Xylanase with improved enzyme activity and preparation method therefor

Abstract

A xylanase and a preparation method therefor, a polynucleotide encoding the xylanase, a recombinant vector comprising the polynucleotide, a host cell expressing the xylanase, a method for preparing an Aspergillus niger strain producing the xylanase, and a use of the xylanase in feed.

Description

Xylanase with enhanced enzyme activity and preparation method thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application number CN202311613123.8, filed on November 28, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to xylanases and polynucleotides encoding the xylanases, as well as recombinant vectors and host cells containing the polynucleotides, and methods for preparing the xylanases and host cells. Background Art

[0004] Xylanases, a type of enzyme that degrades xylan, play an important role in the feed industry. By depolymerizing xylan, an anti-nutritional factor in feed that animals cannot digest and utilize, into oligosaccharides, xylose, and arabinose, anti-nutritional factors are eliminated, further improving feed resource utilization. Furthermore, environmental pollution caused by animal excretion is effectively reduced, which is of vital importance for improving the economic benefits of breeding and developing an environmentally friendly animal husbandry. There are many types of xylanases, but the feed industry requires a high level of thermal stability for xylanases, as they must meet the process requirements for high-temperature pelleting of feed. Among them, the GH11 family xylanases derived from the rumen fungus Neocallimastix patriciarum have high enzyme activity and good heat resistance. Chen et al randomly mutagenized the xylanase from Neocallimastix patriciarum and screened out a recombinant xylanase Xyn-CDBFV with better enzyme activity and alkalinity performance (Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase, 2001 December; 47(12): 1088-94. doi: 10.1139 / w01-118).

[0005] Since then, researchers have conducted extensive research on Xyn-CDBFV to improve its specific enzyme activity and heat resistance. For example, Zhang et al. fused the carbohydrate-binding module (CBM) with CDBFV and expressed it in Pichia pastoris, effectively increasing the rate at which xylanase hydrolyzes reducing sugars. Chinese patent CN 114807093 A significantly improved the thermal stability of the xylanase Xyn-CDBFV by adding a fusion peptide to the C-terminus. Cui et al. fused the N-terminus of lysozyme with the C-terminus of the xylanase Xyn-CDBFV in Pichia pastoris, achieving high-quality production of recombinant lysozyme and xylanase, providing new insights for the industrial application of compound feeds.

[0006] Currently, numerous researchers have studied recombinant xylanases, such as the xylanase Xyn-CDBFV, in Pichia pastoris by adding peptides or through fusion expression. However, yeast expression of some heterologous proteins can present issues such as over-modification and protein folding, which can affect the expression and stability of heterologous proteins. Furthermore, since Pichia pastoris typically requires methanol induction for heterologous protein production, this poses certain risks in actual industrial production. Therefore, developing novel xylanases with high enzymatic activity and excellent thermal stability through novel platforms is of great significance and can effectively reduce production costs. Summary of the Invention

[0007] The present invention provides a xylanase comprising a xylanase Xyn-CDBFV sequence and a fusion peptide segment connected to the C-terminus of the xylanase, wherein the fusion peptide segment comprises the amino acid sequence shown in SEQ ID NO: 1.

[0008] In some embodiments, the fusion peptide further comprises the amino acid sequence shown in SEQ ID NO:2.

[0009] In some embodiments, the fusion peptide segment consists of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0010] In some embodiments, the xylanase Xyn-CDBFV sequence is shown as SEQ ID NO:3.

[0011] In some embodiments, the xylanase comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the xylanase comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the xylanase consists of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the xylanase consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0012] The present invention also provides a polynucleotide encoding the xylanase.

[0013] In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the polynucleotide consists of the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the polynucleotide consists of the nucleotide sequence set forth in SEQ ID NO: 7.

[0014] The present invention also provides a recombinant vector comprising the polynucleotide, and a host cell comprising the polynucleotide or the recombinant vector.

[0015] In some embodiments, the host cell is Aspergillus niger or Pichia pastoris. In some embodiments, the host cell is Aspergillus niger.

[0016] The present invention also provides a method for preparing xylanase, which comprises connecting a fusion peptide segment to the C-terminus of a xylanase Xyn-CDBFV sequence, wherein the fusion peptide segment comprises the amino acid sequence shown in SEQ ID NO: 1.

