CN106434579A - Laccase from Klebsiella pneumoniae, as well as recombinant strain and preparation method thereof - Google Patents
Laccase from Klebsiella pneumoniae, as well as recombinant strain and preparation method thereof Download PDFInfo
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- CN106434579A CN106434579A CN201610899958.8A CN201610899958A CN106434579A CN 106434579 A CN106434579 A CN 106434579A CN 201610899958 A CN201610899958 A CN 201610899958A CN 106434579 A CN106434579 A CN 106434579A
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 108010056929 lyticase Proteins 0.000 description 1
- 229940002712 malachite green oxalate Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- ZMXJAEGJWHJMGX-UHFFFAOYSA-N methyl syringate Chemical compound COC(=O)C1=CC(OC)=C(O)C(OC)=C1 ZMXJAEGJWHJMGX-UHFFFAOYSA-N 0.000 description 1
- YFBSBLHMAWUCJB-UHFFFAOYSA-N methyl syringate Natural products COc1cc(cc(OC)c1O)C(=O)OO YFBSBLHMAWUCJB-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940057059 monascus purpureus Drugs 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000002496 oximetry Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
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- 230000006920 protein precipitation Effects 0.000 description 1
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- PLUQFBYKHVFHMZ-UHFFFAOYSA-N quinoline-6-sulfonic acid Chemical compound N1=CC=CC2=CC(S(=O)(=O)O)=CC=C21 PLUQFBYKHVFHMZ-UHFFFAOYSA-N 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
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- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000004895 subcellular structure Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- ROHGQIQUQCTSOK-UHFFFAOYSA-K trisodium;1-amino-4-[3-[[4-chloro-6-(3-sulfonatoanilino)-1,3,5-triazin-2-yl]amino]-4-sulfonatoanilino]-9,10-dioxoanthracene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=2C(=O)C3=CC=CC=C3C(=O)C=2C(N)=C(S([O-])(=O)=O)C=C1NC(C=1)=CC=C(S([O-])(=O)=O)C=1NC(N=1)=NC(Cl)=NC=1NC1=CC=CC(S([O-])(=O)=O)=C1 ROHGQIQUQCTSOK-UHFFFAOYSA-K 0.000 description 1
- DQTMTQZSOJMZSF-UHFFFAOYSA-N urushiol Natural products CCCCCCCCCCCCCCCC1=CC=CC(O)=C1O DQTMTQZSOJMZSF-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- 239000003643 water by type Substances 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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Abstract
The invention belongs to the technical field of gene engineering of enzymes and relates to a novel laccase from Klebsiella pneumoniae and a preparation method thereof. The invention adopts the technical scheme comprising the following steps of: utilizing a molecular biology method and a gene engineering technology to obtain a plant of Klebsiella pneumoniae capable of generating the laccase by strain screening, amplifying genes of the novel laccase by a PCR technology, and then carrying out expression on the genes of the novel laccase in a bacillus subtillis expression system and a pichia pastoris expression system respectively to obtain a high-stability laccase recombinant strain of bacillus subtillis and a high-stability laccase free expression recombinant strain of pichia pastoris, thereby realizing preparation of the novel laccase. Simultaneously, the novel bacterial laccase has a better decoloring effect for azo and anthraquinone dyes.
Description
Technical field：
The present invention relates to a kind of novel laccase enzyme from Klebsiella pneumonia and its gene, engineering bacteria and preparation side
Method and in particular to the recombinant strains expressing this novel laccase enzyme are obtained by technique for gene engineering and molecular biology method,
And this bacterial laccase albumen, in dye decolored industrial application, belongs to the gene engineering technology field of enzyme.
Background technology：
Laccase (Laccase, E.C.1.10.3.2), also known as urushiol oxidase, blue multicopper oxidase, is a kind of polyphenol of cupric
Oxidase, it with plant in ascorbic acid oxidase, the ceruloplasmin of mammal, cytochrome C oxidase, gallbladder red
Plain oxidase homology, belongs to a member of blue blue multicopper oxidase family.It can be catalyzed multiple phenols and non-phenolic compounds oxygen
Change, be allowed to generate corresponding benzoquinone, simultaneously with the transfer of electronics, by reducing molecular oxygen Cheng Shui and no other in course of reaction
By-product generates.
