CN114591932B

CN114591932B - Acetylxylan esterase mutant and application thereof

Info

Publication number: CN114591932B
Application number: CN202210232551.5A
Authority: CN
Inventors: 宿玲恰; 王蕾; 李志刚; 秦芩; 吴敬
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2023-07-25
Anticipated expiration: 2042-03-09
Also published as: CN114591932A

Abstract

The invention discloses an acetylxylan esterase mutant and application thereof, belonging to the technical field of enzymology engineering. According to the invention, through molecular modification of an acetylxylan esterase gene BPAXE derived from Bacillus pumilus, five mutants with changed enzymatic properties are obtained, and the mutants have certain degradation effects on the mode substrates PVAc and PEA of the adhesive, wherein the K12A has the best effect: in terms of PVAc degradation, turbidity increased by 1.34 times that of wild type and particle size reduced by 1.20 times that of wild type; in the aspect of PEA degradation, the turbidity reduction amount is 1.56 times of that of the wild type, the particle size reduction amount is 1.61 times of that of the wild type, a reaction system which is more stable than that of the wild type can be formed, and the results show that the five acetylxylan esterase mutants have important industrial application potential in degradation of papermaking adhesives.

Description

Acetylxylan esterase mutant and application thereof

Technical Field

The invention relates to an acetylxylan esterase mutant and application thereof, in particular to application of an acetylxylan esterase mutant in degrading papermaking adhesives, and belongs to the technical field of enzymology engineering.

Background

With the annual increase in the utilization rate of waste paper, waste paper has become one of the important raw materials for the paper industry. However, natural wood extractives, artificial synthetic polymers such as coating adhesives, hot melt adhesives and pressure-sensitive adhesives exist in the waste paper, and the waste paper can be continuously flocculated into high-viscosity adhesives in the recycling process and is retained on the surfaces of paper, drying cylinders, forming nets and other equipment, so that the quality of the paper is poor, paper breakage occurs, and meanwhile, the utilization rate of paper machine equipment is reduced. Therefore, how to control the stickies accumulated in the waste paper recycling process is one of the problems to be solved currently. At present, the biological enzyme method attracts attention by the unique advantages of environmental friendliness, high efficiency and specificity, and mainly reduces the viscosity of the adhesive by acting enzyme on the chemical bond of the adhesive, slows down the deposition phenomenon of the adhesive, and becomes an important adhesive control means.

The adhesives can be classified into primary adhesives and secondary adhesives according to the cause of the occurrence of the adhesives. The raw adhesives refer to adhesives which are reserved in paper pulp along with the operation of a waste paper recycling system, and are formed by gathering substances which are gradually dispersed and released from waste paper pulp after being introduced into a production line along with the waste paper pulp and being subjected to recycling processes such as pulp crushing, papermaking and the like. In general, the solid form is mainly water-insoluble, and is characterized by a large size, and most of the water can be removed by the filtering device. The secondary adhesive belongs to an adhesive which is derived from sudden changes of temperature or pH in an environment system after physical and chemical actions. In the pulping process, a plurality of impurities can be dissolved in the pulp water, however, the stability of the adhesive is extremely poor, and a severe destabilization phenomenon can occur after the conditions of external temperature, pH value, surface charge and the like are changed, so that the adhesive is extremely easy to flocculate and form the adhesive with higher viscosity. Because of its uncontrollable nature, the control of secondary adhesives is more difficult in industry than primary adhesives. Currently, precipitation, micro-flotation or ultrafiltration is often used industrially to remove secondary stickies.