[0017] In some embodiments, the fusion peptide further comprises the amino acid sequence shown in SEQ ID NO:2.

[0018] In some embodiments, the fusion peptide segment consists of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0019] In some embodiments, the xylanase Xyn-CDBFV sequence is shown as SEQ ID NO:3.

[0020] The present invention further provides a method for preparing an Aspergillus niger strain that produces xylanase, which comprises introducing the recombinant vector into the Aspergillus niger strain.

[0021] In some embodiments, the recombinant vector comprises an Aspergillus niger expression cassette sequence, which comprises an Aspergillus niger saccharifying enzyme gene gla promoter sequence, an Aspergillus niger amylase signal peptide nucleotide sequence, a xylanase nucleotide sequence as shown in SEQ ID NO: 6 or 7, and an Aspergillus niger saccharifying enzyme gene gla terminator sequence.

[0022] In some embodiments, the method comprises screening the Aspergillus niger strain using a selection marker amdS plate after introducing the recombinant vector.

[0023] The present invention also provides application of the xylanase in feed.

[0024] The beneficial effects of the present invention are as follows: by adding a specific fusion peptide segment to the C-terminus of the GH11 family xylanase Xyn-CDBFV, the problem of Xyn-CDBFV having no enzymatic activity when expressed on the Aspergillus niger platform can be solved; and after the xylanases Xyn-307 and Xyn-309 provided by the present invention are expressed on the Aspergillus niger platform, the enzyme activity levels are significantly improved compared with those when the xylanases Xyn-307 and Xyn-309 are expressed in Pichia pastoris. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 compares the enzyme activity performance of recombinant xylanases Xyn-307 and Xyn-309 produced by the Aspergillus niger platform at the 50L fermenter level.

[0026] Figure 2 compares the enzyme activity performance of yeast platform recombinant xylanases Xyn-307 and Xyn-309 at the 50L fermenter level. DETAILED DESCRIPTION

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0029] The term "about," when used with a numerical variable, generally means that the value of the variable and all values ​​of the variable are within measurement or experimental error (e.g., 95% confidence interval for the mean) or within the broader range of the specified value (e.g., ±5% or ±10%).

[0030] The term "comprise" or its variations such as "comprising," "having," and "including" means that the stated steps or elements are included but any other steps or elements are not excluded. "Consisting of" means that unlisted steps or elements are not included. The term "comprising" of specific steps or elements and its variations also include "consisting of the specific steps or elements" and "consisting essentially of the specific steps or elements."

[0031] When a numerical range is mentioned, it should be considered that the specific values ​​of its upper and lower limits are specifically disclosed, as well as all intermediate ranges included therein, such as intermediate ranges between its upper or lower limit and any intermediate value, or intermediate ranges between any two intermediate values. In addition, any intermediate ranges, subranges and all individual values ​​described in the numerical range can be excluded from the numerical range.

[0032] The term "and / or" should be understood to refer to any one element or any combination of several elements connected by this term.

[0033] The term "xylanase" refers to an enzyme, polypeptide or protein capable of degrading xylan, for example, capable of catalyzing the degradation of β-1,4 glycosidic bonds in xylan.

[0034] The term "recombinant" when applied to a nucleic acid, protein, or vector indicates that the nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by altering a native nucleic acid or protein.

[0035] The term "expression" in the context of the present invention includes any step involved in the production of the xylanase of the present invention including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0036] The term "vector" may be an autonomously replicating vector, i.e., one that replicates independently of a chromosome, such as a plasmid or an artificial chromosome, or may be a vector that is integrated into the genome and replicates together with the chromosome when introduced into a host cell. The expression vector may comprise one or more selectable markers that allow for easy selection of transformed cells, transfected cells, or transduced cells, such as those that provide resistance to antibiotics, heavy metals, and the like. The expression vector may also comprise an origin of replication to allow the expression vector to replicate autonomously in the host cell. The selection of these elements is a common technique in the art, and the method for constructing the recombinant expression vector of the present invention using these elements is well known in the art, and can be found, for example, in J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition.

