A hemicellulose-degrading enzyme system co-expression strain, and a construction method and application thereof

Abstract

The present application relates to a kind of hemicellulose-degrading enzyme system co-expression strain and its construction method and application, belong to genetic engineering technical field.The present application is with xylose metabolic deficiency type escherichia coli as host bacteria, constructs xylosidase Xys, ferulic acid esterase FaeLam, xylanase N20Xyl Multi-enzyme co-expression strain, for enzymolysis bran and xylan, produce xylose and byproduct ferulic acid.However, in the construction process, if the expression order of Xys, FaeLam, N20Xyl is changed, the secretion amount of extracellular enzyme will be different, so the expression order of enzyme is studied.Finally, two hemicellulose-degrading enzyme system co-expression strains with excellent expression effect are screened out, and the expression order is Xys-FaeLam-N20Xyl or FaeLam-Xys-N20Xyl.

Description

Technical Field

[0001] This invention relates to a hemicellulose-degrading enzyme co-expression strain, its construction method, and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] The fusion expression of two or more enzymes using single or multiple plasmids in *E. coli* has been extensively studied. However, when using multiple plasmids for fusion expression, different antibiotics are often added to exert different selection pressures to maintain plasmid stability. Compared with multi-enzyme fusion expression, single-enzyme expression in single cells can reduce the possibility of misfolding and inclusion body formation, while simultaneously increasing enzyme activity.

[0003] Chinese patent document CN 104152392 A (application number 201410374667.8) discloses an engineered bacterium based on hemicellulase and its implementation method. Using the pUC57-XL vector, constructed from the genomic DNA of *Streptomyces griseus* through whole-genome synthesis, as a template, PCR amplification is performed with primers containing restriction enzyme sites to obtain the corresponding nucleotide sequences XL (encoding xylanase) and XO (encoding xylosidase). The amplified gene sequences are then sequentially ligated into the co-expression vector pETDuet-1, ultimately forming the recombinant co-expression vector pETDuet-XL-XO. This recombinant co-expression vector is then transformed into an *Escherichia coli* expression strain to obtain a transgenic co-expression strain. This invention enables the large-scale in vitro expression and synthesis of xylanase and xylosidase using genetic engineering techniques, ultimately achieving rapid in-situ degradation of hemicellulose.

[0004] Chinese patent document CN 102994542 A (application number 201210562444.5) discloses a novel method for constructing an engineered strain GS115 / faeA-xyn11A that co-expresses the ferulic acid esterase A gene and the β-1,4-xylanase gene derived from Aspergillus usamii E001. The constructed Pichia pastoris co-expression system can be used for the industrial production of ferulic acid esterase and β-1,4-xylanase. The two enzymes prepared exhibit strong substrate affinity and high catalytic efficiency, demonstrating significant potential for industrial production and economic value.

[0005] Current research shows that most lignocellulase expressions using single plasmid fusion involve two proteases, while studies on more than two proteases are relatively rare. Furthermore, the effects of different expression sequences on expression levels and enzyme activity when two or more proteases are co-expressed are also little known. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a hemicellulose-degrading enzyme co-expression strain, its construction method, and its applications. This invention constructs a recombinant plasmid in a single plasmid that co-expresses a hemicellulose-degrading enzyme system, including the xylosidase encoding gene Xys, the ferulic acid esterase encoding gene FaeLam, and the xylanase encoding gene N20Xyl. The expression sequence of the enzyme system is optimized, and a hemicellulose-degrading enzyme co-expression strain is constructed, achieving high expression of extracellular enzymes and efficient degradation of lignocellulose.

[0007] The technical solution of the present invention is as follows:

[0008] A recombinant plasmid for co-expressing a hemicellulose-degrading enzyme system, comprising a xylosidase encoding gene Xys, a ferulic acid esterase encoding gene FaeLam, and a xylanase encoding gene N20Xyl, wherein the nucleotide sequence of the xylosidase encoding gene Xys is SEQ ID NO.1, the nucleotide sequence of the ferulic acid esterase encoding gene FaeLam is SEQ ID NO.2, and the nucleotide sequence of the xylanase encoding gene N20Xyl is SEQ ID NO.3.

[0009] According to a preferred embodiment of the present invention, the expression sequence of the hemicellulose degrading enzyme system of the recombinant plasmid is Xys-FaeLam-N20Xyl or FaeLam-Xys-N20Xyl.

[0010] According to a preferred embodiment of the present invention, the vector plasmid of the recombinant plasmid is pETDuet-1.

[0011] A preferred embodiment of the present invention, a method for constructing a recombinant plasmid co-expressing a hemicellulose degrading enzyme system with the expression sequence Xys-FaeLam-N20Xyl, includes the following steps:

[0012] (1) Using plasmids pETDuet-FaeLam and pETDuet-N20Xyl as templates, FaeLam fragment and N20Xyl fragment were obtained by PCR amplification, and FaeLam fragment and N20Xyl fragment were overlapped and spliced ​​to obtain FaeLam-N20Xyl fragment.

[0013] (2) Insert the FaeLam-N20Xyl fragment obtained in step (1) into the plasmid pETDuet-Xys to construct the recombinant plasmid pETDuet-Xys-FaeLam-N20Xyl for co-expression of hemicellulose degrading enzyme system.

[0014] A preferred embodiment of the present invention, a method for constructing a recombinant plasmid co-expressing a hemicellulose-degrading enzyme system with the expression sequence FaeLam-Xys-N20Xyl, includes the following steps:

[0015] 1) Using plasmids pETDuet-Xys and pETDuet-N20Xyl as templates, Xys fragment and N20Xyl fragment were obtained by PCR amplification, and Xys fragment and N20Xyl fragment were overlapped and spliced ​​to obtain Xys-N20Xyl fragment.

[0016] 2) Insert the Xys-N20Xyl fragment obtained in step 1) into the plasmid pETDuet-FaeLam to construct the recombinant plasmid pETDuet-FaeLam-Xys-N20Xyl, which is used to co-express the hemicellulose degrading enzyme system.

[0017] A strain co-expressing a hemicellulose-degrading enzyme system containing a recombinant plasmid co-expressing the above-mentioned hemicellulose-degrading enzyme system.

[0018] According to a preferred embodiment of the present invention, the host bacterium of the hemicellulose degrading enzyme co-expression strain is Escherichia coli.

[0019] More preferably, the Escherichia coli is a xylose-deficient Escherichia coli.

