A method for producing ferulic acid by dual-enzyme catalysis using a deep eutectic solvent as a co-solvent

Abstract

The present invention discloses a method for producing ferulic acid by dual-enzyme catalysis using a deep eutectic solvent as a co-solvent, which comprises the following steps: using a quaternary ammonium salt as a hydrogen bond acceptor and urea, polyol or carboxylic acid as a hydrogen bond donor, mixing them in a molar ratio of 1:(2-3) and performing rotary evaporation to obtain a deep eutectic solvent; dissolving ferulic acid esterase and xylanase in an aqueous solution of the deep eutectic solvent to form a dual-enzyme system; using de-starched wheat bran as a substrate and reacting in the dual-enzyme system to obtain ferulic acid. Compared with the enzymatic production of ferulic acid in an aqueous phase, the thermal stability and pH stability of ferulic acid esterase are improved by using this method, and the amount of ferulic acid produced by enzymatic reaction per unit time and per unit enzyme activity is significantly increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method for producing ferulic acid by double enzyme catalysis using a deep eutectic solvent as a cosolvent. Background Art

[0002] Ferulic acid widely exists in plant cell walls and has a wide range of uses. It is used as a precursor for the production of vanillin, p-hydroxybenzoic acid, and 4-vinylphenol in the food industry; ferulic acid has anti-inflammatory and analgesic effects as a drug in the pharmaceutical industry. Currently, the industrial method for extracting ferulic acid from plant cell walls is usually the alkaline lye extraction method, that is, the spatial structure of the raw material is lysed by alkaline lye at high temperature to release ferulic acid, and then an organic solvent is added for extraction. However, a large amount of wastewater and waste residue are generated during the production process, causing a large amount of environmental pollution. The enzymatic hydrolysis method has mild reaction conditions and is green and environmentally friendly, so it has received extensive attention from researchers.

[0003] Ferulic acid esterase and xylanase are usually used in combination to degrade lignocellulose to produce ferulic acid. The sugar chain structure is cleaved by glycosidase to expose the ferulic acid group, facilitating the action of ferulic acid esterase. However, free enzymes are easily inactivated in an aqueous medium, greatly increasing the production cost. To overcome this defect, researchers have done a lot of work, including immobilized enzymes, raw material pretreatment, etc. However, these methods usually require high investment, limiting their industrial application.

[0004] Solvent engineering has been widely favored by researchers due to its high efficiency and convenience. Commonly used solvents include organic solvents, ionic liquids, supercritical fluids, etc. Deep eutectic solvents (DES) are a new type of green solvent synthesized from natural substances. Compared with other media, they have many advantages such as environmental friendliness, easy large-scale synthesis, and relatively low cost. Due to their excellent properties, they have been widely applied in the fields of enzyme catalysis and biotransformation. Yadav et al. have shown that in choline chloride-based DES aqueous solutions, the thermal and structural stability of α-chymotrypsin is enhanced, and the negative effect of urea is eliminated through the formation of choline chloride-urea (Yadav N, Bhakuni K, Bisht M, et al. Expanding the potential role of deep eutectic solvents toward facilitating the structural and thermal stability of α-chymotrypsin[J]. ACS Sustainable Chemistry & Engineering, 2020, 8(27): 10151-10160.). Daneshjou et al. found that compared with buffers, the stability of chondroitinase ABCI in choline chloride / betaine-glycerol DES is enhanced at -20, 4, and 37 °C (Daneshjou S, Khodaverdian S, Dabirmanesh B, et al. Improvement of chondroitinases ABCI stability in natural deep eutectic solvents[J]. Journal of Molecular Liquids, 2017, 227: 21-25.). However, up to now, deep eutectic solvents have not been applied to the field of enzymatic production of ferulic acid. The present invention for the first time enzymatically prepares ferulic acid in deep eutectic solvents, which has a higher yield compared with aqueous solutions. Summary of the Invention

[0005] Technical problems to be solved: Aiming at the above technical problems, the present invention provides a method for producing ferulic acid by double enzyme catalysis using deep eutectic solvents as co-solvents. Compared with the enzymatic production of ferulic acid in aqueous phase, the thermal stability and pH stability of ferulic acid esterase are improved by using this method, and the amount of ferulic acid produced by enzymatic catalysis per unit time and per unit enzyme activity is significantly increased.

