Xylanase mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a xylanase mutant as well as a preparation method and application thereof. Compared with parent xylanase with an amino acid sequence as shown in SEQ ID NO: 1, the xylanase mutant has sequence identity of not less than 99%; and the compound contains at least one of the following substitutions: N24S, D113G and E251G. Compared with parent xylanase, the xylanase mutant provided by the invention has higher specific activity, has higher relative enzyme activity at the optimal reaction temperature and reaction pH, and is beneficial to industrial application.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a xylanase mutant, its preparation method, and its application. Background Technology

[0002] Xylan is one of the main components of hemicellulose in plant cell walls and is the second largest polysaccharide in nature after cellulose, accounting for about one-third of all renewable organic carbon sources on Earth. The xylan backbone consists of β-D-xylopyranosyl residues linked by β-1,4-glycosyl bonds, which can be substituted to varying degrees by sugars and organic acids, such as glucuronylpyranosyl, 4-O-methyl-D-glucuronylpyranosyl, α-L-arabinofuranosyl, acetyl, ferulic acid, and / or p-coumaryl glycoside groups. Therefore, xylan biodegradation requires the synergistic action of several xylanases. Xylanase, also known as endo-1,4-β-xylanase (EC 3.2.1.8), belongs to the glycoside hydrolase (GH) class and plays a crucial role in catalyzing the breakdown of the β-1-4-linked xylan backbone into xylose.

[0003] Xylanase has a wide range of industrial and biotechnological applications, such as saccharification in the paper and pulp industry, textile industry, biofuel industry, organic waste treatment, food and feed industry, and pharmaceutical industry. Some industrial processes are typically carried out at high temperatures and extreme pH levels; for example, pulp bleaching involves hot water, steam explosion, and alkali pretreatment before enzyme treatment. Using high-temperature alkaline xylanase allows for direct enzymatic treatment, avoiding additional temperature and pH readjustments. This not only saves costs and time but also efficiently reduces the use of chemical bleaching agents in pulp production, improves pulp brightness, and enhances the quality of finished paper. Furthermore, xylanase exhibits activity and stability at alkaline pH levels, and has potential applications in the management of xylan-containing waste in industrial, agricultural, and municipal waste.

[0004] Most alkaline xylanases currently on the market that meet application requirements suffer from low enzyme activity, high addition costs, and difficulty in practical production. Natural alkaline xylanases are mainly derived from bacteria, with a small amount from fungi, but the specific activities of these xylanases are typically low. Therefore, developing alkaline xylanases with high specific activity to reduce production costs is of great significance. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a xylanase mutant, its preparation method, and its applications. The xylanase mutant provided by this invention exhibits higher specific activity under high-temperature alkaline conditions, which is beneficial for industrial applications.

[0006] This invention provides a xylanase mutant.

[0007] Specifically, a xylanase mutant has at least 99% sequence identity with the parent xylanase (TmxB) with an amino acid sequence as shown in SEQ ID NO: 1; and contains at least one of the following substitutions: N24S, D113G, E251G.

[0008] SEQ ID NO.1:SQNVSLRELAEKLNIYIGFAAINNFWSLSDAEKYMEVAR REFNILTPENQMKWDTIHPERDRYNFTPAEKHVEFAEENDMIVHGHTLVWHNQLPGWITGREWTKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDSGTYRESVWYKTIGPEYIEKAFRWAKEADPDAILIYNDYSIEEIN AKSNFVYNMIKELKEKGVPVDGIGFQMHIDYRGLNYDSFRRNLERFAKLGLQIYITEMDVRIPLSGSEEYYLKKQAEVCAKIFDICLDNPAVKAIQFWGFTDKYSWVPGFFKGYGKALLFDENYNPKPCYYAIKEVLEKKIEER.

[0009] In some embodiments of the present invention, the xylanase mutant, relative to the parental xylanase (TmxB) with the amino acid sequence as shown in SEQ ID NO: 1, comprises the following substitutions: N24S, D113G, E251G, or N24S / D113G / E251G.

[0010] The meaning of N24S / D113G / E251G is that it simultaneously includes three substitutions: N24S, D113G, and E251G.

[0011] In some embodiments of the present invention, the parental xylanase is derived from the thermophilic bacterium Thermotoga maritima MSB8.

[0012] The present invention also provides a nucleic acid molecule comprising the nucleotide fragments shown in (a) and / or (b):

[0013] (a) The nucleotide fragment encoding the xylanase mutant described above;

[0014] (b) A nucleotide fragment that is the reverse complementary to (a).