[0020] More preferably, the xylose metabolism-deficient Escherichia coli is a knockout bacterium encoding the xylose isomerase XylA and xylose kinase XylB genes.

[0021] In this invention, the above-mentioned hemicellulose degrading enzyme co-expression strain is obtained by transforming the above-mentioned hemicellulose degrading enzyme co-expression recombinant plasmid into xylose metabolism-deficient Escherichia coli by heat shock method and then screening.

[0022] The application of the above-mentioned hemicellulose degrading enzyme co-expression strains in the preparation of multi-enzyme mixtures containing xylosidase, xylanase and ferulic acid esterase.

[0023] The method for preparing a multi-enzyme mixture containing xylosidase, xylanase, and ferulic acid esterase from the above-mentioned hemicellulose degrading enzyme co-expression strain includes the following steps:

[0024] The hemicellulose-degrading enzyme co-expressing strain was inoculated into SB medium and cultured until OD500. 600 After adjusting the concentration to 0.5–0.9, add lactose to a final concentration of 2–5 g / L, and simultaneously add glycine to a final concentration of 25–100 mM, and Mg2+ metal ions. 2+ Zn 2+ or Fe 2+ The final concentration was 2 mM. After induction of expression at 37°C, the supernatant of the fermentation broth was taken to obtain a multi-enzyme mixture containing xylosidase, xylanase and ferulic acid esterase.

[0025] According to a preferred embodiment of the present invention, cultured to OD 600 It is 0.7.

[0026] According to a preferred embodiment of the present invention, lactose is added to a final concentration of 2 g / L.

[0027] According to a preferred embodiment of the present invention, glycine is added to a final concentration of 100 mM.

[0028] According to a preferred embodiment of the present invention, the added metal ion is Zn. 2+ .

[0029] Application of the above-mentioned hemicellulose degrading enzyme co-expressed strains in the degradation of lignocellulose.

[0030] According to a preferred embodiment of the present invention, the lignocellulose is wheat bran or xylan.

[0031] Application of the above-mentioned hemicellulose degrading enzyme co-expression strains in the preparation of ferulic acid.

[0032] Application of xylan hydrolysate from the above-mentioned hemicellulose degrading enzyme co-expression strains in the preparation of antioxidant products.

[0033] Application of xylan hydrolysate of the above-mentioned hemicellulose degrading enzyme co-expressing strains in the cultivation of probiotics.

[0034] According to a preferred embodiment of the present invention, the probiotics include Lactobacillus pumilus, Enterococcus faecalis, Pediococcus lactis, and Weissella asiatica.

[0035] Beneficial effects:

[0036] 1. This invention uses xylose-deficient *Escherichia coli* as the host bacterium to construct a multi-enzyme co-expression strain of xylosidase Xys, ferulic acid esterase FaeLam, and xylanase N20Xyl, for enzymatic hydrolysis of wheat bran and xylan to produce xylose and the byproduct ferulic acid. However, during the construction process, it was found that changing the expression order of Xys, FaeLam, and N20Xyl caused differences in the secretion of extracellular enzymes, so the expression order of the enzymes was studied. Finally, two hemicellulose-degrading enzyme co-expression strains, *E. coli* BL21 / △xylAB / pETDuet-Xys-FaeLam-N20Xyl(pDⅢ-2) and *E. coli* BL21 / △xylAB / pETDuet-FaeLam-Xys-N20Xyl(pDⅢ-5), were selected with excellent expression effects.

[0037] 2. This invention optimizes conditions such as the type of basal culture medium, the type of inducer, the timing and concentration of inducer addition, glycine concentration, and the type of metal ion addition to achieve higher extracellular enzyme expression and secretion, resulting in better enzymatic hydrolysis during subsequent simultaneous growth and fermentation. Since SB medium is richer in nutrients than LB medium, it can significantly improve protein expression. When lactose is added as an inducer, it has a dual effect, acting as both an inducer and a carbon source for secondary growth to achieve higher cell density, often resulting in better induction than IPTG. Furthermore, lactose is much cheaper than IPTG and is non-toxic, making it suitable for industrial production. The timing of lactose addition also significantly affects extracellular enzyme activity and secretion; if the OD (oxidative stress) is too high when adding the inducer... 600 If the OD is too low, the bacterial cell count will be small, which is not conducive to the accumulation of the target protein; if the OD... 600 Excessive glycine levels can lead to an increase in metabolic products accumulated during bacterial growth, inhibiting the synthesis of target proteins. Glycine improves the secretion of target proteins by enhancing the permeability of the cell membrane. The main mechanism is generally believed to be that glycine can mix into nucleotide-activated peptidoglycan precursors, causing the cross-linked peptidoglycan to become loose.

[0038] 3. This invention utilizes the recombinant strain E. coli BL21 / △xylAB / pDⅢ-2 for the enzymatic hydrolysis of wheat bran and xylan. Complete degradation of xylan requires a well-developed xylanase system, primarily including xylanase and xylosidase. Xylosidase works synergistically with xylanase to completely decompose xylan and is one of the key enzymes in xylan degradation. Ferulic acid esterase can hydrolyze the ester bonds in methyl ferulic acid, oligosaccharide ferulic acid esters, and polysaccharide ferulic acid esters, releasing ferulic acid, which has strong antioxidant properties. However, the amount of wheat bran and xylan added, as well as the fermentation time, both have a certain impact on fermentation. The best enzymatic hydrolysis effect is achieved when wheat bran and xylan are added at concentrations of 10 g / L and 50 g / L, respectively, with optimal fermentation times of 36 h and 48 h, respectively. The reducing sugar yield in the xylan hydrolysis product can reach 33.70 ± 0.46 g / L. The xylan hydrolysate exhibits excellent antioxidant properties, with a hydroxyl radical scavenging capacity of 80.0% and a total antioxidant capacity of 2.289 ± 0.55. By configuring a special MRS culture medium, this invention demonstrates that, compared to ordinary xylose, the active substances contained in the xylan hydrolysate more effectively promote the growth of probiotics.

[0039] 4. This invention enables the efficient production of hemicellulose-degrading enzyme systems in a single host, which has great value in processing lignocellulose to produce biofuels and prebiotics. Attached Figure Description

[0040] Figure 1Agarose gel electrophoresis verification image of PCR amplification products of knockout strain E. coli BL21 / △xylAB; In the image, M: DL5000 DNA Marker; Lane 1: wild-type E. coli BL21; Lane 2: knockout strain E. coli BL21 / △xylAB.

