WO2024055376A1 - Method for treating adhesive in recycled pulp by means of pectin lyase and use - Google Patents

Abstract

A method for treating an adhesive in recycled pulp by means of a pectin lyase and a use. The pectin lyase PMGL-Ba is a pectinase capable of directly acting on highly methylated pectin to effectively cleave α-1,4 glycosidic bonds of polygalacturonic acids by means of a β-elimination reaction, so as to generate pecticoligosaccharides, without generating highly toxic methanol, and has the advantages of good enzyme selectivity, few byproducts, mild reaction conditions, high efficiency and the like. The enzyme can be efficiently expressed in pichia pastoris, and has the characteristics of high expression, simple purification, easy amplification, industrial applicability, environmental friendliness, high conditional adaptation and the like. A plurality of enzyme compounding solutions are provided. When used for treating an adhesive in pulping and papermaking, the pectin lyase can significantly improve the physical properties of paper and reduce the conductivity and turbidity, and is thus a potential novel papermaking complex enzyme preparation.

Description

A method and application of pectin lytic enzyme for treating regenerated pulp stickies Technical field

The invention belongs to the fields of genetic engineering and pulping and papermaking, and particularly relates to a method and application of a pectin lytic enzyme for treating regenerated pulp glue.

Background technique

The global pulp and paper industry continues to grow, with the global pulp market expected to grow to over $600 billion in value by 2027. Waste paper is an important fiber raw material for my country's paper industry, accounting for nearly 60%. Recycled pulp made from waste paper contributes to the recycling of resources and is more conducive to the sustainable development needs of the domestic paper industry. As the number of times waste paper is reused increases, adhesive substances increase sharply, resulting in practical problems such as reduced paper quality and increased frequency of paper machine shutdowns. Solving the problems such as stickiness accumulation during the secondary processing of waste paper is a key technology to improve the quality of recycled pulp.

Sticky substances refer to organic substances that are sticky, soft and hydrophobic that appear during the recycling process of recycled pulp. They are usually a combination of various substances, mainly including resins, adhesives, hot melts, pressure-sensitive substances, etc. Sizing agents and ink residues, etc., the main ingredients are pectin, polyvinyl acetate (PVAc), polyacrylate, styrene-butadiene rubber (SBR), ethylene vinyl acetate (EVA), etc. Adhesives have the characteristics of adhesion, lipophilicity, negative surface charge, and sexual deformation. They are easy to adhere and deposit in corresponding parts of the paper machine system, such as sticky substances blocking the forming wire section and drying cylinders in the drying section. There are sticky sheets or strips of sticky material sticking to the cylinder. This will lead to frequent shutdowns, more frequent scraper replacements, more holes in the paper produced, more black spots on the paper, reduced quality, and shortened replacement time for the forming wire and felt. At present, physical, chemical and biological methods are mainly used to remove and control stickies in slurry. The physical method mainly removes most large stickies and some fine stickies through screening, purification, thermal dispersion, mechanical treatment, etc. However, it has relatively high requirements for the selection of mechanical equipment and the design of the process flow, and there are high requirements. Issues such as energy consumption and low efficiency. The chemical method controls stickiness by adding various chemicals to the slurry. The chemical waste produced accumulates in the recycled white water, which has a negative impact on the normal operation of the paper machine system and the closed cycle of the papermaking white water, and It is easy to produce large amounts of industrial wastewater.

Biological enzymes have significant advantages such as high efficiency and environmental friendliness. Biological papermaking technology based on biological enzymes is an inevitable trend to achieve technological transformation and upgrading and green and healthy development of the papermaking industry. The enzymes currently on the market for adhesive control are mainly esterases and lipases, such as the esterase Optimyze series developed by Buckman Company, the adhesive control enzymes developed by Novozymes

