CN113234742A

CN113234742A - Preparation of novel high-activity ferulic acid esterase and application of novel high-activity ferulic acid esterase in agricultural wastes

Info

Publication number: CN113234742A
Application number: CN202110540431.7A
Authority: CN
Inventors: 辛凤姣; 温博婷; 沈洋; 邵淑丽; 王钰璐; 李�真; 魏雪; 王凤忠; 谷天一
Original assignee: Institute of Food Science and Technology of CAAS
Current assignee: Institute of Food Science and Technology of CAAS
Priority date: 2021-05-18
Filing date: 2021-05-18
Publication date: 2021-08-10

Abstract

The invention discloses a gene for expressing feruloyl esterase, protein coded by the gene, a recombinant expression vector containing the gene, a recombinant genetic engineering bacterium and application thereof, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively. The invention separates a gram-positive obligate anaerobic bacillus-free bacillary bacterium from human feces, screens an amino acid sequence fragment with alpha/beta hydrolase 8 chain center beta folding conformation from a transcriptome library of the bacillary bacterium, reversely transcribes the sequence into cDNA and constructs a recombinant expression vector, successfully expresses heterologously by an escherichia coli host BL21(DE3), purifies a high-concentration and uniform-property target protein, can effectively hydrolyze a model substrate such as methyl ferulate and the like, has the potential of releasing a high value-added product ferulic acid from agricultural residues, and provides a new feasible scheme for industrial application of preparing the ferulic acid by enzymolysis.

Description

Preparation of novel high-activity ferulic acid esterase and application of novel high-activity ferulic acid esterase in agricultural wastes

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene for expressing feruloyl esterase, a protein coded by the gene, a recombinant expression vector containing the gene, a recombinant genetic engineering bacterium and application thereof.

Background

Ferulic Acid (FA) belongs to hydroxycinnamic acid (HGA) in phenolic acid family, is a caffeic acid derivative, and is covalently linked with lignocellulose and other components in plant cell wall through ester bond. Ferulic acid is widely found in vegetables, fruits, herbal medicines and certain beverages such as coffee and beer. The 3-methoxy, 4-hydroxy and carboxyl on the benzene ring enable the ferulic acid to have good oxidation resistance, can effectively remove active oxygen and remove free radicals, and inhibit oxidation reaction caused by ultraviolet rays or radiation, so the ferulic acid is used as a novel antioxidant with strong cell protection activity. Ferulic acid has been used today in the health care and cosmetic industries and has become a potential drug for the treatment of many oxidation related diseases, including alzheimer's disease, cancer, cardiovascular diseases, diabetes and skin diseases, among others.

Lignocellulosic biomass is a currently studied and popular renewable resource that needs to be converted to a low molecular weight carbon source for further utilization. Due to the complex cross-linked network structure, a series of cellulase and hemicellulase are required to act together to degrade the cellulase. Feruloyl esterase (EC 3.1.1.73) can catalyze the hydrolysis of ester bond between hydroxycinnamic acid and backbone glycan, thereby promoting the combination and action of other enzymes and lignocellulose, and releasing ferulic acid with high added value in the process. Compared with the traditional alkaline hydrolysis method, the enzymatic hydrolysis method has higher efficiency and environmental friendliness, has higher specificity, does not damage other valuable chemical substances, and is suitable for industrial production.

The Dorea formicenerans (commercially available from American ATCC germplasm pool: bio-091013 or JCM germplasm pool: bio-104980) related to the invention is a gram-positive obligate anaerobic bacillus-free rod-shaped bacterium separated from human feces. Screening an amino acid sequence fragment with beta-sheet conformation at the 8 chain center of alpha/beta hydrolase from a transcriptome library of the protein has not been reported in any previous literature and is not annotated as ferulic acid esterase. The sequence is reversely transcribed into cDNA and a recombinant expression vector is constructed, preferably escherichia coli host BL21(DE3) is successfully and heterogeneously expressed, the target protein with high concentration and uniform property is purified, the High Performance Liquid Chromatography (HPLC) result shows that the protein can effectively hydrolyze methyl ferulate and other model substrates, the optimal reaction condition of the enzyme is measured and calculated by using a p-nitrobenzene artificial substrate, and then the enzyme is applied to successfully release ferulic acid with high added value from four agricultural residues of DE-starch wheat bran, brewer's grains, corn straws and corncobs, thereby providing a new feasible scheme for the industrial application of preparing ferulic acid by enzymolysis.