[0041] Figure 2 The growth of E. coli BL21 and E. coli BL21 / ΔxylAB in M9 medium.

[0042] Figure 3 This is a schematic diagram of the structure of a multi-enzyme co-expression plasmid.

[0043] Figure 4 SDS-PAGE electrophoresis images of extracellular protein samples from different recombinant strains; In the figure, M: 180kDa Prestained Protein Marker; Lane 1: E. coli BL21 / △xylAB / pDXys; Lane 2: E. coli BL21 / △xylAB / pDFaeLam; Lane 3: E. coli BL21 / △xylAB / pDN20Xyl; Lane 4: E. coli BL21 / △xylAB / pDⅢ-1; Lane 5: E. coli BL21 / △xylAB / pDⅢ-2; Lane 6: E. coli BL21 / △xylAB / pDⅢ-3; Lane 7: E. coli BL21 / △xylAB / pDⅢ-4; Lane 8: E. coli BL21 / △xylAB / pDⅢ-5; Lane 9: E. coli BL21 / △xylAB / pDⅢ-6.

[0044] Figure 5 Bar chart showing the relative enzyme activity of extracellular protein samples from different recombinant strains.

[0045] Figure 6 SDS-PAGE electrophoresis images of extracellular protein samples cultured in different basal media; in the figure, lane 1: LB medium, lane 2: SB medium, lane 3: TB medium, lane 4: 2×YT medium, lane 5: SOB medium.

[0046] Figure 7 Bar chart showing the relative enzyme activity of extracellular protein samples cultured in different basal media.

[0047] Figure 8SDS-PAGE electrophoresis images of extracellular protein samples cultured under different final lactose concentrations; in the figure, lane 1: 0.5 g / L lactose, lane 2: 1 g / L lactose, lane 3: 2 g / L lactose, lane 4: 5 g / L lactose, lane 5: 10 g / L lactose, lane 6: 15 g / L lactose, lane 7: 20 g / L lactose, lane 8: 0.7 mM IPTG.

[0048] Figure 9 This is a bar chart showing the relative enzyme activity of extracellular protein samples cultured under different lactose concentrations.

[0049] Figure 10 For different induction timing (OD) 600 SDS-PAGE electrophoresis image of extracellular protein samples cultured at [location missing]; in the image, lane 1: OD [value missing] 600 0.1, Lane 2: OD 600 0.3, Lane 3: OD 600 0.5, Lane 4: OD 600 0.7, Lane 5: OD 600 0.9.

[0050] Figure 11 For different induction timing (OD) 600 A bar chart showing the relative enzyme activity of extracellular protein samples cultured under [condition].

[0051] Figure 12 SDS-PAGE electrophoresis images of extracellular protein samples cultured at different final glycine concentrations; in the figure, lane 1: 0, lane 2: 25mM, lane 3: 50mM, lane 4: 75mM, lane 5: 100mM, lane 6: 125mM.

[0052] Figure 13 This is a bar chart showing the relative enzyme activity of extracellular protein samples cultured at different final glycine concentrations.

[0053] Figure 14 SDS-PAGE electrophoresis images of extracellular protein samples cultured under different metal ions; in the figure, lane 1: CK, lane 2: Mn 2+ Lane 3: Mg 2+ Lane 4: Zn 2+ Lane 5: Fe 2+ Lane 6: Ca 2+ .

[0054] Figure 15 This is a bar chart showing the relative enzyme activity of extracellular protein samples cultured under different metal ions.

[0055] Figure 16The figure shows the relative enzyme activity changes of extracellular protein samples from strains E. coli BL21 / pDⅢ-2 and E. coli BL21 / △xylAB / pDⅢ-2. In the figure, E. coli BL21 is E. coli BL21 / pDⅢ-2, and E. coli BL21 / △xylAB is E. coli BL21 / △xylAB / pDⅢ-2.

[0056] Figure 17 The curves show the changes in reducing sugar content during the enzymatic hydrolysis of wheat bran (A) or xylan (B) by strain E.coli BL21 / △xylAB / pDⅢ-2 during fermentation.

[0057] Figure 18 The results show the antioxidant capacity of the supernatant from the fermentation broth of xylan fermented by strain E.coli BL21 / △xylAB / pDⅢ-2.

[0058] Figure 19 OD of probiotic cultured from xylan hydrolysate of strain E. coli BL21 / △xylAB / pDⅢ-2 600 Changes in pH. Detailed Implementation

[0059] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the materials and reagents involved in the embodiments are all commercially available products.

[0060] Example 1: Construction of xylose-deficient Escherichia coli

[0061] Xylose metabolism-deficient Escherichia coli was constructed by knocking out the xylose utilization genes (xylose isomerase XylA and xylulose kinase XylB) in E. coli BL21 using Red / ET recombination technology.

[0062] 1. Construction of gene targeting fragments

[0063] Using pKD4 plasmid as a template, the Kan resistance fragment FRT-Kan-FRT was obtained by high-fidelity enzyme amplification using primers KanF and KanR. Using FRT-Kan-FRT as a template, the gene targeting fragment Up-FRT-Kan-FRT-Down, containing the homologous arm of the xylose utilization gene, was obtained by high-fidelity enzyme amplification using primers UpF and DownR. The primers used are listed in Table 1.

[0064] Table 1 Primer sequences

[0065]

[0066] 2. Preparation of Escherichia coli electrocompetent cells

[0067] The pKD46 plasmid, which encodes the Red / ET recombination system, was transformed into E. coli BL21. Transformants were screened and named E. coli BL21 / pKD46. After overnight culture at 30°C, the strain was transferred to LB liquid medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculation ratio and cultured until OD500. 600 When the cytotoxicity reaches 0.2, add 100 μL of 100 g / L L-arabinose and induce at 30 °C for 40 min. Immediately place the bacterial culture in an ice bath for 1 min, gently shaking to ensure thorough cooling. Pre-chill 50 mL centrifuge tubes on ice. Centrifuge the bacterial culture at 4 °C, 6000 rpm for 10 min, discard the supernatant, and recover the cells. Wash and resuspend the cells with 10 mL of ice-chilled CYT buffer (10% (v / v) glycerol, 1.25 g / L yeast extract, 2.5 g / L peptone). Repeat this step three times. Centrifuge the bacterial culture again at 4 °C, 6000 rpm for 10 min, carefully discard the supernatant, and aspirate any residual liquid from the centrifuge tube walls using a pipette. Resuspend the pellet in 4 mL of pre-chilled CYT buffer to obtain E. coli electrocompetent cells. Aliquot the E. coli electrocompetent cells into 40 μL tubes and store at -80 °C.