Laccase is that one kind is acknowledged as eco-friendly " green enzyme ", is widely distributed in nature.It is pressed by laccase
Source can be divided into 4 big class：Plant laccase, animal laccase, fungal laccase and bacterial laccase.Laccase is found in japanese lacquer tree earliest
Juice composition in, in subsequent research, researcher all finds laccase from various plants, but plant laccase was extracting
Substantial amounts of other oxidase is comprised so that purification laccase process is complex in journey.Confirmed later, laccase is except being present in plant again
In thing, it is also present in animal, funguses and antibacterial.
Fungal laccase is primarily present in higher funguses (particularly basidiomycetes), and industrialized commodity laccase mainly comes at present
Come from funguses, but, fungal laccase is easily affected by hydroxyl ion, and activity is very low in the basic conditions or does not almost have,
Heat stability is also poor, and filamentous fungis growth cycle is long, culture medium is required higher, is easily subject to high shear in fermentation tank
The damage of power, and fungal laccase needs to carry out post translational modification, can only rely on screening superior strain oneself expression or using true
Nuclear expression system carries out recombinant expressed, and the shortcomings of there is long the production cycle, high cost, yield poorly, this has had a strong impact on funguses paint
Enzyme application industrially.
Bacterial laccase includes the CotA albumen of Bacilluss, the PpoA albumen of sea Zymomonas mobiliss, escherichia coli CueO albumen
Deng compared with fungal laccase, bacterial laccase can overcome the disadvantages mentioned above of fungal laccase, and has good in the basic conditions
Catalysis activity and higher stability.Bacterial laccase has the advantages that oneself, and some are unique, such as：Need not glycosylation modified, enzyme
Optimum pH scope wide, temperature stability is good, there is Cu2+Resistance etc..The exactly industrial laccase application at present of these properties is required
Want.Therefore, the research of bacterial laccase is of great significance for the extension tool of laccase application.Currently, with respect to thin
Seldom, more bacterial laccase genes with high vigor high stability ability need to excavate further and grind for the research of bacterium laccase
Study carefully.
Because laccase source is different, and there is certain difference in structure and properties in the laccase of separate sources, because
The laccase of this separate sources can show certain difference in terms of its catalysis characteristics.The substrate specificity of laccase and its extensive, energy
Enough 250 gas chromatographies including Polyphenols, diamidogen, arylamine class, carboxylic acidss for the catalysis oxidation aoxidize, are polymerized etc. instead
Should.Because of its special catalytic performance and wide in range substrate specificity, so the application of laccase is also and its extensively, including pollutant fall
Solution, dye decolored removing toxic substances, paper pulp and textile bleaching, oral cavity health teeth whitening, bleaching laundry detergent, flavour of food products change
Good, feed nutrition improves, bioelectronicss are researched and developed etc..For many applications, the oxidation of laccase can be improved by using amboceptor
Ability.The amboceptor being currently known includes：HBT (I-hydroxybenzotriazole), ABTS (double (the 3- ethyl-benzothiazole of 2,2 '-azino
Quinoline -6- sulfonic acid) di-ammonium salts, methyl syringate, NHA (N- hydroxyacetanilide), NEIAA (N- acetyl group-N-Phenylhydroxylamine),
HBTO (3- hydroxyl 1,2,3- phentriazine -4 (3H) ketone), VIO (violuric acid).
Bacillus subtillis belong to gram positive bacteria.Bacillus subtillis expression system has advantages below：1st, can
Efficiently secrete various protein；2nd, the existing considerably long history of use in fermentation industry for many Bacillus subtillis, no
Pathogenic, do not produce any endotoxin；3rd, the background research of Bacilluss microbial geneticss is fully aware of, and grows fast
Speed, to nutrient substance no particular/special requirement the advantages of；4th, codon-bias are inconspicuous；5th, sweat is simple, Ko subtilis bar
Pseudomonas in aerobic bacteria, without anaerobic fermentation equipment, after fermentation ends, simple separation and fermentation liquid and microorganism, you can enter
The separation of destination protein, purification recovery stage；6th, there is resistance, multiple thermostability enzyme preparations can be produced.