Disclosure of Invention

The invention aims to improve the application of the prior art and provides a gene for expressing feruloyl esterase, a protein coded by the gene, a recombinant expression vector containing the gene, a recombinant genetic engineering bacterium and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

one of the purposes of the invention is to provide a gene for expressing feruloyl esterase, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively.

The invention also aims to provide a DSFAE protein encoded by the gene for expressing feruloyl esterase, wherein the amino acid sequence of the DSFAE protein is shown as SEQ ID NO: 1 is shown.

The present invention also provides a recombinant expression vector comprising the ferulic acid esterase gene of claim 1.

Further, the recombinant expression vector was obtained by inserting the gene expressing feruloyl esterase between the restriction sites BamHI and XhoI of the expression plasmid pET-28 a.

The fourth purpose of the invention is to provide a recombinant gene engineering bacterium, which is constructed by transforming the recombinant expression vector into an escherichia coli host BL21(DE 3).

The fifth purpose of the invention is to provide the application of the gene for expressing ferulic acid esterase in-vitro expression of DSFAE protein.

Further, the method for expressing the gene of the ferulic acid esterase to express the DSFAE protein in vitro comprises the following specific steps:

(1) cloning to obtain the gene of claim 1, and inserting the gene between enzyme cutting sites BamHI and XhoI of expression plasmid pET-28a (+) to obtain a recombinant expression vector pET-28a (+) -Dffae;

(2) transforming the recombinant expression vector pET-28a (+) -Dffae into competent cells BL21(DE3) through heat shock, and screening to obtain recombinant genetic engineering bacteria;

(3) culturing the genetically engineered bacteria at 37 deg.C, and culturing to obtain bacterial liquid OD₆₀₀When the value reaches 0.8, cooling to 18 ℃, inducing by using isopropyl thiogalactopyranoside (IPTG) for 14h at 18 ℃, crushing thalli by using a high-pressure homogenization method, and purifying the supernatant of the bacterial liquid by using an affinity chromatography column, an ion column chromatography column and a molecular exclusion chromatography column in sequence to obtain the ferulic acid esterase enzyme liquid.

The sixth purpose of the invention is to provide the application of the gene for expressing the ferulic acid esterase in the production of functional phenolic acid by enzymolysis.

Further, the functional phenolic acid comprises ferulic acid, coumaric acid and sinapic acid, and the raw materials comprise methyl ferulate, methyl coumarate and methyl sinapinate.

Further, the enzymatic production method is to contact the relevant substrate and the protein under the condition of enzymatic reaction, and the condition of enzymatic reaction comprises: the temperature is 20-70 deg.C, pH is 4.5-10.0, and the time is 30 min.

The invention has the advantages that:

(1) the invention expresses the gene sequence of ferulic acid esterase from enterobacteria Dorea formicenerans for the first time in vitro so as to be helpful for researching the functions and regulation mechanism of the protein and have important significance for regulating and controlling the structure of intestinal microflora based on a nutrition strategy to promote human health;

(2) the recombinant expression plasmid of the ferulic acid esterase gene DfFAE is obtained by an in vitro gene cloning technology, can be stably and efficiently expressed after being transferred into escherichia coli, and has the expressed ferulic acid esterase yield of 10mg/mL and the purity of over 95 percent. The ferulic acid esterase produced by the fermentation of the recombinant strain is most suitable for pH8.4, the enzyme activity retention rate is more than 96% after the ferulic acid esterase is treated for 1 hour within the range of pH 8.0-8.4, and the alkali resistance is good; the temperature tolerance is good, the optimum temperature is 40 ℃, and the enzyme activity retention rate is more than 80% after the treatment at 40 ℃ for 6 hours; the metal ions have good tolerance, the sodium ions, the calcium ions and the tin ions can improve the activity of the esterase, and the other metal ions hardly have influence on the enzyme activity of the feruloyl esterase. The invention provides an intestinal flora feruloyl esterase which has good alkali resistance and metal ion tolerance, realizes high-efficiency expression in escherichia coli, has high purity and has very important industrial application value.

Drawings

FIG. 1 is a graph showing the results of HPLC-mode substrate specificity measurement.