[0068] 3. Knockout and verification of the xylose utilization gene in Escherichia coli

[0069] E. coli electroporation competent cells were mixed with the gene targeting fragment Up-FRT-Kan-FRT-Down and added to a pre-chilled electroporation cuvette. The liquid was gently tapped to ensure the suspension reached the bottom of the cuvette, and the mixture was incubated in an ice-water bath for 10 min. The cuvette was dried to remove condensation and mist, and then placed in an electroporator at 1350 V / mm / 25 μF / 200 Ω for electroporation. Immediately after electroporation, 1 mL of LB liquid medium was added, the cells were gently resuspended, and transferred to a 1.5 mL centrifuge tube. The cuvette was then incubated at 30°C with shaking for 1 h. The resuscitation solution was centrifuged at 6000 rpm for 10 min, 900 μL of supernatant was removed, and the resuspended cells were plated onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 30°C.

[0070] The temperature-sensitive pKD46 plasmid in the knockout strain was eliminated by high-temperature culture at 37℃. Then, the mutant strains on LB plates were screened by colony PCR. The size of the gene fragment of the mutant strain was confirmed by PCR amplification using primers UpF and DownR, and the gene fragment was sequenced to obtain the knockout strain E. coli BL21 / △xylAB, which is a xylose metabolism-deficient Escherichia coli.

[0071] The agarose gel electrophoresis results of the above PCR amplification products are as follows: Figure 1 As shown, the PCR products of the knockout strain E. coli BL21 / △xylAB and the wild-type strain E. coli BL21 are approximately 1600 bp and 2900 bp in length, respectively. Furthermore, sequencing results show that the original 2850 bp gene sequence was replaced by a 1496 bp kanamycin resistance gene sequence.

[0072] E. coli BL21 and E. coli BL21 / ΔxylAB were activated in LB liquid medium and then transferred to sugar-free M9 medium and xylose M9 medium at a ratio of 1% (v / v). The media were incubated at 37°C for 12 h, and the OD values ​​were measured and compared. 600 To verify the xylose utilization capacity of E. coli BL21 / △xylAB, three parallel experiments were set up, with the error bar representing the standard deviation of three replicate experiments.

[0073] The sugar-free M9 medium consisted of 11.3 g / L M9, 2 mM magnesium sulfate heptahydrate (sterilized separately and then added), and 2 mM calcium chloride hexahydrate (sterilized separately and then added).

[0074] Xylose M9 medium: 11.3 g / L M9, 2 mM magnesium sulfate heptahydrate (sterilized separately and added), 2 mM calcium chloride hexahydrate (sterilized separately and added), 10 g / L xylose (sterilized separately and added).

[0075] The composition of M9 is as follows: Na2HPO4 6.8g; KH2PO4 3.0g; NaCl 0.5g; NH4Cl 1.0g.

[0076] The growth of E. coli BL21 and E. coli BL21 / ΔxylAB in M9 medium is as follows: Figure 2 As shown. Both E. coli BL21 and E. coli BL21 / ΔxylAB were normally activated in LB medium, but when transferred at a ratio of 1% (v / v) to sugar-free M9 medium and xylose M9 medium, OD values ​​were measured and compared. 600 It can be seen that E. coli BL21 / △xylAB cannot grow normally, indicating that the xylose utilization ability of E. coli BL21 / △xylAB is completely lost, and the encoding genes of xylose isomerase XylA and xylulokinase XylB have been inactivated and replaced by Kan resistance gene fragments.

[0077] Example 2: Construction of multi-enzyme co-expression plasmid

[0078] A multi-enzyme co-expression plasmid containing the xylosidase encoding gene Xys (SEQ ID NO.1), the ferulic acid esterase encoding gene FaeLam (SEQ ID NO.2), and the xylanase encoding gene N20Xyl (SEQ ID NO.3) was constructed.

[0079] Using pET22b plasmid as a vector, recombinant plasmids pET22b-Xys containing the xylosidase encoding gene Xys, pET22b-N20Xyl containing the xylanase encoding gene N20Xyl, and pET22b-FaeLam containing the ferulic acid esterase encoding gene FaeLam were constructed respectively.

[0080] Using recombinant plasmids pET22b-Xys, pET22b-N20Xyl, and pET22b-FaeLam as templates, PCR amplification was performed using high-fidelity enzymes and corresponding primers Xys-F-Nco and Xys-R-Not, Xyl-F-Nco and Xyl-R-Not, and Fae-F-Nco and Fae-R-Not. The amplified gene encoding fragments Xys(Nco / Not), N20Xyl(Nco / Not), and FaeLam(Nco / Not) were purified and stored at -20°C until use. The pETDuet-1 plasmid was digested with restriction enzymes NcoⅠ and NotⅠ and the product was purified. Then, it was ligated with Xys (Nco / Not), N20Xyl (Nco / Not), and FaeLam (Nco / Not) fragments using homologous recombination and transformed into E. coli competent cells. After colony PCR and plasmid extraction PCR verification, plasmids pETDuet-Xys, pETDuet-N20Xyl, and pETDuet-FaeLam were obtained and named pDXys, pDN20Xyl, and pDFaeLam, respectively.

[0081] Using pDN20Xyl as a template, PCR amplification and product purification were performed using a high-fidelity enzyme and primers Xyl-F-Nde and Xyl-R-Xho to obtain the N20Xyl(Nde / Xho) fragment. Using pDFaeLam as a template, PCR amplification and product purification were performed using a high-fidelity enzyme and primers Xyl-Fae-F and Fae-R-Xho to obtain the FaeLam(Xho / Xho) fragment. The N20Xyl(Nde / Xho) fragment and the FaeLam(Xho / Xho) fragment were ligated using an overlap splicing PCR method, and the purified product yielded the N20Xyl-FaeLam(Nde / Xho) fragment. Plasmid pDXys was double-digested with restriction enzymes NdeⅠ and XhoⅠ, and the purified product was ligated with the N20Xyl-FaeLam (Nde / Xho) fragment using homologous recombination and transformed into E. coli competent cells. The cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. Colonies were picked for colony PCR and plasmid extraction PCR verification to obtain plasmid pETDuet-Xys-N20Xyl-FaeLam (pDⅢ-1).