[0006] Technical solution: A method for producing ferulic acid by double enzyme catalysis using deep eutectic solvents as co-solvents, comprising the following steps:

[0007] Step 1. Synthesis of deep eutectic solvent: Using quaternary ammonium salt as hydrogen bond acceptor and urea, polyol or carboxylic acid as hydrogen bond donor, mix them in a molar ratio of 1:(2 - 3) and perform rotary evaporation to obtain the deep eutectic solvent;

[0008] Step 2. Compound double - enzyme system: Dissolve ferulic acid esterase and xylanase in the aqueous solution of the deep eutectic solvent to form a double - enzyme system;

[0009] Step 3. Enzymatic reaction: Using de - starched wheat bran as the substrate, carry out the reaction in the double - enzyme system to obtain ferulic acid.

[0010] Preferably, the hydrogen bond acceptor is choline chloride.

[0011] Preferably, the hydrogen bond donor is urea, glycerol, ethylene glycol or lactic acid.

[0012] Preferably, in Step 1, the deep eutectic solvent is a mixture of choline chloride and glycerol with a molar ratio of 1:2.

[0013] Further, when choline chloride and glycerol are used as the deep eutectic solvent, the volume ratio of the deep eutectic solvent to water is 1:2.

[0014] Preferably, in the double - enzyme system, the enzyme activity ratio of ferulic acid esterase to xylanase is 1:1.

[0015] Preferably, the reaction temperature in Step 3 is 50 °C.

[0016] Preferably, the reaction pH in Step 3 is 8.

[0017] Beneficial effects: The present invention provides a method for the synergistic catalysis of ferulic acid by ferulic acid esterase and xylanase using deep eutectic solvent as a cosolvent. Compared with the enzymatic production of ferulic acid in an aqueous phase, when using the method of the present invention, the thermal stability and pH stability of ferulic acid esterase are both improved, and the amount of ferulic acid produced by enzymatic reaction per unit time and per unit enzyme activity is significantly increased. The specific performance is as follows:

[0018] (1) Thermal stability of ferulic acid esterase

[0019] Ferulic acid esterase has better thermal stability in a solution using choline chloride - glycerol - based DES as a cosolvent than in an aqueous phase. Ferulic acid esterase incubated for 1 h at 30 °C in a solution using choline chloride - glycerol - based DES as a cosolvent can still retain 60% of its activity, and retains 40% of its activity when incubated for 1 h at 50 °C; while in phosphate buffer, it retains 40% of its activity when incubated for 1 h at 30 °C and retains 30% of its activity when incubated for 1 h at 50 °C.

[0020] (2) pH stability of ferulic acid esterase

[0021] Ferulic acid esterase has better pH stability in a solution with choline chloride-glycerol-based DES as a co-solvent than in an aqueous phase. Ferulic acid esterase was incubated for 1 h in a solution with choline chloride-glycerol-based DES as a co-solvent at pH 7 and still retained 75% of its activity. When incubated for 1 h at pH 9, it retained 60% of its activity. While when incubated for 1 h in phosphate buffer at pH 7, it retained 50% of its activity, and when incubated for 1 h at pH 9, it retained 25% of its activity.

[0022] (3)Comparison of the effects of double-enzyme catalysis on the production of ferulic acid

[0023] The best compounding ratio was achieved when the enzyme activity ratio of ferulic acid esterase to xylanase was 1:1, which was used to degrade de-starched wheat bran to produce ferulic acid. About 6 μg of ferulic acid was produced by 6.6 U of total enzyme amount catalyzing for 12 h. Under the same conditions, in a solution with choline chloride-glycerol-based DES as a co-solvent, about 35 μg of ferulic acid could be produced. Description of the drawings

[0024] Figure 1 : Influence of the compounding ratio of ferulic acid esterase and xylanase on the enzymatic reaction;

[0025] Figure 2 : Influence of the type and concentration of DES on the enzymatic reaction;

[0026] Figure 3 : Optimum reaction temperature of ferulic acid esterase in a solution with DES as a co-solvent;