At present, the treatment of adhesives in the domestic paper industry is mainly performed by a traditional chemical method, and the adhesives can play a good role in the paper making process, but the problems of secondary pollution caused by chemicals and the like still exist. Along with further aggravation of global pollution and improvement of environmental protection consciousness, the biological enzyme method is focused on various industrial fields due to the advantages of mild conditions, green environmental protection, strong substrate specificity and the like, so that the biological enzyme method is gradually applied to the field of controlling the stickies in the waste paper recycling process in recent years, thereby not only reducing energy consumption, but also reducing the pollution of chemicals to the environment, and simultaneously achieving the purpose of removing the stickies. The adhesive has a large number of polar groups, the flexibility of a molecular chain is generally low, so that the attraction force between molecules and between molecules of the polymer is enhanced, and the viscosity is increased. The action mechanism of the biological enzyme method is mainly that the main structural components of the adhesive are destroyed by the hydrolysis of chemical bonds in the adhesive by enzyme, so that the viscosity of particles is reduced, the interaction is weakened, and the flocculation phenomenon of the adhesive is effectively weakened.

Esterases degrade adhesives by hydrolyzing ester bonds, and are the most important enzymes for controlling adhesives. The adhesives are hydrolyzed by both enzymes, both oils and synthetic polyesters. The synthetic polyesters in the adhesives are mainly classified into Polyacrylate (PA) and polyvinyl acetate (PVAc). Both polyester backbones are carbon chains, while ester linkages are located in the side chains. The negative charge value of the adhesive polyester is low, the main body is hydrophobic, the carbon-carbon main chain of the adhesive polyester endows the polyester with elasticity, and the side chain ester endows the polyester with viscosity, so that the adhesive polyester is easy to aggregate to form adhesive particles with viscosity.

Acetylxylan esterase is a polyesterase capable of hydrolyzing polysaccharide side chain ester bonds, and the hydrolysis effect on the side chain ester of the adhesive polymer is possibly superior to that of common esterase, so that the acetylxylan esterase can be used as an enzyme for controlling the adhesive.

Disclosure of Invention

In the previous study, the inventor has screened an acetylxylan esterase BPAXE, and experiments prove that the acetylxylan esterase BPAXE has a certain degradation effect on PEA and PVAc, and can be helpful for controlling adhesives in the actual paper industry, but the acetylxylan esterase BPAXE still has a large lifting space. Firstly, because of containing a small amount of free carboxylic acid or hydroxyl groups, the adhesive has weak electronegativity besides the hydrophobic characteristic, and secondly PEA and PVAc are artificially synthesized polyesters, related enzymes or microorganisms are not evolved in the nature, and PEA and PVAc can be efficiently degraded. Therefore, the invention carries out molecular modification on the screened acetylxylan esterase BPAXE from the combination angle of enzyme and substrate, enhances the electropositivity of the catalytic groove of the enzyme through rational analysis, improves the combination efficiency of the BPAXE on the substrate, and improves the efficiency of degrading PEA and PVAc by the BPAXE.

The components of the adhesive which can be treated by esterase or deacetylase are esters or polymer esters, and a small amount of carboxyl and hydroxyl which do not participate in forming ester bonds exist before enzyme treatment, so that the adhesive has electronegativity besides typical hydrophobic characteristics, and the electronegativity of the adhesive is increased along with the progress of enzymolysis reaction and the generation of free carboxyl and hydroxyl. With respect to the characteristics, the inventor respectively constructs BPAXE mutants K12A, K17A, D101N, N K, L and 135K by taking the amino acid sequence of the acetylxylan esterase BPAXE as a starting sequence, and applies the BPAXE mutants to degradation of PEA and PVAc.

The invention provides an acetylxylan esterase mutant, which takes acetylxylan esterase shown in an amino acid sequence SEQ ID NO.1 as a parent enzyme, and substitutes one or more sites of 12 th site, 17 th site, 101 th site, 104 th site and 135 th site of the acetylxylan esterase to obtain the mutant.

In one embodiment, the mutant is any one of the mutants described in (a) to (e):

(a) Substitution of lysine at position 12 to alanine;

(b) Substitution of lysine at position 17 to alanine;

(c) Substitution of aspartic acid at position 101 to asparagine;

(d) Substitution of asparagine at position 104 to lysine;

(e) Leucine at position 135 was replaced with lysine.

In one embodiment, the nucleotide sequence encoding the acetylxylan esterase is shown in SEQ ID NO. 2.

The present invention provides genes encoding the acetylxylan esterase mutants.

The invention provides a recombinant plasmid carrying the gene encoding the acetylxylan esterase mutant.