wait. They are all carboxylate hydrolases, which can break the ester bonds in some adhesives, making the adhesives smaller in size and less efficient in adhesion. It is also difficult for the decomposed adhesives to re-polymerize into large particles. . Existing sticky matter control enzyme patents are also based on esterase and lipase. For example, CN106368035A discloses a method for removing sticky matter from waste paper pulp using high-temperature esterase TTEST; Publication No. CN109666661A provides a method for removing sticky matter from paper pulp. Biological enzyme preparation for sticky substances, modified high-temperature lipase; publication number CN114591931A describes the cloning and expression of an acetyl xylan esterase and its application in the control of papermaking sticky substances; in addition, a multi-enzyme compound degrades sticky substances The patents of biochemicals also mainly use ester enzymes as the main enzyme preparations. For example, CN109957558A introduces the preparation method of neutral esterase and its use in waste paper papermaking process. CN110983849A uses lipase and cutinase as adhesive control enzyme reagents. Although lipase and esterase have certain adhesive removal effects, there are many types of sticky substances in papermaking slurries, and their compositions vary greatly. Relying solely on lipase and esterase to degrade sticky materials, due to the selectivity of biological enzyme substrates, their effectiveness is limited to a certain type or type of sticky material. Therefore, the current biological enzymes have limited control over sticky materials. The effect is not ideal, and new enzymes need to be found to work together in different ways to remove sticky substances.

Contents of the invention

The object of the present invention is to provide a method for treating regenerated pulp glue with pectin lytic enzyme. Pectin lyase PMGL-Ba can withstand medium/high temperature and high alkali conditions, effectively remove stickies, and improve the quality of pulp. It can be combined with a variety of enzymes to control stickies in the production process of recycled pulp. At the same time, it can The method has the characteristics of environmental friendliness, low production cost, simple process, etc., and is suitable for industrial production.

Another object of the present invention is to provide the application of the above method.

The object of the present invention is achieved through the following technical solutions:

A method for treating regenerated pulp glue with pectin lytic enzyme, including the following steps:

Add enzymes to the regenerated pulp and obtain low stickiness pulp after treatment;

The enzyme includes pectin lyase PMGL-Ba, the NCBI database registration number of its amino acid sequence is WP_009329358.1.

The regenerated pulp is recycled from at least one of old corrugated cartons, book and magazine paper, old newspapers, scraps from carton factories, white paper trimmings from printing factories, cement bags, mixed waste paper and miscellaneous waste paper. Pulp; preferably recycled slurry prepared from waste container paper.

The pulp concentration of the regenerated pulp is 2-6%; preferably 2%.

The treatment conditions are pH 5.0-11.0 and temperature 30-90°C; preferably pH 7-8 and temperature 50-60°C; further preferably pH 8.5 and temperature 60°C.

The treatment time is 1 to 3 hours; preferably 3 hours.

The described treatment is a stirring treatment, and the rotation speed is 100-300 rpm; preferably 200 rpm.

The added amount of the pectin lytic enzyme PMGL-Ba is 2 to 50 U/g, based on the absolute dry mass of the regenerated pulp; the preferred added amount is 44 U/g.

The enzyme also includes at least one of lipase, pectic acid lyase (EC 4.2.2.2), amylase, sterolase, xylanase, and esterase; preferably, it also includes lipase and pectic acid. lyase.

The EC number of the lipase is EC 3.1.1.3.

The addition amount of the lipase is 20-30U/g, based on the absolute dry mass of the regenerated pulp.

The Genbank database registration number of the amino acid sequence of the pectate lyase is Genbank: AB428424.1.

The addition amount of the pectate lyase is 2 to 5 U/g, based on the absolute dry mass of the regenerated pulp.

The EC number of the amylase is EC 3.2.1.1.

The addition amount of the amylase is 200-400U/g, based on the absolute dry mass of the regenerated pulp.

The GenBank database registration number of the amino acid sequence of the sterolase is GenBank: AKZ66521.1.

The addition amount of the sterolase is 10 to 30 U/g, based on the absolute dry mass of the regenerated pulp.

The enzyme is a compound compound of lipase, pectate lyase and pectin lyase PMGL-Ba; preferably, it is compound compound according to an enzyme activity ratio of 20-30:2-4:2-50.

The enzyme is obtained by compounding pectate lyase, amylase and pectin lyase PMGL-Ba; preferably, it is compounded according to an enzyme activity ratio of 2-4:200-400:2-50.

The enzyme is obtained by compounding lipase, pectate lyase, amylase, sterolase and pectin lyase PMGL-Ba; preferably the enzyme activity ratio is 20-30:2-4:200-400:10 ～30:2～50 is obtained by compounding.

The low stickiness pulp is pulp with low white water turbidity and low electrical conductivity.

Application of the method for treating regenerated pulp glue with pectin lytic enzyme in papermaking.

The nucleotide sequence of the encoding gene of the pectin lyase PMGL-Ba is shown in SEQ ID NO.1.