[0027] Figure 4 : Optimum reaction pH of ferulic acid esterase in a solution with DES as a co-solvent;

[0028] Figure 5 : Thermal stability of ferulic acid esterase in a solution with DES as a co-solvent;

[0029] Figure 6 : pH stability of ferulic acid esterase in a solution with DES as a co-solvent. Detailed implementation manners

[0030] The ferulic acid esterase involved in the following examples was purchased from Megazyme (Bray, Co. Wicklow, Ireland); ferulic acid (FA) and methyl ferulate (MFA) involved in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd., and xylanase was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0031] Example 1 Determination of ferulic acid esterase activity

[0032] Using 1 mmol / L methyl ferulate (MFA) stock solution as the substrate, the enzyme activity of ferulic acid esterase was determined. 950 μL of methyl ferulate was incubated at 40 °C for 10 min, and then 50 μL of the diluted enzyme solution was added for reaction. The mixture was incubated at 40 °C for 5 min and then boiled for 5 min to terminate the reaction.

[0033] The content of released FA was determined by high performance liquid chromatography. The enzyme activity unit was defined as the amount required to release 1 μmol FA per minute.

[0034] Example 2 Effect of the compounding ratio of ferulic acid esterase and xylanase on the enzymatic reaction

[0035] A compounding experiment of ferulic acid esterase and xylanase was carried out, using de-starched wheat bran as the substrate. 10 mg / mL of wheat bran was reacted with a total of 6.6 U of the compounded enzyme solution. The free ferulic acid esterase and xylanase were compounded at an enzyme activity ratio of 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10. After compounding, the wheat bran decomposition experiment was carried out. The reaction was terminated after culturing in a shaker at 200 rpm and 37 °C for 12 h, and the optimal compounding ratio was determined according to the amount of ferulic acid produced.

[0036] As Figure 1 shown, it can be observed that as the proportion of ferulic acid esterase gradually increases, the release amount of ferulic acid also gradually increases. When the ratio of ferulic acid esterase to xylanase reaches 1:1, the release amount of ferulic acid reaches the maximum. Then, as the proportion of ferulic acid esterase increases again, the release amount of ferulic acid begins to decrease. Therefore, the ratio of ferulic acid esterase to xylanase is determined to be 1:1.

[0037] Example 3 Effect of the type and concentration of DES on the enzymatic reaction

[0038] Aqueous solutions of DES: water with ratios of 5:1, 2:1, 1:1, 1:2, 1:5 were prepared, and the wheat bran decomposition experiment was carried out according to the compounding ratio of free enzymes. The reaction was terminated after culturing in a shaker at 200 rpm and 37 °C for 12 h, and the optimal water content of the best DES was determined according to the amount of ferulic acid produced.

[0039] As Figure 2 shown, all 4 kinds of DES selected as co-solvents can increase the release amount of ferulic acid, and the release amount of ferulic acid first increases and then decreases with the increase of water content. Especially for choline chloride-glycerol-based DES, when the choline chloride-glycerol content (v / v) is 33.3%, the enzyme-catalyzed release amount of ferulic acid reaches the maximum.

[0040] Example 4 Optimal reaction temperature and optimal reaction pH of ferulic acid esterase in the solution with DES as co-solvent

[0041] The optimal temperature was determined by changing the incubation temperature of the enzyme (reacting at 20 - 60 °C for 10 min). The optimal pH was determined by changing the incubation pH of the enzyme, that is, at 40 °C, the pH values of 0.1 M PBS buffer solution and the solution with choline chloride - glycerol - based DES as a co - solvent were increased from 4.0 to 9.0 respectively. The corresponding ferulic acid esterase activity was measured, and the optimal reaction temperature and optimal reaction pH were obtained by comparison.

[0042] As Figure 3 shown, compared with the phosphate buffer solution, the optimal temperature of the enzyme in the solution with DES as a co - solvent changed. The optimal reaction temperature in the phosphate buffer solution was 40 °C, while the optimal reaction temperature of the enzyme in the solution with DES as a co - solvent increased to 50 °C.

