CN110983849A

CN110983849A - Method for degrading adhesive by compounding multiple enzymes and application thereof

Info

Publication number: CN110983849A
Application number: CN201911322345.8A
Authority: CN
Inventors: 吴敬; 宿玲恰; 李光耀
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2020-04-10

Abstract

The invention discloses a method for degrading adhesive by compounding multiple enzymes and application thereof, belonging to the technical field of enzyme engineering. According to the invention, the cutinase and the lipase are compounded, so that the degradation effect of the adhesive is greatly improved; by using the method of the invention, cutinase and lipase are respectively added at 0.8U/g_{Adhesive material}And 1.6U/g_{Adhesive material}The additive amount is added into the crushed pulp generated in the papermaking process containing the stickies for reaction, and the total area of the stickies in the crushed pulp can be reduced by 33.44 percent only by reacting for 30 min.

Description

Method for degrading adhesive by compounding multiple enzymes and application thereof

Technical Field

The invention relates to a method for degrading adhesive by compounding multiple enzymes and application thereof, belonging to the technical field of enzyme engineering.

Background

With the development of economy, the paper making industry in China is also rapidly developed, and in recent years, China has become the largest world-wide paper product producing country and waste paper recycling country. Meanwhile, along with the increase of the protection of forest resources, the environmental awareness of people is continuously improved, in order to protect the environment, the chopping of forest logs is reduced, the ecological balance is kept, and the waste paper recycling receives more and more attention of people.

The waste paper is recycled and reused, and the waste paper is mainly recycled and then processed in a series of ways to produce recycled paper, so that the process not only can save a large amount of plant fiber raw materials and energy and reduce the cost of the paper, but also is beneficial to protecting the environment and reducing the damage to the ecological environment, and the process has economic benefits and good social and environmental benefits.

However, with the continuous improvement of the recycling rate of the waste paper and the continuous increase of the degree of closing of the white water circulation system in the pulping and papermaking, the problem of stickies in the recycling process of the waste paper becomes serious, and the actual production of the recycled paper is seriously influenced.

Depending on the mode of production, adhesives can be divided into primary adhesives (primary adhesives) and secondary adhesives (secondary adhesives). The primary stickies originally exist in the paper pulp, are dispersed in the paper pulp in the pulping process and the subsequent treatment process of papermaking and are dispersed in the paper pulp in a solid form, and because the primary stickies have certain viscosity, the primary stickies are easy to deposit in the paper pulp to cause production accidents; the secondary stickies are from the same source as the primary stickies, but the secondary stickies are dispersed in the pulp in a dissolved form and have no viscosity, and when the physical and chemical environment of the pulp changes, the secondary stickies are unstable to form larger viscous aggregates, so that deposition occurs in the pulp, and production accidents are caused. Studies have shown that the secondary stickies are more concentrated in the pulp than the primary stickies, which indicates that the secondary stickies are more difficult to control and more hazardous than the primary stickies, and thus are particularly important to control of the secondary stickies.

At present, the methods for controlling stickies in pulp are mainly mechanical, chemical and biological. The mechanical method is mainly used for removing most of stickies in the waste paper through process control, and the common methods comprise screening, purification, flotation, washing, dispersion, classification and the like. However, the mechanical method has a great dependence on the design of the process flow and the performance of the equipment, and the quality of the design of the process flow and the quality of the performance of various equipment determine the removal efficiency of the stickies (see the specific references: Wangxu, Zhanhuayu, Chengang. process control technology of stickies in waste paper recycling [ J ]. Chinese paper-making institute, 2002(01): 116-.

The chemical method mainly achieves the purpose of controlling the stickies through the actions of chemical adsorption, modification, dispersion, fixation, surface passivation and the like, and the control objects are mainly regenerated stickies which are difficult to remove by a physical method. However, due to the high cost and unstable use effect, the chemical method is often the last remedy in the production process (see the reference document: Chenjiaxiang. the international research results on the problem of stickies generated when the waste paper is recycled and the current research direction [ J ] paper-making chemicals, 2004(01):1-4+ 12.).

Biological methods mainly decompose and break ether bonds between components of the stickies through enzymolysis, so as to reduce the size of particles of the stickies, reduce the viscosity of the stickies, and further prevent flocculation of the particles of the stickies. However, the enzyme activity of the conventional enzymes for degrading stickies is not so high that the effect of degrading stickies by using these enzymes is not very desirable. Therefore, there is still a need to find a more effective way to degrade stickies.

