[0021] Figure 7 The graph shows the effect of ferulic acid esterase mutant combined with xylanase in promoting the production of ferulic acid from destarched wheat bran. The top row shows the peak position of ferulic acid standard at 4.83 min, and the bottom row shows the release of ferulic acid catalyzed by R164Q and xylanase. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 a part of the embodiments of the present invention, not all of them. 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. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0023] Example 1: Expression and purification of wild-type ferulic acid esterase A ferulic acid esterase gene (BaFAE) derived from archaea groups widely distributed in marine and terrestrial anaerobic sedimentary environments was obtained through metagenomic screening (NCBI accession number: HDO42343.1). The gene sequence is as follows: The gene was constructed into the expression vector pET-28a. After gene sequencing confirmed the correct construction of the recombinant plasmid, the plasmid was transformed into *E. coli* using the heat shock method. Escherichiacoli BL21(DE3) competent cells.

[0024] The specific procedures are as follows: Remove BL21(DE3) competent cells from the -80℃ freezer and immediately thaw them on ice. Add an appropriate amount of recombinant plasmid to 50 μL of competent cells, mix gently, and incubate on ice for 25 min. Then, heat shock the mixture in a 42℃ water bath for 90 s, and quickly transfer it to ice to cool for 2 min. After heat shock, add an appropriate amount of LB liquid medium, mix gently, and revive at 37℃. Spread the revive bacterial culture onto LB solid medium containing kanamycin and incubate overnight at 37℃.

[0025] The following day, a single positive clone was picked and inoculated into LB liquid medium, and cultured with shaking at 37°C and 180 rpm for expansion. When the OD of the culture medium... 600 When the concentration reached approximately 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.3 mM for induction, and the mixture was cultured at 18°C ​​for another 14 h to induce the expression of the target protein.

[0026] The bacterial culture was collected, sonicated, and the target protein was purified by nickel column affinity chromatography. Protein purity was estimated by 15% SDS-PAGE electrophoresis. The protein was recovered using 50 mM Tris-HCl, 150 mM NaCl, and 350 mM imidazole elution buffer (pH=8.0). The protein was then desalted and deimidazole-free using 10 kDa concentration tubes and 50 mM Tris-HCl (pH=8.0) buffer to obtain purified BaPAE enzyme solution (wild-type ferulic acid esterase). Figure 1 ).

[0027] The amino acid sequence of this enzyme, as determined by sequencing, is as follows: MFLIEEKLLLDHYGVLGLIKPVTFENKGQQIIGILHTPDALKSGKKAPGIVMFHGFTGNKTEAHRLFVHVARSLSEAGFIVLRFDFRGSGDSDGEFEDMTVPDEVSDAEKALTFLMEQENVDEERVGILGLSMGGRVAAILASKDR RLKFAILYSPALGPLKERFFSQMSKEKLEKLDSGEPIEVSSGWYLKKKFFETVDYIVPLNIMDRIEVPVLIVHSDKDEIIPLEEALRGYELIKDLNEKNELYVVKGGDHTFSKREHTLEVIRKTLDWINSLNLG (SEQ ID NO: 2).

[0028] Example 2: Expression and purification of mutant ferulic acid esterase This embodiment constructed a BaFAE mutant. Using the wild-type ferulic acid esterase gene from Example 1 as a template, mutants F56Y (phenylalanine at position 56 is mutated to tyrosine) and R164Q (arginine at position 164 is mutated to glutamine) were constructed using site-directed mutagenesis. They were expressed and purified using the same method as in Example 1.

[0029] Example 3: Determination of the heat resistance of ferulic acid esterase The enzymatic properties of ferulic acid esterase were determined by measuring the absorbance change caused by the hydrolysis of the artificial substrate p-nitrophenylferulate (pNPF) to p-nitrophenol (pNP). pNPF was prepared as a 100 mM stock solution in DMSO, stored at -20°C protected from light, and freshly diluted before use. The enzyme activity assay system consisted of 525 μL buffer (pH 8.0 Tris-HCl, containing 2.5% Triton X-100) + 60 μL final concentration 7 mM (pNPF) + 15 μL (enzyme solution). The enzymatic reaction was carried out at 95°C for 10 min, and immediately terminated by adding 600 μL of 2% SDS solution. After termination, the sample was thoroughly mixed and cooled to room temperature, and the absorbance was measured at 410 nm. The amount of pNP generated was calculated using a standard curve plotted under the same termination conditions. All reactions included a heat-inactivated enzyme as a blank control group, and the actual enzyme activity signal was obtained by subtracting background absorbance.

