CN111484988A

CN111484988A - Bifunctional enzyme with xylanase and feruloyl esterase activities, and coding gene and application thereof

Info

Publication number: CN111484988A
Application number: CN201910088638.8A
Authority: CN
Inventors: 袁红莉; 王若楠; 杨金水; 李宝珍
Original assignee: China Agricultural University
Current assignee: Beijing Siliang Technology Co ltd
Priority date: 2019-01-29
Filing date: 2019-01-29
Publication date: 2020-08-04
Anticipated expiration: 2039-01-29
Also published as: CN111484988B

Abstract

The invention discloses a bifunctional enzyme with xylanase and feruloyl esterase activities, and a coding gene and application thereof. The bifunctional enzyme is a protein as follows: a) the amino acid sequence is a protein shown in 29 th to 1030 th positions of the sequence 1; b) the amino acid sequence is a protein shown in a sequence 1; c) a protein comprising a xylanase domain as shown in position 29-423 of SEQ ID NO 1 and a feruloyl esterase domain as shown in position 577-936 of SEQ ID NO 1; d) a fusion protein obtained by connecting a tag to the N-terminus and/or the C-terminus of the amino acid sequence; e) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence; f) and a protein having a homology of 75% or more than 75% with the above-mentioned amino acid sequence and having the same function. The bifunctional enzyme of the invention has the activity of xylanase and feruloyl esterase, and can be used for hydrolyzing substances containing xylan and feruloyl ester bonds.

Description

Bifunctional enzyme with xylanase and feruloyl esterase activities, and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a novel bifunctional enzyme with xylanase and feruloyl esterase activities, and a coding gene and application thereof.

Background

Xylan (xylolan) is one of the main components of plant cell walls, and has a content inferior to cellulose, and is heteropolypolysaccharide, the main chain is connected by a plurality of xylopyranosyl groups through β -1, 4-glycosidic bonds, and the side chains are substituted by a plurality of different groups, such as arabinosyl, acetyl, glucuronic acid residues and the like, and the modification of the side chain groups improves the degradation resistance of the plant cell walls, and greatly hinders the conversion and utilization of biomass.

The xylanase is a generic term for enzymes that degrade xylan into oligosaccharides and xylose β -1, 4-endoxylanase (EC 3.2.1.8) acts on the xylan backbone, randomly cuts xylo-oligosaccharide bonds inside xylan, breaks down it into xylo-oligosaccharide ferulic acid esterase (EC 3.1.1.73) belongs to the class of carboxylesterase, and is capable of catalyzing the cleavage of the ester bonds between ferulic acid and oligosaccharides or polysaccharides, releasing ferulic acid, assisting xylanase to increase the efficiency of biomass degradation, both enzymes are widely used in the food, feed, paper and bio-energy fields (Basit A, L iu J, Rahim K, et al. Thermophilic xylanases: cribe to bottle [ J ] Critical review in Biotechnology,2018: 1; Golan N, Rodrez-oligosaccharide gua L, Sa-vegetable fermentation [ J ] oligosaccharide from J ] dietary supplement, xylose synthase, xylose.

Xylanases and feruloyl Esterases are widely present in microorganisms, plants and animals, in previous studies, a number of xylanases and feruloyl Esterases from fungi, bacteria and actinomycetes have been reported, apart from XynY and XynZ, which are key components of the cellulosome of Clostridium thermocellum (Clostridium thermocellum), only 1 example of a bifunctional enzyme with both xylanase and feruloyl Esterase Activity is currently reported for natural evolution (Blum D L, Kataeva I A, L I X L, et al. feruloyl Esterase Activity of the Clostridium thermocellum cellulosum from said immobilized microorganism under cellulose, 2000,182(5), 1346. about.631. about. xylanase, Ybell M, Yexpressing J. journal of Bacteriology,2000,182(5), and 35. about. xylanase, and xylanase producing a naturally occurring xylanase and a xylanase Substrate of xylanase, and a xylanase, thus, the xylanase and xylanase producing a xylanase of the same oligosaccharide has not been reported (xylanase, and xylanase of the same enzyme), but the naturally occurring xylanase and xylanase of xylanase, and a Substrate for the same time producing of the strain, and xylanase, and a naturally occurring strain, and a xylanase, a strain.

Disclosure of Invention

The invention aims to provide a novel bifunctional enzyme with xylanase and feruloyl esterase activities, and a coding gene and application thereof.

The invention firstly protects the bifunctional enzyme with xylanase and feruloyl esterase activities, which is the protein shown in the following a) or b) or c) or d) or e) or f):

a) the amino acid sequence is a protein shown in 29 th to 1030 th positions of the sequence 1;

b) the amino acid sequence is a protein shown in a sequence 1;

c) a protein comprising a xylanase domain as shown in position 29-423 of SEQ ID NO 1 and a feruloyl esterase domain as shown in position 577-936 of SEQ ID NO 1;

d) a fusion protein obtained by connecting a label to the N end and/or the C end of the amino acid sequence shown in a) or b) or C);

e) protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown by a) or b) or c) or d);

f) a protein having homology of 75% or more than 75% with the amino acid sequence shown by a) or b) or c) or d) or e) and having the same function.

The protein described in the above a) or b) or c) or d) or e) or f) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The protein in c) is specifically a protein of which the amino acid sequence consists of amino acid residues 29 to 423 in the sequence 1, Glu amino acid residues, L eu amino acid residues and amino acid residues 577-936 in the sequence 1 in sequence.

In order to facilitate purification of the protein of d), a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tagⅡ	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of e) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of f) above, wherein the protein has 75% or more homology of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

The invention also protects biological materials related to the protein.

The biomaterial provided by the invention is any one of the following A1) to A8):

A1) nucleic acid molecules encoding the above proteins;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising the recombinant vector of a 4).

In the above biological material, the nucleic acid molecule according to A1) is a gene represented by the following 1) or 2) or 3) or 4) or 5):

1) the coding sequence is a DNA molecule shown in 85 th to 3093 th positions of the sequence 2;

2) the coding sequence is a DNA molecule shown in sequence 2;

3) the coding sequence is a DNA molecule shown in sequence 8;

4) a DNA molecule which has 75% or more identity with the nucleotide sequence defined in 1) or 2) or 3) and encodes the protein;

5) a DNA molecule which hybridizes with the nucleotide sequence defined by 1) or 2) or 3) or 4) under strict conditions and codes the protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence of the present invention encoding the above-mentioned protein can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence isolated in the present invention are derived from and identical to the nucleotide sequence of the present invention as long as they encode the above-mentioned protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that has 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the above-mentioned protein. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above-mentioned biological materials, the expression cassette containing a nucleic acid molecule encoding the above-mentioned protein according to A2) is a DNA capable of expressing the above-mentioned protein in a host cell, and the DNA may contain not only a promoter for promoting transcription of the gene encoding the above-mentioned protein but also a terminator for terminating transcription of the gene encoding the above-mentioned protein. Further, the expression cassette may further include regulatory sequences such as enhancer sequences.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may be pET-30a (+). In one embodiment of the invention, the recombinant vector is specifically pET30a-Xyn10/Fae 1. The pET30a-Xyn10/Fae1 is a vector obtained by replacing a small fragment between the BamHI and SalI cleavage sites of the pET-30a (+) vector with a DNA molecule shown in 85-3093 th site of the sequence 2. In another embodiment of the invention, the recombinant vector is specifically pET30a-Xyn10+ Fae 1. The pET30a-Xyn10+ Fae1 is a vector obtained by replacing a small fragment between the BamHI and SalI cleavage sites of the pET-30a (+) vector with a DNA molecule shown in sequence 8.

