CN114591931A

CN114591931A - Acetylxylan esterase cloning expression and application thereof in papermaking stickies control

Info

Publication number: CN114591931A
Application number: CN202210232539.4A
Authority: CN
Inventors: 宿玲恰; 王蕾; 陈世恒; 秦芩; 吴敬
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-06-07
Anticipated expiration: 2042-03-09
Also published as: CN114591931B

Abstract

The invention discloses acetyl xylan esterase cloning expression and application thereof in papermaking stickers control, belonging to the fields of genetic engineering and enzyme engineering. The acetylxylan esterase ATAXE used in the present invention is derived from Thermococcus (Acetivibrio thermocellus). ATAXE has excellent performance in the aspect of removing stickies in the field of pulping and papermaking, and has good degradation effect on a mode substrate Polyacrylate (PEA). After the solution containing PEA is treated by ATAXE, the turbidity of the solution is obviously reduced, and after 4 hours, the turbidity is reduced by about 50.4 percent; the particle size is reduced by 39.8 percent compared with the commercial enzyme group; the release amount of ethanol of the PEA solution after the side chain treatment by ATAXE is 2.1 times of that of the commercial enzyme group. Therefore, the acetyl xylan esterase ATAXE is applied to industries such as paper making, energy, feed, food and the like, and has great advantages for protecting the environment and reducing the energy consumption.

Description

Acetylxylan esterase cloning expression and application thereof in papermaking stickies control

Technical Field

The invention relates to acetyl xylan esterase cloning expression and application thereof in control of papermaking stickies, belonging to the technical field of genetic engineering and enzyme engineering.

Background

In recent years, recycling of waste paper has become a hot issue in the paper industry due to the continuous consumption of resources, and waste paper has become one of the important raw materials in the paper industry. In actual production, a circulating water system is often adopted in a waste paper recycling production line, and as production is carried out, stickies brought in waste paper can be continuously accumulated in the system as fine stickies, and finally aggregated into large-particle stickies to be deposited on each part of a machine. The natural compound is derived from wood extract in papermaking raw materials, is easy to combine with metal ions in a circulating water system to become insoluble sticky matter, and is deposited on the surface of a machine. The synthetic polymer components are various, mainly including various coating adhesives, hot melt adhesives, residual printing ink and the like, and are introduced into the manufacture and use of paper or paperboard due to different purposes, and are the main components polluted by stickies. At present, the biological method, especially the enzyme method, draws attention of people on the environment-friendly, efficient and specific performance, and the main function of the enzyme method is that the enzyme acts on the chemical bond of the adhesive to cut the adhesive so as to reduce the viscosity of the adhesive and slow down the adhesion phenomenon of the adhesive, and the enzyme method becomes an important adhesive control means.

Two different classification methods have been derived for stickies due to their different classification criteria. One is according to gluing thing size classification, can divide into large granule gluing thing and fine gluing thing according to the size of gluing thing particle diameter size about, mainly distinguishes according to the difference of screening separation condition. In general, a large size of the particle size of a large adhesive with 100 μm as a boundary line means an adhesive trapped in a 100 μm slit sieve under laboratory conditions, and a fine adhesive can pass through a 100 μm or less slit sieve, and is called a fine adhesive. Although the fine stickies are small in size and do not occupy a large number of components, they are easily agglomerated into large particles due to their hydrophobicity and low net charge value, and are deposited on various parts of the paper machine after the filtering device, thereby reducing the paper quality. The second method is classified according to the cause of the adhesive, and the second method is classified into a primary adhesive and a secondary adhesive according to the cause of the adhesive. The raw stickies are stickies which are remained in paper pulp when the waste paper is treated, and then the waste paper pulp is introduced into a production line and gradually aggregated after the processes of pulping, papermaking and the like. In general, it is characterized by being insoluble in water and present in the form of large solid particles, the majority of which are removed by filtration means. The secondary stickies are those which are formed as a result of a change in temperature or pH in the system, belonging to the physicochemical changes. The stability of the adhesive is poor in the pulping process, and severe instability can occur after external conditions such as temperature, pH value and surface charge are changed, so that the adhesive is easy to flocculate to form the adhesive with high viscosity. Because of the uncontrollable nature of secondary stickies, how to solve the problem of controlling the secondary stickies in the papermaking industry is a problem which needs to be solved urgently at present. Currently, precipitation, micro-flotation or ultrafiltration methods are often used in industry to remove secondary stickies.