In one embodiment, the expression vector of the recombinant plasmid includes, but is not limited to, pET series, duet series, pGEX series, pHY300PLK, pPIC3K, or pPIC9K series vectors.

Preferably, pET24a (+) is used as an expression vector.

The present invention provides a microbial cell expressing the acetylxylan esterase mutation, or containing the gene.

In one embodiment, the microbial cell is a prokaryotic cell or a eukaryotic cell.

The invention provides a method for hydrolyzing an adhesive, which is to add the mutant into a reaction system containing the adhesive to hydrolyze the adhesive.

In one embodiment, the mutant is added to the reaction system in an amount of not less than 800U/g substrate.

In one embodiment, the adhesive comprises PEA and PVAc.

In one embodiment, the reaction is carried out at pH 7.0.+ -. 0.5, 45-55 ℃ for not less than 4 hours.

The invention provides the acetylxylan esterase mutant, or a gene encoding the acetylxylan esterase mutant, or application of the method in degrading papermaking adhesives.

In one embodiment, the papermaking adhesive includes, but is not limited to, PEA or PVAc.

The invention has the beneficial effects that:

according to the invention, the acetylxylan esterase gene BPAXE from bacillus pumilus is subjected to molecular transformation, 5 mutants are constructed, and the 5 mutants are applied to PVAc degradation, so that the PVAc degradation effect can be improved, the particle size of PVAc turbid liquid can be reduced, and good stability can be maintained; the 5 mutants can be applied to PEA degradation, can obtain good degradation effect, remarkably reduce particle size of PEA turbid liquid and can keep good stability of PEA turbid liquid.

Drawings

FIG. 1 is a diagram of SDS-PAGE gel electrophoresis analysis of BPAXE and mutants thereof;

FIG. 2 is a graph showing turbidity changes of BPAXE mutant degradation PVAc turbid liquid;

FIG. 3 is a graph showing the particle size change of PVAc turbid liquid degraded by BPAXE mutant;

FIG. 4 is a graph showing the potential change of BPAXE mutant to degrade PVAc turbid liquid;

FIG. 5 is a graph showing turbidity changes of BPAXE mutant degradation PEA turbidity;

FIG. 6 is a graph showing the change in particle size of PEA turbidity degraded by BPAXE mutant;

FIG. 7 is a graph showing the change in potential of BPAXE mutant to degrade PEA turbidity.

Detailed Description

The invention will be further illustrated with reference to specific examples.

1. Coli Escherichia coli JM, E.coli Escherichia coli BL (DE 3) were purchased from Biotechnology (Shanghai) Inc., pET-24a (+) plasmid was purchased from Novagen, PVAc and PEA were purchased from Shanghai sigma, and commercial enzyme CWB-3560 was purchased from Changzyme.

2. Protein mass was determined using the root BCA protein quantification kit (PA 115).

3. The following examples relate to the following media:

LB medium: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of sodium chloride;

TB medium: 12g/L tryptone, 24g/L yeast powder, 5g/L glycerol and KH ₂ PO ₄ 2.31g/L，K ₂ HPO ₄ ·3H ₂ O16.43g/L。

Example 1: construction of mutants

Site-directed mutagenesis was performed on the acetylxylan esterase BPAXE gene (see SEQ ID NO. 2) with the primers shown in Table 1 below, respectively.

Table 1: BPAXE site-directed mutagenesis primer sequence

The specific steps are that a sequence with a nucleotide sequence shown as SEQ ID NO.2 is connected to a polyclonal enzyme cutting site of a pET24a (+) vector, a BPAXE gene connected to the pET24a (+) vector is used as a template, a site-directed mutagenesis primer is used for amplifying the mutated sequence by utilizing a PCR reaction, and the reaction conditions are as follows: pre-denaturation at 98℃for 3min, denaturation at 98℃for 30s, annealing at 55℃for 20s and elongation at 72℃for 6.5min for 30 cycles. And (3) digesting the PCR product by using Dpn I, transferring into escherichia coli JM109, and after a monoclonal is grown, picking a corresponding transformant for sequencing verification to obtain a positive transformant with correct verification.

