Preparation method and application of feruloyl esterase

Abstract

The invention discloses a preparation method and application of feruloyl esterase. The feruloyl esterase provided by the invention is obtained from aspergillus niger, the optimum pH of the feruloyl esterase is 5.5, the optimum temperature of the feruloyl esterase is 40 DEG C, the feruloyl esterase has good metal ion resistance, and the feruloyl esterase still has higher activity after being treated by pepsin and trypsin. The feruloyl esterase and the xylanase cooperatively process the corn byproducts to obtain a certain content of reducing sugar and ferulic acid, so that the conversion of the byproducts is realized. The characteristics are beneficial to the application in food and feed industries, and have wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a preparation method of ferulic acid esterase and application thereof. Background Art

[0002] Cereal feed contains many anti-nutritional factors, which affect its utilization in livestock and poultry feed. Xylan, as a vital hemicellulose component, has a main chain composed of D-pyranose units linearly connected by strong β-1,4-glycosidic bonds. This skeleton forms the basis for the stability of xylan. However, the hemicelluloses in the feed, as well as the hemicelluloses and lignin, are bound together by ferulic acid, p -Coumaric acid and dimerized ferulic acid form ester bonds to form a dense network structure, and bury cellulose molecules and xylan in it, forming a natural barrier. Due to the complexity of the structure, the complete degradation of xylan requires the synergistic action of a series of enzymes, among which xylanase acts on the main chain of xylan in an endo-cutting manner as the main enzyme, and ferulic acid esterase is one of the side chain degrading enzymes, and plays an important role in promoting the degradation of the main chain.

[0003] Ferulic acid esterase (FAE) is a carboxyl esterase that can hydrolyze the ester bond between ferulic acid and polysaccharides, release ferulic acid, reduce the density of plant cell walls, and increase the accessibility of other fiber-degrading enzymes to cell wall polysaccharides, thereby promoting the hydrolysis of cell wall polysaccharides. Ferulic acid esterases are widely available from bacteria, fungi, and plants, and ferulic acid esterases from different sources differ in enzymatic properties. Ferulic acid esterases from bacteria have the best catalytic activity under neutral conditions, while enzymes from fungi have high catalytic activity under acidic conditions, with an optimal catalytic pH of 5.0-5.5. The optimal catalytic reaction temperature of enzymes from different sources also varies greatly, generally between 40-60°C.

[0004] Ferulic acid esterase has many important industrial uses. In the feed industry, adding ferulic acid esterase can enhance the digestion ability of animals to cellulose and hemicellulose in feed, improve the utilization rate of feed, and significantly increase the average daily weight gain of animals, reduce the feed-to-weight ratio, and improve the growth performance of animals. At the same time, compounds such as ferulic acid produced by ferulic acid esterase degradation of feed have multiple physiological functions such as antibacterial, anti-inflammatory, and antioxidant. These substances can play a positive role in animals, improve the immunity and health level of animals, and reduce the occurrence of diseases. Ferulic acid esterase plays an important role in biomass degradation. By hydrolyzing the ferulic acid ester bonds in xylan, it can promote the degradation of xylan and improve the conversion efficiency of biomass. In bioenergy production, ferulic acid esterase can work synergistically with other enzymes to convert biomass into fermentable sugars for the production of biofuels. In the pharmaceutical industry, ferulic acid esterase also has potential application value in the pharmaceutical industry. Ferulic acid has multiple biological activities, such as antioxidant, anti-inflammatory, and antibacterial. Through the action of ferulic acid lipase, ferulic acid can be efficiently extracted from natural products and used in the preparation of medicines and health products.

[0005] Currently, there are few reports on feruloyl esterase, and the feruloyl esterase derived from wild-type strains shows low enzyme activity, and its production cycle is lengthy and inefficient. In addition, when exploring its application potential, the synergistic compound effect between feruloyl esterase and xylanase must be fully considered to optimize the enzymatic efficiency. In view of this, the in-depth development of efficient and high-quality feruloyl esterase resources is of vital scientific significance and reference value for promoting the study of its synergistic effect with xylanase in the biomass conversion process. Summary of the invention

[0006] The purpose of the invention is to provide a preparation method of ferulic acid esterase and application thereof.

