CN109112115B - A triphenylmethane dye decolorizing enzyme and encoding gene derived from metagenomic - Google Patents
A triphenylmethane dye decolorizing enzyme and encoding gene derived from metagenomic Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/308—Dyes; Colorants; Fluorescent agents
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
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Abstract
本发明涉及生物技术和基因工程领域，本发明提供了一种从活性污泥宏基因组文库中筛选具有脱色酶基因克隆子的方法，并公开了一种三苯甲烷类脱色酶基因，其核苷酸序列如SEQ ID NO.1所示；该基因编码的氨基酸序列如SEQ ID NO.2所示。该脱色酶可有效使三苯甲烷类染料色度降低，具有广阔的应用前景。
The invention relates to the fields of biotechnology and genetic engineering. The invention provides a method for screening clones with decolorizing enzyme genes from an activated sludge metagenome library, and discloses a triphenylmethane decolorizing enzyme gene, the nucleoside of the decolorizing enzyme gene. The acid sequence is shown in SEQ ID NO.1; the amino acid sequence encoded by the gene is shown in SEQ ID NO.2. The decolorizing enzyme can effectively reduce the chromaticity of triphenylmethane dyes and has broad application prospects.
Description
Technical Field
The invention relates to the field of biotechnology and bioengineering, in particular to a triphenylmethane dye decolorizing enzyme Tmt derived from a metagenome of printing and dyeing wastewater activated sludge and a coding gene thereoftmt。
Background
A great deal of natural or synthetic dyes are used in the industries of textile, printing and dyeing, papermaking, leather, coating and the like, and triphenylmethane dyes are important dyes and are very commonly used. Most of triphenylmethane dyes have mutagenic, teratogenic or carcinogenic effects, and chromaticity of the triphenylmethane dyes also affects light transmittance of water bodies so as to hinder growth of plants and microorganisms, so that the triphenylmethane dyes attract wide attention.
The biological treatment of dye wastewater is a trend in development because of its low cost, environmental friendliness and low secondary pollution. When the immobilized decolorizing enzyme is applied to the decolorization of dye wastewater, the immobilized decolorizing enzyme can be prevented from being influenced by factors such as aeration condition, nutrition, pH value, temperature, dye toxicity and the like, and has good application potential, so the research focus of treating dyes by a biological method is shifting from the screening of decolorizing bacteria to the screening, cloning and expression of decolorizing enzyme. Currently, the cloned decolorizing enzymes mainly focus on several functional enzymes such as triphenylmethane reductase (TMR), laccase (laccase), azo reductase (azo reductase) and peroxidase (peroxidase). There are reports on the use of immobilized enzymes for decolorizing azo dyes.
When functional enzymes are screened, the screening method based on traditional microorganism culture can only search target genes from culturable microorganisms with the total amount of 1% of the microorganisms in the environment, and the coverage is too narrow. The solution is a metagenome technology, which bypasses the microbial culture link and provides more comprehensive and diversified gene resources for screening target genes.
Disclosure of Invention
The invention aims to provide a triphenylmethane decolorizing enzyme with high activity.
The invention obtains a positive clone with decolorization enzyme gene from an activated sludge metagenome library of a sewage treatment system of a printing and dyeing mill through malachite green enrichment and screening, and finds a gene related to decolorization from the positive clonetmtThe total length of the gene is 333 bp (from the start codon to the stop codon), and 110 amino acids are coded. Cloning to obtain decolorizing enzyme gene by PCR methodtmtThe recombinant plasmid pTMT obtained after being connected with a cloning vector pUC18 can show good decolorization capability in Escherichia coli DH5 alpha. Cloning the strain of the gene recombinant plasmid pTMTEscherichia coliIs preserved in 2017, 6, 8 and 8 days to the China center for type culture Collection at the university of WuhanThe number is CCTCC M2017318.
The first purpose of the invention is to provide a gene encoding the decolorizer genetmtThe nucleotide sequence of the gene is shown as SEQ ID NO. 1.
The second purpose of the invention is to provide a decolorizing enzyme Tmt, the amino acid sequence of which is shown in SEQ ID NO. 2.
It is a third object of the present invention to provide a method of screening clones having a decolorizer gene from a metagenomic library.
The invention also provides a gene containing the decolorizing enzymetmtThe recombinant vector of (1). The expression vector is pUC 18.
The low-temperature alkaline esterase Est18 is derived from an activated sludge metagenome of a printing and dyeing mill, and the amino acid sequence of the low-temperature alkaline esterase Est18 is shown as SEQ ID number 2:
MSAFVYLTMAIVAEVIATTMLKASEGFTRLWPSLLVVLGYGVAFWGLSMVVKSMPLGIVYAIWSGMGIVLVSIAAVFVYQQKLDWPAIVGMGLIIAGVLVINLLSKASVH
drawings
FIG. 1: decolorization of pTMT positive clones is shown.