At present, the treatment of stickies in the domestic paper industry is mainly based on chemical and physical methods, and although the stickies can play a good role in the paper making process, the treatment still has the problems of secondary pollution and the like caused by chemicals. With the further aggravation of global pollution and the improvement of environmental protection consciousness, the biological enzyme method is concerned by various industrial fields because of the advantages of mild conditions, environmental protection, strong substrate specificity and the like, and therefore, the biological enzyme method is gradually applied to the field of controlling the stickies in the waste paper recovery process in recent years, so that the energy consumption is reduced, the pollution of chemicals to the environment is reduced, and the purpose of removing the stickies is achieved. The adhesive has a large number of polar groups, and the flexibility of a molecular chain is generally low, so that the attraction force in and among polymer molecules is enhanced, and the viscosity is increased. The action mechanism of the biological enzyme method is mainly to destroy the main structural components of the adhesive by the hydrolysis of the enzyme on the chemical bonds in the adhesive, so that the particle viscosity is reduced, the interaction is weakened, and the flocculation phenomenon of the adhesive is effectively weakened.

Esterases degrade stickies by hydrolyzing ester bonds, and are the predominant enzyme used to control stickies. The adhesive can be hydrolyzed by esterase and is made of grease and synthetic polyester. After analyzing the composition of the white water in the paper mill, Polyethylacrylate (PEA) was found to be one of the most influential gum compositions for the dry end. However, there are few reports of degrading PEA by esterase, and no good esterase capable of degrading PEA exists.

Disclosure of Invention

Based on the problems existing at present, the invention aims to provide an acetyl xylan esterase which can be efficiently applied to the industry.

The first object of the present invention is to provide a method for degrading a sticker, characterized in that the sticker is hydrolyzed using an acetylxylan esterase having an amino acid sequence shown in SEQ ID NO. 1.

In one embodiment, the acetylxylan esterase is added to a system containing stickies; adding the acetyl xylan esterase into a reaction system according to the addition amount of 40-1000U per gram of the adhesive for reaction.

Preferably, the acetyl xylan esterase is added into the reaction system for reaction according to the addition amount of 88U to 882U per gram of the viscose.

In one embodiment, a 1% (1g/100mL) solution of PEA is prepared, the acetylxylan esterase is diluted and added to the 10mL reaction, and 1mL of PEA solution is added, made up to 10mL with buffer.

In one embodiment, the reaction is carried out at pH7.5-9.0, 40-60 deg.C, 150-250 rpm for not less than 4 h.

Preferably, the reaction is carried out at 200rpm at pH7.5 and 50 ℃ for 4 h.

In one embodiment, the adhesive comprises PEA.

The second purpose of the invention is to provide the application of the acetyl xylan esterase with the amino acid sequence shown as SEQ ID NO.1 in degrading adhesive substances.

In one embodiment, the adhesive comprises PEA.

The third purpose of the invention is to provide an expression vector containing the nucleotide sequence shown in SEQ ID NO.2, a microbial cell expressing the acetyl xylan esterase with the amino acid sequence shown in SEQ ID NO.1 or the application of the acetyl xylan esterase with the amino acid sequence shown in SEQ ID NO.1 in preparing products for degrading adhesive substances.

In one embodiment, the expression vector containing the nucleotide sequence shown as SEQ ID NO.2 uses the expression vector including, but not limited to, pET series, Duet series, pGEX series, pHY300PLK, pPIC3K or pPIC9K series vectors as a starting vector.

In one embodiment, the microbial cells are derived from Escherichia coli.

Preferably, the escherichia coli is escherichia coli BL 21.

