CN111690631B

CN111690631B - Endo-xylanase mutant S23B01, and preparation method and application thereof

Info

Publication number: CN111690631B
Application number: CN202010675785.8A
Authority: CN
Inventors: 张蕊; 周峻沛; 黄遵锡; 柳微; 胡家乐; 沈骥冬; 吴倩; 慕跃林
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2021-02-23
Anticipated expiration: 2040-07-14
Also published as: CN111690631A

Abstract

The invention discloses an endo-xylanase mutant S23B01, a preparation method and application thereof, wherein the mutant S23B01 has an amino acid sequence shown as SEQ ID NO.1, the optimum pH value is 5.5, and the optimum temperature is 65 ℃. FeSO at 10.0mM for the mutant endo-xylanase S23B01 compared to the wild enzyme₄Activity in 5.0 to 25.0% (w/v) NaCl, activity in 3.0 to 30.0% (w/v) KCl, Na in 15.0 to 30.0% (w/v)₂SO₄The activity of (5) is improved. Mutations of the inventionThe endo-xylanase S23B01 can be applied to the biotechnology fields of soy sauce fermentation, food processing in high-salt environment and the like.

Description

Endo-xylanase mutant S23B01, and preparation method and application thereof

Technical Field

The invention relates to an endo-xylanase mutant, and in particular relates to an endo-xylanase mutant S23B01, and a preparation method and application thereof.

Background

Hemicellulose is a renewable resource second only to the cellulose content in nature and accounts for about 30-35% of the dry cell weight. Hemicellulose consists of heteropolysaccharides, xylan is the most abundant polysaccharide in hemicellulose, and is abundant in hardwoods, softwoods, and annual plants. Endo-xylanase, xylanase for short, capable of randomly cleaving the beta-1, 4-glycosidic bond of the backbone of the xylan backbone to produce xylo-oligosaccharides and/or xylose. According to the classification of the Cazy database, the families with endoxylanase activity mainly include

families

5, 7, 8, 10, 11, 26, 30 and 43. Among them, most of the reported xylanases belong to families 10 and 11 (Subramaniayanyan et al.Critical Reviews in Biotechnology,2002,22: 33.).

The xylanase with good resistance to different salts can be suitable for wider biotechnology fields, such as soy sauce fermentation, food processing in high-salt environment and the like; the food is processed or fermented in a high-salt environment, so that the pollution of microorganisms can be prevented, and energy consumed by sterilization and the like can be saved; the preservation time of the enzyme preparation can also be extended in a high salt environment (Warden and Williams, Nature Communications,2015,6: 10278.). The salt concentration in the catalytic environment is high and the salt species are different in different biotechnology fields,for example, the NaCl concentration is high in the soy sauce fermentation process, and a large amount of Na is needed in the paper-making pulping process₂SO₄. Therefore, improving the resistance of xylanase to high concentrations of various salts facilitates its application in biotechnology.

Disclosure of Invention

The invention aims to provide an endoxylanase mutant S23B01, and a preparation method and application thereof, wherein the mutant S23B01 is used for treating NaCl, KCl and Na₂SO₄Has tolerance and higher enzyme activity than that of a wild enzyme rXynAGN16L, and can be applied to the biotechnology fields of soy sauce fermentation, food processing in a high-salt environment and the like.

In order to achieve the aim, the invention provides an endoxylanase mutant S23B01, wherein the mutant S23B01 has an amino acid sequence shown as SEQ ID NO. 1.

The invention also aims to provide a coding gene of the endoxylanase mutant S23B 01.

Preferably, the coding gene s23b01 has a nucleotide sequence shown as SEQ ID No. 2.

Another objective of the invention is to provide a recombinant vector containing the coding gene s23b 01.

Preferably, the recombinant vector adopts an expression vector pEasy-E2.

Another object of the present invention is to provide a recombinant bacterium comprising the coding gene s23b 01.

Preferably, the bacterium adopted by the recombinant bacterium is Escherichia coli BL21-Gold (DE 3).

Another objective of the invention is to provide a preparation method of the endoxylanase mutant S23B01, which comprises the following steps:

connecting the coding gene s23b01 with an expression vector pEasy-E2, and transforming the connection product into escherichia coli BL21-Gold (DE3) to obtain a recombinant strain containing the coding gene s23b 01; and culturing the recombinant strain, and inducing the expression of the endoxylanase mutant S23B 01.