[0082] Using the same methods and corresponding primers (Tables 2 and 3), fragments FaeLam-N20Xyl(Nde / Xho), Xys-FaeLam(Nde / Xho), FaeLam-Xys(Nde / Xho), Xys-N20Xyl(Nde / Xho), and N20Xyl-Xys(Nde / Xho) were obtained. Plasmids pDXys, pDN20Xyl, and pDFaeLam were then double-digested with restriction enzymes NdeⅠ and XhoⅠ to construct xylosidase Xys and xylanase N2. Five other plasmids with different expression sequences of 0Xyl and ferulic acid esterase FaeLam are: pETDuet-Xys-FaeLam-N20Xyl (pDⅢ-2), pETDuet-N20Xyl-Xys-FaeLam (pDⅢ-3), pETDuet-N20Xyl-FaeLam-Xys (pDⅢ-4), pETDuet-FaeLam-Xys-N20Xyl (pDⅢ-5), and pETDuet-FaeLam-N20Xyl-Xys (pDⅢ-6). A schematic diagram of the multi-enzyme co-expression plasmids pDⅢ-1, pDⅢ-2, pDⅢ-3, pDⅢ-4, pDⅢ-5, and pDⅢ-6 constructed above is shown below. Figure 3 As shown.

[0083] Table 2. Amplification primers used to obtain the double enzyme gene fragment.

[0084]

[0085] Table 3 Primer sequences (underlined areas are restriction enzyme sites)

[0086]

[0087]

[0088] Example 3: Construction and screening of multi-enzyme co-expression strains

[0089] The plasmids pDXys, pDN20Xyl, pDFaeLam, pDⅢ-1, pDⅢ-2, pDⅢ-3, pDⅢ-4, pDⅢ-5, and pDⅢ-6 constructed in Example 2 were transformed into E. coli BL21 / △xylAB via a 42°C heat shock method. The transformed plasmids were then plated onto LB agar plates containing 100 μg / mL ampicillin and incubated statically overnight at 37°C. Colonies were picked and cultured overnight at 37°C with shaking in LB liquid medium containing 100 μg / mL ampicillin. The colonies were then transferred at a 1% (v / v) ratio to LB liquid medium containing 100 μg / mL ampicillin and cultured for OD... 600 After reaching a concentration of 0.5, IPTG (final concentration 0.5 mM) was added, and the mixture was continuously induced at 37°C for 36 h.

[0090] After induction, the bacterial culture was centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain extracellular protein samples. The extracellular protein samples obtained were analyzed by 12% SDS-PAGE.

[0091] Weigh 25 mg of wheat bran into a 1.5 mL Eppendorf tube, add 500 μL of buffer A (8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, and 0.27 g potassium dihydrogen phosphate dissolved in 1000 mL ddH2O, adjusted to pH 7.0 with hydrochloric acid and sodium hydroxide), add 500 μL of extracellular protein sample, and incubate at 37 °C for 1 h. Simultaneously, use an equal volume of buffer A as a blank control. After the reaction, centrifuge at 12000 r / min for 5 min, take 150 μL of the supernatant, add 150 μL of DNS reagent to terminate the reaction, and incubate in a boiling water bath for 5 min to induce a colorimetric reaction. After standing at room temperature, measure the absorbance at 562 nm using a microplate reader, calculate the reducing sugar content (calculated as xylose), and calculate the enzyme activity. Compare the enzyme activities of extracellular protein samples from different multi-enzyme co-expression strains. Set up three parallel experiments, with the error bar representing the standard deviation of three replicates.

[0092] SDS-PAGE electrophoresis results of extracellular protein samples from different multi-enzyme co-expression strains are as follows: Figure 4 As shown, the enzyme activity assay results are as follows: Figure 5 As shown in Table 4.

[0093] Table 4. Relative enzyme activities of extracellular protein samples from different multi-enzyme co-expression strains

[0094]

[0095] Note: In the table, strains Xys, FaeLam, N20Xyl, pDⅢ-1, pDⅢ-2, pDⅢ-3, pDⅢ-4, pDⅢ-5, and pDⅢ-6 represent recombinant strains transformed into E. coli BL21 / △xylAB strains with plasmids pDXys, pDFaeLam, pDN20Xyl, pDⅢ-1, pDⅢ-2, pDⅢ-3, pDⅢ-4, pDⅢ-5, and pDⅢ-6, respectively.

[0096] Analysis showed that although the expression levels of single-enzyme strains were significantly higher than those of multi-enzyme co-expression strains, enzyme activity comparisons revealed that the combined enzymatic hydrolysis effect of multi-enzyme co-expression strains was significantly better than that of single-enzyme strains. Furthermore, different expression sequences of xylosidase (Xys), xylanase (N20Xyl), and ferulic acid esterase (FaeLam) resulted in varying extracellular enzyme expression levels and activities. The two strains with the best extracellular enzyme activity were *E. coli* BL21 / △xylAB / pDⅢ-2 and *E. coli* BL21 / △xylAB / pDⅢ-5, which are the co-expression strains of the hemicellulose degrading enzyme system of this invention. The hemicellulose degrading enzyme co-expression strain *E. coli* BL21 / △xylAB / pDⅢ-2, with the best enzyme activity, was selected for subsequent optimization of fermentation conditions.

[0097] Example 4: Optimization of fermentation conditions for strains co-expressing hemicellulose-degrading enzyme systems

[0098] 1. Optimization of culture medium types

[0099] Five conventional culture media (LB, SB, TB, 2×YT, and SOB) were used for basal culture medium screening. Activated bacterial suspensions of strain E. coli BL21 / △xylAB / pDⅢ-2 were transferred at a ratio of 1% (v / v) to each of the five basal media containing 100 μg / mL ampicillin and cultured until OD500. 600 After reaching a final concentration of 0.5 mM, IPTG was added, and the mixture was induced at 37°C for 36 h. The bacterial culture was centrifuged at 12000 r / min for 5 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain an extracellular protein sample. The extracellular protein sample obtained above was analyzed by 12% SDS-PAGE, and the enzyme activity was determined by the DNS method described in Example 3.