In one embodiment, the expression vector containing the nucleotide sequence shown in SEQ ID No.2 is transferred into a host cell to construct a microbial cell expressing the acetyl xylan esterase with the amino acid sequence shown in SEQ ID No. 1.

In one embodiment, the microbial cell expressing the acetylxylan esterase having the amino acid sequence shown in SEQ ID No.1 is cultured and induced to obtain the acetylxylan esterase having the amino acid sequence shown in SEQ ID No. 1.

In one embodiment, the product comprises an enzyme preparation, a microbial preparation, a sewage treatment agent.

In one embodiment, the adhesive comprises PEA.

The fourth purpose of the invention is to provide an expression vector containing the nucleotide sequence shown as SEQ ID NO.2, a microbial cell expressing the acetyl xylan esterase with the amino acid sequence shown as SEQ ID NO.1 or the application of the acetyl xylan esterase with the amino acid sequence shown as SEQ ID NO.1 in papermaking or waste paper recovery.

In one embodiment, the microbial cells are derived from Escherichia coli.

Preferably, the escherichia coli is escherichia coli BL 21.

The invention has the beneficial effects that:

the acetyl xylan esterase ATAXE used by the invention is derived from thermophilic coccus (Acetivibrio thermocellus), the optimum pH of the acetyl xylan esterase ATAXE is 7.5, and the relative enzyme activity of the acetyl xylan esterase ATAXE is kept above 80% within the pH range of 7.5-9.0; the optimal temperature is 70 ℃, and the research on the thermal stability shows that more than 90 percent of relative enzyme activity is reserved in 80 hours when the recombinant ATAXE is incubated at 50 ℃, and the half-life period is 84 hours; the intracellular enzyme activity of the recombinant ATAXE is 234.7U/mL, and the specific enzyme activity of the recombinant ATAXE after purification is 176.3U/mg.

The acetylxylan esterase ATAXE has the effect of removing papermaking stickies, particularly has a good degradation effect on PEA, and is added into a reaction system containing PEA for reaction, and the turbidity of the reaction liquid after the reaction is obviously reduced compared with that of the initial solution and a commercial enzyme group; the Zeta potential of the solution after reaction is improved to a certain extent, and the stability of a reaction system is improved; and the release amount of ethanol in the solution after reaction is more than that of the commercial enzyme, which is beneficial to reducing the flocculation phenomenon of the stickies.

Drawings

FIG. 1 is a diagram of SDS-PAGE gel electrophoresis analysis;

FIG. 2 is a SDS-PAGE gel electrophoresis analysis of ATAXE after purification;

FIG. 3 is a pH optimum diagram of the recombinant acetylxylan esterase ATAXE;

FIG. 4 is a pH stability diagram of the recombinant acetylxylan esterase ATAXE;

FIG. 5 is a temperature optimum diagram of the recombinant acetylxylan esterase ATAXE;

FIG. 6 is a graph of the thermostability of the recombinant acetylxylan esterase ATAXE;

FIG. 7 is a graph of the turbidity of the model substrate PEA as a function of the recombinant acetylxylan esterase ATAXE;

FIG. 8 is a graph showing the change in particle size of a model substrate PEA by recombinant acetylxylan esterase ATAXE;

FIG. 9 is a Zeta potential change diagram of recombinant acetylxylan esterase ATAXE against a model substrate PEA.

FIG. 10 is a graph showing the particle size change of the recombinant acetylxylan esterase ATAXE in the optimization of enzyme addition.

FIG. 11 is a Zeta potential change diagram of the enzyme addition optimization of the recombinant acetylxylan esterase ATAXE.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: escherichia coli (Escherichia coli) JM109, Escherichia coli (Escherichia coli) BL21(DE3) and pET-24a (+) plasmid were obtained from Novagen, Inc., as referred to in the following examples.

