CN111592990B - Recombinant expression strain of feruloyl esterase, preparation method and application thereof - Google Patents

Abstract

The invention provides a Kluyveromyces marxianus recombinant strain capable of being used for preparing feruloyl esterase AnfaeA, which is constructed by cloning an Aspergillus niger feruloyl esterase AnfaeA gene with an optimized sequence onto an expression vector and converting a Kluyveromyces marxianus host strain, wherein the yield of feruloyl esterase obtained by high-density fermentation of the recombinant strain is 12,0000U/ml. The invention also provides a method for preparing ferulic acid by hydrolyzing corn cob meal by using ferulic acid esterase, and ferulic acid can be obtained by efficiently hydrolyzing corn cob meal by using the synergistic effect of ferulic acid esterase AnfaeA prepared by the recombinant strain and xylanase. The ferulic acid esterase provided by the invention can be used for preparing phenolic substances such as ferulic acid, p-coumaric acid and the like by applying agricultural byproducts such as corncobs, rice bran, corn husks and the like through biodegradation, so that the ferulic acid esterase has wide application value in the fields of food, paper making, feed, medicine and the like, and meanwhile, the preparation method is simple in process, high in yield and very suitable for large-scale production.

Description

Recombinant expression strain of feruloyl esterase, preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a method for preparing an enzyme product by using a recombinant yeast strain, in particular to a Kluyveromyces marxianus strain for recombinant expression of ferulic acid esterase, a method for preparing the ferulic acid esterase by using the strain, and application of the prepared ferulic acid esterase in enzyme preparation of ferulic acid.

Background

Feruloyl esterases (EC 3.1.1.73), also known as cinnamates, are a subset of carboxylic ester hydrolases. The ferulic acid esterase can hydrolyze ester bonds formed by interaction of phenolic acids such as ferulic acid and hemicellulose, lignin and the like, break a dense net-shaped structure in plant cell walls, expose cellulose, improve the degradation rate of the cellulose and release antioxidant substances such as ferulic acid, so that the ferulic acid esterase is widely applied to the fields of food, paper making, feed, medicine and the like and has high application value. For example, in the field of bioprocessing, feruloyl esterase is considered to be an essential accessory enzyme for the hydrolysis of lignocellulose when producing bioethanol and the like, and feruloyl esterase is used in combination with other hydrolases and oxidases, which significantly promotes the decomposition of plant materials and increases the availability of fermentable carbohydrates. In the food and pharmaceutical fields, ferulic acid and other hydroxycinnamic acids are phenolic phytochemicals that are widely used in the food and cosmetic industries due to their unique and effective properties, such as being useful as antioxidants, sun protection factors, depigmenting agents, and the like. The hydroxycinnamic acids such as ferulic acid can be used as carriers of vitamin C and E to enhance the photoprotection of skin, and can better permeate into stratum corneum due to stronger lipophilicity. In addition, they have various medicinal effects and health-care functions, such as antibacterial, anti-inflammatory, antidiabetic, antithrombotic, etc. In the pulp and paper industry, whiteness is an important characteristic of wood pulp in order to produce high quality paper. Discoloration of the pulp is caused by residual lignin in the pulp, and bleaching is a key step in the removal of residual lignin to whiten the pulp. Chemical bleaching is typically accomplished industrially using costly and hazardous chemicals such as chlorine dioxide, hydrogen peroxide or ozone, which are environmentally hazardous. When the ferulic acid esterase is mixed with xylanase and lignin oxidase to treat paper pulp, the delignification rate is equivalent to the result of chemical bleaching. Meanwhile, the energy consumption is reduced in the biological bleaching process, the chemical oxygen demand value of the pulping wastewater is obviously reduced, and the method is environment-friendly. Fiber digestibility is an important indicator of animal feed in the feed industry. Dyspepsia can hinder animal growth, cause immune stress reaction, reduce the feed conversion rate of livestock and reduce the breeding income. Ferulic acid and hydroxycinnamic acid are beneficial to the health of animals, ferulic acid of plant cell wall components is one of main inhibiting factors of a ruminant digestive system, ferulic acid esterase is added into feed to accelerate the degradation of the plant cell wall components and improve the fiber digestion of plant nutrients, and meanwhile, the digestion degree of the animals on the feed can be improved, and the growth of the animals is accelerated.

Since the first identification of feruloyl esterase in the 80's of the 20 th century and the first isolation and purification in 1991, more than 80 feruloyl esterases with different molecular weights, substrate preferences and optimal reaction conditions have been obtained from microorganisms. The protein homology, physicochemical properties and action mechanism of ferulic acid esterase from different microorganisms are all obviously different, and the ferulic acid esterase is classified into A, B, C, D, E type based on the biochemical and functional characteristics of enzyme, the recognition and utilization of a substrate containing hydroxycinnamic acid methyl ester (ferulic acid methyl ester, p-coumaric acid methyl ester, caffeic acid methyl ester and sinapic acid methyl ester) and the similarity of amino acid sequences. The type A ferulic acid esterase preferentially selects meta methoxyl substituted phenolic compounds as substrates, especially ferulic acid and sinapic acid, but the activity is only reflected on a substrate containing ferulic acid ester connected with O-5, and when the substrate contains ferulic acid ester connected with O-2 of L-arabinofuranose, the substrate has no catalytic activity; type B feruloyl esterases tend to select substrates containing 1-2 hydroxyl groups, such as p-coumaric acid and caffeic acid, and are active on both ferulic acid ester substrates containing O-5 and O-2 linkages to L-arabinofuranose. Another difference between the two types of ferulic acid esterases is that the A-type ferulic acid esterases have a higher selectivity towards hydrophobic substrates with larger substituents on the phenyl ring. Type C and type D feruloyl esterases differ only in their ability to release ferulic acid dimers, but have broad specificity for artificially synthesized hydroxycinnamic esters. Type A and type D feruloyl esterases produce small amounts of ferulic acid dimers. In addition, phylogenetic analysis also showed that some microbial feruloyl esterases were not in any of these four classifications and were therefore classified as putative E-type. This type of feruloyl esterase can only be identified by its amino acid sequence identity, and no further correlation can be established due to the lack of comparable enzyme activity data.