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides an expression vector, wherein the starting vector is pPICZαA, carrying a ferulic acid esterase gene fragment, and the gene fragment is a DNA sequence optimized according to yeast preferred codons.

[0008] The optimized DNA sequence is shown in SEQ ID NO: 3 or 4.

[0009] Furthermore, the expression vector comprises an optimized α signal peptide sequence, and the full length of the optimized α signal peptide sequence is shown in SEQ ID NO:5.

[0010] In a second aspect, the present invention provides a host cell containing the expression vector, preferably Pichia pastoris.

[0011] In a third aspect, the present invention provides an engineered bacterium that produces ferulic acid esterase, wherein the engineered bacterium is transformed with the expression vector into Pichia pastoris X-33 to obtain a recombinant Pichia pastoris engineered bacterium.

[0012] In a fourth aspect, the present invention provides a method for preparing ferulic acid esterase, the method comprising: fermenting the engineered bacteria, and separating and purifying the ferulic acid esterase from the fermentation product.

[0013] Furthermore, the method comprises the following steps: (1) Inoculate the recombinant Pichia pastoris into 50 mL of BMGY medium and culture at 28°C and 220 rpm in a shaking incubator until the OD 600 When the value is 2-6, the bacteria are collected by centrifugation at 5,000 rpm; (2) Resuspend the collected cells in 50 mL of BMMY medium, ensuring that the final methanol concentration in the medium is 0.5% v / v. Maintain the above methanol concentration for 96 hours and add methanol every 12 hours to maintain its concentration until the end of fermentation.

[0014] The BMGY medium was composed of 1% yeast extract, 2% peptone, pH 6.0 100 mmol / L potassium phosphate solution, 1.34% YNB, 1% glycerol, 4×10 -5 % Biotin.

[0015] The BMMY medium is composed of 1% yeast extract, 2% peptone, pH 6.0 100 mmol / L potassium phosphate solution, 1.34% YNB, 0.5% methanol, 4×10 -5 % Biotin.

[0016] In a fifth aspect, the present invention provides the use of ferulic acid esterase and xylanase in combination in the conversion of corn by-products.

[0017] The ferulic acid esterase in the present invention is derived from Aspergillus niger ( Aspergillus niger ), whose amino acid sequence is shown in SEQ ID NO: 2. Xylanase is derived from Aspergillus sulphuroides ( Aspergillus sulphureus ), whose reference sequence number in NCBI is DQ355509.

[0018] In a sixth aspect, the present invention provides a method for converting corn by-products, comprising mixing xylanase and ferulic acid esterase at an enzyme activity ratio (U:U) of (13-133):1, adding the mixture to a buffer system containing corn by-products, and mixing and incubating the mixture.

[0019] Preferably, the enzyme activity ratio of xylanase to ferulic acid esterase is 133:1, 26:1 or 13:1; Further, the corn by-product is added to a pH 5.5 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and then xylanase and feruloyl esterase are added; wherein 10 mL of the reaction system contains 0.1-0.35 g of corn by-product, 30-300 U of feruloyl esterase, and 4000-6000 U of xylanase; and the above reaction system is incubated at 40-45° C. for 23-25 ​​h (preferably incubated at 40° C. for 24 h).

[0020] In a specific embodiment of the present invention, the reaction system (10 mL) contains 0.1 g of destarched corn husk (or 0.1 g of destarched puffed corn husk or 0.35 g of destarched corn DDGS), 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 5.5), and the enzyme activity ratios of xylanase to recombinant ferulic acid esterase are 133:1, 26:1 and 13:1 (corresponding to 4000 U of xylanase activity in the reaction system).

[0021] In the present invention, the corn by-products include but are not limited to de-starched corn husks, de-starched puffed corn husks or de-starched corn DDGS.

[0022] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The ferulic acid esterase provided by the invention is obtained from Aspergillus niger, has an optimum pH of 5.5, an optimum temperature of 40°C, has good metal ion resistance, and still has high activity after being treated with pepsin and trypsin. The ferulic acid esterase and xylanase synergistically treat corn by-products to obtain a certain content of reducing sugar and ferulic acid, thereby realizing by-product conversion. These characteristics are conducive to its application in the food and feed industries, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SDS-PAGE analysis of the shake flask fermentation supernatant in Example 3 of the present invention.