2. Enzymes and other biochemical reagents:

antibiotics (Kana) were purchased from shanghai bioengineering, inc;

the Exnase II seamless ligation kit was purchased from Nanjing Novozam Biotech, Inc.;

the medium molecular weight protein marker standard and the SDS-PAGE gel electrophoresis kit are purchased from Shanghai Bin Yuntian biotechnology limited company;

p-nitrophenyl phenolate (pNPA), Polyethylacrylate (PEA) were purchased from Sigma-Aldrich;

the PCR primer is synthesized by Wutin-free Tianlin bioengineering company, Inc.;

commercial enzyme CWB-3560 was purchased from Tianjin Chang vitamin science and technology Co., Ltd;

other domestic analytical reagents were purchased from the national pharmaceutical group chemical reagents, Inc.

3. Culture medium:

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

(2) TB culture medium: tryptone 12g/L, yeast powder 24g/L, glycerin 5g/L, KH₂PO₄ 2.31g/L，K₂HPO₄·3H₂O 16.43g/L。

4. Activity assay of acetylxylan esterase ATAXE

The specific method comprises the following steps: the enzyme activity was measured by continuous spectrophotometry at 50 ℃.

The reaction volume was 1.5mL, and the production rate of p-nitrophenol (pNP) was recorded at a wavelength of 405nm, with 30. mu.L of the crude enzyme solution from the fermentation obtained in example 1, 30. mu.L of p-nitrophenol acetate (pNPA) at a concentration of 50mmol/L, and 1.44mL of Tris-HCl buffer (pH 7.0).

The definition of enzyme activity is: the enzyme amount required for the pNPA catalytic hydrolysis to generate 1 mu mol pNP is an enzyme activity unit (U) at 50 ℃ per minute.

5. The commercial enzyme activity detection takes pNPB as a substrate, and the rest steps are the same as the activity analysis of the acetylxylan esterase ATAXE; the enzyme activity of the commercial enzyme is 2500U/mL.

6. 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.

Example 1: preparation of recombinant acetylxylan esterase ATAXE

The gene encoding acetylxylan esterase ATAXE (nucleotide sequence shown in SEQ ID NO. 2) was ligated to the polyclonal cleavage site of pET24a (+) plasmid to obtain recombinant plasmid pET24a (+) -ATAXE containing acetylxylan esterase gene ATAXE, and then the recombinant plasmid was transformed into Escherichia coli JM109 to obtain recombinant Escherichia coli strain JM 109/ATAXE. The recombinant plasmid is transformed into Escherichia coli BL21 to obtain recombinant Escherichia coli strain E.coli BL21(DE3)/pET24a (+) -ataxe.

Coli BL21(DE3)/pET24a (+) -ataxe is inoculated in a liquid LB culture medium (containing 30 ug/mL kanamycin) for 8-10h, the seeds are inoculated into a TB liquid fermentation culture medium (containing 30 ug/mL kanamycin) according to the inoculation amount of 5% (v/v), the shake flasks are placed at 37 ℃ and 200r/min for 2h of isothermal culture, Isopropyl-beta-D-thiogalactopyranoside (IPTG) is added for induction, and the isothermal culture is continued for 24h under the condition of changing to 25 ℃ and 200 r/min. Centrifuging the fermentation liquid for 20min at 8000r/min, collecting thallus, re-dissolving with buffer solution, homogenizing and crushing at high pressure, centrifuging the crushed liquid at 4 deg.C and 12000rpm for 15min, and collecting supernatant to obtain crude fermentation liquid of acetylxylan esterase.

Performing enzyme activity determination and SDS-PAGE protein electrophoresis detection on the supernatant crude enzyme solution (figure 1), purifying by using a nickel affinity column, eluting the target protein by using imidazole (10mmol/L-300mmol/L) with different concentrations, collecting eluates with different concentration gradients, and performing enzyme activity determination and SDS-PAGE protein electrophoresis detection on the eluates respectively to detect the purity. After being purified by a nickel column, SDS-PAGE results show that the recombinant beta-xylosidase is expressed in escherichia coli. The results showed that electrophoretic purity was achieved and the size of the purified protein was approximately 36.8kDa (FIG. 2). The specific activity of acetylxylan esterase was found to be 176.3U/mg.