Although ferulic acid esterase can be synthesized by various microorganisms, it has not been directly used for large-scale production at present due to limited enzyme-producing ability of wild strains. The molecular biology method is utilized to construct the genetic engineering strain, so that the production strain with higher stability and yield becomes a research hotspot. In the prior art, a plurality of encoding genes of the ferulic acid esterase are cloned and expressed heterogeneously, wherein Escherichia coli (Escherichia coli), Pichia pastoris (Pichia pastoris) and Aspergillus niger (Aspergillus niger) are the most common expression systems. Coli expression systems are the most commonly used expression systems, but they have defects, such as lack of post-translational modification required for eukaryotic gene expression, failure to fold correctly after protein expression, easy formation of inactive inclusion bodies, denaturation and purification by affinity chromatography, loss of protein and reduction of enzyme activity, and the defects limit the application of e coli expression systems to some extent.

Disclosure of Invention

The present invention is made to solve the above problems, and aims to provide a method for preparing ferulic acid esterase with low production cost, simple process and higher safety, and an application thereof; the preparation method of the ferulic acid esterase provided by the invention can be applied to the field of industrial related production.

The invention provides a Kluyveromyces marxianus strain for recombinant expression of feruloyl esterase, which is characterized in that the Kluyveromyces marxianus strain is constructed by cloning a feruloyl esterase gene derived from Aspergillus niger into a recombinant expression vector and transforming the Kluyveromyces marxianus strain.

The Kluyveromyces marxianus strain for recombinant expression of feruloyl esterase also has the technical characteristics that the sequence of the feruloyl esterase gene is shown as SEQ ID No.1, or the nucleotide sequence shown as SEQ ID No.1 is obtained by substituting, deleting or adding one or more nucleotides, and the encoded protein has a derivative nucleotide sequence of feruloyl esterase activity.

The Kluyveromyces marxianus strain for recombinant expression of feruloyl esterase also has the technical characteristics that the sequence of the feruloyl esterase gene is a nucleotide sequence shown as SEQ ID No.2, or a derivative nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotides into the nucleotide sequence shown as SEQ ID No.2 and encoded protein of the nucleotide sequence has feruloyl esterase activity.

The Kluyveromyces marxianus strain for recombinant expression of ferulic acid esterase also has the technical characteristics that a vector for transferring the ferulic acid esterase gene into the Kluyveromyces marxianus strain comprises a yeast autonomous replication sequence, an inulase promoter, an ferulic acid esterase gene, an inulase terminator and a screening marker gene sequence, and the specific sequence of the vector is shown as SEQ ID No. 6.

The Kluyveromyces marxianus strain for recombinant expression of feruloyl esterase in the embodiment can also have the technical characteristics that the Kluyveromyces marxianus strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 10621.

As another embodiment, the invention provides a preparation method of feruloyl esterase, which is characterized in that a feruloyl esterase solution is obtained by fermenting and culturing the Kluyveromyces marxianus strain which is used for recombining and expressing feruloyl esterase.

In another embodiment, the present invention provides a method for preparing ferulic acid, which comprises preparing ferulic acid enzyme solution by the above-mentioned method for preparing ferulic acid esterase, and hydrolyzing agricultural byproducts by using the ferulic acid enzyme solution as hydrolase to obtain ferulic acid.

The preparation method of the ferulic acid can also have the technical characteristics that the hydrolase is also added with any one or the combination of more of xylanase, xylosidase and cellulase.

The preparation method of the ferulic acid can also have the technical characteristics that the agricultural byproduct is any one or the combination of more of rice bran, wheat bran, corn bran, corncob and bean pulp.

Action and Effect of the invention

According to the Kluyveromyces marxianus strain for recombinant expression of feruloyl esterase and the method for preparing feruloyl esterase by using the strain, the sequence of the feruloyl esterase gene from Aspergillus niger is optimized, the feruloyl esterase gene is cloned to a recombinant expression vector and is converted into the Kluyveromyces marxianus strain, and the obtained recombinant strain can normally express feruloyl esterase and obtain feruloyl esterase with high enzyme activity, so that the Kluyveromyces marxianus strain has good practical application value and can be used as hydrolase to degrade agricultural byproducts such as rice bran, wheat bran, corn cob and bean pulp and release the agricultural byproducts to obtain ferulic acid.

Drawings

FIG. 1 is an electrophoretogram of a PCR amplification product of the A.niger-derived ferulic acid esterase AnfaeA gene of example 1 of the present invention.

FIG. 2 is a SDS-PAGE analysis of the AntaeA fermentation broth of Aspergillus niger origin in example 5 of the present invention.

FIG. 3 shows the results of enzyme activity detection of the Aspergillus niger-derived feruloyl esterase AnfaeA of example 6 of the present invention.

FIG. 4 is a high density fermentation growth curve of the recombinant strain of Kluyveromyces marxianus, an Aspergillus niger-derived feruloyl esterase, in example 7 of the present invention.

FIG. 5 shows the high-density fermentation enzyme activity of the recombinant strain of Kluyveromyces marxianus, an esterase of Aspergillus niger origin in example 7 of the present invention.

FIG. 6 shows the result of high density fermentation SDS-PAGE analysis of the recombinant strain of Kluyveromyces marxianus, an esterase of Aspergillus niger origin according to the present invention.