Example 2: fermentation expression of acetylxylan esterase mutant, enzyme activity determination and protein purification

(1) Fermentation expression of acetylxylan esterase mutants

The transformant verified to be correct in example 1 was extracted with a plasmid extraction kit, and the extracted plasmid was transferred into E.coli BL21 (DE 3) to construct a recombinant strain E.coli BL21 (DE 3)/pET 24a (+) -axe. E.coli BL21 (DE 3)/pET 24a (+) -axe is inoculated in a liquid LB culture medium (containing 30 mu g/mL kanamycin) to grow for 8-10 h, and seeds are inoculated in a TB liquid fermentation culture medium (containing 30 mu g/mL kanamycin) according to the inoculum size of 5% (v/v); shaking the flask at 37deg.C and 200r.min ^-1 Is cultured at constant temperature for 2 hours under the culture condition of (2), and is added with IPTG with the final concentration of 0.2mM to carry out induction expression for 24 hours at 25 ℃. The obtained bacterial liquid 8000 r.min ^-1 Centrifuging for 20min, discarding supernatant, collecting thallus, redissolving thallus with buffer solution, breaking cell wall with high pressure refiner, centrifuging again, and collecting wall-broken supernatant to obtain crude enzyme solution.

(2) SDS-PAGE protein electrophoresis detection

The crude enzyme of the supernatant was subjected to SDS-PAGE protein electrophoresis (FIG. 1), and SDS-PAGE results showed that recombinant BPAXE and mutants thereof were expressed in E.coli, and that the mutant protein was approximately 36.07kDa in size and substantially identical to the wild-type band.

(3) Crude enzyme liquid enzyme activity determination

The enzyme activity determination method of the acetylxylan esterase comprises the following steps: the enzyme activity was determined by continuous spectrophotometry at 50 ℃. The total reaction volume was 1.5mL, including 30. Mu.L of crude fermentation broth, 30. Mu.L of p-nitrophenol acetate (pNPA) at a concentration of 50mmol/L, and 1.44mL of Tris-HCl buffer (pH 7.0), and the rate of formation of p-nitrophenol (pNP) was recorded at a wavelength of 405 nm.

The definition of enzyme activity is: the amount of enzyme required to produce 1. Mu. Mol of pNP per minute by catalytic hydrolysis of pNPA at 50℃is one enzyme activity unit (U).

Table 2 below shows the enzyme activities of the BPAXE mutant and the wild type thereof at 50 ℃, and the results show that the enzyme activity of the BPAXE mutant N104K, K17A, L K is improved compared with the wild type enzyme activity, wherein the K17A is improved by 52.0% most obviously; K12A and D101N were reduced, with the most significant reduction in K12A being a 83.2% reduction.

Table 2: BPAXE mutant and wild type enzyme activity thereof at 50 DEG C

(4) Protein purification

Purifying the target protein by adopting a nickel column affinity chromatography method. Adding the crude enzyme solution into a nickel column for loading, mixing a binding buffer solution (A solution) and an elution buffer solution (B solution) according to different proportions, pumping into the column for gradient elution, collecting effluent liquid, and performing SDS-PAGE gel electrophoresis. And selecting the optimal concentration according to SDS-PAGE analysis, and obtaining purified protein after continuing ultrafiltration of effluent liquid of the optimal concentration.

The buffers involved are as follows:

binding buffer (solution a): tris-HCl buffer 25mmol/L, naCl 500mmol/L, adjusted to pH8.0 using HCl;

elution buffer (solution B): tris-HCl buffer 25mmol/L, naCl 500mmol/L, imidazole 300mmol/L was adjusted to pH8.0 using HCL.

Example 3: mutant pair of acetylxylan esterasePVAcIs to be degraded by

Preparation of 1% (w/v) PVAc solution: 1g PVAc was dissolved in methanol and fixed to a volume of 100mL.