FIG. 2 is a diagram showing the results of the measurement of the specificity of the p-nitrobenzene artificial substrate.

FIG. 3 is a graph showing the temperature activity of feruloyl esterase.

FIG. 4 shows pH activity curves and pH tolerance curves of feruloyl esterase.

FIG. 5 is a graph showing the temperature tolerance of feruloyl esterase.

FIG. 6 is a graph showing the results of evaluation of the metal ion and organic solvent resistance of feruloyl esterase.

FIG. 7 shows the fit of the equation of Mie for feruloyl esterase.

FIG. 8 is a graph showing the tendency of feruloyl esterase to hydrolyze different natural substrates to release ferulic acid.

Detailed Description

The technical scheme of the invention is further explained by combining the specific examples as follows:

experimental materials and general experimental methods:

1. bacterial strain and carrier:

escherichia coli (Escherichia coli) Trans1-T1, BL21(DE3) from Beijing Quanjin (TransGen Biotech) Inc., and pET-28a (+) plasmid from Beijing Liuhe Huada Gene technology, Inc.

2. Enzymes and other biochemical reagents:

the tool enzyme comprises restriction enzyme, DNA ligase and Taq enzyme, and the DNA extraction, purification and gel recovery kit is purchased from Takara company. Methyl Ferulate (MFA), methyl p-coumarate (MpCA), Methyl Sinapinate (MSA), and Methyl Caffeate (MCA) were purchased from MERDA. Others are made in China (all can be purchased from common biochemical agents).

3. Culture medium:

(1) liquid LB medium: 10g/L of sodium chloride (NaCl), 10g/L of Tryptone (Tryptone) and 5g/L of Yeast powder (Yeast Extract);

(2) solid LB medium: 10g/L of sodium chloride (NaCl), 10g/L of Tryptone (Tryptone), 5g/L of Yeast powder (Yeast Extract) and 20g/L of Agar (Agar);

sterilizing the above culture medium with high pressure steam at 121 deg.C under 103kPa for 20min, cooling the solid culture medium to about 50 deg.C, and pouring into a super clean bench.

Example 1 E.coli expression of Feruloyl esterase Gene

(1) Amplification of a target gene:

the inventors of the present invention found that a ferulic acid esterase DfFAE derived from Dorea formicenerans (commercially available from American ATCC germplasm pool: bio-091013 or Japanese JCM germplasm pool: bio-104980) has an amino acid sequence shown in SEQ ID NO. 1. The esterase nucleotide sequence obtained by the artificial chemical synthesis method is shown as SEQ ID NO.2, and under the condition that the gene sequence is obtained, the gene can be artificially synthesized by the technical personnel in the field according to the prior gene recombination technology, the technology is mature, the technical personnel in the field can realize the synthesis without creative labor, and the details are not repeated. The two ends of the synthesized Dffae gene are also provided with BamHI and XhoI enzyme cutting sites so as to be convenient for connecting with an expression vector. The Dffae gene is amplified by taking the nucleotide sequence as a template, and the primers are as follows:

DSFAE-F：5’-CGCGGATCCATGAACCAGAAAAAAG-3’

DSFAE-R：5’-CCGCTCGAGTCAGTTGGTGTGTTTC-3’

the BamHI and XhoI sites are underlined, respectively.

(2) Constructing a recombinant expression vector:

the DSFAE gene is subjected to double enzyme digestion by BamHI and XhoI, and then is connected with an expression vector pET-28a (+) subjected to enzyme digestion by BamHI and XhoI, an escherichia coli competent cell T1 is transformed, a recombinant plasmid pET-28a (+) -Dffae is constructed, the recombinant plasmid is coated on an LB (Langmycin) flat plate with kanamycin resistance to screen positive clones, a single colony is selected to be reserved and used as colony PCR (polymerase chain reaction), then the colony PCR is sent to Beijing engine for sequencing, the sequencing result shows that the gene has the full length of 837bp and codes 274 amino acids, the recombinant plasmid contains the Dffae gene, and the positive clone plasmid is extracted for later use.