Example 2: determination of enzymatic Properties of recombinant acetylxylan esterase ATAXE

1. Determination of optimum pH and pH stability of recombinant acetylxylan esterase ATAXE

The recombinant acetylxylan esterase purified in example 1 was subjected to enzymatic reactions at different pH to determine its optimum pH.

Selecting a proper pH value range at 50 ℃, and respectively determining the enzyme activities of the recombinase at different pH values. The enzyme activity was measured as described in example 2. The highest enzyme activity is defined as 100%, and the enzyme activities measured under other pH conditions and the highest enzyme activity are converted into relative enzyme activities according to a proportion, so that the optimum pH of the recombinase is explored. The buffers used were: na (Na)₂HPO₄-KH₂PO₄Buffer solution (pH 6.0-8.0), Na2CO₃-NaHCO₃Buffer (pH8.0-9.0).

As shown in FIG. 3, the recombinase has an optimum pH of 7.5 when pNPA is used as a substrate, and the enzyme activity is maintained at 80% or more in the pH range of 7.5 to 9.0.

Incubating the purified enzyme at 37 ℃, selecting the range of pH 5.0-10.0, placing a proper amount of recombinant enzyme liquid in a water bath, preserving the temperature for 72 hours, and then determining the residual enzyme activity. The enzyme activity at the initial time is defined as 100%, and the enzyme activity measured by sampling at different time periods and the initial enzyme activity are converted into relative enzyme activity according to a proportion, so that the pH stability of the recombinase is researched. The buffers used were: na (Na)₂HPO₄-KH₂PO₄Buffer solution (pH 5.0-8.0), Na2CO₃-NaHCO₃Buffer (pH 8.0-10.0). As shown in FIG. 4, the recombinant enzyme had good stability around pH which is slightly acidic and neutral, and the enzyme activity was maintained at 80% or more, with a pH stability range of 5.0 to 8.0, when pNPA was used as the substrate.

2. Determination of optimum temperature and thermal stability of recombinant acetylxylan esterase ATAXE

The optimum temperature of recombinant acetylxylan esterase ATAXE is determined as Na₂HPO₄-KH₂PO₄(pH7.5) in a buffer solution system, and measuring the enzyme activity of the purified enzyme at different temperatures (60-85 ℃). The enzyme activities of the recombinant enzymes at different temperatures were determined separately at 5 ℃ intervals. The highest enzyme activity is defined as 100%, and the enzyme activity and the highest enzyme activity measured under other temperature conditions are converted into relative enzyme activity according to a proportion, so that the optimal reaction temperature of the recombinase is explored. The results of the measurement of the optimal temperature of the enzyme reaction are shown in fig. 5, the optimal temperature of the activity of the recombinant enzyme is 70 ℃, the enzyme activity is remarkably reduced after 70 ℃, and the recombinant enzyme belongs to medium-high temperature enzyme, but the relative enzyme activities of ATAXE at 60 ℃ and 85 ℃ are 67% and 75% respectively, which indicates that the reaction temperature range of the enzyme is wide.

The thermal stability was determined by measuring the thermal stability in Na₂HPO₄-KH₂PO₄(pH7.5) the diluted recombinant enzyme solution was placed in a 50 ℃ water bath shaker at an industrially applicable temperatureIncubation, which was sampled every 10h and the residual enzyme activity was determined under optimal temperature and pH conditions, without any treatment of the enzyme as a control. The enzyme activity at the initial time is defined as 100%, the enzyme activity measured by sampling at different time periods and the initial enzyme activity are converted into relative enzyme activity according to a proportion, so that the temperature stability of the recombinase is researched, and each experiment is carried out in parallel for three times. The thermal stability test of the enzyme shows that the recombinant ATAXE can keep more than 90% of the activity within 80h, and the half-life period is 84 h. (FIG. 6).

Example 3: application of acetyl xylan esterase ATAXE in degradation mode substrate

Preparing a substrate:

(1) 1% (w/v) PEA solution: accurately weighing 1g of PEA, adding an appropriate amount of acetone solution, fully dissolving, and then fixing the volume to 100mL by using a volumetric flask.