FIG. 7 shows the release of ferulic acid by the 24-hour degradation of corncobs by the fermentation broth of a recombinant strain of the Aspergillus niger-derived feruloyl esterase AnfaeA Kluyveromyces marxianus in the presence/absence of xylanase.

Detailed Description

In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is specifically described in the following with the embodiment and the attached drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

In the following examples, reagents, kits and the like of unspecified origin are commercially available in general, and the procedures and conditions of unspecified experimental operation are conventionally performed in the art.

Example 1 Synthesis and amplification of a DNA sequence encoding the AntaeA protein, a feruloyl esterase of Aspergillus niger origin

The nucleotide sequence of the antavosidase AntaeA gene from Aspergillus niger was codon optimized without altering the amino acid sequence, with reference to the codon preference of Kluyveromyces marxianus. Wherein, the nucleotide sequence of the AntaeA gene of the feruloyl esterase from Aspergillus niger is shown as SEQ ID No.1, the nucleotide sequence of the optimized AntaeA gene is shown as SEQ ID No.2, and the corresponding amino acid sequence after optimization is shown as SEQ ID No. 3. In this example, the gene sequence was synthesized by Suzhou Jinzhi Biotechnology, Inc.

The AnfaeA gene is amplified by PCR by using a primer AnfaeA-132-F (shown in SEQ ID No. 4) and a primer AnfaeA-132-R (shown in SEQ ID No. 5) by taking the synthesized AnfaeA DNA as a template. The PCR procedure was performed according to the product instructions of Phanta Super Fidelity DNA Polymerase (Vazyme, cat. No. P505-d1/d2/d 3).

FIG. 1 is an electrophoretogram of a PCR amplification product of the A.niger-derived ferulic acid esterase anfaeA gene of example 1 of the present invention; in FIG. 1, lane 1 is GeneRuler 1kb DNA Ladder (Thermo), and lane 2 is PCR amplification product of AnfaeA gene.

As shown in FIG. 1, the PCR amplification product was analyzed by agarose electrophoresis, confirming that a 783bp fragment of interest was obtained. And (3) recovering the PCR product according to the operation of the specification of a SanPrep column type DNA glue recovery kit (product No. B518131-0050) to obtain the AnfaeA gene fragment.

Example 2 construction of Aspergillus niger-derived Feruloyl esterase AnfaeA Kluyveromyces marxianus recombinant expression vector

The Kluyveromyces marxianus expression vector pUKDN132 is subjected to double enzyme digestion by restriction enzymes Sma I and Not I, and after the enzyme digestion product is subjected to 1% agarose gel electrophoresis, a vector fragment of about 11kb is recovered by using a SanPrep column type DNA gel recovery kit.

A Gibson Assembly traceless connection system (NEB company, product No. E2611S/L) is adopted to connect an AnfaeA gene fragment with a vector fragment to obtain an Aspergillus niger derived ferulic acid esterase AnfaeA Kluyveromyces marxianus recombinant expression vector 132-AnfaeA. The recombinant vector comprises a yeast autonomous replication sequence, an inulinase promoter, a feruloyl esterase anfaeA gene derived from Aspergillus niger, an inulinase terminator and a screening marker gene URA3 sequence, wherein the specific sequence of the recombinant vector is shown as SEQ ID No. 6.

Example 3 construction of Kluyveromyces marxianus recombinant Strain FIM-AnfaeA

The yeast expression host strain adopted in the embodiment is derived from Kluyveromyces marxianus FIM-1 (deposited in China general microbiological culture Collection center with the preservation number of CGMCC No.10621 and the preservation unit code of CGMCC, the preservation unit address of No.3 Homeh No.1 of North Chen West Lu of the south Kogyo area in Beijing, the preservation date of 3 and 13 days in 2015, and the classification name of Kluyveromyces marxianus) and the gene URA3 are knocked out by a homologous recombination method, and the YPD containing 5 fluoroorotic acid (1.5g/L) is utilized to screen and obtain the uracil-deficient expression host strain which is named as Kluyveromyces marxianus FIM-1 (delta URA 3).

132-AntaeA was transferred into FIM-1 (. DELTA.ura 3) by lithium acetate conversion (World Journal of Microbiology & Biotechnology 16:653-654, 2000). The transformed product was spread on SD plates (0.67% amino acid-free yeast nitrogen source, 2% glucose, 2% agar), and the plates were incubated in a 30 ℃ incubator for 2-4 days until colonies were formed.

Example 4 screening of Kluyveromyces marxianus recombinant Strain FIM-AnfaeA Positive clones

Single clones were picked from SD plates, transferred to fresh SD plates, and grown for 1 day. 1/4 match-size strains were picked and suspended in 20ul 0.2% SDS in a 100 ℃ boiling water bath for 5 min. After cooling to room temperature, the mixture was centrifuged at 12,000rpm for 10 seconds. Taking the supernatant as a template, and carrying out PCR reaction by using AnfaeA-132-F and AnfaeA-132-R primers. The PCR process was performed according to the product instructions of Phanta Super Fidelity DNA Polymerase. And (3) performing agarose electrophoresis on the PCR product to obtain a positive clone with a 783bp band, namely an expression recombinant strain of the ferulic acid esterase AnfaeA gene from the aspergillus niger, and naming the positive clone as a Kluyveromyces marxianus recombinant strain FIM-AnfaeA.

Example 5 expression and detection of the Aspergillus niger derived feruloyl esterase AnfaeA.

