CN114480446A - Method for constructing lignin degrading enzyme gene set - Google Patents
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- CN114480446A CN114480446A CN202210164778.0A CN202210164778A CN114480446A CN 114480446 A CN114480446 A CN 114480446A CN 202210164778 A CN202210164778 A CN 202210164778A CN 114480446 A CN114480446 A CN 114480446A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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Abstract
The invention provides a method for efficiently and conveniently constructing a lignin degrading enzyme gene set. A sample containing a large number of microorganisms in a natural or artificial environment is subjected to lignin degradation microorganism enrichment culture by taking lignin as a growth substrate under a laboratory condition. And identifying that the lignin degrading enzymes are produced or the lignin is degraded in the culture process, screening and identifying a plurality of lignin degrading enzymes by means of high-throughput sequencing and metagenomic analysis, and constructing a lignin degrading enzyme gene set. The invention greatly enlarges the source of lignin degrading enzyme screening, provides a large amount of degrading enzymes with excellent performance for constructing a lignin biorefinery, and can also promote the development of a lignocellulose full-component biorefinery.
Description
Technical Field
The invention belongs to the field of biomass lignin resource utilization, and particularly relates to a method for constructing a lignin degrading enzyme gene set by using a metagenomic technology.
Background
Renewable biomass resources are the only resources on the earth which can directly replace petroleum resources in a mode of liquid fuels and organic chemical products, and have important value in converting the biomass resources into bio-oil and aromatic chemicals under the aims of reducing the dependence of petroleum and fossil resources and realizing the environmental protection of carbon peak reaching and carbon neutralization, and the development of a biorefinery technology has economic and environmental protection feasibility. The lignin is one of the main components of lignocellulose, is the renewable aromatic high polymer with the largest source on the earth, mainly comprises C-O ether bonds, and is partially C-C bonds. The biological enzyme degradation method is concerned and paid attention to the efficient process due to the green color, but 99% of microorganisms cannot be cultured in a laboratory, so the types of enzymes excavated by the traditional method for culturing single bacteria and then separating and purifying target enzymes are very limited. The newly developed high-throughput sequencing technology can detect DNA information of all microorganisms in a sample, and can process and analyze DNA data to maximally mine genes of target enzymes.
The method firstly constructs a gene set of lignin degrading enzymes, excavates a plurality of lignin degrading enzymes with excellent performance, and provides a novel and efficient method for developing a green high-performance biocatalyst in a lignin biorefinery.
Disclosure of Invention
The invention aims to provide a method for constructing a lignin degrading enzyme gene set. The invention samples from natural/artificial environment, enriches and cultures lignin-degrading microorganisms in a laboratory by taking lignin as a substrate, obtains all DNA information of microbial communities in the sample by utilizing a high-throughput sequencing technology, screens and identifies genes of lignin degrading enzymes by utilizing a metagenomic analysis means, and constructs a lignin degrading enzyme gene set.
The invention provides a method for constructing a lignin degradation laccase gene set, which comprises the following specific steps:
(1) taking a sample rich in microorganisms from a natural or artificial environment;
(2) taking the sample obtained in the step (1), and enriching lignin degrading microorganisms in the obtained sample under a laboratory condition; the method specifically comprises the following steps: adding natural lignin serving as a microbial growth substrate into the obtained sample, and providing other conditions for maintaining the normal growth of the microbes;
(3) enriching and culturing the product obtained in the step (2) for a period of time, extracting a crude enzyme solution in the product obtained in the step (2), stopping enriching and culturing when detecting that lignin degrading enzyme exists in the crude enzyme solution or detecting that lignin is degraded, and storing an enriched sample at a low temperature;
(3.1) wherein the lignin degrading enzyme is detected by selecting various specific substrates to carry out protein concentration test;
(3.2) the degradation of the lignin is to detect small molecular polymers or monomolecular compounds after the lignin structure is broken through an instrument;
(4) taking the enriched sample in the step (3), and extracting DNA in the sample;
(5) entering the DNA in the step (4) into a high-throughput sequencing platform, and collecting the measured DNA data information;
(6) carrying out metagenomics analysis on the DNA data obtained in the step (5);
(6.1) cleaning data, and removing redundant data and pollution data;
(6.2) splicing the short sequences generated in the step (6.1) into long sequences;
(6.3) transcribing the long DNA sequence produced in step (6.2) into an amino acid sequence;
(6.4) comparing the amino acid sequence in step (6.3) with a carbohydrate database, and screening out sequences having correlation with the lignin degrading enzyme.