In a specific embodiment of the present invention, the recombinant microorganism is recombinant Escherichia coli, specifically E.coli B L21 (DE3) containing pET30a-Xyn10/Fae1 or pET30a-Xyn10+ Fae 1.

The invention also protects any one of the following applications S1) -S7):

s1) the application of the protein in xylanase and/or feruloyl esterase;

s2) the use of the above protein or the above biomaterial in the preparation of xylanase and/or feruloyl esterase;

s3) the application of the protein or the biological material in the preparation of hydroxylated cinnamic acid or xylo-oligosaccharide;

s4) the application of the protein or the biological material in degrading xylan and/or hydroxylated cinnamate compounds;

s5) use of the above protein or the above biomaterial for hydrolysis of a substance containing xylan and hydroxylated cinnamic acid ester bonds;

s6) the use of the above protein or the above biomaterial for catalyzing the cleavage of xylosidic bonds within xylan;

s7) the use of the above protein or the above biomaterial in catalyzing the cleavage of ester bonds between hydroxylated cinnamic acid and oligosaccharide or polysaccharide.

In the application, the hydroxylated cinnamate compound can be hydroxylated methyl cinnamate or hydroxylated ethyl cinnamate.

The hydroxylated cinnamic acid comprises ferulic acid, coumaric acid, caffeic acid and sinapic acid.

The invention finally provides a preparation method of the protein.

The preparation method of the protein protected by the invention comprises the following steps: fermenting and culturing a recombinant microorganism containing a gene encoding the protein, expressing the gene encoding the protein to obtain a recombinant microorganism culture containing the protein, and purifying the recombinant microorganism culture to obtain the protein.

In the above method, the recombinant microorganism containing the gene encoding the protein is obtained by introducing the gene encoding the protein into a recipient microorganism.

Further, a gene encoding the protein is introduced into the recipient microorganism by a recombinant vector. The recombinant vector is obtained by inserting the coding gene of the protein into a multiple cloning site of an expression vector.

Furthermore, the coding gene of the protein is a DNA molecule shown in 85 th to 3093 th positions of a sequence 2 or a DNA molecule shown in a sequence 8.

The expression vector may be a pET-30a (+) vector the recipient microorganism may be E.coli B L21 (DE 3).

In the above method, the fermentation culture method can be performed by inoculating the recombinant Escherichia coli solution into L B liquid medium (containing kanamycin) and culturing until OD₆₀₀When the concentration is 0.8-1.0, IPTG inducer is added, and induction culture is carried out at (16-37) DEG C for 3-24 h.

Further, the fermentation culture method comprises inoculating the recombinant Escherichia coli liquid into L B liquid medium (containing 50 μ g/m L kanamycin) at a volume ratio of 1:100, culturing, and culturing at 37 deg.C and 200rpm in a constant temperature shaking table to OD₆₀₀When the concentration was changed to 0.8 to 1.0, IPTG inducer (final concentration: 0.5mM) was added thereto, and the mixture was subjected to induction culture at 37 ℃ for 3 hours.

Further, Ni column affinity chromatography was used for purification.

Primer pairs for amplifying the full length or any segment of any one of the above genes also belong to the protection scope of the invention.

The primer pair can be specifically a primer pair consisting of a forward primer F shown in a sequence 3 and a reverse primer R shown in a sequence 4.

Experiments prove that the prepared bifunctional enzyme has high enzyme activity in the temperature range of (40-50) DEG C, and the optimal action temperature is 50 ℃; the enzyme has good temperature stability, and the enzyme activity is still kept above 100% after heat preservation for 1h at 40 ℃; the reaction pH can be 5-8, and the optimum pH values of xylanase and feruloyl esterase are 6.0 and 7.0 respectively; the xylanase has excellent pH stability, wherein the xylanase can be kept for 1h under the condition of pH (3-12), the enzyme activity can be kept above 74.4%, and the ferulic acid esterase can be kept for 1h under the condition of pH (4-10), and the enzyme activity can be kept above 96.7%. In addition, the enzyme can release xylooligosaccharide and ferulic acid and coumaric acid when being acted on steam-exploded corncobs.

The invention provides a novel bifunctional enzyme with xylanase and feruloyl esterase activities, which has better pH stability and can act on natural lignocellulose to release xylooligosaccharide, ferulic acid and coumaric acid simultaneously. The bifunctional enzyme provided by the invention can meet the requirements on the pH stability of xylanase and feruloyl esterase in industries such as feed, food and the like, has wide industrial application potential, provides a material for further construction of industrial high-yield engineering strains, and provides a comparable sequence and a referential property for stable xylanase and feruloyl esterase, so that the bifunctional enzyme provided by the invention has very good research prospect and commercial application value.

Drawings

FIG. 1 is an agarose gel electrophoresis of xylanase and feruloyl esterase bifunctional enzyme genes.

FIG. 2 shows the result of SDS electrophoresis of protein purification.

FIG. 3 shows the properties of xylanase and feruloyl esterase.

FIG. 4 shows the results of liquid chromatography detection of steam explosion of corncobs by bifunctional enzymatic hydrolysis of xylanase and feruloyl esterase.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The genomic DNA used in the following examples was extracted from farmyard manure in the kingdom village in the street of mythic, eastern, and metagenomic sequencing was completed.

Escherichia coli (E.coli) DH5 α: Biomed, product No. BC 116-02.

Escherichia coli (E.coli) B L21 (DE3) Biomed, Inc., product number BC 201-02.

pET-30a (+) vector: novagen company, product number: 69909.

ni column

6 Fast Flow: sigma company, product number: GE 17-5318-06.

Zelkova xylan is purchased from Megazyme and has the product number of P-XY L NBE-10G.

Methyl ferulate: purchased from Alfa Aesar, product number: B22657.

xylose: purchased from beijing huacyanine technologies ltd, product number: X1500-500G.

Ferulic acid: purchased from beijing huacyanine technologies ltd, product number: 46278-1 g.

L B the medium is prepared from tryptone 1% (w/v), yeast extract 0.5% (w/v), NaCl 1% (w/v), pH7.0, and sterilizing at 121 deg.C for 20 min.

The preparation method of the corn cob by steam explosion comprises the following steps: crushing corncobs (collected from farmland in Luoyang city, Henan province), sieving with a 50-mesh sieve, maintaining the pressure for 300s under 1.5MPa, suddenly releasing the pressure, performing blasting treatment, and storing the treated sample at 4 ℃ for later use.