Preferably, the recombinant strain is cultured in a medium containing 100. mu.g mL of the recombinant strain^-1The LB culture solution of Amp is used as a culture medium,shaking culture, when OD₆₀₀When the concentration is 0.6-1.0, adding IPTG for induction.

The invention also aims to provide application of the endoxylanase mutant S23B01 in the field of food.

The endo-xylanase mutant S23B01, the preparation method and the application thereof have the following advantages:

the salt adaptation of the mutant endo-xylanase S23B01 of the invention was altered compared to the wild-type enzyme. The optimum pH values of the purified mutant enzyme S23B01, the wild enzymes rXynAGN16L and rXynAHJ3 were 5.5, 5.5 and 6.0, respectively, and the optimum temperatures were 65 ℃, 50 ℃ and 75 ℃, respectively. FeSO at 10.0mM₄In the method, the enzyme activity of the mutant S23B01 is respectively 18% higher than that of the wild enzyme rXynAGN16L and 7% higher than that of the wild enzyme rXynAHJ 3; in 5.0-25.0% (w/v) NaCl, the enzyme activity of the mutant S23B01 is respectively 12-26% higher than that of the wild enzyme rXynAGN16L and 20-26% higher than that of rXynAHJ 3; in KCl of 3.0-30.0% (w/v), the enzyme activity of the mutant S23B01 is respectively 13-41% higher than that of a wild enzyme rXynAGN16L and 1-18% higher than that of rXynAHJ 3; na in the range of 15.0-30.0% (w/v)₂SO₄In the method, the enzyme activity of the mutant S23B01 is 5-21% higher than that of a wild enzyme rXynAGN16L, and 10-12% higher than that of rXynAHJ 3.

The mutant endo-xylanase S23B01 can be applied to the biotechnology fields of soy sauce fermentation, food processing in high-salt environment and the like.

Drawings

FIG. 1 is SDS-PAGE analysis (CK: protein Marker) of recombinant endoxylanases rXynAGN16L, rXynAHJ3 and its mutant S23B01 expressed in E.coli.

FIG. 2 shows the activity of recombinant endoxylanase rXynAGN16L, rXynAHJ3 and its mutant S23B01 in NaCl.

FIG. 3 shows the activity of recombinant endoxylanase rXynAGN16L, rXynAHJ3 and its mutant S23B01 in KCl.

FIG. 4 shows recombinant endoxylanase rXynAGN16L, rXynAHJ3 and its mutant S23B01 in Na₂SO₄Activity in (c).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The materials and reagents adopted in the experiment of the invention are as follows:

bacterial strain and carrier: escherichia coli BL21-Gold (DE3) and expression vector pEasy-E2 were purchased from Beijing Quanyujin Biotechnology Ltd; arthrobacter (Arthrobacter sp.) and varelia sericata (Lechevalieria sp.) are offered by university of mazechu in Yunnan.

Enzymes and other biochemical reagents: DNA polymerase and dNTP were purchased from Beijing Quanjin Biotechnology Ltd, beech xylan was purchased from Sigma, corncob xylan was purchased from Shanghai leaf Biotechnology Ltd, error-prone PCR kit was purchased from Beijing Tianenzur Gene technology Ltd, bacterial genome extraction kit was purchased from GENE STAR, PopCulture^TMCell lysates were purchased from Merck group, Inc., Germany, and were all made in the home (all available from general Biochemical Co., Ltd.).

Culture medium: adopts an LB culture medium, and comprises the following components: peptone (Peptone)10g, Yeast extract (Yeast extract)5g, and NaCl 10g, with distilled water to 1000mL, pH natural (about 7). On the basis of the solid medium, 2.0% (w/v) agar was added.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Experimental example 1 construction of mutant library

The construction process of the mutation library is specifically as follows:

(1) extracting Arthrobacter (Arthrobacter sp.) and Verticillium lecanii (Lechevalieria sp.) genomes according to the instruction of a bacterial genome extraction kit of GENE STAR company;