[0100] SDS-PAGE electrophoresis results of extracellular protein samples cultured in different basal media are as follows: Figure 6 As shown, the enzyme activity assay results are as follows: Figure 7As shown in Table 5.

[0101] Table 5. Relative enzyme activities of extracellular protein samples cultured in different basal cultures

[0102] relative enzyme activity 53.52％ 100％ 73.65％ 25.71％ 56.57％

[0103] Analysis showed that the secretion and activity of soluble extracellular enzymes by strain E. coli BL21 / △xylAB / pDⅢ-2 were significantly higher in SB medium than in other media. Therefore, SB medium was used as the basic fermentation medium for subsequent optimization of fermentation conditions.

[0104] 2. Optimization of lactose inducer concentration

[0105] Lactose, an inexpensive and safe inducer, was used instead of expensive and toxic IPTG. Activated culture of strain E. coli BL21 / ΔxylAB / pDⅢ-2 was transferred at a ratio of 1% (v / v) to SB medium containing 100 μg / mL ampicillin and cultured until OD... 600 After reaching a final concentration of 0.5, lactose was added (final concentrations of 0.5, 1, 2, 5, 10, 15, and 20 g / L, respectively), and IPTG at a final concentration of 0.7 mM was used as a control. Induction was performed at 37°C for 36 h. The bacterial culture was centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter to obtain extracellular protein samples. The extracellular protein samples were analyzed by 12% SDS-PAGE, and enzyme activity was determined by the DNS method.

[0106] SDS-PAGE electrophoresis results of extracellular protein samples cultured under different lactose concentrations are as follows: Figure 8 As shown, the enzyme activity assay results are as follows: Figure 9 As shown in Table 6.

[0107] Table 6. Relative enzyme activities of extracellular protein samples cultured under different lactose concentrations.

[0108] relative enzyme activity 41.69％ 52.62％ 100％ 71.91％ 53.03％ 40.33％ 18.13％

[0109] Analysis showed that as the final lactose concentration increased, the secretion and activity of soluble extracellular enzymes in strain E. coli BL21 / △xylAB / pDⅢ-2 gradually increased, reaching their peak at a final lactose concentration of 2 g / L. However, further increases in lactose concentration led to a decrease in the secretion of soluble extracellular enzymes in strain E. coli BL21 / △xylAB / pDⅢ-2. Furthermore, compared to the results of IPTG induction (final concentration 0.7 mM), the expression levels and activities of extracellular enzymes induced by lactose (final concentration 2 g / L) were significantly higher.

[0110] 3. Optimization of the timing of inducer addition

[0111] In order to find the optimal induction time for adding inducing agents (OD) 600 The activated bacterial culture of strain E. coli BL21 / △xylAB / pDⅢ-2 was transferred at a ratio of 1% (v / v) to SB medium containing 100 μg / mL ampicillin and cultured until OD500. 600 After adjusting the concentrations to 0.1, 0.3, 0.5, 0.7, and 0.9, lactose (final concentration 2 g / L) was added, and the mixture was induced at 37°C for 36 h. The bacterial culture was centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter to obtain extracellular protein samples. The extracellular protein samples were analyzed by 12% SDS-PAGE, and enzyme activity was determined by the DNS method.

[0112] SDS-PAGE electrophoresis results of extracellular protein samples cultured at different induction times are as follows: Figure 10 As shown, the enzyme activity assay results are as follows: Figure 11 As shown in Table 7.

[0113] Table 7. Different induction times (OD) 600 Relative enzyme activity of extracellular protein samples cultured under [condition]

[0114]

[0115]

[0116] Analysis shows that in OD 600 When the OD value reached 0.7, the soluble extracellular enzyme yield and activity of the induced strain E. coli BL21 / △xylAB / pDⅢ-2 were the highest. Further increasing the OD value... 600 If this happens, the yield and enzyme activity will decrease accordingly.

[0117] 4. Optimization of glycine addition amount

[0118] The activated bacterial culture of strain E. coli BL21 / △xylAB / pDⅢ-2 was transferred at a ratio of 1% (v / v) to SB medium containing 100 μg / mL ampicillin and cultured until OD500. 600 After reaching a concentration of 0.7, lactose (final concentration 2 g / L) was added, along with glycine (final concentrations of 0, 25, 50, 75, 100, and 125 mM), and the mixture was induced at 37°C for 36 h. The bacterial culture was centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter to obtain extracellular protein samples. These samples were analyzed by 12% SDS-PAGE, and enzyme activity was determined using the DNS method.

[0119] SDS-PAGE electrophoresis results of extracellular protein samples cultured at different glycine concentrations are as follows: Figure 12 As shown, the enzyme activity assay results are as follows: Figure 13 As shown in Table 8.

[0120] Table 8. Relative enzyme activities of extracellular protein samples cultured at different glycine concentrations

[0121] relative enzyme activity 71.92％ 85.78％ 89.07％ 94.49％ 100％ 84.76％

[0122] Analysis showed that appropriate addition of glycine (25-100mM) could significantly increase the secretion and activity of soluble extracellular enzymes in the strain, but further increase of glycine would affect the secretion and activity of extracellular enzymes in strain E. coli BL21 / △xylAB / pDⅢ-2.

[0123] 5. Optimization of metal ion addition

[0124] The activated bacterial culture of recombinant strain E. coli BL21 / △xylAB / pDⅢ-2 was transferred at a ratio of 1% (v / v) to SB medium containing 100 μg / mL ampicillin and cultured until OD500. 600 After reaching a concentration of 0.7, lactose (final concentration 2 g / L) was added, along with glycine (final concentration 100 mM) and different metal ions (Mn). 2+ Mg 2+ Zn 2+ Fe 2+ Ca 2+ The bacterial culture was induced at 37°C for 36 h at a final concentration of 2 mM. After centrifugation at 12000 r / min for 5 min, the supernatant was filtered through a 0.22 μm filter membrane to obtain extracellular protein samples. The extracellular protein samples were analyzed by 12% SDS-PAGE and enzyme activity was determined by DNS method.

[0125] SDS-PAGE electrophoresis results of extracellular protein samples cultured under different metal ions are as follows: Figure 14 As shown, the enzyme activity assay results are as follows: Figure 15 As shown in Table 9.