After 0.5mL of PVAc solution with a concentration of 1% was taken in a 15mL glass bottle, the protein was purified, and after the concentration of the enzyme protein was diluted to 0.1g/L with Tris-HCl buffer (50 mM, pH 7.0), 0.5mL was taken and added to the glass bottle, 9mL of buffer was added to make the final volume 10mL, and the enzyme activities of wild type and K12A, D101N, N104K, K17A, L135K mutants in the system were 8.73U, 16.38U, 13.09U, 11.08U, 19.41U, 23.55U (i.e., 873U/g substrate, 1638U/g substrate, 1309U/g substrate, 1108U/g substrate, 1941U/g substrate, 2355U/g substrate, respectively), and the enzyme activity assay method was the same as that of example 2, step 3. Placing in a shaking table at 50 ℃ at 200rpm for reaction for 4 hours, and measuring turbidity change, particle size change and ZETA potential change of a reaction system by using a spectrophotometer, a nano particle size and a ZETA potential analyzer.

Commercial enzyme group: 0.5mL of PVAc solution with the concentration of 1% is taken in a 15mL glass bottle, the enzyme activity of commercial enzyme is 2500U/mL (the enzyme activity measuring method is the same as step 3 in example 2, the difference is that the substrate is pNPB), the specific activity is 833.33U/mg, after the concentration of the diluted protein is 0.1g/L, 0.5mL is taken and added into the glass bottle, 9mL of buffer solution is added to make the final volume be 10mL, and the enzyme activity in the system is 41.67U. Placing in a shaking table at 50 ℃ at 200rpm for reaction for 4 hours, and measuring turbidity change, particle size change and ZETA potential change of a reaction system by using a spectrophotometer, a nano particle size and a ZETA potential analyzer.

Buffer instead of enzyme solution served as blank control, three parallel controls were made for each experimental group.

(1) Turbidity change of enzymatic hydrolysis PVAc turbid liquid

The solvent that dissolves PVAc is typically methanol. When it is dissolved in the buffer solution of the enzyme reaction, methanol will be dissolved in water first, so that PVAc cannot be dissolved in methanol continuously, a large amount of particles will be formed in the enzyme reaction system and further flocculated into large particles, and adhere to the inner wall of the reaction vessel, so that the turbidity is low. When enzyme acts on PVAc side chain acetyl to release acetic acid, PVAc hydrophilicity increases, and the PVAc falls off from the inner wall of the reaction vessel and is dispersed into stable small particles from large particles, and the stable small particles exist in the form of suspension. Thus, the degradation effect of the acetylxylan esterase on PVAc can be characterized by measuring turbidity changes.

The turbidity increase indicates that the large particles that are bound together are dispersed after the PVAc is degraded, further slowing down the tendency of PVAc to flocculate. As shown in fig. 2, the turbidity decrease trend of the blank group after 4 hours of reaction was evident with the initial turbidity being 100%. The turbidity decrease trend of the BPAXE wild type group and the turbidity decrease trend of the mutant group are basically consistent, the turbidity is higher than that of the commercial enzyme group, the turbidity of the K12A group is 75 times that of the wild type group, and the turbidity is 1.34 times that of the wild type group, so that the mutant K12A of the BPAXE has the best degradation effect on the PVAc substrate.

(2) Particle size variation of enzymatically hydrolyzed PVAc clouds

Particle size detection is one of the important means for analyzing the aggregation state of fine particles, and the change of particle size is often used for representing the degradation effect of a model substrate in a system. And removing large particles from the PVAc turbid liquid after the enzyme reaction by high-speed centrifugation, and analyzing the particle size of the PVAc turbid liquid.

As shown in FIG. 3, the particle size of the blank group is larger, and the particle sizes of the BPAXE, the mutant thereof and the PVAc turbid liquid after commercial enzyme treatment are reduced to different degrees. The particle size of the wild type BPAXE group is reduced by 63.98% compared with that of a blank group, the particle size of the commercial enzyme group is reduced by 23.55%, and the mutant K12A group is reduced by 77.07%, which shows that the mutant K12A of the BPAXE has the best degradation effect on the substrate PVAc.