[0043] As Figure 4 shown, the pH adaptation region of the enzyme in the solution with DES as a co - solvent was wider, and the optimal pH range of the enzyme increased. At pH 4, the enzyme activity in the phosphate buffer solution was already less than 10%, while the enzyme activity in the solution with DES as a co - solvent still remained above 55%. At pH 9, the enzyme activity in the phosphate buffer solution remained about 50%, while the enzyme activity in the solution with DES as a co - solvent was still above 75%.

[0044] Example 5 Thermal stability and pH stability of ferulic acid esterase in the solution with DES as a co - solvent

[0045] The thermal stability of ferulic acid esterase in different solutions was measured. Ferulic acid esterase was placed in phosphate buffer solution and the solution with choline chloride - glycerol - based DES as a co - solvent respectively, incubated at 30 - 70 °C for 1 h, and then the ferulic acid esterase activity was measured. The pH stability of ferulic acid esterase in different solutions was measured. Ferulic acid esterase was placed in phosphate buffer solution and the solution with choline chloride - glycerol - based DES as a co - solvent respectively, incubated at pH range of 5.0 - 9.0 for 1 h, and then the ferulic acid esterase activity was measured.

[0046] As Figure 5 shown, ferulic acid esterase had better thermal stability in the solution with choline chloride - glycerol - based DES as a co - solvent than in the aqueous phase. Ferulic acid esterase incubated at 30 °C for 1 h in the solution with choline chloride - glycerol - based DES as a co - solvent could still retain 60% of its activity, and retained 40% of its activity when incubated at 50 °C for 1 h; while in the phosphate buffer solution, it retained 40% of its activity when incubated at 30 °C for 1 h and retained 30% of its activity when incubated at 50 °C for 1 h.

[0047] As Figure 6As shown, ferulic acid esterase has better pH stability in a solution with choline chloride-glycerol-based DES as a co-solvent than in an aqueous phase. Ferulic acid esterase incubated for 1 h in a solution with choline chloride-glycerol-based DES as a co-solvent at pH 7 can still retain 75% of its activity, and retains 60% of its activity when incubated for 1 h at pH 9; while when incubated for 1 h in a phosphate buffer at pH 7, it retains 50% of its activity, and retains 25% of its activity when incubated for 1 h at pH 9.

[0048] Example 6 Comparison of the effects of double-enzyme catalysis on the production of ferulic acid

[0049] The best compounding ratio is achieved when the enzyme activity ratio of ferulic acid esterase to xylanase is 1:1, which is used to degrade de-starched wheat bran to produce ferulic acid. About 6 μg of ferulic acid is produced by 6.6 U of total enzyme amount catalyzed for 12 h; under the same conditions, in a solution with choline chloride-glycerol-based DES as a co-solvent, about 35 μg of ferulic acid can be produced.

Claims

1. A method for producing ferulic acid by dual-enzyme catalysis using a deep eutectic solvent as a co-solvent, characterized in that, It includes the following steps: Step 1: Synthesize the deep eutectic solvent: Using choline chloride as the hydrogen bond acceptor and glycerol as the hydrogen bond donor, mix them in a molar ratio of 1:(2 - 3) and perform rotary evaporation to obtain the deep eutectic solvent; Step 2: Compound the double enzyme system: Dissolve ferulic acid esterase and xylanase in the aqueous solution of the deep eutectic solvent according to an enzyme activity ratio of 1:1 to form a double enzyme system; Step 3: Enzymatic reaction: Using de-starched wheat bran as the substrate, carry out the reaction in the double enzyme system to obtain ferulic acid.

2. The method for producing ferulic acid by dual-enzyme catalysis using a deep eutectic solvent as a co-solvent according to claim 1, characterized in that, In the above Step 1, the deep eutectic solvent is a mixture of choline chloride and glycerol with a molar ratio of 1:

2.

3. A method for producing ferulic acid by dual-enzyme catalysis using a deep eutectic solvent as a co-solvent, characterized in that, The volume ratio of the deep eutectic solvent to water is 1:

2.

4. A method for producing ferulic acid by dual-enzyme catalysis using a deep eutectic solvent as a co-solvent, as claimed in claim 1, wherein In the above Step 3, the reaction temperature is 50 °C.

5. A method for producing ferulic acid by double enzyme catalysis using a deep eutectic solvent as a co-solvent, characterized in that, In the above Step 3, the reaction pH is 8.

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