EXAMPLE 2 isolation and purification of Feruloyl esterase

The recombinant plasmid pET-28a (+) -Dffae constructed by the ferulic acid esterase gene is transformed into an Escherichia coli BL21-Gold (DE3) host by heat shock, and the genetic engineering bacteria for expressing the ferulic acid esterase is constructed. Culturing the above genetically engineered bacteria at 37 deg.C to obtain bacterial liquid OD₆₀₀When the value reaches 0.8, isopropyl thiopyran galacto-alcohol is used at 18 DEG CGlycoside (IPTG) is induced for 12h, after the thalli are crushed by a high-pressure homogenization method, the supernatant of the bacterial liquid passes through an affinity chromatography column (nickel column), an ion column (Source15Q) and a molecular sieve (Superdex200) in sequence to prepare the ferulic acid esterase enzyme liquid (10.2 mg/ml). The ferulic acid esterase amino acid sequence is numbered WP _117657856 in the NCBI database.

Example 3 measurement of the Activity of purified Feruloyl esterase and evaluation of the enzymatic Properties

1) Determination of substrate specificity of feruloyl esterase

Four modes of substrate specificity were determined by HPLC: the chromatographic conditions were a Waters Atlantis dC18 chromatographic liquid phase column, the column temperature was 25 ℃, and the mobile phase solution was: ultrapure water (0.1% formic acid) and methanol (0.1% formic acid) in the proportions: 0-9.5min, 40: 60 (v/v); 9.5-18min, 85: flow rate of 15 (v/v): 0.5ml/min, the detection wavelength is: 323 nm. Determining the amount of ferulic acid by retention time and peak area using external standard method. The results show (as shown in FIG. 1) that the enzyme has good activity on MFA, MpCA and MSA, and is consistent with the characteristics of the A-type feruloyl esterase described in the prior literature.

The specificity of the p-nitrobenzene artificial substrate is determined by a spectrophotometric method: the enzyme was reacted with four artificial substrates, i.e., p-nitrophenylacetate (pNPA), p-nitrophenylbutyrate (pNPB), p-nitrophenyloctanoate (pNPO) and p-nitrophenylferulate (pNPF), 1. mu.L of the enzyme solution was added to 284. mu.L of a pH 7.0 potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution containing 2.5% Trixton X-100, 10mM 15. mu.L of the substrate was added to initiate the reaction, and OD was measured after 5min at 30 ℃ and OD was measured₄₀₅The value is obtained. The results show (as shown in fig. 2) that DfFAE has significant activity on pNPA, pNPB and pNPO, and the activity on pNPB is the highest, and then the following operations all use pNPB.

2) pH activity curve of feruloyl esterase

The pH gradient conditions of 5.0, 5.4, 6.0, 7.4, 8.0, 8.4 and 9.0 are set by using a citric acid-sodium citrate buffer solution, a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution and a boric acid-borax buffer solution, and the activity of the purified ferulic acid esterase is measured. The results show (as shown in figure 4) that the optimum action pH of the enzyme is 8.4, and the relative enzyme activity is above 69% in the range of pH 8.0-9.0.

3) Temperature activity curve of feruloyl esterase

The reaction temperature in the enzyme activity determination process is set to 4 ℃, 25 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ respectively, the reaction is carried out for 5min under each gradient condition of the pH8.4 environment, and the activity of the purified ferulic acid ester enzyme is determined. The results show (as shown in figure 3) that the optimal action temperature of the enzyme is 40 ℃, and the relative enzyme activity is above 58% in the range of 25-50 ℃.

4) Assessment of pH stability of Feruloyl esterase

Diluting the original enzyme solution by 3 times with a molecular sieve buffer, adding 1 mu L of the diluted enzyme solution into buffer systems with different pH values of 4.0, 5.0, 6.0, 7.4, 8.0, 8.4 and 9.0, incubating for 1h at 4 ℃, and determining the residual enzyme activity, wherein the result shows (as shown in figure 4) that the enzyme has the best stability in the pH range of 8.0-8.4, and the relative enzyme activity can be kept above 96%.

5) Evaluation of temperature stability of Feruloyl esterase

Diluting the original enzyme solution by 3 times with molecular sieve buffer, incubating at 4 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C and 60 deg.C for 15min, 30min, 1h, 2h, 4h and 6h, respectively, and measuring the residual enzyme activity at each time point, the result shows (as shown in FIG. 5) that the enzyme has good thermal stability at 50 deg.C or lower.