Example 1 preparation of recombinant protein Xyn10/Fae1

Clone of Xyn10/Fae1 gene

1. Design of primers

Designing a forward primer F and a reverse primer R according to the Xyn10/Fae1 gene sequence in the metagenome, wherein the forward primer is added with a BamHI enzyme cutting site and a protective base thereof, and the reverse primer is added with a SalI enzyme cutting site and a protective base thereof, and the primer sequences are as follows:

a forward primer F: 5' -CGGATCCGCAGTGCCAGGTAATG-3' (SEQ ID NO: 3);

reverse primer R: 5' -GCGTCGACTTATTTTACAACCATATTAG-3' (SEQ ID NO: 4).

2. PCR amplification

And (3) performing PCR amplification by using the genome DNA as a template and adopting the forward primer F and the reverse primer R in the step 1 to obtain a PCR product. The PCR product is Xyn10/Fae1 gene, and the nucleotide sequence is shown as sequence 2.

An amplification system: according to

The 25. mu. L reaction was formulated according to the instructions for the ultra-fidelity DNA polymerase (NEB).

Amplification conditions: 30s 1 cycles at 98 ℃, 10s at 52 ℃, 1min30s at 72 ℃, 30 cycles, 10min at 72 ℃ and one cycle.

The PCR product was recovered by cutting the gel using 1% agarose gel electrophoresis, and a DNA fragment of about 3.1kb (as shown in FIG. 1) was recovered according to the gel recovery kit (TIANGEN) instructions.

Second, construction of recombinant expression vector and recombinant bacterium

1. Construction of recombinant expression vectors

And (3) carrying out double enzyme digestion on the PCR product obtained in the step one through BamHI and SalI, cutting the gel, recovering, connecting the PCR product with a pET-30a (+) vector subjected to double enzyme digestion through BamHI and SalI, transferring the PCR product into an E.coli DH5 α competent cell, coating the competent cell on a L B solid plate containing 50 mu g/m L kanamycin, culturing the competent cell at 37 ℃ for 12h, and carrying out colony PCR identification by adopting a forward primer F and a reverse primer R to obtain a positive clone.

2. Sequencing validation

The positive clone which is correctly identified by colony PCR is inoculated into L B liquid culture medium containing 50 mu g/m L kanamycin to be cultured, the plasmid is extracted and sent to Beijing Huada Gen company for sequencing, and the plasmid which is correctly sequenced is named as pET30a-Xyn10/Fae 1.

The sequencing result shows that: pET30a-Xyn10/Fae1 is a vector obtained by replacing a small fragment between the BamHI and SalI cleavage sites of the pET-30a (+) vector with the DNA molecule shown at 85-3093 th position in the sequence 2. DNA coding sequence 1 shown by 85-3093 th nucleotides from 5' end, namely Xyn10/Fae1 protein with signal peptide removed from 29-1030 th amino acid residues from N end. The protein shown in the sequence 1 is Xyn10/Fae1 protein containing a signal peptide sequence.

3. Construction of recombinant bacterium

Transferring a plasmid pET30a-Xyn10/Fae1 with correct sequencing into E.coli B L21 (DE3) competent cells, coating the competent cells on an L B solid plate containing 50 mu g/m L kanamycin, culturing for 12h at 37 ℃, and carrying out colony PCR identification by adopting a forward primer F and a reverse primer R to obtain a recombinant strain B L21 (DE3)/pET30a-Xyn10/Fae 1.

Empty vector pET-30a (+) was transferred into E.coli B L21 (DE3) competent cells, spread on L B solid plates containing 50. mu.g/m L kanamycin, and cultured at 37 ℃ for 12 hours to obtain empty vector control strain B L21 (DE3)/pET30 a.

Thirdly, induction culture of recombinant bacteria and purification of recombinant protein Xyn10/Fae1

1. Induced culture of recombinant bacteria

The recombinant strain B L21 (DE3)/pET30a-Xyn10/Fae1 single colony constructed in the second step is picked up, inoculated into L B culture medium containing 50 ug/m L kanamycin at 5m L, cultured overnight at 37 ℃ at 200rpm, transferred to L B culture medium containing 50 ug/m L kanamycin at 1% inoculum size the next day, and cultured until the OD (bacterial liquid density) of the bacterial liquid concentration₆₀₀When the concentration is 0.8-1.0, IPTG inducer (final concentration is 0.5mM) is added, induction culture is carried out for 3h at 37 ℃, thalli are collected by centrifugation, cells are resuspended by 20mM Tris-HCl buffer solution with the pH value of 8.0, and supernatant is taken by ultrasonic disruption and centrifugation (ultrasonic disruption parameters: working time 2s, intermittent time 4s, total time 10min, power 200W; centrifugation conditions: 4 ℃, 10000rpm, 15min) to obtain intracellular supernatant of the recombinant strain B L21 (DE3)/pET30a-Xyn10/Fae 1.

The empty vector intracellular supernatant was obtained by replacing B L21 (DE3)/pET30a-Xyn10/Fae1 with the empty vector control strain B L21 (DE3)/pET30a according to the above culture method, and the other steps were not changed.

2. Purification of recombinant protein Xyn10/Fae1

Purifying intracellular supernatant of B L21 (DE3)/pET30a-Xyn10/Fae1 by Ni column affinity chromatography to obtain recombinant protein Xyn10/Fae 1. the method comprises the following specific steps of purifying Xyn10/Fae1 in crude enzyme solution after cell breaking by Ni column affinity chromatography (Ni column Ni)

6 Fast Flow) with 15m L eluent 1 and 10m L eluent 2 and 15m L eluent 3, respectively, wherein eluent 1 is composed of Tris-Cl buffer solution with final concentration of 20 mmol/L, sodium chloride with final concentration of 500 mmol/L and 20 mmol/L imidazole, eluent 2 is composed of Tris-Cl buffer solution with final concentration of 20 mmol/L, and 500 mmol/LThe reagent consists of sodium chloride with mmol/L and imidazole with final concentration of 60 mmol/L, the eluent 3 consists of Tris-Cl buffer with final concentration of 20 mmol/L, sodium chloride with final concentration of 500 mmol/L and imidazole with final concentration of 300 mmol/L, the pH of the eluent is 8.0, proteins in different eluents are detected by 12.5 percent SDS-PAGE, and the eluent containing target proteins is marked as Xyn10/Fae1 solution.

The polyacrylamide gel electrophoresis is carried out on the Xyn10/Fae1 solution, and the electrophoresis result shows that the Xyn10/Fae1 solution contains a single band of target protein, the size of the target protein is 118kDa (shown in figure 2), and the target protein is the recombinant protein Xyn10/Fae 1.

Example 2 determination of the enzymatic Activity of the recombinant protein Xyn10/Fae1 as xylanase and Feruloyl esterase

Enzyme activity of recombinant protein Xyn10/Fae1 on model substrate zelkova xylan (BWX)

The enzyme activity was measured as follows (all buffers used were 50mM disodium hydrogen phosphate-citric acid buffer pH 6.0):

1. the zelkova xylan is dissolved in the buffer solution to make the final concentration 10mg/m L, namely BWX solution.