Disclosure of Invention

[ problem ] to

The invention aims to provide a method for degrading stickies with good effect.

[ solution ]

In order to solve the technical problem, the invention provides a method for degrading an adhesive by compounding multiple enzymes, which is to add cutinase and lipase into the adhesive or a mixture containing the adhesive for reaction.

In one embodiment of the invention, the cutinase is:

(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 1; and/or the presence of a gas in the gas,

(b) a protein consisting of an amino acid sequence shown in SEQ ID No. 2; and/or the presence of a gas in the gas,

(c) a protein derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a) and having cutinase activity; and/or the presence of a gas in the gas,

(d) a protein derived from (b) by substituting, deleting or adding one or more amino acids in the amino acid sequence of (b) and having cutinase activity.

In one embodiment of the invention, the lipase is:

(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 3; alternatively, the first and second electrodes may be,

(b) and (b) the protein which is derived from the (a) and has lipase activity, wherein the amino acid sequence in the (a) is substituted, deleted or added with one or more amino acids.

In one embodiment of the invention, the cutinase is:

(c) a protein derived from (a) having cutinase activity and having no less than 60% homology with the amino acid sequence in (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a); and/or the presence of a gas in the gas,

(d) a protein derived from (b) having cutinase activity and having no less than 60% homology with the amino acid sequence in (b) by substituting, deleting or adding one or more amino acids in the amino acid sequence in (b).

In one embodiment of the invention, the lipase is:

(b) and (b) a protein derived from (a) having lipase activity and having not less than 60% homology with the amino acid sequence in (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a).

In one embodiment of the invention, the cutinase is added to the adhesive or the mixture containing the adhesive in an amount of 0.1-3U/g_{Adhesive material}。

In one embodiment of the invention, the cutinase is added to the adhesive or the mixture containing the adhesive in an amount of 0.8U/g_{Adhesive material}。

In one embodiment of the invention, the lipase is present in a mucilage or a mixture containing a mucilageThe amount of the additive is 0.1 to 3U/g_{Adhesive material}。

In one embodiment of the invention, the lipase is added to the stickies or the mixtures containing stickies in an amount of 1.6U/g_{Adhesive material}。

In one embodiment of the present invention, the temperature of the reaction is 40 to 60 ℃.

In one embodiment of the invention, the temperature of the reaction is 50 ℃.

In one embodiment of the present invention, the reaction time is 20 to 40 min.

In one embodiment of the invention, the reaction time is 30 min.

In one embodiment of the present invention, the stickies refer to primary stickies and/or secondary stickies generated during the papermaking process.

In one embodiment of the present invention, the stickies refer to secondary stickies generated during the papermaking process.

In one embodiment of the invention, the stickies-containing mixture is the pulp from a papermaking process.

In one embodiment of the invention, the method comprises the steps of putting crushed pulp generated in the papermaking process into a polyester bag, uniformly kneading, then placing the uniformly kneaded crushed pulp for 30-40 min at the temperature of 40-60 ℃ for preheating, and finally adding cutinase and lipase into the preheated crushed pulp for reaction.

The invention also provides the application of the method in treating the adhesive.

The invention also provides application of the method in papermaking or waste paper recovery.

[ advantageous effects ]

According to the invention, the cutinase and the lipase are compounded, so that the degradation effect of the adhesive is greatly improved; by using the method of the invention, cutinase and lipase are respectively added at 0.8U/g_{Adhesive material}And 1.6U/g_{Adhesive material}Adding the additive into pulp generated in the papermaking process containing stickies for reaction for only 30min,the total area of stickies in the pulp was reduced by 33.44%.

Detailed Description

The crushed pulp generated in the paper making process related in the following examples is from the first four-stage fine screening process of recycled paper manufacturing workshop of Shandong Huatai paper industry Co., Ltd; the pPIC9K vector referred to in the following examples was purchased from semer flyer; pichia pastoris KM71, referred to in the examples below, was purchased from Shanghai Qincheng Biotech, Inc.

The preparation methods referred to in the following examples are as follows:

the cutinase (HiC) whose amino acid sequence is shown in SEQ ID No.1 (the nucleotide sequence of the gene coding for this cutinase is shown in SEQ ID No. 4) is specifically described in the references "Ruoyu Hong, Yirong Sun, Lingqai Su, Lengtao Gu, Fuqing Wang, Jing Wu ＊. High-level expression of Humicola insolens cutting enzyme in Pichia pastoris and out carbon residue and use in biological filtration. journal of Biotechnology,2019,304: 10-15".