The amino acid sequence of the pectin lyase PMGL-Ba is a polypeptide sequence containing at least 90% sequence identity with SEQ ID NO.1; preferably, it has at least 95% with SEQ ID NO.1, most preferably Polypeptides with at least 100% sequence identity; including a polypeptide derived from SEQ ID NO.1 that has been modified by substitution, deletion and/or insertion of one or more (several) amino acids, homology above 90%.

The pectin lyase PMGL-Ba is obtained by constructing a eukaryotic recombinant expression vector and transferring the codon-optimized coding gene nucleotide sequence into Pichia pastoris and then chromatographing the supernatant after fermentation.

The enzyme activity of the pectin lytic enzyme PMGL-Ba supernatant can reach 200-500U/mL, and the protein produced by the engineering bacteria can reach 0.4-0.9g/L. Preferably, the enzyme activity of the supernatant can reach 440.0U/mL, and the protein produced by the supernatant of the engineering bacteria can reach 0.89g/L.

The chromatography includes at least one of cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography and affinity chromatography. Preferably, the chromatography is Ni-NTA affinity chromatography.

Preferably, the starting vector of the eukaryotic recombinant expression vector includes but is not limited to pPICZαA.

Preferably, the Pichia pastoris includes but is not limited to Pichia pastoris X33.

Preferably, using pectin lyase PMGL-Ba alone can achieve the purpose of removing stickies and improving paper quality.

More preferably, when multiple enzymes are used in combination, the adhesive can be better degraded and the physical properties of the paper can be improved.

Compared with the existing technology, the present invention has the following advantages and effects:

Pectin lyase PMGL-Ba is a pectinase that can directly act on highly methylated pectin. It effectively cleaves the α-1,4-glycosidic bond of polygalacturonic acid through β-elimination reaction to produce pectin. Oligosaccharide, and does not produce highly toxic methanol. It has the advantages of good enzyme selectivity, less by-products, mild reaction conditions, and high efficiency. In the pulping and papermaking process, because the adhesive is negatively charged in the slurry system, it can adsorb various cationic substances, reducing the efficiency of the cationic polymer in adsorbing sticky substances, thereby aggravating the deposition of adhesive, so it is also called It is "anionic garbage", that is, dissolved and colloidal substances (DCS), the main substances of which include methylated pectin and polygalacturonic acid. Therefore, pectin lyase improves the efficiency of cationic polymers and reduces the deposition of sticky materials by degrading polygalacturonic acid in DCS. It can also act on polygalacturonic acid substances such as resins in sticky materials to thereby Degrade sticky substances. The present invention aims to develop a medium-alkaline, medium-high-temperature pectin lytic enzyme suitable for use in factory environments, which can specifically degrade methylated pectin, thereby reducing sticky deposits. It works together with esterase and xylanase The combined use of the two methods has better effects, has low production cost and simple process, and is more suitable for industrial production.

The present invention adopts a eukaryotic expression method to obtain pectin lyase derived from recombinant Bacillus, which has the characteristics of high expression level, simple purification, easy amplification, and is suitable for industrial application.

The pectin lytic enzyme PMGL-Ba in the present invention can cope with the medium-high temperature and medium-alkaline environment during the production of regenerated pulp, and can still maintain good sticky removal effect at higher temperatures.

The invention provides a method for treating papermaking adhesives with pectin lytic enzyme. The enzyme can be used alone or in combination with other types of enzymes. It can significantly improve the physical properties of paper and reduce turbidity and conductivity. It is A potential new enzyme preparation for papermaking, specifically,

The pectin lytic enzyme PMGL-Ba obtained by the present invention can effectively degrade polygalacturonic acid substances, improve the efficiency of cationic polymers, and thereby absorb more sticky substances;

Pectin lytic enzyme PMGL-Ba is used in combination with esterase, lipase, xylanase, etc. to hydrolyze the adhesive substances between cellulose and hemicellulose, cellulose and cellulose, effectively separating the fibers and increasing the The permeability of the fiber helps the fiber fully absorb water and swell, and enhances the physical properties of the paper such as tensile strength, tearing, ring pressure, and burst resistance.

Description of drawings

Figure 1 is a diagram of the SDS-PAGE analysis results of the recombinant pectin lyase PMGL-Ba Ni-NTA affinity chromatography in Example 1; wherein, lane M: molecular weight marker; lane 1: protein obtained after purification; lane 2: Pectin lyase PMGL-Ba crude enzyme solution.