Kluyveromyces marxianus recombinant strain FIM-AnfaeA and host bacterium FIM-1 are respectively inoculated into 50ml of YD culture medium (2% yeast extract and 4% glucose), cultured at 30 ℃ and 220rpm for 72h, and centrifuged to collect supernatant. The supernatant was added to 5 XSDS PAGE loading buffer (150mM Tris-HCl (pH 7.0), 12% SDS, 6% mercaptoethanol, 30% glycerol, 0.05% Coomassie Brilliant blue G-250) by ratio and in a boiling water bath for 5 min. After cooling to room temperature, the mixture was centrifuged at 12,000rpm for 1 min. The supernatant was subjected to SDS-PAGE. The steps of SDS-PAGE were performed according to the method of the "molecular cloning protocols".

FIG. 2 is a SDS-PAGE analysis of an AntaeA fermentation broth of Aspergillus niger origin in example 5 of the present invention; in FIG. 2, lane 1 is a Pageruler Prestained Protein ladder (Thermo), lane 2 is a supernatant of fermentation broth of Kluyveromyces marxianus expression host bacterium FIM-1, and lane 3 is a supernatant of fermentation broth of recombinant Kluyveromyces marxianus FIM-1-AnfaeA.

As shown in FIG. 2, the SDS-PAGE result showed that a significant protein band (band marked as "AnfaA" in FIG. 2) appeared around 39kDa in the FIM-AnfaA fermentation broth, compared with the fermentation broth of the host bacterium FIM-1ura 3. delta. and the size of the band was consistent with the theoretical molecular weight of 39kDa in the Aspergillus niger-derived ferulic acid esterase AnfaA protein, thus presuming that the protein band was the Aspergillus niger-derived ferulic acid esterase AnfaA protein.

Example 6 detection of enzyme Activity of Kluyveromyces marxianus recombinant Strain FIM-AnfaeA

The recombinant Kluyveromyces marxianus strain FIM-1-AnfaeA of example 5 was cultured in YD medium (2% yeast extract, 4% glucose) for 72 hours, and the centrifugation supernatant was treated with PBST (80% NaCl, 2% KCl, 14.2% Na)₂HPO₄, 2.7％KH₂PO₄2.5% Triton X-100, pH6.4), mixing 20. mu.L of the diluted enzyme solution with 170. mu.L of PBST and 10. mu.L of 10mM 2-chloro-4 nitrophenol ferulic acid ester ((2-chloro-4-nitrophenyl ferulate, CNPF), reacting in a 37 ℃ water bath for 20min, immediately taking 200. mu.L of the mixture in a flat-bottom 96-well plate, and measuring OD (OD) by using a BioTeck EON microplate reader₄₁₀And (4) light absorption value, OD value after measurement, and CNP concentration generated by reaction according to the CNP standard curve. The enzyme activity unit of 1U is defined as the amount of CNP produced by hydrolysis per minute of 1nM。

FIG. 3 shows the results of enzyme activity detection of the Aspergillus niger-derived feruloyl esterase AnfaeA of example 6 of the present invention. In FIG. 3, top1 delta u-132-AnfaeA-1, top1 delta u-132-AnfaeA-2, top1 delta u-132-AnfaeA-3 and top1 delta u-132-AnfaeA-4 are respectively picked four different transformants for parallel determination of enzyme activity.

As shown in FIG. 3, the enzyme activity detection result shows that the enzyme activity of the ferulic acid esterase AnfaeA is about 680U/mL after the recombinant strain is fermented in the YD culture medium for 72 h.

Example 7 high Density fermentation of Kluyveromyces marxianus recombinant Strain FIM-AnfaeA

Kluyveromyces marxianus genetically engineered bacterium FIM-AnfaeA is inoculated on a YPD plate, and cultured for 2 days at 30 ℃ for activation. Then transferring the culture medium into a synthetic culture medium, wherein the synthetic culture medium contains glucose or sucrose (2-20%), ammonium sulfate (5-15g/L), monopotassium phosphate (3-20g/L), magnesium sulfate (1-5g/L) and other components; then, the seeds are cultured for 18-24h at 30 ℃ and 220rpm with a shaking table at constant temperature to obtain a seed solution.

Inoculating the seed liquid into a 5L fermentation tank containing 5L culture medium (the same formula as the above culture medium) at a ratio of 10% for high density fermentation, ventilating and stirring during fermentation, controlling dissolved oxygen at 20-30%, controlling temperature at 30 deg.C, pH at 5.5, and fermenting for 72 hr.

FIG. 4 is a high density fermentation growth curve of the recombinant strain of Kluyveromyces angustifolia of the Aspergillus niger origin of the Aspergillus niger of example 7 of the present invention, and FIG. 5 is a high density fermentation enzyme activity condition of the recombinant strain of the Kluyveromyces angustifolia of the Aspergillus niger origin of the Aspergillus niger of example 7 of the present invention. FIG. 6 shows the high density fermentation SDS-PAGE analysis of the recombinant strain of Kluyveromyces marxianus, an Aspergillus niger-derived ferulic acid esterase, of example 7 of the present invention.

As shown in FIGS. 4 to 6, the results of high density fermentation showed that the cell density OD was obtained after the recombinant strain was fermented in a 5L fermentor for 72 hours_600nmCan reach about 524 (figure 4), the output of the ferulic acid esterase is about 120,000U/mL (figure 5) calculated by enzyme activity, anda higher level was also achieved in terms of protein content (figure 6).

Example 8 hydrolysis of corn cob by Kluyveromyces marxianus recombinant Strain FIM-1-AnfaeA

Group 1: 2g of ground corn cob was put into a 150mL shaking flask, and 1,000U/g of the feruloyl esterase-containing fermentation supernatant obtained in example 7 was added to the flask to make up to 50mL with 50mM sodium acetate buffer (pH 5.5). The reaction mixture was placed on a shaker and reacted at 37 ℃ and 220rpm for 24 hours. Centrifuging the reaction solution for 3h, 6h, 12h and 24h, respectively, collecting the supernatant, and detecting the content of ferulic acid in the reaction solution by High Performance Liquid Chromatography (HPLC). In HPLC, an Agilent XDB-C18 column (5 μm, 250 mm. times.4.6 mm) was used, and methanol and 1% glacial acetic acid were used as mobile phases, in a ratio of 2: 3, the flow rate is 0.8mL/min, the detection temperature is 30 ℃, the detection wavelength is 318nm, and the content of the ferulic acid is calculated according to the peak height.