[0037] The term "host cell" refers to any cell type susceptible to transformation, transfection, transduction, etc., which can contain a polynucleotide or recombinant vector of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0038] The term "thermostable" refers to the ability of an enzyme to resist irreversible inactivation (usually by denaturation) at relatively high temperatures, meaning that the enzyme retains a specified amount of enzymatic activity after exposure to a specified temperature for a given period of time.

[0039] Unless otherwise indicated, nucleic acids are written herein in 5' to 3' orientation from left to right and amino acid sequences are written left to right in amino-terminus (N-terminus) to carboxyl-terminus (C-terminus) orientation.

[0040] The present invention provides a xylanase comprising a xylanase Xyn-CDBFV sequence and a fusion peptide segment connected to its C-terminus, wherein the fusion peptide segment comprises the amino acid sequence set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2. The amino acid sequence of xylanase Xyn-CDBFV is set forth in SEQ ID NO: 3. In a specific embodiment, the amino acid sequence of the fused xylanase Xyn-307 is set forth in SEQ ID NO: 4 (the underlined portion is the fusion peptide segment SEQ ID NO: 1). In a specific embodiment, the amino acid sequence of the fused xylanase Xyn-309 is set forth in SEQ ID NO: 5 (the underlined portion is the fusion peptide segment sequence SEQ ID NO: 1, and the bold italicized portion is the fusion peptide segment sequence SEQ ID NO: 2).

[0041] Those skilled in the art know that the xylanase can also be linked to other peptide sequences (including but not limited to tag sequences, such as histidine tag sequences) without affecting the functional properties of the xylanase (including but not limited to the enzyme activity, thermal stability and other properties described herein), and such xylanases are also included in the scope of the xylanases described herein.

[0042] The present invention also provides a polynucleotide encoding the xylanase described above. In a specific embodiment, the polynucleotide encodes xylanase Xyn-307, the sequence of which is shown in SEQ ID NO: 6. In a specific embodiment, the polynucleotide encodes xylanase Xyn-309, the nucleotide sequence of which is shown in SEQ ID NO: 7.

[0043] The present invention also provides a recombinant vector comprising the polynucleotide described above. In a specific embodiment, the recombinant vector comprises a polynucleotide encoding xylanase Xyn-307. In a specific embodiment, the recombinant vector comprises a polynucleotide encoding xylanase Xyn-309.

[0044] The present invention also provides a host cell comprising the polynucleotide or recombinant vector as described above, wherein the host cell may be Aspergillus niger or Pichia pastoris. In a specific embodiment, the host cell is Aspergillus niger.

[0045] The present invention also provides a method for preparing a genetically engineered Aspergillus niger bacterium that produces xylanase, comprising the following steps:

[0046] (1) Gene synthesis of a DNA fragment containing the Aspergillus niger saccharifying enzyme gene gla promoter (the nucleotide sequence of which is shown in SEQ ID NO: 8) and terminator (the nucleotide sequence of which is shown in SEQ ID NO: 9);

[0047] (2) The Aspergillus niger amylase signal peptide sequence (its nucleotide sequence is shown in SEQ ID NO: 10) and the nucleotide sequence of xylanase Xyn-307 or Xyn-309 are connected in series between the promoter and the terminator to form an Aspergillus niger expression cassette sequence;

[0048] (3) cloning the Aspergillus niger expression cassette sequence into a plasmid to obtain a recombinant expression plasmid;

[0049] (4) cloning the recombinant expression plasmid into Escherichia coli competent cells DH5α, selecting positive transformants for colony PCR and DNA sequencing, culturing the transformants with correct sequencing, and extracting the recombinant expression plasmid on a large scale;

[0050] (5) After linearization of the extracted recombinant expression plasmid, the plasmid was transformed into Aspergillus niger protoplasts and screened on a plate (0.8 M sucrose + 2 mL acetamide + 200 mL trace elements) using the selection marker amdS (the nucleotide sequence of which is shown in SEQ ID NO: 11);

[0051] (6) The grown transformants were activated using TB3 low-sugar plates and inoculated into enzyme-producing medium for 144 h. The supernatant was centrifuged and subjected to protein electrophoresis identification and enzyme activity detection in the fermentation supernatant. Several transformants with high xylanase activity were screened out and rescreened for confirmation to obtain genetically engineered Aspergillus niger bacteria that produced xylanase Xyn-307 or Xyn-309.