[0030] Enzyme activity unit definition: Under optimal reaction conditions, the amount of enzyme required to produce 1 μmol / min of p-nitrophenol is defined as one enzyme activity unit.

[0031] Optimal temperature and temperature stability determination: The purified enzyme solution was reacted under gradient temperature conditions (50℃, 60℃, 70℃, 80℃, 90℃, 95℃, and 99℃). Using the above enzyme activity reaction system as a baseline, a reaction system was constructed in 1.5mL EP tubes, with an equal volume of heat-inactivated enzyme as a blank control. The tubes were incubated in a metal bath for 10 min, and the reaction stop solution (2% SDS) was added. The mixture was then transferred to an ELISA plate, and the kinetic detection program of the ELISA reader was immediately started to calculate the relative enzyme activity (%). All experimental data were performed in triplicate. Using the activity of the unincubated enzyme solution as 100%, enzyme activities of enzyme solutions with different incubation times were measured in the reaction system to verify the enzyme's thermal stability and calculate the relative enzyme activity. All experiments were performed in triplicate.

[0032] like Figure 2 As shown in Figure A, the wild-type ferulic acid esterase prepared in Example 1 has an optimal temperature as high as 95°C and exhibits high activity between 80°C and 99°C, reaching more than 50% of its optimal enzyme activity. However, at 70°C, it only reaches 19.14% of its maximum enzyme activity, indicating that it is a typical thermostable enzyme. Meanwhile, experiments determining the half-life at different temperatures showed (e.g.) Figure 2 As shown in Figure B, the half-life reaches 71 hours at 75°C and 180 minutes at 99°C. This series of data indicates that the enzyme has extremely high thermal stability and has extremely high commercial application value.

[0033] The high-temperature resistance of the F56Y mutant is as follows: Figure 2 As shown in Figure C. The results showed that during the first 5-hour high-temperature treatment cycle, the F56Y mutant maintained a high activity level overall and did not exhibit rapid inactivation. Notably, centrifugation of the samples after the high-temperature incubation did not result in significant protein precipitation (e.g., ...). Figure 2 (As shown in D), indicating that the enzyme maintains good soluble state under these conditions. These results further demonstrate that the ferulic acid esterase F56Y mutant can maintain a stable structural conformation and catalytic function under conditions of high temperature, low ionic strength, and the absence of additional stabilizers. This excellent thermostability exhibited in simplified buffer systems provides important support for its direct application in the construction of high-temperature crude enzyme systems and in actual biomass conversion processes.

[0034] The high-temperature resistance of the R164Q mutant is as follows: Figure 3As shown. UV scanning revealed that the R164Q mutant catalyzed both methyl and ethyl ferulate hydrolysis in a 10-minute reaction system. Furthermore, using pNPF as a substrate, it exhibited the same thermostability as the wild type. Given that the R164Q mutant maintained high-temperature stability while demonstrating more defined substrate hydrolysis behavior, this mutant was used as a representative for subsequent crude enzyme system construction and performance evaluation, and validation was performed using a crude extraction method involving only ultrasonic disruption. SDS-PAGE analysis results showed ( Figure 3 A) After high-temperature ultrasonic treatment, most non-thermostable proteins from the host were mainly distributed in the precipitate, while the characteristic band corresponding to the target ferulic acid esterase mutant remained clear and stable in the supernatant. This indicates that the high-temperature treatment strategy can achieve relative enrichment of the target functional enzyme in the crude enzyme system without relying on chromatographic purification steps.

[0035] Based on this, the changes in enzyme activity of the obtained R164Q mutant crude enzyme solution after continuous incubation at 75℃ for 5 h were evaluated. The results showed that ( Figure 3 (B) The crude enzyme system maintained over 100% of its initial enzyme activity throughout the incubation process. Notably, after 1-2 hours of incubation, the relative enzyme activity of the R164Q mutant crude enzyme solution showed a certain degree of recovery, approaching or even slightly exceeding the initial level. This phenomenon indicates that in a minimally invasive system containing 50 mM Tris-HCl and 10% glycerol buffer (pH 8.0), this thermostable ferulic acid esterase can maintain good solubility and catalytic function for a long period against a complex protein background. These results further validate the feasibility and engineering application potential of constructing a high-temperature crude enzyme process system based on the enzyme's inherent thermal stability.