Figure 2 is a diagram showing the enzymatic property analysis results of recombinant pectin lyase PMGL-Ba in Example 2; wherein, A: optimal reaction pH; B: pH stability; C: optimal reaction temperature; D: temperature stability.

Figure 3 is a diagram showing the analysis results of the effects of metal ions, reagents, etc. on the enzyme activity of recombinant pectin lyase PMGL-Ba in Example 2; A: the effect of metal ions and reagents on the enzyme activity; B: the effect of commonly used papermaking auxiliaries on the enzyme activity influence on enzyme activity.

Figure 4 is a diagram of the application effect of a single enzyme in the control of regenerated pulp stickiness in Example 3; A: the index results of conductivity, turbidity, and cation demand; B: the index results of tensile and tear strength Test; C: Test results of ring pressure and bursting strength.

Figure 5 is a diagram of the application effect of the compound enzyme in the control of regenerated pulp stickiness in Example 6; where, A: index results of conductivity and turbidity; B: index detection of tensile and tear strength; C : Test results of ring pressure and bursting strength.

Detailed ways

The present invention will be described in further detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

If no specific test conditions are specified in the following embodiments, conventional test conditions or test conditions recommended by the reagent company are usually followed. The materials, reagents, etc. used are all reagents and materials obtained from commercial sources unless otherwise specified.

In the embodiment, the amount of enzyme added was calculated relative to the absolute dry pulp mass, and the experimental results were repeated three times and averaged. The pectin lyase PMGL-Ba used in the present invention is derived from the Bacillus strain. Its amino acid sequence is as shown in the NCBI database ID WP_009329358.1, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 1.

Example 1 A method for producing recombinant pectin lyase PMGL-Ba

The whole gene synthesis method was used to obtain the coding gene of Pichia pastoris codon-optimized pectin lyase PMGL-Ba. The optimized nucleotide sequence of the coding gene is shown in SEQ ID NO: 1, which was synthesized by a commercial company. ; Use restriction endonucleases Eco RI and Not I to double-digest the target gene PMGL-Ba and the vector pPICZαA, use homologous recombination method for ligation, and transform the ligation product into E. coli TOP10 competent cells to obtain recombinant E. coli. The extracted recombinant plasmid pPICZαA-PMBL-Ba was identified and sequenced by Eco RI and Not I double enzyme digestion. The recombinant plasmid is digested with the restriction endonuclease DraI, integrated with the yeast genome, and formed a fusion protein with HIS to obtain a recombinant expression vector. Use electroporation method to transform it into the host cell Pichia pastoris X33 (for specific steps, please refer to the literature High-Level Expression and Biochemical Properties of A Thermo-Alkaline Pectate Lyase From Bacillus sp.RN1 in Pichia pastoris With Potential in Ramie Degumming) , obtain genetically engineered bacteria.

Inoculate the selected Pichia pastoris recombinant into 10 mL BMGY liquid medium, culture it with shaking at 30°C, 250 rpm overnight; centrifuge at 3000g for 2 minutes at room temperature, collect the cells, and transfer them to BMMY medium. The OD ₆₀₀ of the bacterial liquid is 0.5 to 1.0; continue to culture for 24 to 144 hours at 30°C and 250 rpm. During this period, samples are taken every 24 hours and methanol is added to the culture medium to 1.0% v/v. After the culture is completed, solid-liquid separation is performed to obtain fermentation. supernatant to obtain crude enzyme solution. The obtained fermentation supernatant was subjected to Ni-NTA column chromatography using the AKTA protein purification system. Elute with 20mM Tris buffer containing 50mM imidazole, concentrate and collect the target protein solution with an ultrafiltration tube (Millipore), and SDS-PAGE analysis shows that the pure pectin lyase PMGL-Ba target protein can be obtained, and the protein yield can reach 0.89 g/L. The NCBI database ID of the amino acid sequence of the obtained protein is WP_009329358.1. (figure 1).

Example 2 Determination of enzymatic properties of pectin lyase PMGL-Ba

1. Determination of enzyme activity

Take an appropriate amount of the pectin lyase PMGL-Ba solution obtained in Example 1 and centrifuge it at 10,000 rpm for 5 minutes, dilute the supernatant appropriately, and take out 10 μL to 2 mL of a 50 mM pH 8.5 Tris-HCl reaction system (containing 0.5% methylated pectin). , react at 60°C for 10 minutes, add DNS in a boiling water bath to develop color, and measure the absorption value at 540 nm; use the inactivated enzyme solution as a blank control group. Enzyme activity is defined as: under optimal conditions, the amount of enzyme required to hydrolyze the substrate to generate 1 μmoL galacturonic acid per minute is defined as one unit of enzyme activity, U.