In the present invention, the sample in step (1) may be in a solid phase or a liquid phase.
In the present invention, the laboratory enrichment conditions in step (2) may be various conditions that enable normal growth of microorganisms at any temperature.
In the invention, the lignin degrading enzyme in the step (3) is detected by selecting various specific substrates to carry out protein concentration test.
In the invention, the degradation of the lignin in the step (3) is to detect small molecular polymers or monomolecular compounds after the lignin structure is broken through an instrument.
In the present invention, the method for extracting DNA from the sample in step (4) is not limited.
In the invention, the high-throughput sequencing platform in the step (5) is not limited.
In the invention, the metagenomic analysis software type in the step (6) is not limited.
The invention has the beneficial effects that: the invention provides a method for efficiently and conveniently constructing a large amount of lignin degrading enzymes, which does not depend on screening and culturing of strains in a laboratory, greatly expands the sources of the lignin degrading enzymes, provides a large amount of degrading enzymes with excellent performance for constructing a lignin biorefinery, can also promote the development of a lignocellulose full-component biorefinery, and can generate great ecological effect, economic effect and social benefit.
Detailed Description
The following examples are intended to further illustrate the invention and are not intended to limit the invention.
Example 1
The method comprises the steps of taking high-temperature compost residues back from a high-temperature compost treatment unit of a municipal waste treatment plant, mixing the residues with water, placing the mixture in a laboratory constant-temperature incubator, controlling the temperature to be 60-90 ℃, keeping natural ventilation and a certain rotating speed in the incubator, and keeping an aerobic environment in a sample by assisting manual stirring on the basis of the natural ventilation. Supplementing water to the sample to enable the water content to be about 40%, wherein the adding proportion of the lignin is that the lignin: mixed liquor =1:10, while supplementing a certain amount of basic carbon source and nitrogen source. Culturing for 5-10 days. 5ml of the culture medium was taken out of the mixture, and the crude enzyme solution on the upper layer was extracted by stirring and centrifugation and was kept at 60 ℃.
The enzyme activity was determined as ABTS method. Laccase activity is detected in the experimental group, the culture is stopped, and a certain sample is taken for low-temperature storage. Extracting DNA in a sample by using a DNA kit, entering an Illumina high-throughput sequencing platform, and setting the sequencing depth to be more than 10G; collecting a measured original sample DNA sequence read length data set; carrying out primary cleaning, removing redundant and polluted data by using MetaWrap integrated software, and assembling short sequences (reads) into long sequences (contigs); and performing data binning on the long sequence to obtain a plurality of bins. According to default parameters, bins are compared with CAZyme (carbohydrate database) to screen out laccase genes; and classifying all laccase gene sequences into a document to build a gene set of the lignin degrading enzyme laccase.
The result shows that DNA sequences of microbial communities in 20G samples are detected by an Illumina high-throughput sequencing platform, and 64 genes of laccase are obtained through data analysis and processing.
Example 2:
the surface layer muddy water mixture is taken from the natural hot spring, is cultured by a shaking table at the temperature of 60 ℃ and the rpm/min, and is stirred and mixed evenly by hand at intervals of several hours. The concentration of the suspended sludge in the mixed liquid is controlled to be 140-200 mg/L. Water (mixed solution of glucose and ammonium sulfate) is added every 6 to 8 hours. The total amount of lignin added was 20 g. 0.1 mol/L-1 copper sulfate solution 5ml is supplemented every day. A control group was set, and only a mixed solution of glucose and ammonium sulfate was added. The crude laccase solution activity was determined as in example 1. On day 10, the strongest laccase activity was measured at 260. + -. 62.89U/L.
DNA was extracted as in example 1, sequenced, cleaned, assembled and analyzed, and 68 lignin degrading enzyme laccase gene sets were obtained from the 20G DNA data.