(2) designing primers 5'-GTGCAGCCGGAGGAAAAACG-3' (SEQ ID No.5) and 5'-GATGAAGGCAGGATCCGGGGT-3' (SEQ ID No.6) according to the nucleotide sequence JQ863105(SEQ ID No.3) of the Arthrobacter (Arthrobacter sp.) endoxylanase recorded by GenBank, and carrying out PCR amplification by using the genome of the Arthrobacter (Arthrobacter sp.) as a template to obtain an endoxylanase gene xyNAGN 16L; in addition, according to the nucleotide sequence JF745868(SEQ ID No.4) of endoxylanase of the strain Variella denticulata (Lechevalieria sp.) recorded by GenBank, primers 5'-GTCTCGGCCCCGCCGGACGT-3' (SEQ ID No.7) and 5'-GGCTCGCTTCGCCAGCGTGG-3' (SEQ ID No.8) are designed, and the genome of the strain Variella denticulata (Lechevalieria sp.) is used as a template for PCR amplification to obtain the endoxylanase gene xynAHJ 3; the reaction parameters for PCR amplification were: denaturation at 94 deg.C for 5 min; then denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min for 30sec, and heat preservation at 72 ℃ for 10min after 30 cycles;

(3) performing gene mutation by using the PCR product as a template and using an error-prone PCR kit according to a kit instruction;

(4) performing ultrasonic random interruption on the error-prone PCR product by using an ultrasonic interruption instrument Biorupt, and cutting and purifying the interrupted product after 2% agarose gel electrophoresis;

(5) the purified small fragment DNA itself is used as a primer and a template to carry out DNA Family shuffling (DNA Family shuffling) PCR, and the PCR reaction parameters are as follows: denaturation at 96 deg.C for 1min 30 sec; then, denaturation at 94 ℃ for 30sec, annealing at 65 ℃ for 90sec, annealing at 62 ℃ for 90sec, annealing at 59 ℃ for 90sec, annealing at 56 ℃ for 90sec, annealing at 53 ℃ for 90sec, annealing at 50 ℃ for 90sec, annealing at 47 ℃ for 90sec, annealing at 44 ℃ for 90sec, annealing at 41 ℃ for 90sec, elongation at 72 ℃ for 1min for 30sec, and heat-retention at 72 ℃ for 7min after 35 cycles;

(6) taking a purified DNA family reorganization PCR product as a template, carrying out sequence full-length amplification by using amplification primers of endoxylanase genes xynAHJ3 and xynAGN16L and reaction conditions, wherein the amplification product contains a mutation sequence and an unmutation sequence;

(7) connecting the full-length amplification product of the sequence with an expression vector pEasy-E2, and connecting the connection productTransformed E.coli BL21-Gold (DE3) was cultured overnight, and a single colony was picked from the transformed plate and cultured in a medium containing 150. mu.L of liquid LB medium (containing 100. mu.g mL of each solution)^-1Amp, Amp is ampicillin) in a 96-well cell culture plate, quickly shaking and culturing at 37 ℃ for about 16h, adding 50 mu L of 40% (w/w) glycerol into each well, mixing uniformly, and storing at-70 ℃.

Example 2 screening of mutants

The screening process of the mutant is as follows:

(1) mu.L of the bacterial suspension was collected from a 96-well cell culture plate of the stored mutant library, and inoculated into LB medium (containing 100. mu.g mL) containing 200. mu.L/well of the liquid^-1Amp) in a 96 deep well plate, cultured at 37 ℃ and 200rpm with shaking to OD₆₀₀>1.0 (about 20h), add 2mM IPTG and 100. mu.g mL^-1Amp 200 u L liquid LB culture solution, at 20 degrees C, 160rpm overnight induction;

(2) after induction, 40. mu.L/well of PopCulture was added^TMCell lysate was used to lyse cells at 25 ℃ for 30min with shaking.

(3) 50 μ L of McIlvaine buffer (pH 7.0) containing 1.0% (w/v) beech xylan and 50 μ L of cell lysate were reacted in a 96-well plate at 70 ℃ for 2 h. Adding 150 microliter DNS reagent to terminate the reaction after the reaction is finished, preserving the heat in a thermostat at 140 ℃ for more than 20min, cooling to room temperature, and reading OD by using a microplate reader_540nmThe value of (a) was compared with a lysate reaction group of E.coli BL21-Gold (DE3) strain containing only empty vector pEASY-E2;