[0024] Figure 2 The wet weight growth curve and enzyme activity of the fermentation cells in the fermenter in Example 3 of the present invention are shown. Note: a: glycerol culture stage; b: glycerol feeding stage; c: methanol induction stage.

[0025] Figure 3 This is the SDS-PAGE analysis of the fermentation supernatant in the fermenter in Example 3 of the present invention.

[0026] Figure 4 This is the effect of temperature on the activity of recombinant ferulic acid esterase FAE in Example 4 of the present invention.

[0027] Figure 5 This is the effect of pH on the activity of recombinant ferulic acid esterase FAE in Example 4 of the present invention.

[0028] Figure 6 This is the pH stability of the recombinant ferulic acid esterase FAE in Example 4 of the present invention.

[0029] Figure 7 This is the effect of metal ions on the activity of recombinant ferulic acid esterase FAE in Example 4 of the present invention.

[0030] Figure 8 This is the pepsin and trypsin resistance of the recombinant ferulic acid esterase FAE in Example 5 of the present invention.

[0031] Fig. 9 It is the amount of reducing sugar released by the recombinant ferulic acid esterase FAE in Example 6 of the present invention when degrading different substrates. DETAILED DESCRIPTION

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0033] In the present invention, the Pichia pastoris expression vector pPICZαA and strain X-33 used were purchased from Invitrogen.

[0034] The present invention also relates to an improved pPICZαA-N vector, wherein the α signal peptide sequence is optimized (the full length of the optimized α signal peptide sequence is shown in SEQ ID NO: 5), and the full sequence of the vector is shown in SEQ ID NO: 6.

[0035] The competent cells of E. coli Top10 used in the following examples were purchased from Beijing Qingke Xinye Biotechnology Co., Ltd. Eco RI, XB I, ligase T4 and its matching ligation buffer were purchased from New England Biolabs; linear enzyme Sac I was purchased from TaKaRa.

[0036] The pPICZαA-FAE plasmid involved in the following examples is constructed by using the selected restriction enzymes EcoRI and Xba I to double-digest pPICZαA and a DNA fragment carrying the ferulic acid esterase gene FAE, and then connect them and transform them into Escherichia coli, pick a single colony to extract the plasmid and verify it. The construction of the pPICZαA-FAE plasmid was completed by Beijing Qingke Biotechnology Co., Ltd.

[0037] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to the mass percentage. However, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of the solution.

[0038] Example 1 Optimization and synthesis of Aspergillus niger ferulic acid esterase gene Based on the ferulic acid esterase gene sequence in Aspergillus niger (its NCBI accession number is XP_001393337), the optimization design of the gene sequence was carried out. First, the nucleotide sequence with accession number 001393337 (after removing the signal peptide part) was extracted as SEQ ID NO:1; at the same time, the amino acid sequence encoded by 001393337 was used as SEQ ID NO:2. On the basis of not changing the original amino acid sequence, the codons used frequently in Pichia pastoris were used to replace the codons used less frequently, and adjustments were made to eliminate continuous pyrimidine base sequences, while reducing the secondary structure stability of the 5' end of mRNA. After codon optimization, the G+C content of the obtained gene was reduced from the original 55% to 45%, which is closer to the GC content of the Pichia pastoris genome.

[0039] On this basis, two different optimized sequences were designed and generated, namely SEQ ID NO: 3 and 4. These two optimized sequences were synthesized by Beijing Qingke Xinye Co., Ltd., and Eco RI and XB I restriction endonuclease cleavage site. In order to further improve the expression efficiency, these optimized sequences were screened.

[0040] The optimized feruloyl esterase gene sequence (SEQ ID NO:3, 4) was constructed into the vector pPICZαA and introduced into Escherichia coli. The recombinant plasmid was extracted and purified after linearization. The purified plasmid was introduced into Pichia pastoris X-33 competent cells to obtain recombinant feruloyl esterase engineered bacteria. The fermentation measured the induced enzyme activity in shake flasks of 2.3 U / mL before optimization of the feruloyl esterase gene sequence, and the induced enzyme activity in shake flasks after sequence optimization was 4.52 U / mL (corresponding to SEQ ID NO:3) and 4.63 U / mL (corresponding to SEQ ID NO:4).