The preparation of cutinase (TfC) whose amino acid sequence is shown in SEQ ID No.2 (the nucleotide sequence of the gene coding for this cutinase is shown in SEQ ID No. 5) is described in detail in the references Lingqia Su, RonaldW Woodard, Jian Chen, Ju, excellular location of Thermobifidaf ash enzyme expressed in Escherichia coli BL21(DE3) with output of a signal peptide, Applied and Environmental Microbiology,2013,79(14):4192 4198 ".

The preparation method of the lipase (TlL) with the amino acid sequence shown as SEQ ID No.3 (the nucleotide sequence of the gene coding the lipase is shown as SEQ ID No. 6) related in the following examples is as follows:

chemically synthesizing a gene which has a nucleotide sequence shown as SEQ ID No.6 and codes lipase (an amino acid sequence shown as SEQ ID No. 3); carrying out enzyme digestion on the obtained gene and a pPIC9K vector by using restriction enzymes EcoR I and Not I, and then connecting products obtained by enzyme digestion to obtain a recombinant plasmid pPIC 9K-TlL; electrically transferring the recombinant plasmid pPIC9K-TlL into pichia pastoris KM71 to obtain a transformation product; coating the transformation product on an MD solid culture medium, and performing inverted culture at 30 ℃ for 24 hours; selecting positive transformants, extracting plasmids for sequencing verification, and obtaining recombinant pichia pastoris KM71/pPIC9K-TlL after successful verification; selecting a single colony of recombinant pichia pastoris KM71/pPIC9K-TlL, inoculating the single colony into a seed culture medium, and culturing at 30 ℃ and 200rpm for 24 hours to obtain a seed solution; inoculating the seed solution into a fermentation medium in an inoculation amount of 5% (v/v), carrying out summer culture at 30 ℃ and 200rpm for 24h, adding methanol in an addition amount of 1% (v/v) into the fermentation medium, and carrying out induction culture at 30 ℃ and 200rpm for 96-144 h to obtain a fermentation liquid; centrifuging the fermentation liquor at 12000rpm for 20min, removing thallus, and collecting the supernatant as crude enzyme solution of lipase (TlL);

wherein, the MD solid culture medium: YNB 13.4g/L, biotin 4X 10^-4g/L, glucose 20g/L and agar 20 g/L;

seed culture medium: peptone 20g/L, yeast powder 5g/L, glycerol 30g/L and YNB 13.4 g/L;

fermentation medium: 0-20 g/L potassium sulfate, 12-18 g/L magnesium sulfate heptahydrate, 0.5-1.5 g/L calcium sulfate dihydrate, 3-6 g/L potassium hydroxide, 20-30 mL/L phosphoric acid and 20-40 g/L glycerol.

The detection methods referred to in the following examples are as follows:

determination of cutinase enzyme activity:

continuous spectrophotometry was used. The reaction system is 1.5mL and comprises 30 mu L of cutinase acetase liquid and 1470 mu L of Tris-HCl buffer solution (pH 8.0) containing 50mmol/L of sodium thiodeoxycholate and 50mmol/L of p-nitrobenzoate (pNPB); the reaction system was reacted at 37 ℃ and the absorbance of the reaction solution at 405nm was measured by an ultraviolet spectrophotometer during the reaction to record the production rate of p-nitrophenol (see specifically: Ruoyu Hong, Yirong Sun, Lingqai Su, Lengao Gu, Fuqing Wang, Jing Wu. High-level expression of Humicola insolens cutting enzyme in Pichia pastoris with out carbon residue standing and its use of luminescence surface biological biosourcing. journal of Biotechnology,2019,304: 10-15).

The cutinase enzyme activity is defined as: the enzyme amount for catalyzing the hydrolysis of p-nitrobenzyl butyrate to generate 1 mu mol of p-nitrophenol per minute is an enzyme activity unit (1U) at 37 ℃.