Degradation experiment steps:

acetyl xylan esterase ATAXE group: the purified acetylxylan esterase enzyme solution was diluted to 17.63U/mL in advance, and 8.5mL of a preheated 10min buffer (pH7.5), 0.5mL of the enzyme solution (equivalent to 881.5U/g substrate), and finally 1mL of the substrate PEA, 10mL in total, were added to each of 15mL clear glass vials. Then the mixture is placed in a water bath shaker at 50 ℃ and 200r/min for reaction for 4 h.

② commercial enzyme group: the enzyme activity of the commercial enzyme is 2500U/mL, the specific activity is 833.3U/mg, after the protein concentration is diluted to 0.1g/L (83.33U/mL), 8.5mL of buffer solution which is preheated for 10min is respectively added into a 15mL transparent glass bottle, 0.5mL of pre-diluted commercial enzyme solution (equivalent to 4166.5U/g substrate) is added, and finally 1mL of substrate PEA is added, wherein the total amount is 10 mL. Then the mixture is placed in a water bath shaker at 50 ℃ and 200r/min for reaction for 4 h.

(2) Turbidity Change measurement

And (2) respectively measuring the light absorption value at the wavelength of 600nm by using a spectrophotometer at 0.5h, 1h, 2h and 4h in the reaction process of the step (1) to characterize the change of turbidity in the degradation experiment process. The turbidity at the beginning of the reaction was defined as 100%, and the turbidity measured during the reaction was converted to relative turbidity in proportion to the initial turbidity. To reduce experimental error, three replicates of each group were run.

In the paper industry, PEA has good adhesiveness, so PEA is widely applied to various paper adhesives. In the process of recycling the used paper, the used paper is continuously retained on the production line of the paper making machine, thereby causing the quality of the subsequent paper to be reduced and increasing the production cost. PEA itself is soluble in an organic solvent such as acetone, and forms a suspension in an aqueous system. After the enzyme solution is added into the system, ester bonds of PEA side chains are degraded along with the continuous reaction, so that the hydrophilicity of the PEA side chains is enhanced, the aggregation tendency of PEA is inhibited, and the turbidity is reduced to a certain extent. FIG. 7 shows the turbidity change curve of ATAXE against PEA as a model substrate, and the results show that the turbidity of ATAXE is reduced remarkably, and after 4 hours of reaction, the turbidity is reduced by about 50.4%; after 4 hours, the turbidity of the commercial enzyme group is reduced by about 25.5 percent. Therefore, ATAXE has obvious PEA degradation effect, better than that of the commercial enzyme group, and better industrial applicability.

(3) Measurement of particle diameter and Zeta potential

And (3) stopping the reaction by adopting a method of boiling in hot water for 10min after the degradation experiment is finished, cooling at room temperature, taking out a proper amount of reaction liquid from the system, centrifuging at 12000r/min, and filtering the supernatant by using an organic filter head with the pore diameter of 0.22 mu m. And (3) sucking 1mL of filtrate into a measuring vessel by using an injector, detecting by using a nanometer particle size and Zeta potential analyzer, and taking the average value of three parallel controls on the detection results of the particle size and the Zeta potential.

The particle size detection is one of important means for analyzing the agglomeration state of fine particles, and the change of the particle size is often used for representing the degradation effect of a substrate in a system. And (3) removing relatively large particles from the PEA turbid solution after the enzyme degradation through high-speed centrifugation, and analyzing the particle size of the PEA turbid solution. FIG. 8 shows the change of the particle size of the PEA as a model substrate by acetylxylan esterase ATAXE, the particle size of the PEA as a control group is 502nm, the particle size of the PEA after the commercial enzyme is used for acting on the PEA is slightly reduced to 460nm, and the change range is small. The particle size of PEA turbid liquid treated by ATAXE is smaller than 277nm, the degradation effect is optimal, and the particle size of ATAXE compared with the commercial enzyme group is reduced by 39.8%. In conclusion, the ATAXE is shown to destroy the ester bond structure of the polymer in the adhesive, and has a certain effect on relieving the viscosity of the adhesive.