Group 2: xylanase is used to replace the fermentation supernatant, wherein the addition of xylanase is 100U/g, and other operation processes and conditions are the same as those in group 1.

Group 3: adding xylanase at 100U/g while adding fermentation supernatant, and performing the same operation and conditions as those in group 1.

FIG. 7 shows the release of ferulic acid by the 24-hour degradation of corncobs by the fermentation broth of a recombinant strain of the Aspergillus niger-derived feruloyl esterase AnfaeA Kluyveromyces marxianus in the presence/absence of xylanase. In FIG. 7, "control" is a control group (without addition of fermentation supernatant or xylanase), "faeA" is group 1, "xyn" is group 2, and "faeA + xyn" is group 3.

As shown in figure 7, the enzymolysis result shows that after the corncob is degraded for 24 hours, the ferulic acid esterase can release 1.81mg/g of ferulic acid from the corncob, and after the corncob is degraded for 24 hours by matching with 100U/g of xylanase, the release amount of the ferulic acid reaches 2.8mg/g (figure 7), which is improved by 50% compared with the release amount of the ferulic acid esterase which is singly used.

Effects and effects of the embodiments

From the above embodiments, it can be seen that, after the ferulic acid esterase gene derived from aspergillus niger is subjected to sequence optimization, cloned to a recombinant expression vector and transformed into a kluyveromyces marxianus strain, the obtained recombinant strain can normally express ferulic acid esterase, and the enzyme activity of the obtained ferulic acid esterase can reach 680U/mL, so that the ferulic acid esterase has good practical application value, for example, can be used as hydrolase for degrading agricultural byproducts and releasing ferulic acid.

Further, according to example 8, when ferulic acid esterase, which is used as a hydrolase to degrade agricultural byproducts, is prepared, if xylanase is added thereto to form a mixed enzyme, hydrolysis efficiency can be improved, so that more ferulic acid is released in a short time, thereby improving the application value of ferulic acid esterase.

In addition, as can be seen from the point that the hydrolysis efficiency is improved after the xylanase is added, the effect of improving the hydrolysis efficiency can be achieved by replacing the xylanase in the mixed enzyme with other similar enzymes, that is, the mixed enzyme contains not only ferulic acid esterase but also any one or a combination of more of xylanase, xylosidase and cellulase, and the prepared hydrolase still has good hydrolysis capacity.

Furthermore, the hydrolytic enzyme has good hydrolytic capacity for corncobs, so that the hydrolytic enzyme can effectively hydrolyze a plurality of similar agricultural byproducts including the corncobs, such as any one or combination of corncobs, rice bran, wheat bran, corn husks and bean pulp.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

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Met Ala Ser Thr Gln Gly Ile Ser Glu Asp Leu Tyr Asn Arg Leu Val

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Glu Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asp Leu Cys Asn Ile

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Pro Ser Thr Ile Ile Lys Gly Glu Lys Ile Tyr Asn Ala Gln Thr Asp

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Ile Asn Gly Trp Ile Leu Arg Asp Asp Thr Ser Lys Glu Ile Ile Thr

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Tyr Thr Leu Thr Pro Phe Asp Thr Leu Pro Gln Cys Asn Asp Cys Glu

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Val His Gly Gly Tyr Tyr Ile Gly Trp Ile Ser Val Gln Asp Gln Val

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Glu Ser Leu Val Lys Gln Gln Ala Ser Gln Tyr Pro Asp Tyr Ala Leu

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Thr Val Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala Leu Thr Ala

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Ala Gln Leu Ser Ala Thr Tyr Asp Asn Val Arg Leu Tyr Thr Phe Gly

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Glu Pro Arg Ser Gly Asn Gln Ala Phe Ala Ser Tyr Met Asn Asp Ala

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Phe Gln Val Ser Ser Pro Glu Thr Thr Gln Tyr Phe Arg Val Thr His

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Ser Asn Asp Gly Ile Pro Asn Leu Pro Pro Ala Asp Glu Gly Tyr Ala

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Thr Phe Val Cys Thr Gly Asp Glu Val Gln Cys Cys Glu Ala Gln Gly

225 230 235 240

Gly Gln Gly Val Asn Asp Ala His Thr Thr Tyr Phe Gly Met Thr Ser

245 250 255

Gly Ala Cys Thr Trp

260

<210> 4

<211> 52

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ttacaagaga gacggtgacc ccggggcctc tacccaaggt atctccgaag ac 52

<210> 5

<211> 54

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atcaaagctt gcggccttaa gcggccgctt accaagtaca agcaccggag gtca 54