(4) taking 10 mu L of mutant cell lysate with endoxylanase activity, taking 90 mu L of McIlvaine buffer solution (pH 7.0) containing 0.5% (w/v) beech xylan, adding 10% (w/v) and 25% (w/v) NaCl, and reacting in a 96-deep-hole plate at 70 ℃ in an incubator for 10 min;

(5) adding 150 mu LDNS reagent to terminate the reaction after the reaction is finished, preserving the heat in a thermostat at 140 ℃ for more than 20min, cooling to room temperature, and reading OD by using a microplate reader_540nmThe corresponding mutant lysate reaction group without NaCl was used as a control;

(6) comparing the enzyme activity of the mutant with that of wild recombinant enzymes rXynAGN16L and rXynAHJ3 to obtain 1 mutant with the serial number of S23B01, wherein the 1 mutant has the enzyme activity improved in 10% (w/v) and 25% (w/v) NaCl, the amino acid sequence of the mutant is shown as SEQ ID No.1 and is a shuffling heterozygote of two wild enzymes, and the nucleotide sequence of the mutant is shown as SEQ ID No. 2.

Example 3 preparation of the mutant S23B01 and the wild enzymes rXynAGN16L and rXynAHJ3

The recombinant strains containing the mutant S23B01, the wild enzymes rXynAGN16L and rXynAHJ3 were inoculated to LB (containing 100. mu.g mL of plasmid) at an inoculum size of 0.1% respectively^-1Amp) in the culture medium, the mixture was rapidly shaken at 37 ℃ for 16 hours.

Then, the activated bacterial suspension was inoculated into fresh LB (containing 100. mu.g mL) at an inoculum size of 1%^-1Amp) culture solution, rapidly shaking and culturing for about 2-3 h (OD)₆₀₀0.6-1.0) was reached, IPTG (isopropyl thiogalactoside) was added to a final concentration of 0.1mM for induction, and the shaking culture was continued at 20 ℃ for about 20 hours. Centrifugation was carried out at 12000rpm for 5min to collect the cells. After suspending the cells in an appropriate amount of Tris-HCl buffer (pH 7.0), the cells were sonicated in a low temperature water bath.

Centrifuging the crude enzyme solution concentrated in the cells at 13,000rpm for 10min, sucking the supernatant, and respectively carrying out affinity and purification on the target protein by using Nickel-NTA Agarose and 0-500 mM imidazole.

The SDS-PAGE results (see FIG. 1) show that the mutant enzyme S23B01, the wild enzymes rXynAGN16L and rXynAHJ3 were purified, and the product was a single band.

Example 4 determination of the Properties of the purified enzymes of mutant S23B01 and the wild enzymes rXynAGN16L and rXynAHJ3

1. Activity analysis of the mutant S23B01 and the purified enzymes of the wild enzymes rXynAGN16L and rXynAHJ3

The activity determination method adopts a 3, 5-dinitrosalicylic acid (DNS) method: dissolving the substrate in a buffer solution to a final concentration of 0.5% (w/v); the reaction system contains 100 mu L of proper enzyme solution and 900 mu L of substrate; preheating a substrate at a reaction temperature for 5min, adding an enzyme solution, reacting for 10min, adding 1.5mL of DNS (Domain name System) to terminate the reaction, boiling in boiling water for 5min, cooling to room temperature, and measuring an OD (optical Density) value at a wavelength of 540 nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down the substrate to produce 1. mu. mol reducing sugars (calculated as xylose) per minute under the given conditions.

2. Determination of pH Activity and pH stability of purified enzymes of mutant S23B01 and wild enzymes rXynAGN16L and rXynAHJ3

Determination of the optimum pH of the enzyme: and (3) placing the enzyme solution in a buffer solution with the pH value of 4.0-12.0 at 37 ℃ for enzymatic reaction. Determination of the pH stability of the enzyme: the enzyme solution was placed in a buffer solution with a pH of 3.0 to 12.0, treated at 37 ℃ for 1 hour, and then subjected to an enzymatic reaction at a pH of 7.0 and 37 ℃ with the untreated enzyme solution as a control. The buffer solution is as follows: McIlvaine buffer (pH is 3.0-8.0) and 0.1M glycine-NaOH (pH is 9.0-12.0). And (3) taking beech xylan or corncob xylan as a substrate, reacting for 10min, and determining the enzymatic properties of the purified endo-xylanase.