[0015] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0016] The present invention also provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0017] Preferably, the recombinant cells comprise bacterial or fungal cells.

[0018] More preferably, the fungal cell is a yeast cell.

[0019] This invention also provides a method for preparing the above-mentioned xylanase mutant, comprising the following steps:

[0020] (a) Constructing a recombinant expression vector containing a gene encoding the xylanase mutant of the present invention;

[0021] (b) The recombinant expression vector was introduced into recombinant cells;

[0022] (c) Inducing recombinant cells containing the recombinant expression vector to express xylanase.

[0023] This invention also provides the application of the above-mentioned xylanase mutant in degrading xylan under high temperature and alkaline conditions.

[0024] In some embodiments, the xylanase mutant of the present invention can be applied in industries such as textiles, food, feed, and papermaking.

[0025] Specifically, the above-mentioned xylanase mutant is applied in papermaking. For example, applying the xylanase mutant to the pulp bleaching process can not only significantly reduce the amount of chloride used, thus mitigating the environmental pollution caused by the papermaking industry, but also help improve paper quality, such as increasing paper whiteness and strength.

[0026] Specifically, the application of the above-mentioned xylanase mutant in waste paper deinking.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The xylanase mutant provided by this invention has a higher specific activity than the parent xylanase, and a higher relative enzyme activity at the optimal reaction temperature and pH, which is beneficial for industrial applications. Moreover, the optimal reaction temperature is as high as 90℃, which is higher than that of the parent xylanase, making it more conducive to high-temperature processing environments. The xylanase mutant provided by this invention can be directly used for enzyme treatment, avoiding additional temperature and pH adjustments, and saving production costs and time.

[0029] (2) The xylanase mutant of the present invention is particularly advantageous for application in the papermaking industry. During the pulp bleaching process, the xylanase mutant of the present invention can adapt to high temperatures without the need for additional temperature adjustment. Detailed Implementation

[0030] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0031] Unless otherwise specified, the biological materials, reagents, or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0032] One embodiment of this invention provides a xylanase mutant, which is based on the xylanase (TmxB) gene (GenBank: MT032174.1) of the thermophilic bacterium *Thermotoga maritima* MSB8. After multiple mutations, combinatorial mutations, and high-throughput screening, a xylanase mutant with improved thermostability was obtained through expression (the amino acid sequence of the corresponding parental xylanase is shown in SEQ ID NO. 1). The xylanase mutant of this invention possesses xylanase activity, including but not limited to hydrolytic enzyme activity, such as the ability to hydrolyze glycosidic bonds present in xylan, such as catalyzing the hydrolysis of intra-β-1,4-xylosidic bonds; it has higher enzyme activity than the parental xylanase shown in SEQ ID NO. 1; and it also possesses better thermostability (higher optimal reaction temperature) than the parental xylanase shown in SEQ ID NO. 1.

[0033] SEQ ID NO.1: SQNVSLRELAEKLNIYIGFAAINNFWSLSDAEKYMEVARREFNILTPENQMKWDTIHPERDRYNFTPAEKHVEFAEENDMIVHGHTLVWHNQLPGWITGREWTKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDSGTYRESVWYKTIGPEYIEKAFR WAKEADPDAILIYNDYSIEEINAKSNFVYNMIKELKEKGVPVDGIGFQMHIDYRGLNYDSFRRNLERFAKLGLQIYITEMDVRIPLSGSEEYYLKKQAEVCAKIFDICLDNPAVKAIQFWGFTDKYSWVPGFFKGYGKALLFDENYNPKPCYYAIKEVLEKKIEER

[0034] The xylanase mutant of this invention comprises an amino acid sequence having at least 99% and less than 100% sequence identity with the parental xylanase of SEQ ID NO:1, obtained by substituting one or more amino acids(s). For the purposes of this invention, sequence identity between two amino acid sequences is determined using the Needle algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later). The parameters used are a vacancy open penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) replacement matrix. The output of Nieder, labeled "Longest Identity" (obtained using the non-simplification option), is used as the identity percentage and calculated as follows:

[0035] (identical residues × 100) / (alignment length - total number of vacancies in alignment).

[0036] Introducing a vector containing a polynucleotide sequence encoding a xylanase mutant into recombinant cells and inducing the recombinant cells to express the xylanase mutant can be used in the processing of food, pharmaceuticals, feed, nutritional additives, textiles, detergents, and paper products.