(3) Zeta potential change of enzymatic hydrolysis PVAc turbid liquid

The Zeta potential is mainly used for characterizing the stability of particles in an enzyme hydrolysis system after reaction by measuring the electrification condition of fine particles. The Zeta potential reflects the aggregation and dispersion of proteins and electrolytes during the reaction, while a higher absolute value of Zeta potential indicates that the measured system is more stable in the natural state, called the charge stabilization of the colloidal system.

As shown in FIG. 4, the potential absolute value of the BPAXE and the mutant group thereof after the reaction is obviously increased compared with that of the blank group and the commercial enzyme group, the potential absolute value of the BPAXE wild type group is increased by 8.1 compared with that of the blank group, the potential absolute value of the mutant group is increased by 6.0-8.6 compared with that of the blank group, wherein the potential absolute value of the K12A group is highest, and the potential absolute value of the mutant group is increased by 54.43% compared with that of the blank group by 15.8, which indicates that the enzyme reaction system formed after the action of the mutant K12A of the BPAXE is the most stable, and the charge balance of the enzyme reaction system is not easily destroyed, and the condition of repeated flocculation occurs.

Example 4: mutant pair of acetylxylan esterasePEAIs to be degraded by

1% (w/v) PEA solution: accurately weighing 1g of PEA, dissolving in methanol and fixing the volume to 100mL.

Acetylxylan esterase and mutant group thereof: 1mL of 1% PEA solution was placed in a 15mL glass bottle, the protein was purified, the enzyme protein was diluted to 0.1g/L with Tris-HCl buffer (50 mM, pH 7.0), 0.5mL of the diluted solution was placed in the glass bottle, and 8.5mL of the diluted solution was added to give a final volume of 10mL, and the enzyme activities of the wild-type and K12A, D101N, N104K, K17A, L K mutant systems were 8.73U, 16.38U, 13.09U, 11.08U, 19.41U, 23.55U (i.e., 873U/g substrate, 1638U/g substrate, 1309U/g substrate, 1108U/g substrate, 1941U/g substrate, 2355U/g substrate, respectively) and the enzyme activity was measured in the same manner as in step 3 of example 2. Placing in a shaking table at 50 ℃ at 200rpm for reaction for 4 hours, and measuring turbidity change, particle size change and ZETA potential change of a reaction system by using a spectrophotometer, a nano particle size and a ZETA potential analyzer.

Commercial enzyme group: 1mL of PEA solution with the concentration of 1% is taken in a 15mL glass bottle, the enzyme activity of commercial enzyme is 2500U/mL (the enzyme activity measuring method is the same as step 3 in the example 2, the difference is that the substrate is pNPB), the specific activity is 833.33U/mg, after the concentration of protein is diluted to 0.1g/L, 0.5mL is taken and added into the glass bottle, 8.5mL of buffer solution is added to make the final volume be 10mL, and the enzyme activity of the system is 41.67U. Placing in a shaking table at 50 ℃ at 200rpm for reaction for 4 hours, and measuring turbidity change, particle size change and ZETA potential change of a reaction system by using a spectrophotometer, a nano particle size and a ZETA potential analyzer.

(1) Turbidity change of enzymatic hydrolysis PEA turbid liquid

PEA itself is soluble in organic solvents such as acetone to form a suspension in an aqueous system. After enzyme solution is added into the system, ester bonds of PEA side chains are degraded along with the continuous progress of the reaction, so that the hydrophilicity of the ester bonds is enhanced, the aggregation tendency of PEA is inhibited, the turbidity is reduced to a certain extent, and therefore, the degradation effect of the acetyl xylan esterase on the PEA can be represented by measuring the reduction of the turbidity.

As shown in FIG. 5, with the initial turbidity being 100%, the turbidity of the commercial enzyme group and the turbidity of the BPAXE wild type and the mutant group thereof show a decreasing trend, the turbidity of the BPAXE wild type is reduced by 30.3% compared with the blank group, the turbidity of the mutant group is reduced by 22.5-47.2% compared with the blank group, wherein the turbidity of the K12A group is reduced by 47.2%, which indicates that the mutant K12A of the BPAXE has the best degradation effect on the substrate PEA.