The results show that: the optimum pH values of the mutant enzyme S23B01, the wild purified enzymes rXynAGN16L and rXynAHJ3 are 5.5, 5.5 and 6.0 respectively; the mutant S23B01, the wild enzyme rXynAGN16L and rXynAHJ3 were stable at pH 5.5-10.

3. Determination of the thermal Activity and thermal stability of the purified enzymes of mutant S23B01 and the wild enzymes rXynAGN16L and rXynAHJ3

Determination of the thermal activity of the enzyme: carrying out an enzymatic reaction at 0-90 ℃ in a buffer solution with a pH of 7.0. Determination of the thermostability of the enzyme: treating the enzyme solution with the same amount of enzyme at 37 deg.C for 0-60 min, and performing enzymatic reaction at pH7.0 and 37 deg.C, wherein untreated enzyme solution is used as control. And (3) taking beech xylan or corncob xylan as a substrate, reacting for 10min, and determining the enzymatic properties of the purified endo-xylanase.

The results show that: the optimal temperatures of S23B01, rXynAGN16L and rXynAHJ3 are 65 ℃, 50 ℃ and 75 ℃ respectively, and the enzyme activities of 80.2%, 17.7% and 97.7% are respectively carried out at 70 ℃; S23B01 and rXynAHJ3 were stable at 50 ℃ and rXynAGN16L was very unstable at 50 ℃.

4. Influence of different metal ions and chemical reagents on the activity of the purified enzyme of the mutant S23B01 and the wild enzymes rXynAGN16L and rXynAHJ3

10.0mM of metal ions and chemical reagents were added to the enzymatic reaction system to examine the effect on the enzymatic activity. The enzyme activity was determined at 37 ℃ and pH7.0 using beech xylan or corncob xylan as substrate.

The results (as shown in table 1) indicate that: 10.0mM HgCl₂Can completely inhibit S23B01, rXynAGN16L and rXynAHJ 3; FeSO at 10.0mM₄In the mutant S23B01, the enzyme activity is respectively 18% higher than that of the wild enzyme rXynAGN16L and 7% higher than that of the wild enzyme rXynAHJ 3.

TABLE 1 Effect of Metal ions and chemical reagents on the viability of mutant S23B01 and wild enzymes rXynAGN16L and rXynAHJ3

5. Purified enzymes of mutant S23B01 and wild enzymes rXynAGN16L and rXynAHJ3 in NaCl, KCl and Na₂SO₄Activity of (1)

(1) Determination of the Activity of enzymes in NaCl

3.0-30.0% (w/v) NaCl is added into the enzymatic reaction system, and enzymatic reaction is carried out at pH7.0 and 37 ℃. And (3) taking beech xylan or corncob xylan as a substrate, reacting for 10min, and determining the enzymatic properties of the purified endo-xylanase.

The results show that: in 5.0-25.0% (w/v) NaCl, the enzyme activity of the mutant S23B01 is respectively 12-26% higher than that of the wild enzyme rXynAGN16L and 20-26% higher than that of rXynAHJ3 (see figure 2).

(2) Determination of enzyme Activity in KCl

Adding 3.0-30.0% (w/v) KCl into the enzymatic reaction system, and performing enzymatic reaction at pH7.0 and 37 ℃. And (3) taking beech xylan or corncob xylan as a substrate, reacting for 10min, and determining the enzymatic properties of the purified endo-xylanase.

The results show that: in KCl of 3.0-30.0% (w/v), the enzyme activity of the mutant S23B01 is respectively 13-41% higher than that of a wild enzyme rXynAGN16L and 1-18% higher than that of rXynAHJ3 (see figure 3).

(3) The enzyme is in Na₂SO₄Activity assay in (1)

Adding 3.0-30.0% (w/v) Na into an enzymatic reaction system₂SO₄The enzymatic reaction was carried out at pH7.0 and 37 ℃. And (3) taking beech xylan or corncob xylan as a substrate, reacting for 10min, and determining the enzymatic properties of the purified endo-xylanase.

The results show that: na in the range of 15.0-30.0% (w/v)₂SO₄In the mutant S23B01, the enzyme activity is 5-21% higher than that of the wild enzyme rXynAGN16L, and 10-12% higher than that of rXynAHJ3 (see figure 4).