[0037] This invention also provides a method for preparing a xylanase mutant, comprising the following steps:

[0038] (a) Constructing a recombinant expression vector containing a gene encoding the xylanase mutant of the present invention;

[0039] (b) The recombinant expression vector was introduced into recombinant cells;

[0040] (c) Inducing recombinant cells containing the recombinant expression vector to express xylanase.

[0041] This invention also provides a method for producing xylanase with improved heat resistance, specifically including the following steps:

[0042] The recombinant strain was cultured under suitable conditions to produce a xylanase mutant;

[0043] Purify the xylanase mutant;

[0044] And optionally, the xylanase mutant is processed.

[0045] If the xylanase mutant is secreted into the nutrient medium, it can be recovered directly from the medium. If the expressed xylanase mutant is not secreted into the nutrient medium, it can be recovered from the cell lysate.

[0046] Xylanase mutants can be expressed in a variety of expression systems, and correspondingly, appropriate downstream processing and purification steps must be selected. In some embodiments of the invention, xylanase mutants can be expressed in bacterial hosts, and the protein is secreted into the periplasm or extracellular space. The expression organism is cultured according to standard fermentation methods in appropriate volumes. In a preferred embodiment, cells are grown in a fermenter, and growth conditions such as pH, temperature, oxygen, and / or nutrient supply are optionally controlled. The first step of purification involves separating the cells from the supernatant using one or more of several techniques such as sedimentation, microfiltration, centrifugation, or flocculation. In a preferred embodiment, microfiltration is a suitable method. If expressed intracellularly, the cells are treated to release the protein from the intracellular space. These treatments may include pressurization, enzymatic stimulation, osmotic shock, freezing, sonication, or other treatments to produce a cell extract, which may or may not be further purified.

[0047] In some preferred methods, the affinity-labeled protein is purified by affinity chromatography with a metal chelating agent to obtain a high-purity target protein. In other preferred embodiments, the high-purity target protein is obtained by HPLC purification.

[0048] In a further embodiment of the present invention, the fermentation cell suspension containing the expressed xylanase mutant is dried as a whole using methods including but not limited to fluidized bed drying, conveyor drying, spray drying, or drum drying or any combination thereof.

[0049] The xylanase mutants provided in this invention can be used for a variety of industrial applications. In particular, the xylanase mutants of this invention are suitable for applications involving the degradation of xylan under high-temperature alkaline conditions.

[0050] In some embodiments, the xylanase mutant of the present invention can be applied in industries such as textiles, food, feed, and papermaking.

[0051] In one aspect, the xylanase mutant provided by this invention, when used in the pulp bleaching process, can significantly reduce the amount of chloride used, thereby mitigating environmental pollution from the paper industry. The xylanase mutant provided by this invention also helps improve paper quality, such as increasing paper whiteness and strength. In another aspect, the xylanase mutant provided by this invention is used in waste paper deinking.

[0052] The following describes a more specific embodiment.

[0053] The following are the main experimental materials, reagents, and methods used in the examples:

[0054] 1. Strains and vectors: Strains containing xylanase (TmxB) gene and expression plasmid, Escherichia coli strain Top10, Pichia pastoris strain X33, and pPICZαA vector were all purchased from Invitrogen.

[0055] 2. Enzymes and kits: Ultra-fidelity 2×Master Mix PCR polymerase, restriction endonucleases, etc. were purchased from NEB, plasmid extraction kit and purification kit were purchased from Beijing Tiangen Company, and PCR amplification primers were synthesized from Shanghai Sangon Biotech Co., Ltd.

[0056] 3. Culture medium

[0057] The culture medium for *E. coli* was LB medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0). LB+Amp medium consisted of LB medium with ampicillin added to a final concentration of 100 μg / mL, and LB+Zeo medium consisted of LB medium with Zeocin added to a final concentration of 25 μg / mL.

[0058] The yeast culture medium was YPD medium (1% yeast extract, 2% peptone, 2% glucose). The yeast selection medium was YPD+Zeo medium (YPD+Zeo medium is YPD medium with Zeocin added to a final concentration of 100 μg / mL);

[0059] Yeast-induced expression of BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v / v)) and BMMY (except that 0.5% methanol was used instead of glycerol, the other components were the same as BMGY).