Detailed Description
Example 1: screening of clones harboring a decolorizer Gene
An activated sludge sample is collected from a sewage treatment system of a certain dye house at the bottom of 2014, a metagenome Fosmid Library is constructed by adopting a Library construction Kit CopyControl Library Kit (epicentre), a cloning vector is pCC1FOS, and a plating strain is Phage T-1 resist EPI 300-T1RE. coliAnd detecting by enzyme digestion electrophoresis, wherein the size of the insert fragment is 20-45 kb.
The strain with the decolorizing enzyme gene is screened from the constructed metagenome library of the activated sludge by adopting a limited culture medium enrichment method.
1. The prepared sterilized liquid M9 minimal medium was removed, 100. mu.l of 50 mg/ml chloramphenicol (final concentration: 50. mu.g/ml), 1 ml LB medium (final concentration: 1%) and 100. mu.l 10 mg/ml malachite green (final concentration: 10. mu.g/ml) were added to each 100 ml of the liquid M9 medium, and the mixture was shaken and mixed well. Subpackaging the limited nutrient enrichment medium into test tubes, adding 5 ml of the limited nutrient enrichment medium into each test tube, and marking;
2. adding 10 mul of library bacterial liquid into a test tube, and making two parallel library bacteria for each library bacterial liquid;
3. placing the test tube into a shaking table at 37 ℃ and 170 rpm for culturing for a period of time, and observing whether the color of the dye in the test tube changes or not;
4. screening of positive clones:
1) respectively diluting bacterial liquid in the test tubes with color change to 10-1、10-2、10-3Coating 100 mul of the diluent on LB solid culture medium added with chloramphenicol (the final concentration is 50 mug/ml) and malachite green (the final concentration is 50 mug/ml), coating three culture mediums on each dilution concentration of each test tube, and marking;
2) inverting the culture dish, and then putting the culture dish into an incubator at 37 ℃ for culturing until bacterial colonies can be observed;
3) and (3) carrying out streak separation on the single colony with the transparent circle on an LB solid medium flat plate which is also added with chloramphenicol and malachite green, inverting and placing in an incubator at 37 ℃ for culturing for a period of time until a single colony grows out, and verifying whether the dye degradation transparent circle appears around the single colony.
Example 2: decolorizing enzyme genetmtCloning of (2)
The strain with transparent circle around is inoculated into a liquid culture medium of 5 ml LB added with chloramphenicol (final concentration is 50 mug/ml), and is cultured for more than 12 h at 37 ℃ and 180 rpm in a shaking way, and a plasmid extraction kit is adopted to extract fosmid plasmid containing decolorizer gene. By usingSau3AI carries out partial enzyme digestion on the fosmid plasmid, cuts the rubber, recovers a 2-5 kb DNA fragment, clones the DNA fragment to the pUC18 plasmid, and transformsE.coliThe strain DH5 alpha was spread on LB plates supplemented with ampicillin (100. mu.g/ml) and cultured overnight at 37 ℃ in an inverted state. The single colony grown was transferred to a screening plate of malachite green (100. mu.g/ml) containing ampicillin (100. mu.g/ml), and positive clones with a clearing circle appearing around the periphery were selected after culturing. And extracting the plasmid after the streaking repeated verification and selecting the plasmid with the proper size of the inserted segment for sequencing to obtain the base sequence of the inserted segment.
ORF analysis (http:// www.ncbi.nlm.nih.gov/gorf. html) was performed based on Open Reading Frame Finder at NCBI, and it was found that one of the ORFs can decolorize malachite green by cloning and functional verification of the putative ORF. The ORF has a length of 333 bp and is namedtmtThe nucleotide sequences of the bases are shown as SEQ ID No.1, and the base compositions are 56A (16.8%), 71C (21.3%), 112G (33.6%) and 94T (28.2%).tmtEncodes a protein Tmt with the size of 110 amino acid residues, and the amino acid sequence of the protein Tmt is shown as SEQ ID No. 2. The gene was found by aligning the sequences by Blast (http:// Blast. ncbi. nlm. nih. gov /)And Serratia marcocensThe 5217200-5217532 fragment in the CAV1492 whole genome has 96% identity, and the encoded protein haveSerratia ureilyticaThe multidrug DMT transporter (WP-046687127) contains an EamA superfamily, which is a family of proteins related to multidrug metabolism and drug resistance. Most of the traditional decolorizing enzymes are oxidation-reduction enzymes, and no report exists at present for indicating that the genes are related to dye decolorization.