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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tcccagccaa ccccggatcc tgccttcatc cccggcggcg ccgccaaccc gacagcgacg 1200

ccgatcgcag catcccgcgg caccggcaac tccgtggcgc tcaccttcga tgacgggccc 1260

gagcccggcg aaaccacagc tgtcctcgat ttcctcaagg acaagggcat cactgccacc 1320

ttctgcgtca tcggagcgaa catccaggcc cccggcggag ccgagctggt gaagcgcatg 1380

gtcgaggagg gccacacgct gtgcaaccac ggcaccacgt atgcggacat gggttcgtgg 1440

acccaggaac agattaaggc cgacctggtg gaaaacctcc gcatcatccg tgaagccgcc 1500

ggcacgcctg atctgcaggt cccctatttc cgggcaccga acggaagctg gggagtcacg 1560

ggcgaagtag ccgcagcgct tggtatgcag ccgctgggcc tgggcaatgt catctttgac 1620

tgggacggca atgacctcag cgaagccacc ctcacggcaa acctccgtgc cgcgttcacc 1680

cccggcgcgg tggtgctggc gcacgtcggc ggcggtgacc ggaccaacac agtgaaggca 1740

gttacgacgg tcgtgaccga aaagctcgcc caggggtgga cgttcgccct tccgcagggc 1800

ggtgccccgg aggaaccttc cggcggtgtg ccctcggact tcgagaccgg aaccgacggc 1860

tggaccgcgc gcggggactc agtggcggtc aacctcagct ccgacgcccg caccggatcc 1920

ggaagcctgc tggtcacgaa ccggacccag gactggcacg gtgccgcact cgacgtcacg 1980

ggcgccctac cggtcggctc ggccgtaaag atgtccgtct gggccaagct cgcccccggg 2040

cagcagccgg cggcactgaa aatgtccgtt cagcgggaca acggcggcgg gagtgcctat 2100

gaaggcgttg ccggagccgg ggcttcggtc accgccgacg gctggaccga acttgccggg 2160

acttacaccc tcggcgcagc agcggacaaa gcccaggtgt acatcgaagg tgctgtcggc 2220

gtggggttcc tgctcgatga cttcagcctc gccgcatatg ttgagcctcc ccttcaggag 2280

gacatacccg ggttgaaaga cgtccttggc ctgcagggca tcgagcacgt gggagcagca 2340

atcgacgcac gcgagacagc gggcaccgca gcgaacctcc tgcggaaaca cttcaatgcc 2400

ttcactcccg agaacgccgg caggcccgag agcgtgcagc cggtggaggg tcagttcacc 2460

cttacccagc tggaccagct gctggacttc gcagccgcca acaatgtcaa ggtgtacgga 2520

catgtgctgg tctggcattc ccagacccct gagtggttct tcaaggacgg gacccgggac 2580

ctgaccggca accggtccga ccgggcgctg ctgagggcac gcatggaggc acatatcaag 2640

ggcatcgcag atcacatcaa tgcccgctac ccggaggggg acagccccat ttgggcctgg 2700

gacgttgtca acgagaccat tgcggacggt gacacggcca acccgcacga catgcgggac 2760

agccgctggt tccaggtcct cggtgaacgt tttgtcgatg atgccttccg tctcgcggac 2820

aagtacttcc cggaggcaaa gctcttcatc aacgactaca acaccgagat gccccagaaa 2880

cgggccgact atctcgagct gattcgtgcc ctggaagccc ggggcgtacc catcgacggc 2940

gtgggccacc aggcgcacgt cgacgtggca cgtccggtgc agtggctcga ggactcgatc 3000

aaggccgttg agaaggtcaa tcctgacctg atgcaggcga tcactgagct cgacgtgaac 3060

gcgtccaccg agaatcaggg cgcggacgtg gacggtgccc cggtggatcc gtaccagccg 3120

gcattcggga acgacgggga cgccgccgcg gaagtcggat actactaccg cgacttgttc 3180

gccatgctgc gcaagcacag ttcggctatt gattcggtga ccgtctgggg catcagcaac 3240

gcccgcagct ggctgcggac ctggccgatg gcccggccct gggagcagcc gcttccattc 3300

gacgatgatc tgcaggctgc accggcctac tggggaatcg tggatcccgc gaaactgccg 3360

gcccggcctg ccgacgtgct ggcaccccgc atcgccgatc agccggacgt ggtggccttt 3420

tcaaagcgcg ccggacgggt gaaggtggct tacccgttgc cctcggcgat cgacaccctc 3480

gacggcaaag tgccggtgga ctgttctccg cgccgcggca gcacctttgc cgtggggacc 3540

actgcggtca cctgcacggc cacggatgcc gccggcaaca cgaggaccag cagcttcgac 3600

gtggtggtga agaagcaccg gcaccacgga aggcactga 3639

<210> 4