Pichia sp. is a kind of unicellular lower eukaryotes, and condition of culture is common, and growth and breeding speed is rapid.Finish red ferment
When mother system is used for expressing gene engineering product, production cost can be effectively reduced with large-scale production.Pichia anomala expression system
System has lot of advantages when expressing external source destination protein, has broad prospects, suitable industrialization and large-scale production, this table
The advantage reaching system is mainly manifested in：1st, expression is high：The expression vector of pichia yeast expression system, is with alcohol oxidase gene
Promoter start expression, the expression of its external source destination protein is very high, and the expression of P.pastoris is than General Expression system
Even 100 times of high 10 times of the expression (expression of conventional expression system is typically in milligram level) of system.2nd, stability is high：Finish red ferment
The expression vector of female expression system is existed by being incorporated on the chromosome of yeast, and replicates with the duplication of chromosome,
It is not presented in plasmid self replication, the stability of therefore recombinant bacterial strain is very high.3rd, hypersecretion expression：Pichia sp. table
Reaching a factor leaders in the expression vector of system is a secretory signal sequence with good secretion effect, Neng Goutong
Crossing certain approach makes expression product be secreted into extracellular, alleviates the metabolic burden of host cell simultaneously, is conducive to cell
Continued propagation.4th, the processing after destination protein translation and modification：Pichia sp. has the complete subcellular structure of eukaryote, energy
Enough carry out the processing modification after eukaryotic protein translation (as glycosylation, fat phthalein, phosphorylation, protein cleavage, folding with
And the formation of disulfide bond etc.), so that the structure of secretory protein is closer to native protein, and degree of glycosylation is suitable, fits
In Clinical practice.5th, genetic background research is clearer, can realize the high level of destination protein by gene expression regulation mechanism
Expression.6th, it is easy to carry out industrialized production, Pichia sp. belongs to unicellular microorganism, there is low production cost, nutritional requirement letter
Single (carbon source of yeast is generally glycerol, glucose and methanol etc.), fermentation technology is simple, can carry out high density fermentation etc. excellent
Point, yeast has the potentiality that large scale fermentation produces foreign protein, and the foreign protein of self secretion of yeast expression system is seldom,
Purification for destination protein is favourable.Pichia yeast expression system become modern molecular biology study most important instrument and
Model, is the ideal instrument of expression alien gene.
In the present invention, novel laccase enzyme encoding gene derives from Klebsiella pneumonia, and it belongs to antibacterial, to kerekou pneumonia
In primary Salmonella genome, novel laccase enzyme encoding gene is cloned, and novel laccase enzyme gene in Bacillus subtillis expression system and is finished
Expressed in red yeast expression system, respectively obtain Bacillus subtillis high stability laccase recombinant bacterial strain and Pichia sp. is high
Stability laccase dissociates and expresses recombinant bacterial strain, after recombinant bacterial strain fermentation, processes through corresponding, can obtain high stability laccase
Catalyst, and this new restructuring laccase can decolour to Anthraquinones and azo dyes.
Content of the invention：
It is an object of the invention to overcoming and avoiding the industrial at present weak point producing existing for laccase, and provide one
Plant the novel bacterial laccase encoding gene from Klebsiella pneumonia and the engineering bacteria expressing this novel bacterial laccase gene
Strain.
The technology path that the present invention realizes purpose is as follows：With the genome of Klebsiella pneumonia as template, and according to
The Klebsiella pneumonia laccase maturation peptide gene of report, analyzes its conserved sequence, the laccase maturation peptide gene of the design present invention
Amplimer P1 and P2, P3 and P4, wherein, forward primer P1 and downstream primer P2 be used to amplification in Bacillus subtillis
The genes of interest of expression, upstream and downstream primer introduces restriction enzyme site BamH I, Hind III respectively；Forward primer P3 and
Downstream primer P4 is used to expand the genes of interest of expression in Pichia pastoris GS115, and upstream and downstream primer introduces restricted respectively
Restriction enzyme site EcoR I, Not I.The laccase gene Lac obtaining Klebsiella pneumonia is cloned by PCR, by itself and pBSA43
After carrier and pPIC9K carrier connect respectively, construction recombination plasmid pBSA43-Lac and pPIC9K-Lac；Conversion escherichia coli
JM109, obtains recombinant bacterial strain JM109/pBSA43-Lac and JM109/pPIC9K-Lac.Correct recombiant plasmid will be verified again
PBSA43-Lac and pPIC9K-Lac, successful expression in Bacillus subtillis WB600 and Pichia pastoris GS115, obtains respectively
Produce the recombinant bacterial strain of high stability novel laccase enzyme, obtain the new paint of high stability of high yield further by fermentation technology optimization
Enzyme.