[0041] Example 2 Construction of a recombinant ferulic acid esterase expression strain Escherichia coli containing pPICZαA-N-FAE (i.e., FAE on the vector pPICZαA-N) and pPICZαA-N plasmid ( Escherichia coli , E. coli) were inoculated into 10 mL of LB liquid medium containing Zeocin (100 μg / mL) and cultured at 37°C for 12 h. The recombinant clone plasmid was extracted from E. coli using a plasmid extraction kit (Omega, USA). Eco RI and XB I double-digested the extracted recombinant plasmid. At the same time, the vector pPICZαA-N was also digested with the same enzyme. The digested vector was ligated with the ferulic acid esterase gene fragment using T4 ligase. The ligation product was then transferred into TOP 10 competent cells, and the plasmid was extracted again and sequenced for verification. After sequencing confirmation, the plasmid was cloned and cloned into the pPICZαA-N vector. Sac The recombinant plasmid was linearized with endonuclease I. The linearized product was purified using a DNA purification kit (Omega, USA). The purified linearized plasmid was introduced into Pichia pastoris X-33 competent cells by electroporation. The transformed Pichia pastoris cells were spread on YPDS solid plate medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 1 mol / L sorbitol, 20 g / L agar, autoclaved at 115℃) containing Zeocin (100 μg / mL) and incubated upside down at 28℃. After a single colony grew on the plate, a single colony was selected and streaked on YPD solid medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, autoclaved at 115℃) containing Zeocin (100 μg / mL) and cultured at 28℃. The resulting strain was the recombinant ferulic acid esterase FAE Pichia pastoris engineering strain.

[0042] Example 3 Fermentation of recombinant ferulic acid esterase engineered strain The recombinant Pichia pastoris strain was inoculated in 50 mL BMGY (1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate solution (pH 6.0), 1.34% YNB, 1% glycerol, 4×10 -5 % biotin), and cultured in a shaking incubator at 28°C and 220 rpm until OD 600 The value was 2-6, and the cells were collected by centrifugation at 5,000 rpm. The collected cells were resuspended in 50 mL BMMY (1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate solution (pH 6.0), 1.34% YNB, 0.5% methanol, 4×10 -5% biotin), ensuring that the final concentration of methanol is 0.5% (v / v). The induction culture was carried out for 96 hours under the condition of maintaining the above methanol concentration, during which methanol was added every 12 hours to maintain its concentration. After the above-mentioned induction culture, the shake flask expression level of the recombinant ferulic acid esterase was measured. The expression level was measured using p-nitrophenolferulate as the substrate, and the expression level reached 6.81 U / mL (the shake flask induced enzyme activity was 4.52 U / mL or 4.63 U / mL before the optimization of the α signal peptide sequence and after the codon optimization). SDS-PAGE electrophoresis analysis confirmed that the molecular weight of the expressed recombinant ferulic acid esterase was approximately 37 kDa ( Figure 1 ).

[0043] Ferulic acid esterase activity assay: The reaction system consisted of 100 μL of 10 mmol / L ferulate p-nitrophenolate (Sigma, USA) in DSMO solution, 800 μL of 0.1 mol / L sodium phosphate buffer (pH 6.5) containing 2.5% (V / V) Triton X-100 (Sigma, USA), and 100 μL of enzyme solution. The reaction was incubated at 35°C for 20 min, and the reaction was terminated on ice for 5 min. The absorbance OD was measured using a microplate reader. 410 OD was obtained according to the p-nitrophenol standard curve. 410 The relationship between the concentration of the substrate p-nitrophenol and the concentration of p-nitrophenol after degradation by ferulic acid esterase was calculated, and then the enzyme activity of ferulic acid esterase was calculated.