<211> 1104

<212> DNA

<213> JF745868

<400> 4

atgaggtcgg ctcgtctggt catcgctttg ttcgctgccg tggcgttgtc ggcgccaccg 60

gcttcggcgg tctcggcccc gccggacgtg agcggccaca aacagacgtt gcgctcggca 120

gcgcccaagg gtttccacat cggcacggcc gtcgcgggcg gcggccacca cgagaaccag 180

ccgtacccgg accccttcac ctcggacagc gagtaccgga aggtgctggc cgcggagttc 240

aactcggtct cgcccgagaa ccagatgaag tgggagtaca tccacccgga gcgcggccgg 300

tacaacttcg gcatggccga cgccatcgtc cggttcgcca agcagaaccg gcaggtggtc 360

cgcgggcaca ccctgatgtg gcacagccag aacccggagt ggctggagca gggcgacttc 420

accgcggccg aactgcgcga gatcctgcgc gagcacatca tgaccgtggt cggccggtac 480

aagggcaagg tccagcagtg ggacgtggcc aacgagatct tcaccgacgc cggcgctctg 540

cggaccacgg agaacatctg gatccgtgaa ctcggtccgg gcatcgtggc ggacgcgttc 600

cgctgggcgc accaggccga ccccaaggcg aagctgttct tcaacgacta caacgtcgaa 660

agcgtcaacg cgaagagcga cgcgtactac gcgctgatca aggagctgcg cgccgcgggt 720

gtgcccgtgc acggcttctc cgcccaggcg cacctcagcc tggactacgg cttcccggac 780

gacctggagc gcaacctgaa gcggttcgcc gacctccggc tggagaccgc gatcaccgag 840

ctcgacgtgc ggatgaccct gcccgcgagc ggcgtgccga cggcggccca gctgcagcag 900

caggccgact actaccagcg cacgctcgcg gcctgcctga aggtcaggac ctgcaagtcg 960

ttcaccatct ggggcttcac cgacaagtac tcgtgggtgc cggtcttctt ccaggggcag 1020

ggtgcggcca cggtgatgtg gaacgacttc ggtcgcaagc aggcgtacta cgcgctgcgg 1080

tccacgctgg cgaagcgagc ctga 1104

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

gtgcagccgg aggaaaaacg 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 6

gatgaaggca ggatccgggg t 21

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

gtctcggccc cgccggacgt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

ggctcgcttc gccagcgtgg 20

Claims

1. An endoxylanase mutant S23B01, wherein the amino acid sequence of the mutant S23B01 is shown as SEQ ID NO. 1.

2. An endoxylanase mutant S23B01 encoding gene S23B01 as claimed in claim 1, wherein the nucleotide sequence of the encoding gene S23B01 is shown as SEQ ID No. 2.

3. A recombinant vector comprising the gene s23b01 according to claim 2.

4. The recombinant vector according to claim 3, wherein the recombinant vector employs an expression vector pEasy-E2.

5. A recombinant bacterium comprising the coding gene s23b01 of claim 2.

6. The recombinant strain according to claim 5, wherein the strain adopted by the recombinant strain is Escherichia coli BL21-Gold (DE 3).

7. A method for preparing the endoxylanase mutant S23B01 of claim 1, comprising:

connecting the coding gene s23b01 and an expression vector pEasy-E2 as claimed in claim 2, and transforming the connection product into Escherichia coli BL21-Gold (DE3) to obtain a recombinant strain containing the coding gene s23b 01;

and culturing the recombinant strain, and inducing the expression of the endoxylanase mutant S23B 01.

8. The method according to claim 7, wherein the recombinant strain is cultured in a medium containing 100. mu.g-mL^-1Amp LB culture solution, shaking culture, OD₆₀₀When the concentration is 0.6-1.0, adding IPTG for induction.

9. The application of the endoxylanase mutant S23B01 in the field of food products, as claimed in claim 1.