Determination of lipase activity:

continuous spectrophotometry was used. The reaction system is 1mL, and comprises 80 μ L of crude enzyme solution of lipase and 920 μ L of Tris-HCl buffer solution (pH 8.0) containing 2.5mmol/L of p-nitrophenyllaurate (pNPL); the reaction system was reacted at 37 ℃ and during the reaction, the absorbance of the reaction solution at 405nm was measured by an ultraviolet spectrophotometer to record the production rate of p-nitrophenol (see in particular references: Relihua, Zhenren Tou, willow Queen, et al. Thermomyces lanuginosus ZJB09222 lipase gene cloning and expression in E.coli [ J ]. food and fermentation industries, 2012,38(5): 56-60.).

Lipase activity is defined as: the enzyme amount for catalyzing the hydrolysis of the p-nitrophenyllaurate to generate 1 mu mol of p-nitrophenol per minute at 37 ℃ is an enzyme activity unit (1U).

Example 1: influence of enzyme type on degradation effect of mucilage

The method comprises the following specific steps:

(1) taking crushed pulp, carrying out suction filtration on the crushed pulp, and drying the crushed pulp in an oven at 115 ℃ for 45min to obtain dried crushed pulp;

(2) placing the dried crushed pulp into a polyester bag, uniformly kneading, and placing the uniformly kneaded crushed pulp for 30min at the temperature of 50 ℃ for preheating to obtain preheated crushed pulp;

(3) dividing the preheated crushed pulp into 6 groups, namely a blank control group, an HiC independent treatment group, a TfC independent treatment group, a TlL independent treatment group, a HiC + TlL compound treatment group and a TfC + TlL compound treatment group, wherein the preheated crushed pulp of the blank control group is not added with any crude enzyme solution, and the preheated crushed pulp of the HiC independent treatment group is added with 0.8U/g_{Adhesive material}Dry weight of HiC crude enzyme solution, 0.8U/g addition to preheated broke of TfC separate treatment group_{Adhesive material}Crude enzyme solution of TfC (dry weight), TlL Pre-heated pulped separately treated group, was added at 1.6U/g_{Adhesive material}(dry weight) TlL crude enzyme solution, HiC + TlL pre-heated pulping of combined processing group, 0.8U/g_{Adhesive material}HiC crude enzyme solution (dry weight)And 1.6U/g_{Adhesive material}(dry weight) TlL crude enzyme solution, 0.8U/g was added to preheated pulp from TfC + TlL complex treatment group_{Adhesive material}Crude enzyme solution of TfC (dry weight) and 1.6U/g_{Adhesive material}After the crude enzyme solution of TlL (dry weight) is added, all groups of preheated crushed pulp react for 30min at the temperature of 50 ℃ to obtain treated crushed pulp;

(4) filtering the treated crushed pulp, and diluting the crushed pulp with deionized water until the concentration of the adhesive is 1% (v/v) to obtain diluted crushed pulp; placing the diluted crushed pulp into a Pul Mac Master Screen sizing instrument, and separating large stickies in the diluted crushed pulp, wherein the used filter paper is black special filter paper;

(5) after separation, taking out the filter paper with the adhesive from a Pul Mac Master Screen sizing instrument, covering the filter paper with an anti-sticking paper with an organic silicon coating surface, drying at 94 ℃ and 95kPa for 10min, and removing the anti-sticking paper;

(6) immersing the filter paper with the anti-sticking paper removed in the step (5) into black water-based ink for dyeing to eliminate the influence of a small amount of pulp fibers, taking out the filter paper which is completely soaked by the black water-based ink, sucking the redundant ink on the filter paper, covering the filter paper with the anti-sticking paper with an organic silicon coating surface, drying at 94 ℃ under 95kPa for 10min, and removing the anti-sticking paper;

(7) uniformly scattering a layer of alumina powder on the surface of the filter paper with the anti-sticking paper removed in the step (6), clamping the filter paper between two paper boards, drying at 94 ℃ and 95kPa for 10min to uniformly adhere the alumina powder to the sticky spot area, removing the paper boards and the anti-sticking paper, and slightly sweeping the non-adhered alumina powder on the filter paper by using a soft brush to obtain the filter paper with the stickies;

(8) after the image was obtained on the scanner with the filter paper with the stickies, the image was analyzed with the Verity IA Master-Screen image analysis software to determine the total area (mm) of the stickies remaining²Kg) the degradation effect of the different groups of stickies is characterized (see Table 1 for the degradation effect).

As can be seen from Table 1, the effect of the combination of cutinase and lipase on the degradation of adhesive is far better than that of the single action of the cutinase and the lipase.