2. Uniformly mixing a diluted recombinant protein Xyn10/Fae1 solution (the concentration is 0.007mg/m L) of 100 mu L with a BWX solution of 100 mu L, incubating at 50 ℃ for 10min, adding 150 mu L DNS, uniformly mixing, boiling water bathing for 5min, immediately placing in ice water to stop reaction, adding 350 mu L H, and stirring₂O, detection of OD_540nmThe absorbance value of (c). Meanwhile, an equal volume of buffer solution was used as a negative control instead of the enzyme solution.

3. Dissolving xylose with different concentrations in buffer solution to obtain standard solutions with different concentrations, and detecting OD of the standard solutions with different concentrations_540nmThe absorbance value of (D) is OD taking the concentration of xylose as the abscissa_540nmAnd making a standard curve for the ordinate for enzyme activity determination.

The amount of protein required to release 1. mu. mol xylose per minute was defined as the xylanase activity (U/mg) of the recombinant protein Xyn10/Fae 1.

According to the enzyme activity detection method, the Xyn10/Fae1 solution is replaced by the empty vector intracellular supernatant to be used as a contrast.

The experimental results show that no xylanase enzyme activity was detected in the empty vector supernatant. Under the conditions, the xylanase enzyme activity of the recombinant protein Xyn10/Fae1 is 37.51U/mg.

II, enzyme activity of recombinant protein Xyn10/Fae1 on model substrate ferulic acid methyl ester (MFA)

The enzyme activity was measured as follows (all buffers used were 50mM disodium hydrogen phosphate-citric acid buffer pH 7.0):

1. methyl ferulate was dissolved in methanol to give a final concentration of 100mM, and diluted 100-fold with buffer to give a 1mM MFA solution.

2. The diluted recombinant protein Xyn10/Fae1 solution (the concentration is 0.07mg/m L) of 10 mu L and the MFA solution of 190 mu L are mixed uniformly, incubated at 50 ℃ for 10min and then added with 100 mu L acetonitrile to stop the reaction, meanwhile, equal volume of buffer solution is used as a negative control to replace enzyme solution, and HP L C detects the peak area of ferulic acid.

3. Dissolving ferulic acid in methanol, diluting with a buffer solution to prepare ferulic acid solutions with different concentrations, detecting peak areas corresponding to the ferulic acid with different concentrations by using HP L C, and making a standard curve by taking the concentration of the ferulic acid as a horizontal coordinate and the corresponding peak area as a vertical coordinate, wherein the standard curve is used for measuring the hydrolysis capacity of the recombinant protein Xyn10/Fae1 on MFA.

The amount of protein required to release 1. mu. mol ferulic acid per minute was defined as the feruloyl esterase enzyme activity (U/mg) of the recombinant protein Xyn10/Fae 1.

The HP L C analysis conditions comprise high performance liquid chromatography (SHIMADZU L C-15C), analysis column ACE Excel 5C18-Amide (250mm × 4.6.6 mm), mobile phase water acetonitrile and formic acid 4:6:0.01, flow rate 1.0m L/min, detector SHINMADZU SPD-15C, detection wavelength 322nm, column temperature 40 ℃ and sample injection amount 20 mu L.

The experimental results show that no feruloyl esterase enzyme activity was detected in the empty vector supernatant. Under the conditions, the ferulic acid esterase enzyme activity of the recombinant protein Xyn10/Fae1 is 11.19U/mg.

Example 3 Properties of the recombinant protein Xyn10/Fae1

First, optimum pH measurement

The enzyme activity determination method is the same as the steps in example 2, and the recombinant protein Xyn10/Fae1 prepared in example 1 is used as a solution to be determined. Except that the following buffers (at 50mM each) were used: disodium hydrogen phosphate-citric acid buffer (pH5.0), disodium hydrogen phosphate-citric acid buffer (pH 6.0), disodium hydrogen phosphate-citric acid buffer (pH 7.0), and disodium hydrogen phosphate-citric acid buffer (pH 8.0).

The xylanase activity of the recombinant protein Xyn10/Fae1 is highest in a buffer solution with the pH value of 6.0, the ferulic acid esterase activity is highest in a buffer solution with the pH value of 7.0, the highest enzyme activity is taken as 100%, and the relative enzyme activities under other pH values are calculated.

II, measuring pH stability

The recombinant protein Xyn10/Fae1 (control protein concentration of 0.7mg/m L) was treated with glycine-hydrochloric acid buffer (pH 2.2), glycine-hydrochloric acid buffer (pH3.0), sodium acetate buffer (pH 4.0), sodium acetate buffer (pH5.0), NaHPO, at different pH buffers (all at 50mM)₄-NaH₂PO₄Buffer (pH 6.0), NaHPO₄-NaH₂PO₄Buffer (pH 7.0), NaHPO₄-NaH₂PO₄Buffer (pH 8.0), glycine-sodium hydroxide buffer (pH 9.0), glycine-sodium hydroxide buffer (pH 10.0), NaH₂PO₄NaOH buffer (pH 11.0), NaH₂PO₄NaOH buffer (pH 12.0). After 1 hour at 4 ℃ the enzyme activity was determined as in example 2.

The enzyme activity of the untreated recombinant protein Xyn10/Fae1 is taken as 100%, and the relative enzyme activity of the recombinant protein Xyn10/Fae1 after treatment with different pH values is calculated, and the result is shown in FIG. 3 (a). The xylanase has excellent pH stability, wherein the xylanase can be kept for 1h under the condition of pH3-12, the enzyme activity can be kept above 74.4%, and the ferulic acid esterase can be kept for 1h under the condition of pH 4-10, and the enzyme activity can be kept above 96.7%.

Third, optimum reaction temperature

The enzyme activity determination method is the same as the steps in example 2, and the recombinant protein Xyn10/Fae1 prepared in example 1 is used as a solution to be determined. The differences lie in the following reaction temperatures: 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃. The result shows that the recombinant protein Xyn10/Fae1 has the highest activity at 50 ℃, the highest enzyme activity is recorded as 100%, and the relative enzyme activities at other reaction temperatures are calculated.

Fourthly, measuring the temperature stability

After the recombinant protein Xyn10/Fae1 was incubated at 4 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C and 70 deg.C for 1h, the solution was ice-washed for 10min as a solution to be tested, the protein concentration was controlled at 0.7mg/m L, and the enzyme activity was determined according to the procedure of example 2.

The enzyme activity of the untreated recombinant protein Xyn10/Fae1 is taken as 100%, and the relative enzyme activity of the recombinant protein Xyn10/Fae1 after treatment at different temperatures is calculated, and the result is shown in figure 3 (b). The enzyme has good temperature stability, and the enzyme activity is still kept above 100% after heat preservation for 1h at 40 ℃.

Example 4 recombinant protein Xyn10/Fae1 hydrolysis steam explosion of corncobs

1. Preparation of hydrolysis reaction System

Uniformly mixing substrate steam explosion corncobs, recombinant protein Xyn10/Fae1 and 50mM disodium hydrogen phosphate-citric acid buffer solution (pH 6.0) to obtain a hydrolysis reaction system (the total volume is 1m L), wherein the mass fraction of the corncobs in the system is 2%, and the final concentration of the recombinant protein Xyn10/Fae1 in the system is 400 nM.