Claims (8)
1. A method for constructing a lignin-degrading laccase gene set is characterized by comprising the following specific steps:
(1) taking a sample rich in microorganisms from a natural or artificial environment;
(2) taking the sample obtained in the step (1), and enriching lignin degrading microorganisms in the obtained sample under a laboratory condition; the method specifically comprises the following steps: adding lignin as a microorganism growth substrate in the obtained sample, and providing other conditions for maintaining the normal growth of the microorganism;
(3) enriching and culturing the product obtained in the step (2) for a period of time, extracting a crude enzyme solution from the product obtained in the step (2), stopping enriching and culturing when detecting that lignin degrading enzyme exists in the crude enzyme solution or detecting that lignin is degraded, and storing an enriched sample at a low temperature;
(3.1) wherein the lignin degrading enzyme is detected by selecting various specific substrates to carry out protein concentration test;
(3.2) the degradation of the lignin is to detect small molecular polymers or monomolecular compounds after the lignin structure is broken through an instrument;
(4) taking the enriched sample in the step (3), and extracting DNA in the sample;
(5) entering the DNA in the step (4) into a high-throughput sequencing platform, and collecting the measured DNA data information;
(6) carrying out metagenomics analysis on the DNA data obtained in the step (5);
(6.1) cleaning data, and removing redundant data and pollution data;
(6.2) splicing the short sequences generated in the step (6.1) into long sequences;
(6.3) transcribing the long DNA sequence produced in step (6.2) into an amino acid sequence;
(6.4) comparing the amino acid sequence in step (6.3) with a carbohydrate database, and screening out sequences having correlation with the lignin degrading enzyme.
2. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the sample in step (1) may be in solid phase or liquid phase.
3. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the laboratory enrichment conditions in step (2) can be any conditions that allow the normal growth of microorganisms at any temperature.
4. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the lignin-degrading enzymes detected in step (3) are protein concentration tests with specific substrates.
5. The method for constructing lignin-degrading laccase gene set according to claim 1, wherein in the step (3), the lignin is degraded by detecting small molecule polymer or single molecule compound after lignin structure breakage by instrument.
6. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the DNA extraction method in the sample in step (4) is not limited.
7. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the high throughput sequencing platform in step (5) is unlimited.
8. The method for constructing a lignin-degrading laccase gene set according to claim 1, wherein the metagenomic analysis software in step (6) is not limited in kind.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101652381A (en) * | 2007-01-30 | 2010-02-17 | 维莱尼姆公司 | Be used to handle the enzyme of lignocellulose, encode their nucleic acid and methods for making and using same thereof |
| CN105462999A (en) * | 2015-12-08 | 2016-04-06 | 江西省农业科学院农业应用微生物研究所 | Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology |
| CN105821033A (en) * | 2016-05-11 | 2016-08-03 | 清华大学 | Extracting method of cellulose degradation flora metagenome |
| CN107653243A (en) * | 2017-11-15 | 2018-02-02 | 中国农业科学院农业基因组研究所 | A kind of method that microorganism macro genome DNA is extracted from intestinal contents |
| CN109658980A (en) * | 2018-03-20 | 2019-04-19 | 上海交通大学医学院附属瑞金医院 | A kind of screening and application of excrement gene marker |
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- 2022-02-23 CN CN202210164778.0A patent/CN114480446A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101652381A (en) * | 2007-01-30 | 2010-02-17 | 维莱尼姆公司 | Be used to handle the enzyme of lignocellulose, encode their nucleic acid and methods for making and using same thereof |
| CN105462999A (en) * | 2015-12-08 | 2016-04-06 | 江西省农业科学院农业应用微生物研究所 | Method for screening beta-glucosaccharase gene from mildewed sugarcane leaves based on metagenomic technology |
| CN105821033A (en) * | 2016-05-11 | 2016-08-03 | 清华大学 | Extracting method of cellulose degradation flora metagenome |
| CN107653243A (en) * | 2017-11-15 | 2018-02-02 | 中国农业科学院农业基因组研究所 | A kind of method that microorganism macro genome DNA is extracted from intestinal contents |
| CN109658980A (en) * | 2018-03-20 | 2019-04-19 | 上海交通大学医学院附属瑞金医院 | A kind of screening and application of excrement gene marker |
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