[0060] 4. Chemical reagents:

[0061] Xylanase standard and xylan were purchased from Sigma-Aldrich, and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0062] 5. Method for determining xylanase activity

[0063] Take 2.00 mL of diluted enzyme solution, add 2.0 mL of 100 mg / mL xylan solution, and incubate at 85 °C for 30 min. Add 5 mL of DNS reagent to terminate the enzymatic reaction. Heat in a boiling water bath for 5 min. Cool to room temperature with tap water, add water to a final volume of 25 mL, and measure the absorbance at 540 nm. Replace the crude enzyme solution with an equal volume of buffer solution as a blank control; all other procedures are the same. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from a 5 mg / mL xylan solution at 85 °C and pH 8.5.

[0064] Example 1: Synthesis of xylanase (TmxB) gene and construction of vector

[0065] The xylanase (TmxB) gene (GenBank: MT032174.1) of the extreme thermophilic bacterium Thermotoga maritima MSB8 has the amino acid sequence shown in SEQ ID NO:1.

[0066] EcoRI and XbaI restriction sites were introduced at the 5' and 3' ends of the xylanase TmxB gene and ligated into the Puc57-amp vector. The vector was transformed into E. coli TOP10 competent cells to obtain positive clones of Puc57-TmxB strain. The clones were then inoculated into LB+Amp medium and cultured for 18 hours. The plasmid was extracted, digested with EcoRI and XbaI, and the target gene fragment was purified by gel extraction and ligated into the expression vector pPICzαA to obtain the expression vector pPICzαA-TmxB.

[0067] Example 2: Error-prone PCR random mutation

[0068] Using pPICzαA-TmxB as a template, a mutation was randomly introduced into the xylanase TmxB gene using a fault-prone PCR method. The fault-prone PCR primers are as follows:

[0069] TmxB-F: 5'-gaaaagagaggctgaagctgaattc-3' (SEQ ID NO: 2);

[0070] TmxB-R: 5'-gcggccgccagctttctagaacaaa-3' (SEQ ID NO: 3).

[0071] PCR amplification products were digested with EcoRI and XbaI, and the size of the PCR bands was verified by 1% agarose gel electrophoresis. The target PCR product was purified and recovered. The template was digested with the restriction endonuclease DpnI and ligated into the expression vector pPICzαA, which had been digested with EcoRI and XbaI. The vector was then transformed into *E. coli* Top10 competent cells using the heat shock method. The transformed cells were plated on LB+Zeo plates, and recombinant transformants were picked for PCR validation. Sequencing was used to verify the mutation rate of error-prone PCR. The recombinant transformants grown on the plates were washed off, and the plasmid was extracted. The plasmid was linearized with PmeI, and the linearized fragment was recovered and purified. The fragment was then transformed into *Pichia pastoris* X33 competent cells by electroporation, plated on YPD+Zeo plates, and selected to obtain yeast recombinant transformants.

[0072] Example 3: High-throughput screening of high specific activity mutant strains

[0073] Yeast recombinant transformants obtained in Example 2 were transferred to 24-well plates containing 2 mL of BMGY medium and cultured at 30°C with shaking at 250 rpm for 24 h. The supernatant was then removed by centrifugation. 2 mL of BMGY medium was added to each well, and the plates were cultured at 30°C with shaking at 250 rpm, with 0.5% methanol added daily for induction. After 72 h of culture, the supernatant was collected by centrifugation for xylanase activity assay. The relative specific activity of the mutant was calculated by dividing the mutant activity by the parental xylanase activity. Yeast recombinant transformants with increased specific activity were streaked onto plates. Five single colonies from each transformant were selected and screened repeatedly using the above steps to confirm the high specific activity of the yeast recombinant transformants. The genome was extracted, and the mutant sequence of the target gene was determined to obtain the high specific activity xylanase mutant.

[0074] Table 1. Relative activity of parental xylanase TmxB and mutant.

[0075]

[0076]

[0077] As shown in Table 1, xylanase mutants of single-point mutant strains N24S, D113G and E251G were obtained through screening. Compared with the parent xylanase TmxB of wild-type TmxB, their relative specific activities were increased by 41.5%, 75.8% and 68.2%, respectively.

[0078] Example 4: Combinatorial Mutation and Screening

[0079] The high-specific-activity xylanase mutants obtained in Example 3 were subjected to two-site or multi-site combined mutations, and the high-throughput method described in Example 3 was used for screening. Through multiple rounds of combined mutations and screening, the optimal combination of high-specific-activity xylanase mutants was finally selected. The relative specific activity of the combined mutants was calculated by dividing the mutant enzyme activity by the parent xylanase activity.