Meanwhile, the inactivated recombinant protein Xyn10/Fae1 in boiling water bath for 30min is used as a substrate control.

2. Hydrolysis reaction

And (3) reacting the hydrolysis reaction system for 48h under the condition of 200rpm of a shaking table at 40 ℃, then carrying out boiling water bath for 10min, then carrying out high-speed centrifugation at 12000rpm for 10min, removing precipitates, and filtering supernatant to obtain a sample to be detected.

3. HP L C detection of hydrolysates

The supernatant was filtered through a 0.22 μm filter and then ferulic acid and coumaric acid were measured by the method described in example 2.

HP L C analysis conditions comprise high performance liquid chromatography, SHIMADZU L C-15C, analytical column ACE Excel 5C18-Amide (250mm × 4.6.6 mm), mobile phase water acetonitrile, formic acid 4:6:0.01, flow rate 1.0m L/min, detector SHINMASZU SPD-15C, detection wavelength 322nm, column temperature 40 ℃ and sample injection 20 mu L.

The results are shown in FIG. 4. As can be seen from the figure, the recombinant protein Xyn10/Fae1 can effectively hydrolyze ferulic acid and coumaric acid ester bonds in steam explosion corncobs to generate ferulic acid and coumaric acid.

Example 5 preparation of recombinant protein Xyn10+ Fae1 and enzyme Activity determination thereof

Preparation of recombinant protein Xyn10+ Fae1

1. Clone of Xyn10+ Fae1 gene

1) Design of primers

According to the Xyn10/Fae1 gene sequence, except the primer F in example 1, a forward primer F ', a reverse primer R ' -1 (primer plus SacI enzyme cutting site and its protective base) and a reverse primer R ' -2 primer (primer plus SalI enzyme cutting site and its protective base) of the fusion protein Xyn10+ Fae1 are designed, and the primer sequences are as follows:

forward primer F': 5' -CGAGCTCCCTGCTACAACTTCAGTTCC-3' (SEQ ID NO: 5);

reverse primer R' -1: 5' -CGAGCTCTTCTATTTTTCCAGTTGTAG-3' (SEQ ID NO: 6);

reverse primer R' -2: 5' -GCGTCGACTTATACACCTGTTTGAGA-3' (SEQ ID NO: 7).

2) PCR amplification

Two independent PCR runs were performed using genomic DNA as a template. One round of PCR amplification is carried out by adopting the forward primer F and the reverse primer R ' -1 in the step 1 of the embodiment 1, and the other round of PCR amplification is carried out by adopting the forward primer F ' and the reverse primer R ' -2, thus obtaining a PCR product.

An amplification system: according to

Amplification conditions: 30s 1 cycles at 98 ℃, 10s at 55 ℃, 45s at 72 ℃, 30 cycles, 10min at 72 ℃ and one cycle.

The PCR product was recovered by cutting the gel using 1% agarose gel electrophoresis, and DNA fragments of about 1.2kb and 1.1kb were recovered by the method described in reference to gel recovery kit (TIANGEN).

2. Recombinant expression vectors and identification

1) Construction of recombinant expression vectors

Carrying out double enzyme digestion on the PCR product of about 1.2kb obtained in the step one by BamHI and SacI, carrying out double enzyme digestion on the obtained PCR product of about 1.1kb by SacI and SalI, cutting and recovering gel, connecting the obtained PCR product with a pET-30a (+) vector subjected to double enzyme digestion by BamHI and SalI, transferring the obtained PCR product into E.coli DH5 α competent cells, coating the competent cells on a L B solid plate containing 50 mu g/m L kanamycin, culturing the competent cells at 37 ℃ for 12h, and carrying out PCR identification by using a forward primer F and a reverse primer R' -2 colony to obtain a positive clone.

2) Sequencing validation

The positive clone which is correctly identified by colony PCR is inoculated into L B liquid culture medium containing 50 mu g/m L kanamycin to be cultured, the plasmid is extracted and sent to Beijing Huada Gen company for sequencing, and the plasmid which is correctly sequenced is named as pET30a-Xyn10+ Fae 1.

The sequencing result shows that: pET30a-Xyn10+ Fae1 is a vector obtained by replacing a small fragment between the BamHI and SalI cleavage sites of the pET-30a (+) backbone vector with a DNA molecule A shown in sequence 8. The DNA molecule A sequentially consists of a DNA molecule shown in 85 th-1269 th sites from the 5 'end of the sequence 2, a SacI enzyme cutting site GAGCTC, a DNA molecule shown in 1729 th-2808 th sites from the 5' end of the sequence 2 and a termination codon TAA.

The amino acid sequence of the recombinant protein Xyn10+ Fae1 coded by the DNA molecule A sequentially consists of 29 th to 423 th amino acid residues from the N terminal of the sequence 1, Glu amino acid residues, L eu amino acid residues and 577 th and 936 th amino acid residues from the N terminal of the sequence 1.

3. Construction of recombinant bacterium

The construction method of the recombinant strain is the same as 3 in step two of example 1.

4. Induced culture of recombinant bacteria and purification of recombinant protein Xyn10+ Fae1

The induction culture of the recombinant bacteria and the purification method of the recombinant protein Xyn10+ Fae1 are the same as the third step of the example 1.

Secondly, enzyme activity determination of recombinant protein Xyn10+ Fae1 as xylanase and feruloyl esterase

The method for measuring the enzyme activity of the recombinant protein Xyn10+ Fae1 as xylanase and feruloyl esterase is the same as that in example 2.

The xylanase enzyme activity of the recombinant protein Xyn10+ Fae1 is 147.81U/mg, and the enzyme activity of the ferulic acid esterase is 22.13U/mg.

Thirdly, hydrolyzing the recombinant protein Xyn10+ Fae1 and performing steam explosion on corncobs

The method for hydrolyzing the recombinant protein Xyn10+ Fae1 and steam blasting the corncobs is the same as that in example 4.

The results are shown in FIG. 4. As can be seen from the figure, the recombinant protein Xyn10+ Fae1 can effectively hydrolyze ferulic acid and coumaric acid ester bonds in steam explosion corncobs to generate ferulic acid and coumaric acid.