To achieve these goals, one of technical scheme of present invention offer is：A kind of novel bacterial laccase, described laccase
From one plant of Klebsiella pneumonia through inventor's screening, the SEQ ID No in its aminoacid sequence such as sequence table：2 institutes
Show；
When described laccase is with ABTS for substrate mensure novel laccase enzyme zymologic property, optimum temperature is 35 DEG C, the most suitable work
It is 4 with pH；During with DMP for substrate mensure novel laccase enzyme zymologic property, optimum temperature is 70 DEG C, and the most suitable action pH is 8；
Meanwhile, pH stability and the heat stability of this novel laccase enzyme are measured with DMP for substrate, result shows：This novel fine
Bacterium laccase has good stability in the range of pH5～9, is embodied in and is incubated 35h in pH5, pH6 and pH9, and remnant enzyme activity exists
More than 60%, it is incubated 35h in pH7 and pH8, remnant enzyme activity is more than 85%；Good in 30～60 DEG C of heat stability, it is embodied in
It is incubated 5h at 30～40 DEG C, remnant enzyme activity is more than 95%, is incubated 4h at 50～60 DEG C, remnant enzyme activity is more than 50%；With
The laccase from Klebsiella pneumonia of report is compared, the pH stability of the claimed novel laccase enzyme of this patent and temperature
Stability is more preferable；
The encoding gene of described laccase is Lac, the SEQ ID No in base sequence such as sequence table：Shown in 1；
To achieve these goals, the two of the technical scheme of present invention offer are：Said gene is rebuild restructuring carry
Body, and the high efficient expression in Bacillus subtillis WB600 and Pichia pastoris GS115, obtain producing high stability novel laccase enzyme
Recombinant bacterial strain, obtains the high stability novel laccase enzyme of high yield further by fermentation technology optimization；
Host cell for expressing described novel bacterial laccase is Bacillus subtillis WB600, and expression vector is
pBSA43；
Host cell for expressing described novel bacterial laccase is Pichia pastoris GS115, and expression vector is pPIC9K；
The experimental procedure of the present invention is summarized as follows：
1st, a kind of novel laccase enzyme gene from Klebsiella pneumonia, the recombinant bacterial strain of this novel laccase enzyme of construction expression
(Bacillus subtillis WB600/pBSA43-Lac) and its preparation process of this novel bacterial laccase comprise the steps：
(1) one plant of bacterial strain that can produce laccase, i.e. Klebsiella pneumonia are obtained by high flux bacterial screening；
(2) with the genome of Klebsiella pneumonia as template, according to reporting Klebsiella pneumonia laccase mature peptide
Gene, is analyzed its conserved sequence, amplimer P1 and P2 of the laccase maturation peptide gene of the design present invention, is obtained by PCR clone
Obtain the laccase gene Lac of Klebsiella pneumonia, it is connected with E. coli-B bacilluss shuttle plasmid pBSA43
Afterwards, construction recombination plasmid pBSA43-Lac, converts e. coli jm109, obtains recombinant bacterial strain JM109/pBSA43-Lac；Obtain
Klebsiella pneumonia novel laccase enzyme encoding gene；
(3) recombiant plasmid pBSA43-Lac is transformed into Bacillus subtillis WB600, builds and obtain recombinant bacterial strain WB600/
pBSA43-Lac；
(4) recombinant bacterial strain is carried out fermentation preparation high stability novel bacterial laccase；
(5) prepare high stability novel laccase enzyme.
2nd, a kind of novel laccase enzyme gene from Klebsiella pneumonia, builds the free restructuring expressing this novel laccase enzyme
Bacterial strain (Pichia pastoris GS115/pPIC9K-Lac) and its preparation process of this novel bacterial laccase comprise the steps：
(1) one plant of bacterial strain that can produce laccase, i.e. Klebsiella pneumonia are obtained by high flux bacterial screening.