[0080] Table 2. Relative activity of parental xylanase TmxB and combined mutant.

[0081] name Compared to living Wild-type TmxB 100.0％ N24S / D113G / E251G 276.0％

[0082] As shown in Table 2, the three-point mutant combination N24S / D113G / E251G xylanase mutant obtained through screening showed a relative activity increase of 176.0% compared to the wild-type parent xylanase TmxB.

[0083] Example 5: Optimal reaction pH of parental xylanase TmxB and its mutant

[0084] The xylanase activity was measured at 85℃ and at pH 5.0, 6.0, 7.0, 8.0, 8.5, 9.0, and 10.0, and the results are shown in Table 3. The xylanase activity measured at pH 6.0 was taken as 100%, and the relative enzyme activity of each enzyme under different pH conditions was calculated.

[0085] Table 3

[0086]

[0087]

[0088] As shown in Table 3, the optimal reaction pH for the xylanase mutant was pH 6.0, and the relative enzyme activity under different pH conditions was significantly higher than that of the parent xylanase TmxB.

[0089] Example 6: Optimal reaction temperature of parental xylanase TmxB and its mutant

[0090] The xylanase activity was measured at pH 8.5 and at 60℃, 70℃, 80℃, 85℃, and 90℃, and the results are shown in Table 4. The xylanase activity measured at the optimum temperature was taken as 100%, and the relative enzyme activity of each enzyme under different temperature conditions was calculated.

[0091] Table 4

[0092]

[0093] Table 4 shows that the optimal reaction temperatures of the xylanase point mutants N24S and E251G are similar to those of the parent xylanase TmxB, both at 85℃. However, the optimal reaction temperature of the mutant combination N24S / D113G / E251G and the point mutant D113G is 90℃, which is higher. Furthermore, the relative enzyme activities of mutants N24S and E251G at 90℃ are also higher than those of the parent xylanase TmxB. Therefore, the xylanase mutants of this invention are more suitable for high-temperature processing environments, avoiding additional temperature and pH adjustments, and saving production costs and time.

[0094] Example 7: Application of parental xylanase TmxB and its mutant in paper bleaching

[0095] Parental xylanase TmxB and mutant samples were diluted to the same enzyme activity and added at 500 ppm to 10% pulp. Enzyme treatment was performed at pH 8.5 and 85℃ for 2 hours. Subsequently, magnesium sulfate 0.2%, EDTA 0.3%, sodium hydroxide 1.5%, and hydrogen peroxide 3% were added sequentially, and the reaction was carried out at 70℃ for 2 hours. Papermaking was then performed, and the paper whiteness was measured after drying. The results are shown in Table 5. The whiteness improvement data is the difference between the whiteness of the experimental sample and the blank sample (without enzyme addition); the larger the difference, the higher the paper whiteness.

[0096] Table 5

[0097]

[0098] As shown in Table 5, the whiteness improvement effect of the three single-point mutants of xylanase is better than that of the wild-type TmxB mutant. The three-point combination mutant N24S / D113G / E251G has a better whiteness improvement effect, which is 2.1 times that of the wild type.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A xylanase mutant, characterized in that The amino acid sequence of the parent xylanase is not less than 99% identical to that of SEQ ID NO: 1, and the xylanase comprises at least one of the following substitutions: N24S, D113G, and E251G.

2. The xylanase mutant according to claim 1, characterized in that The parent xylanase is derived from the thermophilic bacterium Thermotoga maritima MSB8.

3. A nucleic acid molecule, characterized in that Containing the nucleotide fragments shown in (a) and / or (b): (a) a nucleotide fragment encoding the xylanase mutant according to claim 1 or 2; (b) A nucleotide fragment that is the reverse complement of (a).

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that The recombinant cell comprises the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4.

6. The recombinant cell according to claim 5, characterized in that The recombinant cells include bacterial or fungal cells.

7. The recombinant cell according to claim 6, characterized in that The fungal cells are yeast cells.

8. The method for preparing the xylanase mutant according to claim 1 or 2, characterized in that: The following steps are involved: (a) constructing a recombinant expression vector comprising a gene encoding the xylanase mutant according to claim 1 or 2; (b) introducing the recombinant expression vector into a recombinant cell; (c) inducing the recombinant cell comprising the recombinant expression vector to express xylanase.

9. Use of the xylanase mutant according to claim 1 or 2 in degrading xylan under a high temperature alkaline environment.

10. Use of the xylanase mutant according to claim 1 or 2 in textile, food, feed and papermaking.

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