6) Feruloyl esterase tolerance assessment for ions and organic solvents

The relative activity of the enzyme was determined after diluting the original enzyme solution 3 times with molecular sieve buffer and incubating in metal ions and organic solvent at 0.1M or 1% (as shown in FIG. 6) for 15min, and the results showed that Mg²⁺Has remarkable promoting effect on the enzyme, Na⁺、Ca²⁺、Sn²⁺Ions and EDTA have obvious promotion effect, SDS has obvious inhibition effect on the enzyme, and other ions and reagents have little or no influence.

7) K of feruloyl esterase_m、k_catValue measurement and calculation

The Mie constant K of the purified feruloyl esterase (shown in FIG. 7) was measured by using p-nitrophenyl butyrate (pNPB) as a substrate_mValue 0.38mM, conversion number k_catThe value is 1.57s^-1，k_cat/K_mThe value was 4.11s^-1mM^-1。

EXAMPLE 4 preparation of Ferulic acid by enzymatic hydrolysis

Substrate pretreatment:

1. pulverizing testa Tritici into powder, soaking in 0.25% potassium acetate at 95 deg.C for 10min, washing with large amount of water until pH is 7, repeating the steps twice, oven drying, sieving with 60 mesh sieve, and weighing.

2. Pulverizing brewer's grains, soaking in 85% ethanol, boiling for 10min, cooling, filtering, repeating the steps twice, sequentially washing with anhydrous ethanol and diethyl ether, oven drying, sieving with 60 mesh sieve, and weighing.

3. The corn cob is dried at 105 ℃ after being subjected to steam explosion treatment, and weighed after being screened by a 60-mesh sieve.

4. The corn stalks are dried at 105 ℃ after being subjected to mechanical shearing type superfine grinding treatment, and weighed after being screened by a 60-mesh sieve.

And (3) carrying out enzymolysis reaction: the enzyme catalysis system was carried out in a 50mL closed vessel in a volume of 10 mL. Weighing 10 wt% of pretreatment substrate, adding 1% of xylanase for carrying out enzymolysis for 24h in advance, and then adding 1% of ferulic acid esterase for carrying out enzymolysis for 24h under the conditions of pH8.4, water bath at 40 ℃ and shaking table rotating speed of 200 rmp. Sampling at 0, 2, 4, 6, 8, 12, 16, 20 and 24h, heating for 5min to terminate the reaction, and detecting the ferulic acid release amount in the sample by HPLC. The same procedure was performed with inactivated enzyme solution as a blank.

And (3) product analysis: as shown in FIG. 8, the final amount of FA released by using wheat bran as a substrate is 2.4mg/g, and the conversion rate is 94.86%; the final FA release amount by taking the brewer's grain as a substrate is 3.8mg/g, and the conversion rate is 80.85 percent; the final release amount of the corn cob serving as a substrate is 6.8mg/g, and the conversion rate is 62.96%; the final release amount of the corn straw used as a substrate is 2.65mg/g, and the conversion rate is 94.64%.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute for agricultural product processing of Chinese academy of agricultural sciences

<120> preparation of novel high-activity ferulic acid esterase and application thereof in agricultural wastes