Sequence listing

<110> university of agriculture in China

<120> bifunctional enzyme with xylanase and feruloyl esterase activities, and coding gene and application thereof

<160>8

<170>PatentIn version 3.5

<210>1

<211>1030

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Met Lys Lys Ile Leu Ser Ala Phe Leu Ser Leu Ala Leu Val Leu Ser

1 5 10 15

Leu Ile Thr Ile Ala Pro Ala Ala Lys Val Glu Ala Ala Val Pro Gly

20 25 30

Asn Ala Asn Leu Leu Asn Thr Tyr Gly Arg Ser Phe Gly His Ile Gly

35 40 45

Thr Cys Val Thr Pro Tyr Gln Trp Asn Asp Ser Asn Thr Arg Asn Phe

50 55 60

Ile Lys Gly Glu Tyr Asn Ser Ile Thr Met Glu Asn Glu Met Lys Pro

65 70 75 80

Asp Ala Val Leu Asn Ser Ser Thr Met Ser Val Ala Gln Ala Lys Gln

85 90 95

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

100 105 110

Leu Asn Phe Ser Thr Ile Asp Ser Met Met Lys Asn Ala Tyr Asp Asn

115 120 125

Gly Leu Gln Ile Arg Tyr His Thr Leu Val Trp His Ser Gln Thr Pro

130135 140

Asp Trp Tyr Phe Arg Ser Gly Tyr Ser Ser Asn Gly Gly Tyr Val Ser

145 150 155 160

Lys Ser Gln Met Asn Ala Arg Met Glu Phe Tyr Ile Lys Ser Val Met

165 170 175

Asn His Val Tyr Ser Ser Gln Tyr Ala Ser Thr Val Tyr Cys Trp Asp

180 185 190

Val Val Asn Glu Tyr Met His Ala Thr Asn Ser Gly Trp Gln Lys Ile

195 200 205

Tyr Gly Ala Val Asn Thr Arg Pro Asp Phe Val Lys Leu Ala Phe Gln

210 215 220

Tyr Ala Tyr Glu Thr Leu Glu Tyr Phe Asn Leu Glu Asn Ser Val Ser

225 230 235 240

Leu Phe Tyr Asn Asp Tyr Asn Thr Tyr Ile Asp Cys Asp Lys Ile Ile

245 250 255

Ser Met Ile Asn Tyr Ile Asn Ala Glu Lys Lys Ile Cys Ser Gly Val

260 265 270

Gly Met Gln Ser His Leu Ser Ser Thr Tyr Pro Ser Val Ser Tyr Tyr

275 280 285

Lys Ala Ala Leu Asp Lys Phe Ile Lys Gln Gly Tyr Glu Val Gln Ile

290 295300

Thr Glu Leu Asp Ala Lys Gly Asn Asn Asn Ser Asp Gln Ala Asn Tyr

305 310 315 320

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

325 330 335

Asn Ile Thr Ala Ile Thr Trp Trp Gly Leu His Asp Gly Ala Ser Trp

340 345 350

Arg Arg Gly Asp Asn Pro Leu Leu Phe Ser Ser Leu Gly Val Lys Lys

355 360 365

Gln Ser Tyr Thr Ser Val Leu Glu Ala Tyr Tyr Glu Ala Gly Phe Pro

370 375 380

Ile Ser Pro Val Thr Pro Thr Ala Ser Ile Lys Pro Ser Val Asn Pro

385 390 395 400

Ser Thr Ala Pro Ser Asn Asn Pro Ser Thr Val Pro Thr Gln Ser Val

405 410 415

Ala Thr Thr Gly Lys Ile Glu Asn Gly Val Tyr Tyr Ile Lys Asn Val

420 425 430

Asn Ala Gln Lys Tyr Leu Gln Val Ala Gly Asn Thr Gly Ala Asp Ala

435 440 445

Gln Asn Val Glu Leu Gly Ser Gly Thr Gly Val Ala Gly Gln Lys Trp

450 455460

Glu Val Thr Asn Asn Ser Asp Gly Thr Val Thr Leu Lys Ser Ala Leu

465 470 475 480

Gly Ser Phe Ser Leu Asp Val Ala Asn Ala Ala Asp Glu Asp Gly Ala

485 490 495

Asn Val Gln Ile Tyr Thr Ser Tyr Asp Gly Asp Ala Gln Lys Phe Phe

500 505 510

Ile Lys Glu Thr Ala Thr Asp Gly Ile Tyr Gln Ile Ala Thr Lys Ala

515 520 525

Ser Ser Gly Thr Lys Asn Leu Asp Gly Ser Asn Tyr Gly Thr Glu Asp

530 535 540

Gly Thr Asn Ile Gln Gln Trp Ser Asn Thr Thr Asn Thr Asn Gln Gln

545 550 555 560

Trp Ile Phe Glu Lys Ile Gly Gly Ser Ser Ser Thr Thr Thr Gln Ala

565 570 575

Pro Ala Thr Thr Ser Val Pro Val Val Thr Ser Ser Ala Pro Ser Thr

580 585 590

Val Pro Thr Gln Ser Thr Gln Thr Asn Gly Ser Met Thr Asp Thr Ala

595 600 605

Lys Ala Tyr Met Ala Lys Met Asn Val Val Asn Lys Cys Pro Ser Gly

610 615 620

Ala Asp Gln Lys Gln Ala Gly Arg Thr Tyr Pro Ala Ala Thr Lys Lys

625 630 635 640

Thr Tyr Tyr Ser Thr Thr Thr Gly Ser Asn Arg Ser Cys Asn Val Phe

645 650 655

Leu Pro Ala Asn Tyr Ser Ser Ser Lys Lys Tyr Pro Val Leu Tyr Leu

660 665 670

Leu His Gly Ile Met Gly Asn Glu Asp Ser Met Leu Gly Asn Ala Ile

675 680 685

Glu Ile Pro Thr Asn Leu Ala Ala Gln Gly Lys Ala Lys Glu Met Ile

690 695 700

Ile Val Leu Pro Asn Glu Tyr Ala Pro Ala Pro Gly Thr Ser Val Ala

705 710 715 720

Ala Gly Leu Asn Gln Ala Tyr Phe Asp Gly Tyr Asp Asn Phe Ile Asn

725 730 735

Asp Leu Thr Lys Asp Leu Met Pro Tyr Ile Glu Lys Asn Tyr Ser Val

740 745 750

Ala Thr Gly Arg Asp Asn Thr Ala Ile Ala Gly Phe Ser Met Gly Gly

755 760 765

Arg Asn Ala Leu Tyr Ile Gly Tyr Ala Arg Pro Asp Leu Phe Gly Tyr

770 775 780

Val Gly Ala Phe Ser Pro Ala Pro Gly Val Thr Pro Gly Gln Asp Tyr

785 790 795 800

Ser Gly Phe His Lys Gly Leu Phe Ser Glu Asn Asp Phe Arg Ile Lys

805 810 815

Asp Glu Arg Tyr Val Pro Tyr Val Thr Leu Ile Ser Cys Gly Thr Asn

820 825 830

Asp Ser Val Val Gly Thr Phe Pro Lys Ser Tyr His Asp Ile Leu Thr

835 840 845

Arg Asn Asn Gln Pro His Ile Trp Phe Glu Val Pro Gly Ala Asp His

850 855 860

Asp Asn Asn Ala Ile Ala Ala Gly Tyr Tyr Asn Phe Val Ser Ala Ala

865 870 875 880

Phe Gly Val Leu Gly Ile Asp Asn Thr Thr Pro Ser Thr Gln Pro Ser

885 890 895

Thr Gln Pro Ser Val Ala Pro Ser Val Glu Pro Ser Val Glu Pro Ser

900 905 910

Thr Gln Pro Ser Thr Glu Pro Ser Thr Gln Pro Ser Val Thr Pro Ser

915 920 925

Gln Thr Pro Ser Gln Thr Gly Val Thr Cys Glu Tyr Asn Val Val Ser

930 935 940

Asp Trp Gly Ser Ser Phe Gln Gly Glu Ile Val Ile Thr Asn Asn Ser

945 950 955 960

Gly Lys Thr Ile Asn Gly Trp Thr Leu Thr Cys Asp Tyr Asn Cys Glu

965 970 975

Ile Val Asn Leu Trp Asn Ala Asp Phe Val Gly Gln Thr Gly Thr Lys

980 985 990

Val Thr Val Lys Asn Pro Ser Trp Asp Ala Asn Leu Pro Asp Gly Lys

995 1000 1005

Ser Val Thr Ile Ser Phe Ile Ala Asn Gly Thr Asp Lys Ser Ala

1010 1015 1020

Pro Thr Asn Met Val Val Lys

1025 1030

<210>2

<211>3093

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atgaaaaaaa tattaagcgc gtttttatct cttgccttag tgttaagcct gattacaatc 60