2. Determination of optimal reaction conditions

According to the enzyme activity measurement method in step 1, place the pectin lytic enzyme PMGL-Ba enzyme solution (0.0089g/L, based on the amount of protein) at different pH (6.0~11.0, 60℃) and different temperatures (50~70 ℃, pH8.5), the enzyme activity of pectin lyase PMGL-Ba was determined to determine its optimal reaction pH and optimal reaction temperature. The results are shown in Figure 2A-B.

Afterwards, the enzyme solution (0.0089g/L) was placed in buffers with different pH (5.0~11.0) and treated at room temperature for 12 hours. The residual enzyme activity was measured according to the method in step 1 to study the pH of pectin lyase PMGL-Ba. stability. After the enzyme solution (0.0089g/L) was incubated at different temperatures (30°C ~ 90°C) for 1 hour, the residual enzyme activity was measured according to the method in step 1 to determine its temperature stability. The results are shown in Figure 2C~D Show. The buffer used in the above experiment used 50mM acetic acid-sodium acetate buffer for pH 5.0 to 6.0, 50mM Tris-HCl buffer for pH 7.0 to 9.0, and 50mM glycine-sodium hydroxide buffer for pH 10.0 to 11.0.

The results show that the optimal reaction pH of pectin lyase PMGL-Ba is 8.5 (Figure 2A); when treated for 12 hours in the pH range of 5.0 to 11.0, more than 80% of the enzyme activity can be maintained (Figure 2B); the optimal reaction temperature is 60°C (Figure 2C); when treated for 1 hour at a temperature lower than 60°C, the recombinant pectin lyase PMGL-Ba can maintain more than 80% of the enzyme activity (Figure 2D). Therefore, the pectin lytic enzyme PMGL-Ba of the present invention is suitable for the papermaking process environment.

3. Effects of metal ions and papermaking additives on enzyme activity

According to the above enzyme activity determination method, the effects of 2mM/5mM metal ions, surfactant SDS, metal ion chelating agent EDTA and 0.01%, 0.001% of different papermaking additives on the catalytic activity of recombinant pectin lyase PMGL-Ba were studied. ; Refer to the method in step 2, add 2mM/5mM different metal ions/papermaking aids to the enzyme solution (0.0089g/L), treat at room temperature for 12 hours, and measure the residual enzyme activity according to the method in step 1. The metal ions include Ca ²⁺ , K ⁺ , Na ⁺ , Cu ²⁺ , Fe ³⁺ , and Mg ²⁺ ; the eight common papermaking additives and their functions are:

CPAM, cationic polyacrylamide: paper enhancer, retention and drainage aid;

APAM, anionic polyacrylamide: retention and drainage aid;

NPAM, nonionic polyacrylamide: additive;

PEO, polyethylene oxide: retention and filter aid, dispersant;

CS, cationic starch: retention and drainage aid;

OS, oxidized starch: adhesive, sizing agent;

PAE, polyamide epichlorohydrin resin: wet strength agent;

AKD, polyalkyl ketene: sizing agent.

The usage concentration of the above-mentioned additives is generally 8~10KG/ton (absolutely dry pulp).

The results are shown in Figure 3. The experimental data show that Ca ²⁺ , Fe ³⁺ and SDS at a concentration of 5mM significantly inhibit the enzyme activity, among which Fe ³⁺ can reduce 85% of the enzyme activity; Na ⁺ , 2mM Ca ²⁺ can promote Enzyme activity, among which Ca ²⁺ can enhance the enzyme activity to 116%, other metal ions or reagents have a slight promotion effect or basically have no effect on pectin lyase PMGL-Ba (Figure 3A). None of the eight papermaking additives will damage the enzyme activity, and CPAM, PEO, and APAM at a concentration of 0.01% can significantly enhance the enzyme activity (Figure 3B). Therefore, the pectin lytic enzyme PMGL-Ba prepared in the present invention is active and stable in the presence of commonly used papermaking auxiliaries.