② the principle of measuring the Zeta potential is to characterize the stability of the particles in the system after the enzyme reaction by measuring the charging condition of the fine particles. It has been shown that the Zeta potential reflects the aggregation and dispersion of the protein and electrolyte phases during the process, and that a higher absolute value of the Zeta potential indicates a more stable system in nature, which is called the charge stability of the colloidal system.

FIG. 9 shows the Zeta potential change of acetyl xylan esterase ATAXE on PEA as a model substrate, and the results of using a blank group without enzyme solution as a control show that the potential absolute values of ATAXE and a commercial enzyme group are increased and are both 26.5mv, and the potential of the control group is 23.7mv, which indicates that the reaction system of ATAXE and the commercial enzyme group is more stable, and the colloid charged substance is relatively stable and is not easy to generate flocculation.

(4) Determination of acetic acid and ethanol content

The analysis of the acetic acid and ethanol content was performed by gas chromatography. The acetic acid determination was by common injection method and the ethanol determination was by headspace method. Gas phase conditions: RTX-WAX chromatographic column (30m × 0.32mm × 0.25 μm), with air, hydrogen and tail gas flow rates of 400 mL/min^-1，40mL·min^-1And 30 mL. min^-1The split ratio is 3:1, the injection inlet temperature is 150 ℃, and the injection amount is 1 mu L.

Since the side chain ester bond of the basic structure of PEA is broken by the action of enzyme in the process of degrading PEA to release ethanol, the release amount of ethanol after the enzyme reaction can be used as one of the measurement standards of PEA degradation effect. And measuring the ethanol content by a gas chromatograph by adopting a headspace sampling method. As a result, as shown in Table 1, the blank group had a trace amount of ethanol generated, probably because PEA moieties were destroyed during the termination reaction, and ethanol was released by cleavage of ester bonds due to high temperature. The ethanol release amount of the ATAXE and the PEA treated by the commercial enzyme is 0.48mmol/L and 0.23mmol/L respectively, and the ethanol release amount of the ATAXE is 2.1 times of that of the commercial enzyme group. From the product perspective, it can be speculated that the recombinase has higher side chain cleavage degree on PEA than the commercial enzyme, and can reduce the occurrence of the flocculation phenomenon of stickies in the paper industry.

TABLE 1 recombinant enzyme degradation PEA ethanol Release amount

Example 4

As shown in the same manner as in example 3, the potential, particle size and turbidity change of PEA by the enzyme were measured, except that the amount of ATAXE added was changed to 25. mu.L (corresponding to 44.075U/g substrate), 50. mu.L (corresponding to 88.15U/g substrate), 100. mu.L (corresponding to 176.3U/g substrate) and 200. mu.L (corresponding to 352.6U/g substrate), and that the amounts of commercial enzymes added were 50. mu.L, 100. mu.L and 200. mu.L, and the results showed that the amount of added enzyme had a certain effect. The potential change is shown in FIG. 10, which is the particle size change of the mode substrate PEA by acetylxylan esterase ATAXE and commercial enzyme, the particle size of the control group PEA is 637.9nm, the particle size of ATAXE and commercial enzyme is gradually reduced along with the increase of enzyme adding amount, and when the enzyme adding amount is 50 μ L, the particle size of ATAXE and commercial enzyme after treatment is 447.7nm and 533.7nm respectively. The change range of the particle size of the PEA turbid solution treated by low enzyme dosage ATAXE is larger, and the degradation effect is better than that of a commercial enzyme group. FIG. 11 shows the Zeta potential change of acetyl xylan esterase ATAXE and commercial enzyme for optimizing the enzyme addition amount of a mode substrate PEA, and the result of taking a blank group without enzyme solution as a control shows that the potential absolute values of ATAXE and commercial enzyme groups are increased compared with the control group, and the PEA treated by ATAXE and commercial enzyme is not easy to flocculate. In the review, the enzyme adding amount has certain influence on the degradation of PEA, and the performance change of the PEA is increased along with the increase of the enzyme adding amount, which shows that the acetylxylan esterase ATAXE and the commercial enzyme have certain potential on the degradation of the stickies and are suitable for industrial application.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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 south of the Yangtze river