TABLE 1 Total residual adhesive area for different groups

Group of	Enzyme solution addition amount (U/g)_{Adhesive material})	Total area of remaining adhesive (mm)²/kg)
			Blank control	0	369.95
HiC independent processing	0.8	318.8
			Tfc alone treatment	0.8	311.08
TlL independent processing	1.6	351.95
			HiC + TlL compound treatment	0.8+1.6	246.25
TfC + TlL compound treatment	0.8+1.6	264.59

Example 2: influence of compounding ratio of enzyme on degradation effect of mucilage

The method comprises the following specific steps:

based on example 1, the compounding ratio of the HiC crude enzyme solution and TlL crude enzyme solution in the HiC + TlL compound treatment group was adjusted, and the specific adjustment method and the effect on the degradation effect of the stickies after adjustment are shown in table 2.

On the basis of example 1, the compounding ratio of the crude enzyme solution of TfC and crude enzyme solution of TlL in the TfC + TlL compounded treatment group was adjusted, and the specific adjustment method and the effect on the degradation effect of the stickies after adjustment are shown in table 3.

As is clear from tables 2 to 3, the amount of cutinase added was 0.8U/g_{Adhesive material}The amount of lipase added was 1.6U/g_{Adhesive material}The compound proportion has the best degradation effect on the adhesive when being compounded.

TABLE 2 Total residual adhesive area for different groups

Group of	Enzyme solution addition amount (U/g)_{Adhesive material})	Total area of remaining adhesive (mm)²/kg)
			Blank control	0	369.95
HiC + TlL compound treatment	0.8+1.6	246.25
			HiC+TlL*2	0.8+3.2	287.45
HiC*2+TlL	1.6+1.6	284.10
			HiC+TlL/2	0.8+0.8	293.13
HiC/2+TlL	0.4+1.6	330.02

TABLE 3 Total residual adhesive area for different groups

Group of	Enzyme solution addition amount (U/g)_{Adhesive material})	Total area of remaining adhesive (mm)²/kg)
			Blank control	0	369.95
TfC + TlL compound treatment	0.8+1.6	264.59
			TfC+TlL/2	0.8+0.8	312.73
TfC/2+TlL	0.4+1.6	291.73

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.

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gttggtaaca gagcttttgc tgaatttttg actgtccaaa ctggtggtac tttgtaccgt 600

attactcata ctaacgacat cgttccaaga ttgccaccta gagaatttgg ttactctcat 660

tcttctccag aatactggat taagtctggt actttggttc cagttactag aaatgatatt 720

gtcaagatcg agggtatcga tgctactggt ggtaacaatc aaccaaatat tcctgatatt 780

ccagcccatt tgtggtattt tggtttgatt ggtacttgct tg 822

Claims

1. The method for degrading the adhesive by compounding the multiple enzymes is characterized in that the cutinase and the lipase are added into the adhesive or a mixture containing the adhesive for reaction.

2. The method for degrading adhesive substances by compounding multiple enzymes according to claim 1, wherein the cutinase is:

3. The method for degrading adhesive substances by compounding multiple enzymes according to claim 1 or 2, wherein the lipase is:

4. The method for degrading adhesive substances by compounding multiple enzymes according to any one of claims 1 to 3, wherein the addition amount of the cutinase in the adhesive substances or the mixture containing the adhesive substances is 0.1-3U/g_{Adhesive material}。

5. The method for degrading adhesive substances by compounding multiple enzymes according to any one of claims 1 to 4, wherein the addition amount of the lipase in the adhesive substances or the mixture containing the adhesive substances is 0.1-3U/g_{Adhesive material}。

6. The method for degrading adhesive substances by compounding multiple enzymes according to any one of claims 1 to 5, wherein the reaction temperature is 40-60 ℃.

7. The method for degrading adhesive substances by compounding multiple enzymes according to any one of claims 1 to 6, wherein the reaction time is 20-40 min.

8. The method for degrading stickies by multi-enzyme combination according to any one of claims 1 to 7, wherein the stickies-containing mixture is the pulp from paper making; the method comprises the steps of putting crushed pulp generated in the papermaking process into a polyester bag, uniformly kneading, then placing the uniformly kneaded crushed pulp for 30-40 min at the temperature of 40-60 ℃ for preheating, and finally adding cutinase and lipase into the preheated crushed pulp for reaction.

9. Use of the method according to any one of claims 1 to 8 for treating adhesives.

10. Use of the method of any of claims 1-8 in paper manufacture or waste paper recovery.