4. Substrate specificity analysis

According to the above enzyme activity assay method, under the condition of 50mM Tris-HCl buffer, 0.5% (w/v) polygalacturonic acid-containing substances from different sources are used as substrates, such as plant pectin, polygalacturonic acid from different sources. Galacturonic acid (PGA), methylated pectin, different regenerated pulps, etc. were reacted for 10 minutes at 60°C and pH 8.5 to test the substrate specificity of the recombinant pectin lyase PMGL-Ba (0.0089g /L). The results show that the pectin lytic enzyme PMGL-Ba can degrade different pectins, and can also degrade methylated pectin, and also has certain activity on different regenerated pulps. Therefore, the pectin lytic enzyme PMGL-Ba of the present invention is suitable for Used in the production process of recycled pulp.

Table 1 Analysis results of substrate specificity of pectin lyase PMGL-Ba

0.5% substrate	Enzyme activity (U/mL)	Relative enzyme activity (%)
PGA	15.24	3.04
Apple pectin	18.56	3.71
Citrus pectin	42.51	8.49
Esterified pectin	500.47	100.00
Pectin	1220.41	243.85
old newspaper	0.02	-
Corrugated box-core paper	142.25	28.42
Corrugated box-inner paper	88.22	17.66

Example 3 Treatment of adhesives using biological enzymes alone

In this example, the regenerated slurry is a regenerated slurry prepared from recycled OCC boxboard paper. Weigh an OCC pulp board with a dry weight of 20g, add deionized water to dilute the slurry to 2%, and add different amounts of biomass. The enzymes are as follows: pectin lyase PMGL-Ba 44U/g (relative to absolute dry pulp mass); pectate lyase PEL 3U/g; endocellulase EG1 3U/g; xylanase XYN 175U/g ; Laccase LacTT 0.05U/g; Sterol esterase CHE 20U/g; Amylase Amy-K 300U/g. React for 3 hours under the optimal temperature and pH conditions of each enzyme, with a rotation speed of 300 rpm. The specific temperatures and pH are as follows: the temperature of pectin lyase PMGL-Ba is 60°C and pH 8.5; the temperature of pectate lyase PEL is 80°C and pH 10.0; the temperature of endocellulase EG1 is 60°C and pH 5.0; The temperature of xylanase XYN is 70°C and pH 9.0; the temperature of laccase LacTT is 90°C and pH 7.0; the temperature of sterol esterase CHE is 50°C and pH 7.0; the temperature of amylase Amy-K is 50°C and pH 6.0. After the reaction, collect the slurry, balance the moisture, and use the Kaiser papermaking system to make paper. The paper weight is 80g/m ² and the white water circulation mode is used. Each slurry sample has 3 sets of parallel samples. White water is collected at the last set of parallel samples and passed through The turbidity, cation demand (CD) and conductivity of white water indirectly indicate the content of fine sticky substances. The examples used L&W tensile strength tester, L&W bursting strength tester, L&W crush tester, and L&W tear tester in accordance with national standards GB/T 12914-2008, GB/T 454-2002, TAPPI T810om-98, respectively. TAPPI T414om-12 measures the tensile strength, bursting strength, ring crush strength and tear strength of paper. The results of each test are the average of four groups of parallel samples. Sticky matter detection uses a turbidity meter to measure the turbidity of white water, a conductivity meter to measure the particle size, and a PCD-03PH particle charge analyzer to measure the cation demand to indirectly characterize the content of fine sticky matter. Among them, turbidity can indirectly reflect the CS (colloidal substances) content in the test sample; cation demand/conductivity can reflect the strength of the negative charge of colloidal substances and dissolved substances, thereby inferring the DS (dissolved substances) in the white water sample. substance) content. Without adding enzyme, waste paper pulp was treated under the same conditions, and the experimental measurement results were for the raw material group.

The results shown in Figure 4 show that when a single enzyme is used in the treatment of regenerated pulp, except for the obvious decrease in paper tear strength after amylase and sterolase treatment, other paper properties are improved compared to the blank control; for adhesives In terms of removal effect, after treatment with pectin lyase PMGL-Ba, pectate lyase, sterolase and amylase, the content of fine sticky substances in white water was significantly reduced. Compared with pectate lyase (EC 4.2.2.2), pectin lyase PMGL-Ba is applied to paper pulp and can greatly improve the tear strength of paper by specifically degrading methylated polygalacturonic acid. , and reduce the content of fine adhesives even less. Therefore, the pectin lytic enzyme PMGL-Ba of the present invention has good application potential in pulping and papermaking.