<210> 6

<211> 11388

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 60

catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 120

attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 180

ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 240

ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 300

aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 360

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 420

gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 480

gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 540

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 600

ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 660

ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 720

tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 780

tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 840

ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 900

aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 960

ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 1020

tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 1080

atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 1140

aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 1200

ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 1260

tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 1320

ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 1380

tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 1440

aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 1500

gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 1560

tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 1620

cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 1680

tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 1740

ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 1800

cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 1860

actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 1920

ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 1980

aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 2040

ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 2100

atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 2160

tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 2220

gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 2280

ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 2340

gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 2400

actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt 2460

ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc 2520

aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt 2580

aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact 2640

acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg 2700

gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag 2760

aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac 2820

gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg 2880

ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 2940

aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 3000

tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 3060

ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcatc 3120

actaatgaaa agcatacgac gcctgcgtct gacatgcact cattctgaag aagattctgg 3180

gcgcgtttcg ttctcgtttt cctctgtata ttgtactctg gtggacaatt tgaacataac 3240

gtctttcacc tcgccattct caataatggg ttccaattct atccaggtag cggttaattg 3300

acggtgctta agccgtatgc tcactctaac gctaccgttg tccaaacaac ggaccccttt 3360

gtgacgggtg taagacccat catgaagtaa aacatctcta acggtatgga aaagagtggt 3420

acggtcaagt ttcctggcac gagtcaattt tccctcttcg tgtagatcag aggctatata 3480

catgccgagg tattcgatca ctctacgatg acggtctgtt agctcaacaa cttcttctaa 3540

atgctccata accgtaacgt aagaagcata actgtcaata ctgaagtcat cccagtttat 3600

tggtgctcct gttgaacagt catccactat atgttcgaat agcccaggat cacgaggagg 3660

tcctacaaac ggatacggta cagtcttctt tttatagtct gcaaattcta gaatagcatt 3720

ttttatccaa tagtgtcgaa tcgtcctggc cgttctaccg ataaaggatc caatgtgatt 3780

attagctcca ctacacgata tgttaagttt gatcgatgtc ttgttaacaa acgctaaact 3840

caagttcggc atttccaaca gcgagaagaa atcatcaatt ccatcggcta tctcttgata 3900

agtcattaga tcatatacct tctcgggatg tcgttgagtt actttatgac tagaaatctt 3960

caggttatca tcaacgtaat tgttctccaa tagctctgga gagggacata acaatacttt 4020

gattttttcc atggcctgga cttgtttccg taggaaatac ttgttctttt gtagacgttc 4080

catgatgagt ttgtatacct ctgctggaga tatccattct agatctttga tataagtttg 4140

gtatggtaaa gagttgattt tgtaggacac gtaaatctgc gctagataag tacattgtgc 4200

aaatgcctct ggtacttcgt aagacccatg ctgcgtaatt atagtattat tgagtggatc 4260

ataagcgttg tactcgtttt tgaatttaaa actgtctaat aaggccctgt aaatctctct 4320

gacttgttgt acacctttct gctcttcggg actgagatcg gatagcaatg gagcagcagt 4380

ttctgagctt tctgatgggg ctgacatggc agatgcctat tcaatgctgc cttttgtttg 4440

ggaggttatg aaatgcatct gtttacattg tatgtaatac ccttactagg caatgttata 4500

agcaaaaatc ctttgatcac atggaatatc actttatacg tgttgaaata tgcaaaaaaa 4560

cagtccccct gagctcaggg ggtggtttac gcttttgagg ctcagcagcg cgaattctct 4620

cttggggctg aagtgaaatt taaaaaagtc gcttgaggct cagccggaat tataaaacat 4680