According to the sequence of the ORF gene and the multiple cloning site of the expression vector, the designed expression primer:
DEC48-2F: 5’- CG GGATCC ATCATGAGCGCATTTGTG
DEC48-2R: 5’- CC AAGCTT ATAACACTCAGTGCACCG
wherein GGATCC isHinA dIII restriction site, AAGCTTBamHI enzyme cutting site, the length of the target fragment is about 350 bp. The amplified DNA fragment was digested simultaneously with a double enzyme, ligated with plasmid vector pUC18 after the digestion to obtain recombinant plasmid pTMT, and transformed into pTMTE.coliDH5 α. The positive clones from pTMT obtained were plated on LB plates containing ampicillin and malachite green and cultured upside down for a period of time, with a discolored transparent circle around the colony and no transparent circle around the empty vector control transferred into pUC18 (as shown in FIG. 1).
By verification, the transformant with the pTMT can decolorize triphenylmethane dyes such as brilliant green and the like.
SEQUENCE LISTING
<110> Wuhan university of light industry
<120> triphenylmethane dye decolorizing enzyme derived from metagenome and encoding gene thereof
<130> 2017318
<160> 2
<170> PatentIn version 3.5
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gcgatttggt cgggcatggg catcgtgctg gtgtcgatcg cggcggtatt tgtttatcag 240
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Met Val Val Lys Ser Met Pro Leu Gly Ile Val Tyr Ala Ile Trp Ser
50 55 60
Gly Met Gly Ile Val Leu Val Ser Ile Ala Ala Val Phe Val Tyr Gln
65 70 75 80
Gln Lys Leu Asp Trp Pro Ala Ile Val Gly Met Gly Leu Ile Ile Ala
85 90 95
Gly Val Leu Val Ile Asn Leu Leu Ser Lys Ala Ser Val His
100 105 110
Claims (1)
1. The application of the triphenylmethane dye decolorizing enzyme Tmt in removing triphenylmethane dyes is disclosed, wherein the amino acid sequence of the triphenylmethane dye decolorizing enzyme Tmt is shown as SEQ ID NO: 2.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870968A (en) * | 2010-05-18 | 2010-10-27 | 南京大学 | Triphenylmethane Dye Decolorizing Enzyme and Its Application |
| WO2010143871A9 (en) * | 2009-06-08 | 2011-03-03 | 한국생명공학연구원 | Method for screening and quantifying various enzyme activities using a genetic enzyme screening system |
| EP2530148A1 (en) * | 2011-05-30 | 2012-12-05 | B.R.A.I.N. Biotechnology Research And Information Network AG | Novel beta-galactosidases useful for the production of lactose depleted milk products |
| CN103361374A (en) * | 2013-07-23 | 2013-10-23 | 扬州日兴生物科技股份有限公司 | Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene |
| CN104805098A (en) * | 2015-05-22 | 2015-07-29 | 厦门大学 | Peroxidase of degradable malachite green from mangrove sediments and preparation method for peroxidase |
-
2017
- 2017-06-23 CN CN201710484095.2A patent/CN109112115B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010143871A9 (en) * | 2009-06-08 | 2011-03-03 | 한국생명공학연구원 | Method for screening and quantifying various enzyme activities using a genetic enzyme screening system |
| CN101870968A (en) * | 2010-05-18 | 2010-10-27 | 南京大学 | Triphenylmethane Dye Decolorizing Enzyme and Its Application |
| EP2530148A1 (en) * | 2011-05-30 | 2012-12-05 | B.R.A.I.N. Biotechnology Research And Information Network AG | Novel beta-galactosidases useful for the production of lactose depleted milk products |
| CN103361374A (en) * | 2013-07-23 | 2013-10-23 | 扬州日兴生物科技股份有限公司 | Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene |
| CN104805098A (en) * | 2015-05-22 | 2015-07-29 | 厦门大学 | Peroxidase of degradable malachite green from mangrove sediments and preparation method for peroxidase |
Non-Patent Citations (5)
| Title |
|---|
| Multidrug DMT transporter [Serratia ureilytica];Genbank Database;《Genbank Database》;20150506;Accession No. WP_046687127 * |
| Screening and expression of genes from metagenomes;Leis B等;《Adv Appl Microbiol》;20131231;第83卷;1-68 * |
| Serratia marcescens strain WVU-006 chromosome，complete genome;Ranjbaran,A等;《GenBank Database》;20190708;Accession No. CP041129.1 * |
| 孔雀石绿降解菌的分离筛选及其降解基因的克隆与表达;李泰联;《中国博士学位论文全文数据库工程科技Ⅰ辑》;20160815(第08期);摘要第2、4、5段，第26页第2-4、6段 * |
| 海洋沉积物中可降解孔雀石绿菌株和酶基因的挖掘及其降解特性研究;曲武;《中国学位论文全文数据库》;20160831;全文 * |
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