Example 4 Glue treatment of factory pulp by pectin lytic enzyme PMGL-Ba

The regenerated pulp in this example is taken from the pulp storage tank of a paper mill in Dongguan. The waste paper pulp has gone through the pulping and flotation processes, but no wet end additives have been added. Weigh the factory slurry with a dry weight of 20g, add deionized water to control the slurry concentration to 2%, add 10U/g of pectin lyase PMGL-Ba enzyme solution, the treatment temperature is 60°C, the pH is 7.2, and the treatment time is 1 hour, the stirring speed is 200rpm. After completing the reaction, place it in boiling water for 5 minutes to inactivate the enzyme. Use a 200-mesh pulp bag to clean the pulp, filter it, and maintain a balanced humidity for 24 hours for later use. Use the Kaiser method automatic papermaking machine to make handsheets with a basis weight of 80g/ ^m2 , and dry them at 90°C for 10 minutes after forming. The handsheets were placed in a constant temperature and humidity laboratory (temperature (23±1)°C, relative humidity (50±2)%) for 12 hours and then the paper strength was tested. At the same time, the white water used for papermaking treated with different biological enzymes was taken from the Kaiser method automatic papermaking machine and stored at 4°C.

Various indicators were measured according to the measurement method of Example 3. The results show that compared with the original pulp, the application of pectin lyase PMGL-Ba can enhance various physical properties of paper, including tensile strength that can be enhanced by 125%, tear strength by 124%, and ring compressive strength by 157%. %, the bursting strength can be enhanced by 132%; the use of pectin lytic enzyme PMGL-Ba also reduces the content of fine gums, in which the turbidity can be reduced to 47% and the conductivity can be reduced to 97%. Therefore, the pectin lytic enzyme PMGL-Ba of the present invention can also be used alone to treat sticky matter in regenerated pulp and enhance paper quality.

Example 5

The slurry used in this example comes from the same source as Example 4. Weigh the factory slurry with a dry weight of 20g, add deionized water to control the slurry concentration to 2%, and add 5U/g pectin lyase PMGL-Ba and 5U /g of xylanase react together, the treatment temperature is 60°C, the pH is 7.2, the treatment time is 1 hour, and the stirring speed is 200 rpm. Subsequently, various values were tested according to Example 3.

The results show that compared with the original pulp, composite enzyme treatment can also improve the physical properties of paper, including tensile strength that can be enhanced by 113%, tear strength by 107%, ring crush strength by 130%, and bursting strength by 106%. %; it also reduces the content of fine sticky substances, and the turbidity can be reduced to 74%. Compared with the single use of pectin lyase PMGL-Ba, the combined use of biological enzymes can significantly reduce the conductivity of white water to 84% .

Therefore, the pectin lytic enzyme PMGL-Ba provided by the present invention can be used in factory pulp in conjunction with xylanase, which can significantly improve the physical properties of paper and better reduce the conductivity of white water.

Example 6 Treatment of adhesives using a combination of biological enzymes

In this embodiment, the recycled slurry is recycled OCC container paper, which is from the same source as in Example 3. Weigh an OCC pulp plate with a dry weight of 20g, add deionized water to dilute the pulp to 2%, and add different enzyme solutions for compound processing. Specifically: the addition amount of pectin lytic enzyme PMGL-Ba is 44U/g (relative to (Based on absolute dry pulp mass), lipase ARL (EC 3.1.1.3) 26.2U/g, pectate lyase PEL (GenBank Accession No.AB428424.1) 3U/g, amylase Amy-K (EC 3.2.1.1 ) 300U/g, sterolase CHE (GenBank: AKZ66521.1) 20U/g. The treatment temperature is 50°C, the pH is 7.2, the treatment time is 3 hours, and the stirring speed is 200 rpm. Subsequent detection indicators and methods were characterized according to Example 3. The results of using multiple enzymes to control adhesives are shown in Figure 5.

Among them, compound enzyme formula A refers to: lipase ARL, pectate lyase PEL, amylase Amy-K;

Compound enzyme formula B refers to: lipase ARL, pectate lyase PEL, sterolase CHE;

Compound enzyme formula C refers to: lipase ARL, pectate lyase PEL, pectin lyase PMGL-Ba;

Compound enzyme formula D refers to: pectate lyase PEL, amylase Amy-K, sterolase CHE;

Compound enzyme formula E refers to: pectate lyase PEL, amylase Amy-K, pectin lyase PMGL-Ba;

Compound enzyme formula F refers to: lipase ARL, pectate lyase PEL, amylase Amy-K, sterolase CHE;

Compound enzyme formula G refers to: lipase ARL, pectate lyase PEL, amylase Amy-K, sterolase CHE, pectin lyase PMGL-Ba.