(2) with the genome of Klebsiella pneumonia as template, according to reporting Klebsiella pneumonia laccase mature peptide
Gene, is analyzed its conserved sequence, amplimer P3 and P4 of the laccase maturation peptide gene of the design present invention, is obtained by PCR clone
Obtain the laccase gene Lac of Klebsiella pneumonia, after it is connected, structure with escherichia coli-Pichia sp. shuttle plasmid pPIC9K
Build recombiant plasmid pPIC9K-Lac, convert e. coli jm109, obtain recombinant bacterial strain JM109/pPIC9K-Lac；Obtain pneumonia
Klebsiella novel laccase enzyme encoding gene；
(3) recombiant plasmid pPIC9K-Lac is transformed into Pichia pastoris GS115, builds and obtain recombinant bacterial strain GS115/
pPIC9K-Lac；
(4) recombinant bacterial strain obtaining is screened through Geneticin, in conjunction with the enzyme activity determination of laccase, obtain producing high stability paint
The recombinant bacterial strain of enzyme；
(5) superior strain is carried out fermentation preparation high stability novel bacterial laccase；
(6) prepare high stability novel laccase enzyme.
Beneficial effect：
1st, the present invention passes through specifically to produce the high flux bacterial strain screening of laccase, obtains one plant and can produce laccase
Bacterial isolateses, i.e. Klebsiella pneumonia, the laccase gene of this bacterial strain being obtained by PCR amplification is new for one section through sequencing
Base sequence.
2nd, express novel laccase enzyme recombinant bacterial strain by Bacillus subtillis in the present invention and the free expression of Pichia sp. is new
After the fermentation of laccase recombinant bacterial strain, the new restructuring laccase of preparation has the advantages that following zymologic property：Measured with ABTS for substrate
During novel laccase enzyme zymologic property, optimum temperature is 35 DEG C, and the most suitable action pH is 4；Novel laccase enzyme enzyme is measured for substrate with DMP
When learning property, optimum temperature is 70 DEG C, and the most suitable action pH is 8；This novel bacterial laccase is in the range of pH5～9 simultaneously
Stable, good in 30～60 DEG C of heat stability.
3rd, this novel bacterial restructuring laccase has preferable decolorizing effect to azo and anthraquinone dyes.Therefore, this is heavy
Group laccase has larger application potential in actual applications.
Brief description
Fig. 1 is the PCR amplification electrophoretogram of novel laccase enzyme maturation peptide gene of the present invention；
Wherein：M is DNA Marker, and 1,2 are respectively laccase maturation peptide gene Lac；
Fig. 2 is recombiant plasmid pBSA43-Lac digestion verification figure of the present invention；
Wherein：M is DNA Marker, 1 be recombiant plasmid pBSA43-Lac through BamHI single endonuclease digestion figure, 2 is recombiant plasmid
PBSA43-Lac is through BamHI and Hind III double digestion figure；
Fig. 3 is recombiant plasmid pPIC9K-Lac digestion verification figure of the present invention；
Wherein：M is DNA Marker, and 1 is to recombinate plasmid pPIC9K-Lac through EcoR I single endonuclease digestion figure, and 2 is recombiant plasmid
PPIC9K-Lac is through EcoR I and Not I double digestion figure.
Specific embodiment
With reference to example, the technology contents of the present invention are described further, but the present invention is not limited solely to these enforcements
Example is it is impossible to limit protection scope of the present invention with following embodiments.