[0052] The present invention also provides a shake flask culture method for a recombinant strain of Aspergillus niger producing xylanase Xyn-307 or Xyn-309, comprising inoculating the screened positive transformants of Xyn-307 or Xyn-309 into a shake flask of Aspergillus niger screening culture medium, culturing on a shaker, collecting the supernatant by centrifugation after the fermentation is completed, and measuring the enzyme activity of the xylanase.

[0053] In some embodiments, the incubation temperature of the shaker is 30-35° C. In a preferred embodiment, the incubation temperature of the shaker is 34° C.

[0054] In some embodiments, the rotation speed of the shaking table is 180-250 rpm. In a preferred embodiment, the rotation speed of the shaking table is 220 rpm.

[0055] In some embodiments, the period of the shaking culture is 5-7 days. In a preferred embodiment, the period of the shaking culture is 6 days.

[0056] The present invention will be further described below in conjunction with specific embodiments:

[0057] Experimental materials and reagents

[0058] (1) Strains and vectors

[0059] Escherichia coli DH5α was purchased from Invitrogen, Aspergillus niger CICC2462 strain was purchased from China Industrial Microorganism Culture Collection CICC, and plasmid pUC57 was purchased from GenScript Biotech Co., Ltd.

[0060] (2) Enzymes and related molecular biological reagents

[0061] Agarose Gel DNA Purification Kit and DNA Fragment Purification Kit Ver. 2.0 were purchased from TaKaRa. T4 DNA ligase was purchased from Fermetas. Other biochemical reagents were purchased from Shanghai Sangon Biotechnology Service Co., Ltd.

[0062] (3) Culture medium

[0063] LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride; adjust the pH to 7.0 with NaOH; add 1.5% (w / v) agar powder (solid medium).

[0064] TZ liquid medium: 0.8% beef extract powder, 0.2% yeast extract, 0.5% peptone, 0.2% NaCl, 3% sucrose, pH 5.8.

[0065] STC solution: 1 M D-Sorbitol, 50 mM CaCl2, 10 mM Tris, pH 7.5.

[0066] Acetamide medium: 3% sucrose, 0.05% KCl, 0.1% K2HPO4·3H2O, 0.001% FeSO4, 0.0244% MgSO4, 0.06% acetamide, 0.34% CsCl.

[0067] Aspergillus niger enzyme production culture medium: glucose: 12%; corn steep liquor powder: 1.4%; soybean cake powder: 3%; ammonium sulfate: 1%; potassium dihydrogen phosphate: 1%; magnesium sulfate: 0.02%; calcium carbonate: 0.6%; citric acid: 0.3%; zinc sulfate: 0.005%; ferrous sulfate: 0.005%.

[0068] Determination of xylanase activity

[0069] All determinations of xylanase activity in this article used the standard method specified in the national standard "GB / T23874-2009 Determination of xylanase activity in feed additives - Spectrophotometric method", that is, the reducing oligosaccharides and monosaccharides produced by the catalytic degradation of xylan by xylanase undergo a color reaction with 3,5-dinitrosalicylic acid (DNS) reagent under boiling water bath conditions. The enzyme activity of the tested xylanase is calculated based on the difference between the absorbance of the enzyme reaction sample at 540nm and that of the blank control sample.

[0070] The specific calculation formula of enzyme activity is as follows:

[0071] The activity of xylanase in the diluted enzyme solution used for enzymatic hydrolysis reaction was calculated according to formula (1) and formula (2):

[0072] Where:

[0073] X D ——Activity of xylanase in diluted enzyme solution, U / mL;

[0074] A E ——Absorbance of enzyme reaction solution;

[0075] A B ——Absorbance of enzyme blank sample;

[0076] K – slope of the standard curve;

[0077] C o ——intercept of the standard curve;

[0078] M——molar mass of xylose, M(C5H 10 O5)=150.2g / mol

[0079] t——enzymatic reaction time, min;

[0080] 1000——Conversion factor, 1mmol=1000μmol

[0081] X D The value should be between 0.04U / mL and 0.1U / mL. If it is not within this range, the dilution ratio of the enzyme solution should be adjusted before analysis and determination. D ×D f (2)

[0082] Where:

[0083] X——xylanase activity in the sample, U / g or U / mL;

[0084] D f ——Dilution multiple of the sample.