The comprehensive results show that in the multi-enzyme combined treatment of regenerated pulp, compound enzyme B has the best effect in terms of conductivity treatment, followed by C, D and E, which can also reduce the conductivity; in terms of turbidity treatment, compound enzyme C has the best effect. Well, secondly, the combination of D and F also has a certain effect (Figure 5A). Figure 5B shows the analysis of paper tensile and tear strength. The results show that in terms of tensile strength, compound enzyme treatment reduced this index; for tear strength, except for E, all other enzyme treatments enhanced this index, among which F , G compound has the best effect. Figure 5C shows the analysis of ring pressure and bursting strength. Treatment with complex enzymes E, F, and G can slightly increase the ring pressure strength; treatment with enzymes A, B, and C slightly reduces the bursting strength. In comparison, pectin lyase PMGL-Ba compounded with lipase and pectate lyase is suitable for treating sticky materials, while pectin lyase PMGL-Ba compounded with lipase, pectate lyase, amylase, and sterols Esterase enzymes can be used to enhance paper physical properties. Therefore, the pectin lytic enzyme PMGL-Ba of the present invention is suitable for use with a variety of enzymes to treat regenerated pulp, reduce the sticky content and improve paper performance.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. A method for treating regenerated pulp glue with pectin lytic enzyme, which is characterized by comprising the following steps:

   Add enzymes to the regenerated pulp and obtain low stickiness pulp after treatment;

   The enzyme includes pectin lyase PMGL-Ba, the NCBI database registration number of its amino acid sequence is WP_009329358.1.
2. The method according to claim 1, characterized in that:

   The enzyme also includes at least one of lipase, pectate lyase, amylase, sterolase, xylanase, and esterase.
3. The method according to claim 1, characterized in that:

   The enzyme is lipase, pectic acid lyase and pectin lyase PMGL-Ba, which are compounded according to an enzyme activity ratio of 20-30:2-4:2-50.
4. The method according to claim 1, characterized in that:

   The enzyme is a compound of pectate lyase, amylase and pectin lyase PMGL-Ba at an enzyme activity ratio of 2-4:200-400:2-50.
5. The method according to claim 1, characterized in that:

   The enzymes are lipase, pectate lyase, amylase, sterolase and pectin lyase PMGL-Ba according to the enzyme activity ratio 20~30:2~4:200~400:10~30:2~50 Remixed.
6. The method according to claim 1, characterized in that:

   The regenerated pulp is recycled from at least one of old corrugated cartons, book and magazine paper, old newspapers, scraps from carton factories, white paper trimmings from printing factories, cement bags, mixed waste paper and miscellaneous waste paper. slurry;

   The pulp concentration of the regenerated pulp is 2 to 6%;

   The treatment conditions are pH 5.0-11.0 and temperature 30-90°C;

   The processing time is 1 to 3 hours;

   The described treatment is a stirring treatment, and the rotation speed is 100-300 rpm.
7. The method according to claim 1, characterized in that:

   The addition amount of the pectin lytic enzyme PMGL-Ba is 2 to 50 U/g, based on the absolute dry mass of the regenerated pulp.
8. The method according to claim 1, characterized in that:

   The nucleotide sequence of the encoding gene of the pectin lyase PMGL-Ba is shown in SEQ ID NO.1;

   The amino acid sequence of the pectin lyase PMGL-Ba has at least 90% sequence identity with the sequence shown in SEQ ID NO.1;

   Further, the amino acid sequence of the pectin lyase PMGL-Ba has at least 95% sequence identity with SEQ ID NO.1;

   Further, the amino acid sequence of the pectin lyase PMGL-Ba has at least 100% sequence identity with SEQ ID NO.1;

   Further, the pectin lyase PMGL-Ba includes a polypeptide derived from SEQ ID NO. 1, which is modified by substituting, deleting and/or inserting one or more amino acids, and the homology is at More than 90.
9. The method according to claim 1, characterized in that:

   The pectin lyase PMGL-Ba is obtained by constructing a eukaryotic recombinant expression vector and transferring the codon-optimized coding gene nucleotide sequence into Pichia pastoris and then chromatographing the supernatant after fermentation.
10. Application of the method of treating regenerated pulp glue with pectin lytic enzyme according to any one of claims 1 to 9 in papermaking.

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