[0126] Table 9. Relative enzyme activities of extracellular protein samples cultured under different metal ions

[0127] relative enzyme activity 71.48％ 71.08％ 86.81％ 100％ 92.95％ 26.69％

[0128] Analysis shows that, compared to other metal ions, the addition of Zn... 2+ The addition of Ca significantly increased the secretion and activity of soluble extracellular enzymes in strain E. coli BL21 / △xylAB / pDⅢ-2, while... 2+ It significantly inhibited the normal expression and secretion of extracellular enzymes.

[0129] 6. Fermentation culture under optimized conditions

[0130] The enzyme activity changes of extracellular protein samples from *E. coli* BL21 / pDⅢ-2 and *E. coli* BL21 / ΔxylAB / pDⅢ-2 in SB medium were determined under optimized fermentation conditions. Activated cultures of *E. coli* BL21 / pDⅢ-2 and *E. coli* BL21 / ΔxylAB / pDⅢ-2 were transferred at a ratio of 1% (v / v) to SB medium containing 100 μg / mL ampicillin and cultured until OD500 reached the target value. 600 After reaching a concentration of 0.7, lactose (final concentration 2 g / L) was added, along with glycine (final concentration 100 mM) and Zn. 2+ (Final concentration 2mM) was induced at 37℃ for 54h. During the induction process, the bacterial culture was collected, centrifuged at 12000r / min for 5min, and the supernatant was filtered through a 0.22μm filter membrane to obtain extracellular protein samples. The reducing sugar content was determined by the DNS reagent method and the enzyme activity was calculated.

[0131] The enzyme activity change curve of the extracellular protein sample is shown in the figure. Figure 16 As shown, after induction with lactose (final concentration 2 g / L), the extracellular enzyme activities gradually increased, reaching a maximum at 30 h, and then gradually decreased. The multi-enzyme expression capabilities of E. coli BL21 / pDⅢ-2 and E. coli BL21 / △xylAB / pDⅢ-2 were almost identical.

[0132] Example 5: Enzymatic hydrolysis of wheat bran and xylan by hemicellulose-degrading enzyme co-expression strains

[0133] Wheat bran and xylan were added to SB medium at different concentrations (10-60 g / L). Activated bacterial cultures of E. coli BL21 / ΔxylAB / pDⅢ-2 were transferred at a 1% (v / v) ratio to SB medium containing either wheat bran or xylan, and cultured until OD... 600 To a final concentration of 0.7, add lactose (2 g / L), glycine (100 mM), and Zn. 2+ (Final concentration 2mM) was used to induce expression and simultaneously ferment at 37℃. Fermentation broth was collected at 12, 24, 36, 48, 60, 72, and 84 hours of fermentation. After centrifugation at 12000 r / min for 5 min, the reducing sugar content of the supernatant was determined using the DNS reagent method to compare the optimal amounts of wheat bran and xylan added with the optimal fermentation time. The fermentation supernatant was then used for subsequent antioxidant capacity determination.

[0134] Figure 17The figures show the changes in reducing sugar content during enzymatic hydrolysis of wheat bran (A) or xylan (B) during fermentation. When wheat bran was used as the fermentation substrate, within the range of 10-40 g / L, the reducing sugar content in the fermentation broth decreased with increasing wheat bran concentration. When the wheat bran concentration reached 50 g / L, the reducing sugar content increased slightly. Therefore, the optimal substrate concentration for wheat bran was 10 g / L. At a wheat bran concentration of 10 g / L, the reducing sugar content in the fermentation broth gradually increased with increasing fermentation time, reaching a maximum of 0.23 ± 0.01 g / L at 36 hours. When xylan was used as the fermentation substrate, within the range of 10-50 g / L, the reducing sugar content in the fermentation broth gradually increased with increasing xylan concentration. However, when the concentration reached 60 g / L, the reducing sugar content decreased slightly. Therefore, the optimal substrate concentration for xylan was 50 g / L. When the amount of xylan added was 50 g / L, the degree of enzymatic hydrolysis of xylan gradually increased with the increase of fermentation time, reaching the highest level at 48 h, and the reducing sugar content of the fermentation broth was 33.70 ± 0.46 g / L.

[0135] Example 6: Determination of the antioxidant capacity of xylan hydrolysate from strains co-expressing hemicellulose-degrading enzyme systems

[0136] The antioxidant capacity of the supernatant of the fermentation broth after fermentation was completed when the xylan addition amount was 50 g / L in Example 5 was determined.

[0137] 1. Determination of DPPH free radical scavenging ability

[0138] Take 1 mL of fermentation broth supernatant into a light-protected centrifuge tube, add 2 mL of DPPH solution (0.5 mM), mix well, and let stand in the dark for 30 min. Then centrifuge at 12000 r / min for 10 min, and measure the absorbance at 517 nm using a spectrophotometer. The blank group consisted of 1 mL of enzyme hydrolysate with 2 mL of anhydrous ethanol, and the control group consisted of 1 mL of sterile water with 2 mL of DPPH solution. Ascorbic acid (Vc) was used as a positive control. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0139]

[0140] Where AS is the absorbance of the sample group, A0 is the absorbance of the control group, and A is the absorbance of the blank group.

[0141] 2. Determination of superoxide anion free radical scavenging capacity

[0142] Mix 1 mL of 150 mM Tris-HCl solution, 1 mL of 3 mM DTPA solution, 1 mL of 1.2 mM pyrogallol solution, and 0.5 mL of fermentation broth supernatant thoroughly. Incubate at 25 °C for 10 min, centrifuge at 10000 r / min for 10 min, and measure the absorbance at 325 nm. Ascorbic acid (Vc) was used as a positive control. The formula for calculating the superoxide anion radical scavenging rate is as follows:

[0143]

[0144] Wherein, A0 is the absorbance of 1 mL Tris-HCl solution and 1 mL DTPA solution; A1 is the absorbance of 1 mL Tris-HCl solution, 1 mL DTPA solution and 1 mL pyrogallol solution; A2 is the absorbance of 1 mL Tris-HCl solution, 1 mL DTPA solution and 0.5 mL fermentation broth supernatant; A3 is the absorbance of 1 mL Tris-HCl solution, 1 mL DTPA solution, 0.5 mL fermentation broth supernatant and 1 mL pyrogallol solution.