gcacctgctg caaaagtaga agctgcagtg ccaggtaatg caaacttatt aaacacttac 120

ggcagatctt ttggacacat cggaacatgt gtcacacctt atcaatggaa tgatagtaat 180

acaagaaact ttattaaggg agaatacaac agcattacaa tggaaaatga aatgaaacct 240

gatgctgttt tgaattcctc aactatgtct gttgctcagg caaaacagca aggttattac 300

ataccaagca gctatacaga aagtactgta cctaagttaa acttcagtac aatcgacagt 360

atgatgaaaa atgcttatga caacggtctt cagatccgtt accatacctt agtttggcat 420

agccagactc cagactggta tttccgttcc ggttactctt caaacggcgg atatgtaagc 480

aaatcccaga tgaatgccag aatggaattc tacatcaaat ccgttatgaa tcatgtatac 540

agcagccagt atgcaagcac agtttactgc tgggacgttg taaatgaata catgcatgct 600

actaattcag gatggcagaa aatctacggt gctgtaaata caagaccaga ctttgtaaaa 660

ttagcattcc agtatgctta cgaaacatta gagtacttta acttagagaa ctctgtttcc 720

ttattctaca acgattacaa tacatatatt gactgcgaca aaatcatttc tatgattaac 780

tacatcaatg cagaaaagaa aatctgctct ggtgtcggca tgcagtctca cttaagttct 840

acatatccta gtgtttctta ttacaaagct gcattagata aatttattaa acaaggttac 900

gaagttcaga ttacagaatt agatgcaaaa ggtaacaata acagcgacca ggcaaattac 960

tgcaaacaga ttatggctgc tattctttct gctaagaaag caggcggaaa cattactgca 1020

attacctggt ggggacttca cgatggagct tcctggagaa gaggagacaa tccattatta 1080

ttctctagcc ttggcgtgaa aaaacaatcc tatacatctg tactggaagc ttattatgaa 1140

gcaggattcc caatcagtcc agtgactcca actgcttcta ttaaaccatc agtaaatcct 1200

tcaacagcac cttctaataa tccttctact gttccaactc agtcagtagc tacaactgga 1260

aaaatagaaa acggtgtata ttacatcaaa aatgtaaacg ctcagaaata cttacaggtt 1320

gccggtaaca ctggtgcaga cgctcagaat gttgagcttg gttctggtac aggtgttgca 1380

ggtcagaaat gggaagttac aaataactct gatggtactg taacattaaa gagtgcttta 1440

ggaagcttca gtttagacgt tgcaaatgca gctgatgaag atggcgcaaa tgttcagatt 1500

tacacttctt atgatggaga tgctcagaaa ttcttcatta aagaaactgc aactgatggc 1560

atttaccaga ttgcaacaaa agcaagcagc ggtacaaaga acttagacgg aagcaactac 1620

ggtacagaag atggtacaaa cattcagcag tggtccaaca ctactaatac aaaccagcag 1680

tggatctttg aaaagattgg cggaagttct tctactacaa cacaggctcc tgctacaact 1740

tcagttcccg ttgttacatc cagtgctcca agtactgtac caactcagtc tactcagaca 1800

aacggcagca tgacagatac tgcaaaagca tacatggcaa aaatgaatgt tgtaaataaa 1860

tgcccatctg gtgctgatca gaaacaggct ggcagaactt atcctgctgc aacaaagaaa 1920

acttattatt caacaacaac aggttcaaac cgttcctgta atgtattcct tcctgcaaac 1980

tattcttctt ccaagaaata tcctgtactt tacttattac atggtattat gggaaatgaa 2040

gattctatgt taggaaatgc aattgaaatc cctactaact tagctgcaca gggaaaagca 2100

aaagaaatga ttatcgtact tccaaatgaa tatgcaccag ctcctggtac ttctgtagct 2160

gctggattaa atcaggcata tttcgatgga tatgataact ttatcaacga tttaacaaaa 2220

gacttaatgc catacatcga aaagaactac tccgttgcaa ctggcagaga caatacagca 2280

atcgccggtt tctcaatggg tggacgtaac gctctttata tcggatatgc aagaccagac 2340

ttatttggtt atgttggagc attttctcca gctcctggtg taacacctgg acaggattat 2400

tccggtttcc acaaaggatt attctcagaa aatgatttcc gtatcaaaga tgaaagatat 2460

gtaccatatg taacattaat cagctgtgga accaatgact ctgttgttgg aacattccct 2520

aagagctatc atgatatttt aacaagaaac aatcagccac atatctggtt tgaagtacct 2580

ggtgcagacc atgacaacaa cgcaatcgct gctggatact ataactttgt atctgctgca 2640

tttggagttt taggcattga caatacaaca ccatctacac agcctagcac acagccttct 2700

gtagctccta gcgttgagcc ttcagtagaa cctagtacac agcctagtac agaacctagc 2760

acacagcctt ctgtaactcc tagccagaca ccttctcaaa caggtgtaac atgtgagtac 2820

aatgttgtaa gtgactgggg ttcatccttc cagggtgaaa ttgtaattac taacaactct 2880

ggcaaaacta taaatggctg gactttaaca tgtgactaca actgcgaaat cgtaaactta 2940

tggaatgctg atttcgttgg acagactgga acaaaagtta cagttaaaaa tccatcctgg 3000

gatgcaaatc ttcctgatgg aaaatctgtt acaatcagct tcattgcaaa tggaactgat 3060

aagagtgctc caactaatat ggttgtaaaa taa 3093

<210>3

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cggatccgca gtgccaggta atg 23

<210>4

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gcgtcgactt attttacaac catattag 28

<210>5

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

cgagctccct gctacaactt cagttcc 27

<210>6

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

cgagctcttc tatttttcca gttgtag 27

<210>7

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gcgtcgactt atacacctgt ttgaga 26