[0085] Example 1: Construction of expression plasmids for Aspergillus niger platform xylanases Xyn-CDBFV, Xyn-307, and Xyn-309

[0086] (1) Linearize the pUC57 plasmid using vector-F and vector-R primers;

[0087] (2) GenScript Company synthesized a DNA fragment containing the promoter and terminator of the Aspergillus niger glucoamylase gene gla and the selection marker amdS;

[0088] (3) First, primers amdS-F and amdS-R, and gla-F and gla-R were used to amplify the amdS gene with the recombination arms and the DNA fragment containing the gla promoter and terminator by PCR. The linearized pUC57 plasmid, the amdS gene, and the gla promoter and terminator DNA fragment were recombined using the Gibson Master Mix Kit (E2611, New England Biolabs) to obtain the pGla-amdS plasmid. The sequence was confirmed to be correct by sequencing. This plasmid can be linearized at the AflII site and then used to insert the xylanase Xyn-CDBFV, Xyn-307, and Xyn-309 gene sequences.

[0089] (4) The expression plasmids for xylanase Xyn-CDBFV, Xyn-307, and Xyn-309 were constructed as follows: 1. The Xyn-CDBFV gene with the recombination arms was amplified by PCR using primers Xyn-F and Xyn-R1. The Xyn-CDBFV gene was then recombined with the linearized pGla-amdS plasmid using the Gibson Master Mix Kit (E2611, New England Biolabs) to obtain the Xyn-CDBFV-amdS plasmid, and the sequence was confirmed by sequencing. This plasmid can be linearized at the Hind III site and then used for protoplast transformation. 2. The Xyn-307 gene with the recombination arms was amplified by PCR using primers Xyn-F and Xyn-R2. The Xyn-307 gene with the linearized pGla-amdS plasmid was then recombined with the Gibson Master Mix Kit (E2611, New England Biolabs) to obtain the Xyn-307-amdS plasmid, and the sequence was confirmed by sequencing. This plasmid can be linearized at the HindIII site and used for protoplast transformation. 3. The Xyn-309 gene with recombination arms was amplified by PCR using primers Xyn-F and Xyn-R3. The Xyn-309 gene was then recombined with the linearized pGla-amdS plasmid using the Gibson Master Mix Kit (E2611, New England Biolabs) to generate the Xyn-309-amdS plasmid. The sequence was confirmed by sequencing. This plasmid can be linearized at the HindIII site and used for protoplast transformation.

[0090] The relevant primer sequences are as follows:

[0091] Table 1 Primers in the present invention

[0092] Example 2: Transformation and integration of Aspergillus niger platform xylanase Xyn-CDBFV, Xyn-307, and Xyn-309 expression plasmids

[0093] The linearized xylanase Xyn-CDBFV, Xyn-307, and Xyn-309 expression plasmids were introduced into the Aspergillus niger CICC2462 strain using the protoplast transformation method. The specific steps are as follows:

[0094] (1) Preparation of protoplasts: Aspergillus niger mycelium was inoculated into nutrient-rich TZ liquid medium and cultured for 48 h. The mycelium was collected by filtration using Mira-cloth (Calbiochem) and washed with 0.7 M NaCl (pH 5.8). After the mycelium was filtered dry, it was transferred to an enzymatic solution (pH 5.8) containing 1% cellulase (Sigma), 1% snailase (Sigma) and 0.2% lyticase (Sigma) and hydrolyzed at 30°C and 65 rpm for 3 h. The enzymatic solution containing the protoplasts was then placed on ice and filtered through four layers of lens paper. The filtrate was gently centrifuged at 3000 rpm and 4°C for 10 min, and the supernatant was discarded. The protoplasts attached to the tube wall were washed once with STC solution (1 M D-Sorbitol, 50 mM CaCl2, 10 mM Tris, pH 7.5). Finally, the protoplasts were resuspended in an appropriate amount of STC solution.