[0145] 3. Determination of hydroxyl radical scavenging ability

[0146] Mix 1 mL of buffer A, 0.5 mL of 2.5 mM o-phenanthroline solution, 0.5 mL of 2.5 mM ferrous sulfate solution, 0.5 mL of 20 mM hydrogen peroxide, and 0.5 mL of fermentation broth supernatant. Incubate at 37 °C for 1 h. After centrifugation at 10000 r / min for 10 min, measure the absorbance A at 536 nm. The control group used an equal volume of ddH2O instead of the fermentation broth supernatant, and the blank group used an equal volume of ddH2O instead of both hydrogen peroxide and the fermentation broth supernatant. Ascorbic acid (Vc) was used as a positive control. The formula for calculating the hydroxyl radical scavenging capacity is as follows:

[0147]

[0148] 4. Total antioxidant capacity determination

[0149] Mix 3 mL of ammonium molybdate system (28 mM sodium phosphate, 4 mM ammonium molybdate tetrahydrate, and 0.6 M concentrated sulfuric acid, each prepared separately, and 200 μL of each mixture thoroughly) with 0.3 mL of fermentation broth supernatant. React at 95 °C for 90 min. After cooling to room temperature, centrifuge at 10000 r / min for 10 min. Then measure the absorbance at 695 nm wavelength. Use ascorbic acid (Vc) as a positive control. Compare the total antioxidant capacity based on the absorbance values.

[0150] The results of the antioxidant capacity determination of the fermentation broth supernatant are as follows: Figure 18As shown, the various antioxidant indicators of the fermentation supernatant are also quite good. The hydroxyl radical scavenging capacity can reach 80.0%, and the total antioxidant capacity can reach 2.289±0.55, mainly because there is free ferulic acid in the fermentation supernatant.

[0151] Example 7: Probiotic culture of xylan hydrolysate from hemicellulose-degrading enzyme co-expression strains

[0152] Xylan was added to SB medium at the optimal substrate addition level of 50 g / L, and strain E. coli BL21 / △xylAB / pDⅢ-2 was inoculated into this medium and cultured for OD... 600 To a final concentration of 0.7, add lactose (2 g / L), glycine (100 mM), and Zn. 2+ (Final concentration 2mM), after induction of expression at 37℃ and simultaneous fermentation for 48h, the fermentation broth was centrifuged at 10000r / min for 5min, and the supernatant was collected into a new centrifuge tube to obtain xylan hydrolysate. The xylan hydrolysate was mixed with 2× sugar-free MRS medium at a 1:1 ratio and then autoclaved at 115℃ for 30min to prepare a special MRS medium with xylose hydrolysis as the carbon source, used for probiotic culture. Probiotics such as *Lactobacillus pumilus*, *Enterococcus faecalis*, *Pediococcus lactis*, and *Westernella* were activated in ordinary MRS liquid medium and transferred at a 1% (v / v) volume ratio to the special MRS medium with xylose hydrolysis as the carbon source, and cultured statically at 37℃ for 24h. A sugar-free MRS medium supplemented with 10g / L xylose was used as a control group. The OD of the bacterial culture after 24h static culture was measured. 600 With changes in pH.

[0153] The measurement results are as follows Figure 19 As shown, compared to xylose-based MRS medium, the special MRS medium promotes the growth of probiotics more effectively, with OD... 600 The pH of the medium was slightly higher than that of the xylose-containing MRS medium, indicating that xylose and other components in the xylan hydrolysate promoted the growth of probiotics.

Claims

1. A recombinant plasmid for co-expressing a hemicellulose-degrading enzyme system, characterized in that, The recombinant plasmid co-expressed with the hemicellulose-degrading enzyme system includes the xylosidase encoding gene. Xys ferulic acid esterase encoding gene FaeLam and xylanase encoding gene N20Xyl The xylosidase encoding gene Xys The nucleotide sequence is SEQ ID NO.1, and the gene encoding the ferulic acid esterase is described. FaeLam The nucleotide sequence is SEQ ID NO.2, and the xylanase encoding gene is described. N20Xyl The nucleotide sequence is SEQ ID NO.3, and the expression sequence of the hemicellulose-degrading enzyme system of the recombinant plasmid is as follows: Xys-FaeLam-N20Xyl or FaeLam- Xys-N20Xyl The vector plasmid for the recombinant plasmid is pETDuet-1.

2. A hemicellulose-degrading enzyme co-expression strain containing the recombinant plasmid of the hemicellulose-degrading enzyme system according to claim 1.

3. The hemicellulose-degrading enzyme co-expression strain as described in claim 2, characterized in that, The host bacterium of the hemicellulose degradation enzyme co-expression strain is Escherichia coli.

4. The hemicellulose-degrading enzyme co-expression strain as described in claim 3, characterized in that, The Escherichia coli in question is a xylose-deficient Escherichia coli.

5. The hemicellulose-degrading enzyme co-expression strain as described in claim 4, characterized in that, The xylose metabolism-deficient Escherichia coli is a knockout bacterium encoding the xylose isomerase XylA and xylulose kinase XylB genes.

6. The use of the co-expressed strain of the hemicellulose degrading enzyme system according to claim 2 in the preparation of a multi-enzyme mixture containing xylosidase, xylanase and ferulic acid esterase.

7. The method for preparing a multi-enzyme mixture containing xylosidase, xylanase, and ferulic acid esterase by the hemicellulose-degrading enzyme co-expression strain according to claim 2, characterized in that, Includes the following steps: The hemicellulose-degrading enzyme co-expressing strain was inoculated into SB medium and cultured until OD500. 600 After adjusting the concentration to 0.5-0.9, add lactose to a final concentration of 2-5 g / L, and simultaneously add glycine to a final concentration of 25-100 mM, and the metal ion Mg. 2+ Zn 2+ or Fe 2+ The final concentration was 2 mM. After induction of expression at 37 °C, the supernatant of the fermentation broth was taken to obtain a multi-enzyme mixture containing xylosidase, xylanase and ferulic acid esterase.

8. The method as described in claim 7, characterized in that, Cultured to OD 600 It is 0.

7.

9. The method as described in claim 7, characterized in that, Add lactose to a final concentration of 2 g / L.

10. The method as described in claim 7, characterized in that, Add glycine to a final concentration of 100 mM.

11. The method as described in claim 7, characterized in that, The added metal ion is Zn 2+ .

12. The application of the hemicellulose-degrading enzyme co-expression strain according to claim 2 in the degradation of lignocellulose.

13. The application as described in claim 12, characterized in that, The lignocellulose is wheat bran or xylan.

14. The application of the hemicellulose-degrading enzyme co-expression strain according to claim 2 in the preparation of ferulic acid.

Images