cacctgagtc ttgagagcgc tttcactcac ctgaggctca gctgaaattt caaaaagtca 4740

cttgagccca gaaggagtgt ttcaccccct gaggctataa cgttcgttat tttaatacct 4800

aaataaacaa aaatatatgg tacaggaacg cgaggcaacg cgccgataca gggtcaatgg 4860

gtacacgaga gggtgacact aggcgtagaa agtcattagt ataaaataca gtggtatata 4920

gtagatattt agtttgtttt ccttttcttt ttctccaaaa cgatatcaga catttgtctg 4980

ataatgaagc attatcagac aaatgtctga tatcgttttt caataataat atacatcatc 5040

acaaaacaaa caaacatagc atcgcaagcc ccatcatgcc accaccgtcc gctgtgatcg 5100

caactcatgt ttccggcggt attctgcaat gaattggaga acctcgtctg agataattcc 5160

atgccattgt tcgaacaact ggaggctagg atgagctgag aaggattgag cgaccaagcg 5220

cggacttgac ggtgggctga gtggtgggct accagggctg ttaccctcct cttcaagtag 5280

ctcctcgcga gataaaggtt tattagaagg atccttcaaa acatatattt cactgcccaa 5340

tggggcttcc ttgtaaaaac ctgatataaa ggcaaataca cggtcatcta cagtcacact 5400

accatgactg tagtgtgatc tagccactgc actttgaatt tcacggtccc cagcccaatt 5460

gcccagtgag gagacatatt ttcccttctc agatctcgat aaaaaggtcg ccattaaatg 5520

tcttccaaaa tgagacttcg ggccgttcca gatcttgaag actggttcat cgacatgctg 5580

ggtaagaaac ctagagaacg ttctggccaa tgactctggt aaaaactgat gagtttgatt 5640

agttggtcta ttactggata ctgttttttc aataggcgag caaacacgca aatagtcgta 5700

taatgatatc agaagatcgc aatcaccatt cacaggatag aagttaacgt accgttcagt 5760

tctgcttttc gtttctgtca cagtagcacg cacaattggg cccagaaatg aattgttgta 5820

gatctcaaaa gtccttggat ctagattctt cagatcgctg tatctgcagc aatttccaac 5880

agctcccaga agtagcaatc ggtattccgc tcgttttgta gtggttacgc aggactgatc 5940

gaagaagcag gcaatcctgg agacaatctt ccaaatatct ttttcttttg acagaatatt 6000

agtgaattgt aatccaacca tagaagcatc gtatttatgt gtttcctcgt agcgatcaaa 6060

caaggaaact tcttgatttt taaatgggct aacaacaacc ttgtaagaag gcaatgctga 6120

ttcgatatcc ttttgcagag actctgtctt tcttagtcta acagtgaatt tgataatttt 6180

gtcatcctta tcaaaagaca gagatttgcc aattgcgctc ttgtaagagc ggtaggtatt 6240

gattttcatc tcgcgtcgga tagatagcga ctgcattgtc aagatagaga atagggacgc 6300

cagcttattt ctaatttctt tcgcatttat attaaaggtg tcagattcca gaatttcatt 6360

aatttcatta gcacactgat gaggtgtgag gtgagcagcc tccgcaaagg tagacatagg 6420

ggcattggtt ggaggccttt gaggtaccac tagagtgctg caaacatagc accgttcgag 6480

actttaaaat cttcagtttt aaaattatga aaaaaaacat cgtcctgagt tgaaacggtc 6540

gtttcaacct ccgtgtacag aaagatacat agcatatggc aagctgcacg cagcgtaaac 6600

atgccggaca actgtcattt cgtcagatca gttgatctac tctctgtgat actgcttcgt 6660

ttgtccacgg aggtcggact aactctcacc acgcttccac ggcattcgaa agaactaata 6720

ttgtatcatt gtacatatga ggaacacgca gttgaactga gcaaaccagg actcaggaaa 6780

gcaggaggta agtgctcgct tttcgtggat ccagaggaac gtgaaaattc gccttctcct 6840

cctataccgc cgtatcagat atcagagatg ccccttcatg aacttctcga gtcaggcaat 6900

gctaaattgg ttccaaatcc cgagtttgat ctaactgatc cagacgactt tcataagtgt 6960

ttctcggtca cctattcagc attatcttta atggtaccat atctgcccag agctgctcta 7020

aaggctgctc gagtgttttg taaagatcat tcaatattaa caacggatat gcttgatttg 7080

aattatcttg aagagctaat tgagttctca aaggaaactg tgaacaaaat cccagctaga 7140

atccctatag aggacatgct tctcgagcgg ggatatgtgc taccatgggt tcatggtggt 7200

acagtgaagg gaggaaagct actgaccccc aacgattgat tctttaccga atcattgcat 7260

aattcattgc ataattcatt gcagaatacc gccggaaaca tgagttgcga tcacagcgga 7320

cggtggtggc atgatggggc ttgcgatgct atgtttgttt gttttgtgat gatgtatatt 7380

attattgaaa aacgatatca gacatttgtc tgataatgct tcattatcag acaaatgtct 7440

gatatcgttt tggagaaaaa gaaaaggaaa acaaactaaa tatctactat ataccactgt 7500

attttatact aatgactttc tacgcctagt gtcaccctct cgtgtaccca ttgaccctgt 7560

atcggcgcgt tgcctcgcgt tcctgtacca tatatttttg tttatttagg tattaaaatt 7620

tactttcctc atacaaatat taaattcacc aaacttctca aaaactaatt attcgtagtt 7680

acaaactcta ttttacaatc acgtttattc aaccattcta catccaataa ccaaaatgcc 7740

catgtacctc tcagcgaagt ccaacggtac tgtccaatat tctcattaaa tagtctttca 7800

tctatatatc agaaggtaat tataattaga gatttcgaat cattaccgtg ccgattcgca 7860

cgctgcaacc gcggcacaaa cacaaacaca aacacaaaaa cgctaaatta tgcacacaag 7920

ggccggcggg gctgccggaa aaaaaaaggg aaaaatacac agacgagcgc gcacagatgg 7980

ggttaccact gcaagttaca agttgcaagt tgcacgctgg aatcagaatt ggaatcagaa 8040

ttggaattgg aattagaatt agaattaaac ttggggtagc cacgggaacg ggataactca 8100

ggaatcgctc gcaggcgtct ccgtctaggc aatcccaagg taagcctagg cactcccaca 8160

ggggaaagaa cggttgaagg caaagtagtg ctaacaattg gtaacgaatg gtaacaagtg 8220

tgtccgtctc cacctgacat ttgctagagc tggggattcc acattcttgt gctctgaatt 8280

ctcaaaccga aatggggcgt tgttacccca ggtatccggt tgtagttggc actggggatg 8340

gaaaaaaatg atgttgatgt tgagttagtt gggttgagtc aattagtgcg tgaaagtatc 8400

accacttttg tcatccggcg tttctgtgcg aatcacacac acacacacag tttattggag 8460

cacttgtttc tggcgtattc gtaattgttc tgcggtgcgg ttctgtgtgc atttttcctg 8520

gggtgtctgc cgcacctact catcacccac gccgtgggtt tgagccatgg cggaggtacg 8580

actgactggc tgcctgcctg cctgactgac tgcctgactg caggaaaaga gggtttcgaa 8640

ggaaaaactt ttcctgtgtt aatccggccg tgcgccgctg ctccaaaatc caccttcatg 8700

agaaggagtt tgaaaaaaca aaaaaattca catataaaaa gcgtatctcg agatctcaaa 8760

gtctcccttg aatcgtgttt gccagttgta actcatcctt tattcttcta ttctatctct 8820

ctctttcctt cccctaatca gcaattaaat ccggggtaag gaagaattac tactgtgtgt 8880

aacggttata tttcgttttt tatttttttt ttccattgcc atagagaaag aaaaaaaaaa 8940