(2) Particle size variation of enzymatic hydrolysis PEA turbid liquid

The PEA particle size was measured as in example 3. As shown in FIG. 6, after enzyme treatment, turbidity of the commercial enzyme group and the BPAXE wild type and mutant group thereof all show a decreasing trend, particle size of the commercial enzyme group is reduced by 178nm compared with that of the blank group, the BPAXE wild type group is reduced by 208nm compared with that of the blank group, the BPAXE mutant group is reduced by 150-335 nm compared with that of the blank group, wherein the reduction amount of the mutant K12A group is at most 335nm, which indicates that the mutant K12A of the BPAXE has a better degradation effect on the substrate PEA.

(3) Zeta potential change of enzymatic hydrolysis PEA turbidity

Detection of Zeta potential of PEA was the same as in example 3. As shown in fig. 7, after BPAXE mutant reaction, the absolute value of the potential was significantly increased compared to the blank, all mutant groups were higher than the commercial enzyme group, and K12A, K17A, N104K group was higher than the wild type group. The absolute value of the potential of the wild type is increased by 21.6% compared with that of the blank group, and the absolute value of the potential of the mutant is increased by 19.7-34.1% compared with that of the blank group, which proves that the enzyme reaction system formed after the action of the mutant K12A, K17A, N K of the BPAXE is the most stable.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> BAA220059A

<130> Acetylxylan esterase mutant and use thereof

<160> 2

<170> PatentIn version 3.3

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Met Gln Leu Phe Asp Leu Ser Leu Glu Glu Leu Lys Lys Tyr Lys Pro

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Glu Glu Leu Arg Gln Val Glu Ala Glu Pro Thr Leu Glu Ser Tyr Asp

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Tyr Pro Val Lys Gly Val Lys Val Tyr Arg Leu Thr Tyr Gln Ser Phe

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Gly His Ser Lys Ile Glu Gly Phe Tyr Ala Val Pro Asp Gln Thr Gly

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Pro His Pro Ala Leu Val Arg Phe His Gly Tyr Asn Ala Ser Tyr Asp

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Gly Gly Ile His Asp Ile Val Asn Trp Ala Leu His Gly Tyr Ala Thr

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Phe Gly Met Leu Val Arg Gly Gln Gly Gly Ser Glu Asp Thr Ser Val

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Thr Pro Gly Gly His Ala Leu Gly Trp Met Thr Lys Gly Ile Leu Ser

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Lys Asp Thr Tyr Tyr Tyr Arg Gly Val Tyr Leu Asp Ala Val Arg Ala

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Val Ile Gly Gly Ser Gln Gly Gly Ala Leu Ala Ile Ala Ala Ala Ala

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Leu Ser Asp Ile Pro Lys Val Val Val Ala Asp Tyr Pro Tyr Leu Ser

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taa 963

Claims

1. An acetylxylan esterase mutant characterized in that acetylxylan esterase represented by the amino acid sequence SEQ ID No.1 is used as a parent enzyme, lysine at position 12 is substituted with alanine, lysine at position 17 is substituted with alanine, aspartic acid at position 101 is substituted with asparagine, asparagine at position 104 is substituted with lysine, or leucine at position 135 is substituted with lysine.

2. A gene encoding the mutant acetylxylan esterase according to claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A microbial cell expressing the acetylxylan esterase mutant of claim 1 or comprising the gene of claim 2.

5. The microbial cell of claim 4, wherein the microbial cell is a prokaryotic cell or a eukaryotic cell.

6. A method for hydrolyzing an adhesive, characterized in that the acetylxylan esterase mutant according to claim 1 is added into a reaction system containing the adhesive to hydrolyze the adhesive.

7. The method of claim 6, wherein the reaction is not less than 4h in an environment of ph7.0±0.5 and 45 to 55 ℃.

8. The method of claim 6 or 7, wherein the adhesive comprises PEA and PVAc.

9. Use of an acetylxylan esterase mutant according to claim 1 or a gene according to claim 2 or a method according to any of claims 6 to 7 for degrading papermaking adhesives.