<210>8

<211>2274

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gcagtgccag gtaatgcaaa cttattaaac acttacggca gatcttttgg acacatcgga 60

acatgtgtca caccttatca atggaatgat agtaatacaa gaaactttat taagggagaa 120

tacaacagca ttacaatgga aaatgaaatg aaacctgatg ctgttttgaa ttcctcaact 180

atgtctgttg ctcaggcaaa acagcaaggt tattacatac caagcagcta tacagaaagt 240

actgtaccta agttaaactt cagtacaatc gacagtatga tgaaaaatgc ttatgacaac 300

ggtcttcaga tccgttacca taccttagtt tggcatagcc agactccaga ctggtatttc 360

cgttccggtt actcttcaaa cggcggatat gtaagcaaat cccagatgaa tgccagaatg 420

gaattctaca tcaaatccgt tatgaatcat gtatacagca gccagtatgc aagcacagtt 480

tactgctggg acgttgtaaa tgaatacatg catgctacta attcaggatg gcagaaaatc 540

tacggtgctg taaatacaag accagacttt gtaaaattag cattccagta tgcttacgaa 600

acattagagt actttaactt agagaactct gtttccttat tctacaacga ttacaataca 660

tatattgact gcgacaaaat catttctatg attaactaca tcaatgcaga aaagaaaatc 720

tgctctggtg tcggcatgca gtctcactta agttctacat atcctagtgt ttcttattac 780

aaagctgcat tagataaatt tattaaacaa ggttacgaag ttcagattac agaattagat 840

gcaaaaggta acaataacag cgaccaggca aattactgca aacagattat ggctgctatt 900

ctttctgcta agaaagcagg cggaaacatt actgcaatta cctggtgggg acttcacgat 960

ggagcttcct ggagaagagg agacaatcca ttattattct ctagccttgg cgtgaaaaaa 1020

caatcctata catctgtact ggaagcttat tatgaagcag gattcccaat cagtccagtg 1080

actccaactg cttctattaa accatcagta aatccttcaa cagcaccttc taataatcct 1140

tctactgttc caactcagtc agtagctaca actggaaaaa tagaagagct ccctgctaca 1200

acttcagttc ccgttgttac atccagtgct ccaagtactg taccaactca gtctactcag 1260

acaaacggca gcatgacaga tactgcaaaa gcatacatgg caaaaatgaa tgttgtaaat 1320

aaatgcccat ctggtgctga tcagaaacag gctggcagaa cttatcctgc tgcaacaaag 1380

aaaacttatt attcaacaac aacaggttca aaccgttcct gtaatgtatt ccttcctgca 1440

aactattctt cttccaagaa atatcctgta ctttacttat tacatggtat tatgggaaat 1500

gaagattcta tgttaggaaa tgcaattgaa atccctacta acttagctgc acagggaaaa 1560

gcaaaagaaa tgattatcgt acttccaaat gaatatgcac cagctcctgg tacttctgta 1620

gctgctggat taaatcaggc atatttcgat ggatatgata actttatcaa cgatttaaca 1680

aaagacttaa tgccatacat cgaaaagaac tactccgttg caactggcag agacaataca 1740

gcaatcgccg gtttctcaat gggtggacgt aacgctcttt atatcggata tgcaagacca 1800

gacttatttg gttatgttgg agcattttct ccagctcctg gtgtaacacc tggacaggat 1860

tattccggtt tccacaaagg attattctca gaaaatgatt tccgtatcaa agatgaaaga 1920

tatgtaccat atgtaacatt aatcagctgt ggaaccaatg actctgttgt tggaacattc 1980

cctaagagct atcatgatat tttaacaaga aacaatcagc cacatatctg gtttgaagta 2040

cctggtgcag accatgacaa caacgcaatc gctgctggat actataactt tgtatctgct 2100

gcatttggag ttttaggcat tgacaataca acaccatcta cacagcctag cacacagcct 2160

tctgtagctc ctagcgttga gccttcagta gaacctagta cacagcctag tacagaacct 2220

agcacacagc cttctgtaac tcctagccag acaccttctc aaacaggtgt ataa 2274

Claims

1. The protein is the protein shown in a) or b) or c) or d) or e) or f) as follows:

b) the amino acid sequence is a protein shown in a sequence 1;

2. The protein-related biomaterial according to claim 1, which is any one of the following a1) to A8):

A1) a nucleic acid molecule encoding the protein of claim 1;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising the recombinant vector of a 4).

3. The related biological material according to claim 2, wherein: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3) or 4) or 5):

2) the coding sequence is a DNA molecule shown in sequence 2;

3) the coding sequence is a DNA molecule shown in sequence 8;

4) a DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) or 3) and encoding the protein of claim 1;

5) a DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in 1) or 2) or 3) or 4) and which encodes a protein according to claim 1.

4. Any one of the following S1) -S7):

s1) use of the protein of claim 1 as xylanase and/or feruloyl esterase;

s2) use of the protein of claim 1 or the biomaterial of claim 2 or 3 for the preparation of a xylanase and/or a feruloyl esterase;

s3) use of the protein of claim 1 or the biomaterial of claim 2 or 3 for the preparation of hydroxylated cinnamic acid or xylo-oligosaccharides;

s4) use of the protein of claim 1 or the biomaterial of claim 2 or 3 for degrading xylan and/or hydroxylated cinnamate compounds;

s5) use of the protein of claim 1 or the biomaterial of claim 2 or 3 for the hydrolysis of a substance containing xylan and hydroxylated cinnamate linkages;

s6) use of the protein of claim 1 or the biomaterial of claim 2 or 3 for catalyzing the cleavage of xylosidic bonds within xylan;

s7) use of the protein of claim 1 or the biomaterial of claim 2 or 3 for catalyzing the cleavage of ester bonds between hydroxylated cinnamic acid and oligosaccharide or polysaccharide.

5. Use according to claim 4, characterized in that: the hydroxylated cinnamate compound is hydroxylated methyl cinnamate or hydroxylated ethyl cinnamate;

alternatively, the hydroxylated cinnamic acids include ferulic acid, coumaric acid, caffeic acid, and sinapic acid.

6. The method for producing the protein according to claim 1, comprising the steps of: fermenting and culturing a recombinant microorganism containing a gene encoding the protein of claim 1, expressing the gene encoding the protein of claim 1 to obtain a recombinant microorganism culture containing the protein, and purifying the recombinant microorganism culture to obtain the protein.

7. The method of claim 6, wherein: the recombinant microorganism containing a gene encoding the protein of claim 1, which is obtained by introducing a gene encoding the protein of claim 1 into a recipient microorganism;

or, the gene encoding the protein of claim 1 is introduced into the recipient microorganism by a recombinant vector;

alternatively, the recombinant vector is obtained by inserting a gene encoding the protein of claim 1 into a multiple cloning site of an expression vector.

8. Method according to claim 6 or 7, characterized in that: the protein of claim 1, wherein the gene encoding the protein is a DNA molecule represented by 85 th to 3093 th of SEQ ID NO. 2 or a DNA molecule represented by SEQ ID NO. 8.

9. The method according to claims 6-8, characterized in that: the fermentation culture condition is (16-37) DEG C induction culture (3-24) h.

10. A primer set for amplifying the full length of the gene according to claim 3 or any fragment thereof.