[0044] The ferulic acid esterase expressing strain was selected and inoculated into YPD medium containing Zeocin antibiotic (100 μg / mL) and cultured overnight at 28℃ 220 rpm. Then, 100 μL of the bacterial solution after the overnight culture was transferred into 100 mL YPD liquid medium and cultured overnight at 28℃ 220 rpm. 600 When it reaches 2-6, the culture is inoculated into a 30 L fermenter containing 12 L of fermentation basal medium for cultivation. When the wet cell weight in the fermenter reaches 90 to 100 g / L (this indicates that the basic carbon source has been exhausted), enter the feeding stage and add 50% (w / v) glycerol. After feeding, continue culturing until the wet cell weight reaches more than 215 g / L, and then stop adding glycerol. Allow the cells to undergo a period of starvation until the dissolved oxygen level reaches more than 90%, and then begin induction with methanol. By adjusting the rotation speed of the fermenter, air flow rate and methanol feeding rate, the relative dissolved oxygen level is maintained above 20%. Take samples from the fermenter every 12 hours to measure the wet cell weight and enzyme activity ( Figure 2 ), and the supernatant was subjected to 12% SDS-PAGE electrophoresis analysis ( Figure 3), confirming that the size of the expression product was approximately 37 kDa, which was consistent with the previous shake flask fermentation results. When the wet bacterial weight and enzyme activity reached a peak and remained stable or began to show a downward trend, the fermentation was stopped. The supernatant was collected by centrifugation and freeze-dried. After freeze-drying, the enzyme activity was measured and the result was 583.9 U / g.

[0045] Example 4 Analysis of Enzymatic Properties of Recombinant Ferulic Acid Esterase The enzyme activity of the diluted enzyme solution was measured at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C, and the corresponding enzyme activity values ​​were calculated. The optimal catalytic temperature of the enzyme was determined by comparing the enzyme activity values ​​at different temperatures.

[0046] The enzyme solution was diluted with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5. At the same time, the substrate solution was also prepared with the buffer of the corresponding pH value. The enzyme activity at the above different pH values ​​was measured at a constant temperature of 40°C.

[0047] The test results show that the optimum catalytic temperature of FAE is 40℃ ( Figure 4 In addition, the optimum pH value of the enzyme is 5.5, and in the pH range of 4.5-8.5, the enzyme maintains a high relative activity, showing good pH stability ( Figure 5 and Figure 6 ).

[0048] Using sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH value of 5.5, various metal ion solutions and EDTA with a concentration of 20mM were prepared. Subsequently, the enzyme solution and the metal ion solution were mixed in equal volumes to make the final concentration of the metal ion reach 10mM. After treating at room temperature for 1 hour, the enzyme activity was measured under optimal conditions. At the same time, the enzyme solution without adding any metal ions was set as a blank control group. The experimental results showed that NH4 + , Cu 2+ Metal ions such as EDTA and NH4 have an inhibitory effect on the activity of ferulic acid esterase. + The inhibitory effect of Mg was the most significant, while the relative activity of the enzyme after treatment with other ions remained above 80%. 2+ 、Na + , Mn 2+ , K + Metal ions such as Figure 7 ).

[0049] Example 5 Effect of recombinant ferulic acid esterase on gastric trypsin resistance When ferulic acid esterase acts in animals, it will be affected by enzymes in the body (pepsin and trypsin purchased from Sigma). The present invention explores the enzymatic activity of recombinant ferulic acid esterase after treatment with pepsin (trypsin). The recombinant ferulic acid esterase solution diluted to 10,000 times was mixed with pepsin or trypsin at a concentration of 5 mg / mL, and the treatment time was 30 minutes, 60 minutes, 90 minutes and 120 minutes, respectively. Subsequently, the enzyme activity of each treatment group was determined under the optimal reaction conditions, and the untreated enzyme solution was used as a blank control. The experimental results showed that after 90 minutes of pepsin treatment, the enzyme still maintained nearly 60% of its activity; and after 80 minutes of trypsin treatment, its activity remained above 50% ( Figure 8 ). This result shows that the recombinant ferulic acid esterase has a strong tolerance to pepsin and trypsin, indicating that it can maintain a certain enzyme activity in the animal body and shows good application potential in the field of feed additives.