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 274

<212> PRT

<213> Dorea formicigenerans

<400> 1

Met Asn Gln Lys Lys Glu Cys Met Asp Val His Val Asn Gly Leu Lys

1 5 10 15

Phe His Val Gln Thr Phe Gly Ser Gly Val Pro Ile Ile Met Ile Met

20 25 30

Gly Leu Gly Ala Pro Gly Asp Lys Trp Lys His Asn Tyr Glu Leu Leu

35 40 45

Ser Lys Trp Phe Trp Cys Ile Val Pro Asp Asn Arg Gly Ala Gly Leu

50 55 60

Ser Asp Lys Pro Glu Ala Glu Ser Tyr Thr Thr Glu Gln Met Ala Asp

65 70 75 80

Asp Ile Ile Gly Ile Met Asp Ala Leu Asp Ile Lys Lys Ala His Val

85 90 95

Met Gly Val Ser Met Gly Gly Ala Ile Ala Gln Gln Val Ala Leu Lys

100 105 110

Val Pro Asp Arg Val Leu Ser Leu Ile Leu Thr Ser Thr Phe Ala Ser

115 120 125

Val Ser Pro Ala Phe Lys Lys Ala Leu Asn Leu Ile Cys Glu Leu Lys

130 135 140

Glu Asp Thr Asp Pro Ala Val Leu Lys Gln Leu Asn Leu Trp Met Thr

145 150 155 160

Tyr Gly Gln Tyr Thr Gln Ile His His Pro Glu Lys Ile Glu Lys Ser

165 170 175

Ile Glu Glu Asp Ala Ala Tyr Pro Tyr Pro Met Pro Val Tyr Ala Tyr

180 185 190

Lys Ala Gln Cys Gly Ala Cys Leu Ser His Asn Thr Ala Gly Arg Leu

195 200 205

His Glu Leu Lys Met Pro Val Leu Ile Ala Ala Gly Ala Lys Asp Leu

210 215 220

Phe Met Asn Ile Glu Lys Thr Met Glu Leu Val His Gly Ile Pro Gln

225 230 235 240

Ala Glu Phe Tyr Leu Ala Pro Glu Gly Gly His Val His Gln Trp Glu

245 250 255

Tyr Pro Glu Pro Tyr Asp Ser Val Val Val Gly Phe Leu Met Lys His

260 265 270

Thr Asn

<210> 2

<211> 837

<212> DNA

<213> Artificial sequence

<400> 2

ggatccatga accagaaaaa agaatgcatg gacgtgcatg tgaacggcct gaaatttcat 60

gtgcagacct ttggtagcgg cgtgccgatt attatgatta tgggcttagg cgcgcctggc 120

gataaatgga aacacaacta tgaactgctg agcaaatggt tttggtgcat tgtgccggat 180

aatcgcggtg cgggcttaag cgataaaccg gaagcggaaa gctataccac cgaacagatg 240

gcggatgata ttattggcat tatggacgcg ctggatatta aaaaagcgca tgtgatgggc 300

gttagcatgg gtggtgcgat tgcacagcag gttgcgttaa aagtgccgga tcgcgtgtta 360

agcctgattc tgaccagcac ctttgcgtca gttagcccgg cgtttaaaaa agcgctgaac 420

ctgatttgcg aactgaaaga agataccgat ccggcggtgc tgaaacaact gaacctgtgg 480

atgacctatg gccagtatac ccagattcat cacccggaga aaatcgaaaa gagcatcgaa 540

gaagatgcgg cgtatccgta tccgatgccg gtgtatgcgt ataaagcgca atgcggcgcg 600

tgcttaagcc ataataccgc gggtcgcctg catgaactga aaatgccggt gttaattgcg 660

gcgggcgcga aagatctgtt tatgaacatc gagaagacca tggaactggt gcatggcatt 720

ccgcaggcgg aattttatct ggcgccggaa ggcggtcatg ttcatcagtg ggaatatccg 780

gaaccgtatg atagcgtggt ggtgggcttt ctgatgaaac acaccaactg actcgag 837

Claims

1. A gene for expressing feruloyl esterase, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively.

2. A DfFAE protein encoded by a ferulic acid esterase expression gene according to claim 1, wherein the amino acid sequence of the DfFAE protein is as set forth in SEQ ID NO: 1 is shown.

3. A recombinant expression vector comprising the ferulic acid esterase according to claim 1.

4. The recombinant expression vector according to claim 3, wherein the recombinant expression vector is obtained by inserting the gene expressing feruloyl esterase between the cleavage sites BamHI and XhoI of expression plasmid pET-28 a.

5. A recombinant genetically engineered bacterium, wherein the engineered bacterium is constructed by transforming the recombinant expression vector of claim 3 or 4 into an Escherichia coli host BL21(DE 3).

6. Use of the ferulic acid esterase according to claim 1 for in vitro expression of a DfFAE protein.

7. The use of claim 6, wherein the method for expressing the gene of ferulic acid esterase to express DfFAE protein in vitro comprises the following steps:

8. Use of the ferulic acid esterase expression gene of claim 1 for producing functional phenolic acid by enzymatic hydrolysis.

9. The use of claim 8, wherein the functional phenolic acid comprises ferulic acid, coumaric acid, sinapic acid, and the material comprises methyl ferulate, methyl coumarate, methyl sinapinate.

10. The use according to claim 8, wherein the enzymatic production process comprises contacting the relevant substrate with the protein of claim 2 under enzymatic reaction conditions comprising: the temperature is 20-70 deg.C, pH is 4.5-10.0, and the time is 30 min.