[0095] (2) Protoplast transformation: 10 μL (concentration of 1000 ng / μL) of the xylanase expression plasmid linearized with HindⅢ was added to 100 μL of the protoplast suspension, mixed and allowed to stand at room temperature for 25 min, then 900 μL of PEG solution was added three times, mixed and allowed to stand at room temperature for 25 min, and then centrifuged at room temperature for 10 min at 3000 rpm. The supernatant was discarded, and the protoplasts attached to the tube wall were resuspended in 1 mL of STC solution, mixed with acetamide medium that had been cooled to about 45°C in advance and spread on a plate. After the plate solidified, it was placed in a 34°C incubator for 4-5 days. The transformants were picked to a new acetamide medium plate and placed in a 34°C incubator for another 4-5 days. The grown transformants were called positive transformants.

[0096] The linearized xylanase Xyn-CDBFV, Xyn-307, and Xyn-309 expression plasmids were transformed into Aspergillus niger strains using the above-mentioned protoplast transformation method to obtain xylanase-positive transformants.

[0097] Example 3: Shake flask culture of recombinant Aspergillus niger expression strains producing xylanases Xyn-CDBFV, Xyn-307, and Xyn-309

[0098] Positive transformants of the xylanases Xyn-CDBFV, Xyn-307, and Xyn-309 were inoculated into shake flasks containing 50 mL of Aspergillus niger enzyme-producing medium. The culture was shaken at 34°C and 220 rpm for 6 days. The fermentation supernatant was collected by centrifugation and assayed for xylanase activity. The enzyme activity of the fermentation supernatant after 144 hours of fermentation is shown in Table 2.

[0099] Table 2 Enzyme activity of Aspergillus niger platform xylanase Xyn-CDBFV, Xyn-307, and Xyn-309 fermentation supernatants after 144 h of fermentation

[0100] Example 4: Fermentation of xylanase Xyn-307 and Xyn-309 recombinant Aspergillus niger expression strains in a 50 L fermentor

[0101] Fermentation experiments were conducted using genetically engineered Aspergillus niger strains containing positive transformants of the aforementioned xylanases Xyn-307 and Xyn-309 in a 50-L fermentor. The actual volume of the enzyme-producing culture medium was 30 L. Fermentation conditions were 34°C, agitation at 800 rpm, and aeration at 25 LPM. Fermentation lasted approximately 186 hours, with enzyme activity monitored every four hours. The activity data are shown in Table 3, and the activity curves are shown in Figure 1. The whole-cell enzyme activity of Xyn-309 reached a maximum of 256,000 u / g, while that of Xyn-307 reached a maximum of 168,000 u / g.

[0102] Table 3 Enzyme activity records of Aspergillus niger platform xylanase Xyn-307 and Xyn-309 fermentation

[0103] Example 5: High-efficiency expression of xylanases Xyn-307 and Xyn-309 in Pichia pastoris

[0104] (1) Strains and vectors

[0105] Pichia pastoris X-33 was obtained from GenScript Biotech Co., Ltd., and the vector pPicZaA was purchased from Invitrogen.

[0106] (2) Enzymes and related molecular biological reagents

[0107] Endonucleases and ligases were purchased from TaKaRa, as were the Agarose Gel DNA Purification Kit and the DNA Fragment Purification Kit Ver. 2.0. T4 DNA ligase was purchased from Fermetas. Other biochemical reagents were purchased from Shanghai Sangon Biotechnology Service Co., Ltd.

[0108] (3) Culture medium

[0109] LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride; adjust the pH to 7.0 with NaOH; add 1.5% (w / v) agar powder (solid medium).

[0110] Yeast MD screening medium: 2% glucose, 1.5% agar powder, 0.00004% biotin, 1.34% YNB; MM medium: 0.5% methanol (volume fraction), 1.5% agar powder, 0.00004% biotin, 1.34% YNB; RDB medium: 1 M sorbitol, 2% glucose, 0.00004% biotin, 1.34% YNB, 0.005% amino acids.

[0111] Yeast BMGY induction medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (volume fraction).

[0112] Yeast BMMY induction medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 0.5% methanol (volume fraction).