aaaagagagt ttgtgaagat cttccattcg aatcccataa gtgacacatt taattttttt 9000

tttgttagat atgaagttag catactccct cttgcttcca ttggcaggag tcagtgcttc 9060

agtgatcaat tacaagagag acggtgaccc cgggactagt gcggccgctt aaggccgcaa 9120

gctttgatct gatctgctta ctttactaac gacaaaaaaa aatcaaaaaa aaaaaaacaa 9180

tcagtccttc tcttcttacg atatgatatg attaaatgat gctatgaaat catcttcttc 9240

ttaactttct taaatcttac gcgtcactta ctctatatac ccgtttagct ttgcctggtc 9300

acagcgacat tttatataag tgtacgtatt ttcttttttt ttttaaaaat ttctattcta 9360

accttagaaa agtgcccttt aaaccagctg tcctggcact atatctttat catgtgccgg 9420

tcgctttccc tttccgtttc ccttttcctt tcaattggtg gcctggaatt ccgaactcat 9480

tttcgcatct gaaactaatt ctcgaaacct ttaacatcaa acaattgaaa agatcatcat 9540

caccagaaat aagaaaaaga tcaacacaac agctaataac agtacgaaag aaagatcgct 9600

cgagtgaaaa ggcagccaag aaaggtcatt cgatttgggt ctagactgat tatagacata 9660

ccaattgcac tcagtaagaa aatgagtttc aaatttgacg atgacggtgt ggtaaaagaa 9720

tttcacggca acaccatcat atgccatatt cctcaacaaa ccgaattctt caacaaattg 9780

ttggacttct accgttttgc gaaacgactt tccttctacg acaagatcac cctacttcct 9840

ccttcaagct accacgttac gatcatgaat tgctgccacg aacacgatcg ttctgagggc 9900

cactggccca aaggaatcga tccggacaca agcatgctgc ggtgtacatc acatctgacc 9960

aacattctag gatcggtcga attctgattg gaaagaccat tctgctttac ttttagagca 10020

tcttggtctt ctgagctcat tatacctcaa tcaaaactga aattaggtgc ctgtcacggc 10080

tcttttttta ctgtaccttt gacttccttt cttatttcca aggatgctca tcacaatacg 10140

cttctagatc tattatgcat tataattaat agttgtagct acaaaaggta aaagaaagtc 10200

cggggcaggc aacaatagaa atcggcaaaa aaaactacag aaatactaag agcttcttcc 10260

ccattcagtc atcgcatttc gaaacaagag gggaatggct ctggctaggg aactaaccac 10320

catcgactga ctctatgcac taaccacgtg actacatata tgtgatcgtt tttaacattt 10380

ttcaaaggct gtgtgtctgg ctgtttccat taattttcac tgattaagca gtcatattga 10440

atctgagctc atcaccaaca agaaattcta ccgtaaaagt gtaaaagttc gtttaaatca 10500

tttgtaaact ggaacagcaa gaggaagtat catcagctag ccccataaac taatcaaagg 10560

aggatgtcga ctaagagtta ctcggaaaga gcagctgctc atagaagtcc agttgctgcc 10620

aagcttttaa acttgatgga agagaagaag tcaaacttat gtgcttctct tgatgttcgt 10680

aaaacagcag agttgttaag attagttgag gttttgggtc catatatctg tctattgaag 10740

acacatgtag atatcttgga ggatttcagc tttgagaata ccattgtgcc gttgaagcaa 10800

ttagcagaga aacacaagtt tttgatattt gaagacagga agtttgccga cattgggaac 10860

actgttaaat tacaatacac gtctggtgta taccgtatcg ccgaatggtc tgatatcacc 10920

aatgcacacg gtgtgactgg tgcgggcatt gttgctggtt tgaagcaagg tgccgaggaa 10980

gttacaaaag aacctagagg gttgttaatg cttgccgagt tatcgtccaa ggggtctcta 11040

gcgcacggtg aatacactcg tgggaccgtg gaaattgcca agagtgataa ggactttgtt 11100

attggattta ttgctcaaaa cgatatgggt ggaagagaag agggctacga ttggttgatc 11160

atgacgccag gtgttggtct tgatgacaaa ggtgatgctt tgggacaaca atacagaact 11220

gtggatgaag ttgttgccgg tggatcagac atcattattg ttggtagagg tcttttcgca 11280

aagggaagag atcctgtagt ggaaggtgag agatacagaa aggcgggatg ggacgcttac 11340

ttgaagagag taggcagatc cgcttaagag gggtaccgag ctcgaatt 11388

Claims (7)

1. A Kluyveromyces marxianus strain for recombinant expression of feruloyl esterase is characterized in that the Kluyveromyces marxianus strain is constructed by cloning a feruloyl esterase gene from Aspergillus niger to a recombinant expression vector and converting the Kluyveromyces marxianus strain,

wherein the sequence of the ferulic acid esterase gene is a nucleotide sequence shown as SEQ ID No. 2.

2. The Kluyveromyces marxianus strain recombinantly expressing feruloyl esterase according to claim 1, characterized in that:

wherein, the carrier for transferring the ferulic acid esterase gene into the Kluyveromyces marxianus strain comprises a yeast autonomous replication sequence, an inulase promoter, the ferulic acid esterase gene, an inulase terminator and a screening marker gene sequence,

the specific sequence of the vector is shown as SEQ ID No. 6.

3. The Kluyveromyces marxianus strain recombinantly expressing feruloyl esterase according to claim 1, characterized in that:

the Kluyveromyces marxianus strain is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 10621.

4. A preparation method of feruloyl esterase, which is characterized in that feruloyl esterase solution is obtained by fermenting and culturing Kluyveromyces marxianus strain which is used for recombining and expressing feruloyl esterase according to any one of claims 1 to 3.

5. A process for producing ferulic acid, which comprises preparing a ferulic acid enzyme solution by the ferulic acid esterase preparation process of claim 4, and hydrolyzing an agricultural byproduct with the ferulic acid enzyme solution as a hydrolase to obtain ferulic acid.

6. The method for producing ferulic acid according to claim 5, comprising:

wherein, the hydrolase is also added with any one or a combination of more of xylanase, xylosidase and cellulase.

7. The method for producing ferulic acid according to claim 5, comprising:

wherein the agricultural by-product is any one or combination of rice bran, wheat bran, corn bran, corncob and soybean meal.

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