Embodiment 1：The acquisition of Klebsiella pneumonia novel laccase enzyme maturation peptide gene
1st, the Klebsiella pneumonia that novel laccase enzyme mature peptide gene source goes out in this laboratory screening, extracts its genome
DNA, the extraction step of wherein Klebsiella pneumonia genomic DNA is as follows：
(1) from glycerol tube, inoculation lines in LB solid plate, 37 DEG C of quiescent culture 12h；
(2) from the flat board of culture thalline, picking one single bacterium colony is inoculated in LB liquid medium containing 5mL, in 220r/
Min, cultivates 12h under the conditions of 37 DEG C；
(3) bacterium solution is dispensed in the 1.5mL microcentrifugal tube of sterilizing, 12000r/min is centrifuged 1min collects thalline, abandons
Supernatant；
(4) precipitation is resuspended in the solution I of 200 μ L pre-coolings and repeatedly blows and beats mixing with pipette tips, and add the 50mg/ of 50 μ L
ML lysozyme, 37 DEG C of water-bath 1h；
(5) E.C. 3.4.21.64 of the 10%SDS and 10 μ L of 20 μ L, 65 DEG C of water-bath 2-3h are added；
(6) solution II adding 250 μ L newly to join, covers tightly the mouth of pipe, leniently spins upside down the EP pipe of 1.5mL 6～8 times,
The thalline in EP pipe is made fully to crack；
(7) add the solution III of 350 μ L pre-coolings, leniently the EP pipe of 1.5mL is spun upside down 6～8 times immediately, now
White flock precipitate occurs in EP pipe；
(8) 10min is centrifuged with 12000r/min, supernatant is transferred in another EP pipe, plus isopyknic Tris saturated phenol/
Chloroform (1: 1) mixed solution, after mix homogeneously, 12000r/min is centrifuged 10min, then supernatant is transferred in another EP pipe；
(9) repeatedly extract 2 times, then with equal-volume chloroform 1 time, removal Determination of Trace Phenol；
(10) add the dehydrated alcohol of 2 times of volumes, mix, place 30min in -20 DEG C.12000r/min centrifugation 10min receives
Collection precipitation；
(11) precipitated 2～3 times with 70% washing with alcohol, discard residual liquid；
(12) air drying 20-30min, with the ddH2O dissolution precipitation of 30 μ L sterilizings；
2nd, pass through NCBI gene library lookup, according to the Klebsiella pneumonia laccase maturation peptide gene of report, analyze it
Conserved sequence, the amplimer of the laccase maturation DNA encoding peptide of the design present invention is as follows：
Forward primer P1 (SEQ ID NO.3)：5’—CGCGGATCCTCAACGTCGAGACTTCCTCA—3’
Downstream primer P2 (SEQ ID NO.4)：5’—AAAACTGCAGTTAAACCGTGAACCCCAAC—3’
Forward primer P3 (SEQ ID NO.5)：5’—CCGGAATTCCAACGTCGAGACTTCCTCAA—3’
Downstream primer P4 (SEQ ID NO.6)：5’—ATAAGAATGCGGCCGCTTAAACCGTGAACCCCAAC—3’
Wherein, forward primer P1 and downstream primer P2 is used to expand the genes of interest of expression in Bacillus subtillis,
Upstream and downstream primer introduces restriction enzyme site BamH I, Hind III respectively；Forward primer P3 and downstream primer P4 is used to
Amplification in Pichia pastoris GS115 expression genes of interest, upstream and downstream primer introduce respectively restriction enzyme site EcoR I,
Not I.
Amplification template is Klebsiella pneumonia genomic DNA, and the reaction condition of its amplification is：
Amplification condition is：95 DEG C of denaturations 10min；95 DEG C of degeneration 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 1min40s are anti-
Answer 30 follow bad；72 DEG C of extension 10min.Pcr amplification product, through 0.8% agarose gel electrophoresiies, obtains the band (figure of 1600bp
1), reclaim PCR primer with miniprep dna glue reclaim test kit, and carry out double digestion and purification recovery, obtain the pneumonia gram of the present invention
Thunder primary Salmonella novel laccase enzyme maturation DNA encoding peptide Lac, is shown in sequence 1.
Embodiment 2：The structure of Bacillus subtillis high stability novel laccase enzyme recombinant bacterium
1st, the structure of expression vector pBSA43
PBSA43 is with E. coli-B bacilluss shuttle cloning vector pBE2 as skeleton, be cloned into one strong
Bacilluss constitutive promoter P43, and recombiant protein direct secretion levansucrase signal in culture medium can be made
Sequence sacB and obtain.It carries AmprGene, can be by the use of amicillin resistance as selection markers in escherichia coli；
Also there is Km simultaneouslyrGene, can be by the use of kalamycin resistance as screening mark in Bacillus subtillis, bacillus licheniformis
Note.
2nd, the structure of novel laccase enzyme expression vector pBSA43-Lac
By the novel laccase enzyme gene (Lac) reclaiming through PCR amplification and after BamH I and Hind III double digestion with same
The Bacillus subtillis expression vector pBSA43 ligase of double digestion is attached, and connection product is converted escherichia coli
In JM109 competent cell, through Amp resistance screening, select positive transformant, extract transformant plasmid, and carry out single, double enzyme action
Checking and sequencing, determine and build the correct recombinant bacterial strain JM109/pBSA43-Lac of acquisition.