<120> BAA220058A

<130> acetyl xylan esterase cloning expression and application thereof in control of papermaking stickies

<160> 2

<170> PatentIn version 3.3

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acggaggcca gcgtgaccat caaaagtggc ggcgagaact ggttccaagc catcgtggat 180

ggtaacccgc tgccaccgtt tagcgtgaat gcgaccacca gtacggtgaa actggtgagc 240

ggtctggcgg aaggtgcgca tcatctggtt ctgtggaaac gcaccgaagc gagtctgggc 300

gaagtgcagt ttctgggttt cgatttcggt agcggtaaac tgctggcggc cccaaaaccg 360

ctggagcgca agatcgagtt catcggtgat agcatcacgt gcgcctatgg caatgaaggt 420

acgagcaagg agcagagctt cacgccgaag aacgagaaca gctacatgag ctacgccgcc 480

attaccgccc gtaatctcaa cgccagcgcg aacatgatcg cgtggagcgg tatcggtctg 540

accatgaatt acggtggtgc cccgggcccg ctgattatgg accgctatcc gtacacgctg 600

ccatatagcg gtgttcgctg ggacttcagc aaatacgtgc cgcaagttgt ggttatcaat 660

ctgggcacca acgatttcag caccagcttt gccgacaaga cgaagttcgt gaccgcctac 720

aagaatctga tcagcgaagt gcgtcgcaac tacccggatg cccacatctt ctgctgcgtg 780

ggtccgatgc tctggggtac gggtctggat ctgtgccgca gttatgtgac cgaagtggtg 840

aacgactgca atcgcagcgg tgatctgaag gtgtacttcg tggagttccc acagcaagat 900

ggtagcaccg gttacggcga agattggcac ccgagcatcg ccacgcatca gctgatggcg 960

gaacgtctga cggcggagat caaaaacaaa ctgggctggc tggagtaa 1008

Claims

1. A method for degrading an adhesive, characterized in that the adhesive is hydrolyzed by acetylxylan esterase having an amino acid sequence shown in SEQ ID No. 1.

2. The method according to claim 1, wherein the acetylxylan esterase is added to a system containing stickies; adding the acetyl xylan esterase into a reaction system according to the addition amount of 40-1000U per gram of the adhesive for reaction.

3. The method according to claim 1 or 2, wherein the reaction is carried out at a pH of 7.5 to 9.0, 40 to 60 ℃ and 150 to 250rpm for not less than 4 hours.

4. The method of claim 3, wherein the adhesive comprises PEA.

5. The application of acetyl xylan esterase with an amino acid sequence shown as SEQ ID NO.1 in degrading adhesive substances.

6. Use according to claim 5, wherein the glue comprises PEA.

7. An expression vector containing a nucleotide sequence shown as SEQ ID NO.2, a microbial cell expressing an acetyl xylan esterase with an amino acid sequence shown as SEQ ID NO.1 or an application of the acetyl xylan esterase with an amino acid sequence shown as SEQ ID NO.1 in preparing products for degrading adhesive substances.

8. The use according to claim 7, wherein the expression vector containing the nucleotide sequence shown in SEQ ID No.2 is a starting vector selected from the group consisting of pET series, Duet series, pGEX series, pHY300PLK, pPIC3K and pPIC 9K; the microbial cells take escherichia coli as an initial strain.

9. Use according to claim 7 or 8, wherein the product comprises an enzyme preparation, a microbial preparation, a sewage treatment agent.

10. An expression vector containing a nucleotide sequence shown as SEQ ID NO.2, a microbial cell expressing an acetyl xylan esterase with an amino acid sequence shown as SEQ ID NO.1 or an application of the acetyl xylan esterase with an amino acid sequence shown as SEQ ID NO.1 in papermaking or waste paper recovery.