[0036] Example 4: Determination of the catalytic activity of ferulic acid esterase on different substrates When determining the hydrolytic activity of the wild-type ferulic acid esterase prepared in Example 1 against p-nitrophenyl ester substrates of different chain lengths, Tris-HCl buffer (pH 8.0, containing 2.5% Triton X-100) was used as the reaction system. The total reaction volume was 600 μL, including 525 μL of buffer and 60 μL of substrate working solution (respectively...). p NPC2 p NPC4 p NPC8 or p NPC12 (to a final concentration of 10 mM) and 15 μL of enzyme solution were added. The reaction mixture was placed in a 95°C metal bath and reacted for 10 min. Immediately afterwards, 600 μL of 2% SDS was added to terminate the reaction. After inverting and mixing, the OD was measured. 410A blank control without enzyme was set up to subtract the background of spontaneous substrate hydrolysis, while a heat-inactivated enzyme was used as a control to eliminate non-specific light absorption interference. Relative enzyme activities were calculated, and all experiments were conducted in triplicate. Figure 4 As shown, this wild-type ferulic acid esterase exhibits significant differences in substrate selectivity for p-nitrophenyl esters of different chain lengths. p The catalytic activity of NPC8 was set to 100%, and the enzyme was used to... p NPC4 exhibited moderate catalytic activity, while... p NPC2 and p The low catalytic activity of NPC12 indicates that the enzyme can recognize fatty acid ester substrates of different chain lengths, has a relatively wide substrate adaptability, and is consistent with the catalytic characteristics of typical esterases.

[0037] Example 5: Effects of common metal ions and organic solvents on enzyme activity The effect of metal ions on enzyme activity: Purified wild-type BaFAE enzyme solution was mixed with various common metal ions and incubated at the optimum temperature (95℃) for 10 min. The effect of metal ions on enzyme activity was measured using the enzyme activity reaction system as a baseline and the enzyme activity without metal ions as 100%. The enzyme reaction system was constructed in 1.5 mL EP tubes, with heat-inactivated ferulic acid esterase as a blank control. The tubes were incubated at 95℃ for 10 min, then transferred to a microplate, and reaction stop solution (2% SDS) was added. The microplate reader kinetic detection program was immediately started, and the absorbance (OD value) was recorded once at a wavelength of 410 nm. The background absorbance value of the blank control was subtracted, and the relative enzyme activity (%) was calculated. All experimental data were performed in triplicate. Effects of organic solvents on enzyme activity: The effects of dimethyl sulfoxide (DMSO), ethanol, acetone, methanol, and n-hexane on enzyme activity were determined at final concentrations of 10% and 25%. Residual enzyme activity was measured after the enzyme solution was incubated at 95°C for 10 min. Relative enzyme activity (%) was calculated without adding any organic solvents as a control sample. Figure 5 As shown, the wild-type ferulic acid esterase prepared in Example 1 can tolerate high concentrations of 10 mM Ca. 2+ K + and Na + ion( Figure 5 -A), capable of tolerating low concentrations of 1 mM Fe 3+ Al 3+ Ca 2+ K + And interference from Na+, but high concentrations of metal ions still have a strong inhibitory effect on enzyme activity. Figure 5 Except for Tween 80 and Triton X-100, other organic solvents showed strong inhibitory effects on ferulic acid esterase and mutants. Figure 5-C,D).

[0038] Example 6: pH dependence of ferulic acid esterase The pH dependence of the enzyme was further investigated in the substrate reaction system of Example 1. Experimental results showed that ( Figure 6 This wild-type ferulic acid esterase exhibits the highest catalytic activity under weakly alkaline conditions, with an optimal pH range of approximately 7.5–8.0, and maintains a high relative activity level within this pH range. Enzyme activity gradually decreases as the pH deviates from this range. These results indicate that the enzyme has good compatibility with commonly used process systems based on Tris-HCl or phosphate buffer.