[0113] (4) Construction of recombinant plasmid and expression in Pichia pastoris

[0114] Synthetic xylanase genes Xyn-307 and Xyn-309 were used as templates and ligated into the expression vector pPicZaA, respectively. The xylanase genes were inserted downstream of the signal peptide sequence of the expression vector, forming the correct reading frame with the α-MF signal peptide. The yeast expression vectors pPicZaA-Xyn-307 and pPicZaA-Xyn-309 were constructed and transformed into competent E. coli DH5α cells. Positive transformants were sequenced, and those with correct sequences were used for large-scale production of recombinant plasmids. Approximately 5 μg of the recombinant expression plasmid, linearized with the restriction endonuclease BglII, was electroporated into competent yeast X-33 cells, plated on Zeocin-resistant plates, and cultured at 30°C for 2-3 days. Transformants growing on the plates were selected for further expression experiments.

[0115] (5) Screening of transformants with high xylanase activity in Pichia pastoris

[0116] Using a sterilized pipette tip, single colonies were picked from the plates containing transformants and numbered onto Zeocin plates. Plates containing transformants were incubated in a 30°C incubator for 1–2 days until colonies emerged. Colony PCR verification and sequencing of the Xyn-307 and Xyn-309 expression cassettes were performed. Once sequencing confirmed, transformants were selected from the plates and inoculated into shake flasks containing BMGY medium. Cultures were shaken at 30°C and 250 rpm for approximately 48 hours. After 48 hours of induction, the culture was centrifuged at 3,000 × g for 15 minutes, the supernatant removed, and the culture was switched to BMMY medium containing 0.5% methanol at 30°C and 260 rpm for induction. After 48 hours of induction, the culture was centrifuged at 3,000 × g for 5 minutes, and the supernatant was used for enzyme activity testing to identify transformants with high xylanase activity.

[0117] (6) Enzyme activity expression levels of Pichia pastoris genetically engineered strains Xyn-307 and Xyn-309 in a 50 L fermenter

[0118] Fermentation tanks: The positive transformants of the xylanase Xyn-307 and Xyn-309 were fermented in 50L fermenters, with an actual volume of 30L of enzyme-producing culture medium. Fermentation conditions were 30°C, agitation at 800 rpm, and aeration at 25 LPM. Fermentation lasted approximately 210 hours, with enzyme activity monitored throughout. The activity data are shown in Table 4, and the activity curves are shown in Figure 2. The whole-cell enzyme activity of Xyn-309 reached a maximum of 72,000 u / g, while that of Xyn-307 reached a maximum of 65,000 u / g.

[0119] Table 4 Yeast platform xylanase Xyn-307, Xyn-309 fermentation whole bacteria enzyme activity record

[0120] By comparing Example 4 with Example 5, it can be seen that the enzyme activities of the xylanases Xyn-307 and Xyn-309 from the Aspergillus niger platform were significantly improved compared to those from the yeast platform. The enzyme activity of Xyn-307 increased from 65,000 u / g to 168,000 u / g, an increase of 158.5%; the enzyme activity of Xyn-309 increased from 72,000 u / g to 256,000 u / g, an increase of 255.6%. Secondly, in the Aspergillus niger platform, the enzyme activity expression level of Xyn-309 was significantly improved compared to Xyn-307, reaching a maximum of 256,000 u / g, which is more than 50% higher than that of Xyn-307.

[0121] Example 6

[0122] To test the actual effect of xylanase expressed by the Aspergillus niger platform in feed applications, we tested the water bath heat resistance of Xyn-307 and Xyn-309 from the Aspergillus niger platform and Xyn-307 from the yeast platform. After preparing pelletized products, we tested the stability of feed pellets and the enzymatic hydrolysis effect in vitro. The results are shown in Tables 5, 6, and 7, respectively.

[0123] Table 5 Water bath heat resistance test

[0124] Table 6 Stability test of feed pelleting

[0125] Table 7 In vitro enzymatic hydrolysis effect

[0126] The sequence involved in the present invention is as follows:

[0127] Fusion peptide amino acid sequence 1, SEQ ID NO: 1: SRTKTHLRRGSE

[0128] Fusion peptide amino acid sequence 2, SEQ ID NO: 2:

[0129] The amino acid sequence of xylanase Xyn-CDBFV is SEQ ID NO: 3:

[0130] The amino acid sequence of xylanase Xyn-307 is SEQ ID NO: 4:

[0131] The amino acid sequence of xylanase Xyn-309 is SEQ ID NO: 5:

[0132] The nucleotide sequence of xylanase Xyn-307 is SEQ ID NO: 6:

[0133] The nucleotide sequence of xylanase Xyn-309 is SEQ ID NO: 7:

[0134] Aspergillus niger saccharifying enzyme gene gla promoter nucleotide sequence SEQ ID NO: 8:

[0135] Aspergillus niger saccharifying enzyme gene gla terminator nucleotide sequence SEQ ID NO: 9:

[0136] Aspergillus niger amylase signal peptide nucleotide sequence SEQ ID NO: 10:

[0137] The nucleotide sequence of the selectable marker amdS is SEQ ID NO: 11:

[0138] ***

[0139] Although the above content refers to a particular preferred embodiment, it should be understood that the present invention is not limited thereto. Those skilled in the art will recognize that various modifications can be made to the disclosed embodiments, and these modifications are also intended to fall within the scope of the present invention.

[0140] All publications, patent applications, and patents mentioned in this specification are incorporated herein by reference in their entirety.

Claims

1. A xylanase comprising a xylanase Xyn-CDBFV sequence and a fusion peptide segment connected to its C-terminus, wherein the fusion peptide segment comprises the amino acid sequence shown in SEQ ID NO:

1. 2 . The xylanase according to claim 1 , wherein the fusion peptide segment further comprises the amino acid sequence shown in SEQ ID NO:

2. 3 .

3. The xylanase according to claim 1 or 2, wherein the fusion peptide segment consists of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO:

2. The xylanase according to claim 1 , wherein the xylanase Xyn-CDBFV sequence is shown as SEQ ID NO:

3.

5. The xylanase according to claim 1, comprising the amino acid sequence shown in SEQ ID NO:

4.

6. The xylanase according to claim 1 or 2, comprising the amino acid sequence shown in SEQ ID NO:

5.

7. The xylanase according to claim 1, which consists of the amino acid sequence shown in SEQ ID NO:

4.

8. The xylanase according to claim 1 or 2, which consists of the amino acid sequence shown in SEQ ID NO:

5.

9. A polynucleotide encoding the xylanase of claim 1. 10 . The polynucleotide according to claim 9 , comprising the nucleotide sequence shown in SEQ ID NO:

6. The polynucleotide according to claim 9 , comprising the nucleotide sequence shown in SEQ ID NO:

7.

12. The polynucleotide according to claim 9, which consists of the nucleotide sequence shown in SEQ ID NO:

6. The polynucleotide according to claim 9, which consists of the nucleotide sequence shown in SEQ ID NO:

7.

14. A recombinant vector comprising the polynucleotide according to any one of claims 9 to 13.

15. A host cell comprising the polynucleotide according to any one of claims 9 to 13 or the recombinant vector according to claim 14. The host cell according to claim 15 , wherein the host cell is Aspergillus niger or Pichia pastoris. The host cell according to claim 16 , which is Aspergillus niger.

18. A method for preparing xylanase, comprising connecting a fusion peptide segment to the C-terminus of a xylanase Xyn-CDBFV sequence, wherein the fusion peptide segment comprises the amino acid sequence shown in SEQ ID NO:

1.

19. The method according to claim 18, wherein the fusion peptide segment further comprises an amino acid sequence as shown in SEQ ID NO:

2.

20. The method according to claim 18 or 19, wherein the fusion peptide segment consists of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO:

2.

21. The method according to claim 18, wherein the xylanase Xyn-CDBFV sequence is shown as SEQ ID NO:

3.

22. A method for preparing an Aspergillus niger strain producing xylanase, comprising introducing the recombinant vector of claim 14 into the Aspergillus niger strain.

23. The method according to claim 22, wherein the recombinant vector comprises an Aspergillus niger expression cassette sequence, and the Aspergillus niger expression cassette sequence comprises an Aspergillus niger saccharifying enzyme gene gla promoter sequence, an Aspergillus niger amylase signal peptide nucleotide sequence, a xylanase nucleotide sequence as shown in SEQ ID NO: 6 or 7, and an Aspergillus niger saccharifying enzyme gene gla terminator sequence.

24. The method according to claim 22 or 23, comprising screening the Aspergillus niger strain using a selection marker amdS plate after the introduction of the recombinant vector.

25. Use of the xylanase according to any one of claims 1 to 8 in feed.

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