[0050] Example 6 Study on the degradation of corn DDGS, corn husks and puffed corn husks by ferulic acid esterase FAE Lyophilized ferulic acid esterase and xylanase (NCBI number DQ355509, from Aspergillus sulphureus) were synergistically reacted on three substrates: corn DDGS, corn husks, and puffed corn husks. The substrates were crushed and de-starched. To evaluate the synergistic effect, a blank control group with only xylanase added and treated under the same conditions was set up. The reaction system (10 mL) contained 0.1 g de-starched corn husks (or 0.1 g de-starched puffed corn husks or 0.35 g de-starched corn DDGS), 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 5.5), and the enzyme activity ratios of xylanase to recombinant ferulic acid esterase were 133:1, 26:1, and 13:1 (corresponding to 4000 U of xylanase in the reaction system). The reaction was incubated at 40 ° C for 24 h and centrifuged at 12,000 rpm for 10 min. The amount of reducing sugars produced by the three substrates was determined by the DNS method ( Fig. 9 At the same time, the content of ferulic acid in the enzymatic supernatant was determined by liquid chromatography (Table 1).

[0051] Table 1 Ferulic acid release from different substrates degraded by recombinant ferulic acid esterase FAE and xylanase (mg / L)

[0052] From the above experimental results, it can be seen that compared with the degradation of three substrates by xylanase alone, the recombinant ferulic acid esterase FAE and xylanase have a better synergistic effect, release more reducing sugars and ferulic acid, realize the conversion of by-products, and have broad application prospects.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An expression vector, characterized in that The starting vector is pPICZαA, which carries a ferulic acid esterase gene fragment, and the gene fragment is a DNA sequence optimized according to yeast preferred codons; The optimized DNA sequence is shown in SEQ ID NO: 3 or 4.

2. The expression vector according to claim 1, characterized in that The expression vector comprises an optimized α signal peptide sequence, and the full length of the optimized α signal peptide sequence is shown in SEQ ID NO:

5.

3. A host cell containing the expression vector according to claim 1 or 2; Preferably, the host cell is Pichia pastoris.

4. An engineered bacterium producing ferulic acid esterase, characterized in that: The engineered bacteria are obtained by transforming Pichia pastoris X-33 with the expression vector described in claim 1 or 2 to obtain recombinant Pichia pastoris engineered bacteria.

5. A method for preparing ferulic acid esterase, characterized in that: The method comprises: fermenting and culturing the engineered bacteria according to claim 4, and separating and purifying ferulic acid esterase from the fermentation product.

6. The method according to claim 5, characterized in that The following steps are involved: (1) Inoculate the recombinant Pichia pastoris into 50 mL of BMGY medium and culture at 28°C and 220 rpm in a shaking incubator until the OD 600 When the value is 2-6, the bacteria are collected by centrifugation at 5,000 rpm; (2) Resuspend the collected bacteria in 50 mL BMMY medium, ensure that the final concentration of methanol in the medium is 0.5% v / v, and induce the culture for 96 hours under the condition of maintaining the above methanol concentration. During this period, methanol is added every 12 hours to maintain its concentration until the end of fermentation; The BMGY medium was composed of 1% yeast extract, 2% peptone, pH 6.0 100 mmol / L potassium phosphate solution, 1.34% YNB, 1% glycerol, 4×10 -5 %Biotin; The BMMY medium was composed of 1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate solution at pH 6.0, 1.34% YNB, 0.5% methanol, 4×10 -5 % Biotin.

7. Application of ferulic acid esterase and xylanase in the conversion of corn by-products; in, The amino acid sequence of the ferulic acid esterase is shown in SEQ ID NO: 2; The reference sequence number of the xylanase in NCBI is DQ355509.

8. A method for converting corn byproducts, characterized in that: Mixing xylanase and ferulic acid esterase at an enzyme activity ratio of (13-133):1, adding to a buffer system containing corn byproducts, and mixing and incubating; Preferably, the enzyme activity ratio of xylanase to ferulic acid esterase is 133:1, 26:1 or 13:1; Wherein, the ferulic acid esterase and xylanase are the same as those described in claim 7.

9. The method according to claim 8, characterized in that The corn by-product is added to a pH 5.5 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and then xylanase and ferulic acid esterase are added; wherein 10 mL of the reaction system contains 0.1-0.35 g of corn by-product, 30-300 U of ferulic acid esterase, and 4000-6000 U of xylanase; and the above reaction system is incubated at 40-45°C for 23-25 ​​h.

10. The method according to claim 8 or 9, characterized in that: The corn by-product is selected from destarched corn husks, destarched puffed corn husks or destarched corn DDGS.

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