[0039] Example 7: Ferulic acid esterase mutant combined with xylanase promotes ferulic acid production from destarched wheat bran. In an enzymatic reaction system using destarched wheat bran as a natural substrate, 7.5 wt% destarched wheat bran was added to a system with 20 mM phosphate buffer as the reaction medium, the pH of which was 8.0. Under the above conditions, commercial xylanase (enzyme activity 100,000 U / g, purchased from Maclean's, CAS No.: 9025-57-4) at a final concentration of 5 mg / mL was used for pretreatment at 50°C for 1 h. At the beginning of the pretreatment, ferulic acid esterase (F56Y mutant) prepared in Example 2 at a final concentration of 25 μL / mL was added. During the pretreatment, the ferulic acid esterase had already begun to catalyze the reaction. Then, the temperature was increased to 75°C, which is more suitable for industrial applications, for a more efficient catalytic reaction. The reaction was then monitored (…). Figure 7 The highest concentration of ferulic acid released in the reaction solution can reach 211.37±6.07μg / mL, corresponding to a unit substrate ferulic acid release of approximately 2.21±0.04mg / g.

[0040] The above results demonstrate that the ferulic acid esterase described in this invention maintains stable catalytic activity under alkaline and high-temperature conditions, directly acting on real plant cell wall substrates such as destarched wheat bran to promote the cleavage of ester bonds between ferulic acid and polysaccharides, thereby achieving effective release of ferulic acid. The reaction conditions are highly compatible with commonly used industrial high-temperature pretreatment and continuous reaction processes.

[0041] In existing technologies, although some literature reports that ferulic acid esterases derived from thermophilic microorganisms possess a certain degree of heat resistance, their high-temperature stability is generally still in the range of minutes or low hours. For example, publicly reported ferulic acid esterases derived from... Thermoanaerobactertengcongensis The ferulic acid esterase has a half-life of approximately 50 minutes at 80°C and can only maintain catalytic activity for a short time at 75°C. Although the above enzymes are considered to be thermostable, their ability to operate continuously at high temperatures is still insufficient to meet the long-term stable operation requirements of high-solids systems or high-temperature process windows.

[0042] Compared with the prior art, the ferulic acid esterase described in this invention has a half-life of approximately 71 hours at 75°C, exhibiting a significantly extended level of high-temperature stability, with the stability time increasing from minutes to hours or even days. This performance difference is not simply an increase in the optimal temperature value, but a comprehensive manifestation of maintaining structural integrity and catalytic activity under long-term high-temperature operation. Due to this significantly enhanced thermal stability, this invention achieves for the first time the selective thermal denaturation and precipitation of host proteins under high-temperature conditions, while the target enzyme remains soluble and active, thus constructing a system for direct application of the crude enzyme without chromatographic purification. This technical effect has not been publicly reported in the prior art, nor could it be reasonably expected by those skilled in the art based on existing literature on thermostable enzymes; therefore, it possesses significant and unexpected technical benefits.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A thermostable feruloyl esterase, characterized in that, The ferulic acid esterase is selected from any of the following: (a) A protein with the amino acid sequence shown in SEQ ID NO:2; (b) A derivative protein of SEQ ID NO:2 having a mutant with one or more amino acid residues substituted, deleted or added, and having ferulic acid esterase activity, and having a heat-inactivating half-life of not less than 10 hours at 75°C.

2. The thermoresistant ferulic acid esterase according to claim 1, characterized in that, Its thermal deactivation half-life at 75℃ is not less than 20 hours.

3. The thermoresistant ferulic acid esterase according to claim 1, characterized in that, Its thermal deactivation half-life at 75℃ is not less than 40 hours.

4. The thermoresistant ferulic acid esterase according to claim 1, characterized in that, Its relative enzyme activity is not less than 30% after incubation at 99℃ for 180 min.

5. The thermoresistant ferulic acid esterase according to claim 1, characterized in that, After reacting continuously at 75°C for 5 hours, it maintained no less than 50% of its initial enzyme activity and did not form visible protein flocculent precipitate.

6. The thermostable ferulic acid esterase according to claim 1, characterized in that, The mutant is either the F56Y mutant or the R164Q mutant.

7. A method for promoting the release of ferulic acid from plant materials, characterized in that, The reaction was catalyzed using the thermostable ferulic acid esterase according to any one of claims 1-6.

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