3rd, recombinant expression carrier pBSA43-Lac conversion Bacillus subtillis WB600
The pBSA43-Lac recombiant plasmid of 1 μ L (50ng/ μ L) is added to the Bacillus subtillis WB600 competence of 50 μ L
In cell and mix, transfer in the electric revolving cup (1mm) of pre-cooling afterwards, after ice bath 1-1.5min, electric shock once (25uF, 200
Ω, 4.5-5.0ms).After electric shock finishes, add 1mL recovery medium (LB+0.5mol/L Sorbitol+0.38mol/L immediately
Mannitol).After 37 DEG C of shaking table concussion and cultivates 3h, recovery thing is coated on the LB flat board containing kanamycin, 37 DEG C of cultures
12-24h, picking positive transformant, and carry out single, double digestion verification, determine and obtain Bacillus subtillis recombinant bacterial strain WB600/
pBSA43-Lac.
Embodiment 3：Pichia sp. high stability novel laccase enzyme dissociates and expresses the structure of recombinant bacterium
1st, the structure of novel laccase enzyme Expression vector pPIC9K-Lac
Novel laccase enzyme gene (Lac) and the equally double enzymes that will reclaim through PCR amplification and after EcoR I and Not I double digestion
The yeast expression vector pPIC9K ligase cut is attached；Connection product is converted e. coli jm109 competence
In cell, through Amp resistance screening, select positive transformant；Extract positive transformant plasmid, shake through 37 DEG C and extract matter after pipe culture
Grain, and carry out single, double enzyme action preliminary identification, and correct for digestion verification recombiant plasmid is named as pPIC9K-Lac；By enzyme action
Verify that correct positive colony delivers to the sequencing of Beijing Hua Da Gene science limited company, to further ensure that genes of interest
Correctness, final determination builds and obtains correct recombinant bacterial strain JM109/pPIC9K-Lac.
2nd, the screening of the structure of novel laccase enzyme recombinant bacterial strain and novel laccase enzyme high expression recombinant bacterial strain
(1) preparation of linearisation recombinant expression plasmid pPIC9K-Lac
Recombiant plasmid, be first by the recombinant expression plasmid building pPIC9K-Lac before electricity conversion Pichia pastoris GS115
Carry out linearisation to improve integration efficiency on Pichia chromosome for the recombiant plasmid.Conversion needs linearization plasmid every time
DNA 15 μ g, and plasmid is purer, transformation efficiency is higher.Carry out linearized enzyme digestion with this 2 kinds of restricted enzyme of Sac I and Sal I.
After enzyme action is complete, reclaim linearisation digestion products with miniprep dna QIAquick Gel Extraction Kit.
(2) linearization plasmid pPIC9K-Lac electricity conversion Pichia pastoris GS115, the identification of positive transformant and novel laccase enzyme
The screening of high expression strain
A, the recombinant plasmid dna (15 μ g) of linearized recovery is added to the 100 μ L Pichia sp. preparing in advance
In GS115 competent cell, mix, after ice bath 20min, the reactant liquor of mixing is added in advance in the electric revolving cup of ice bath.
, equipped with the conversion cup 5min of conversional solution, according to the parameter that electric rotary device is recommended, (voltage 2500V, during electric shock for b, ice bath
Between 5ms), carry out Pichia sp. electricity conversion.
After c, pulse, add the sorbitol solution of the 1mol/L of 1mL pre-cooling immediately in electric revolving cup, conversional solution is retransferred
To in the centrifuge tube of a new 1.5mL.
D, 30 DEG C of quiescent culture 1-2h, absorption Pichia pastoris GS115 electricity turns liquid 200 μ L and is coated on MD culture medium flat plate.
E, 30 DEG C of quiescent culture are until transformant occurs.
F, picking transformant single bacterium colony are dissolved in 10 μ L deionized waters, take 2 μ L bacterium solution, add Lyticase wall breaking enzyme,
30 DEG C of reaction 10min, reactant liquor is immediately placed in -80 DEG C of refrigerator freezing 10min, so that yeast cell wall is fully cracked, the base of release
Because group enters performing PCR as template.To proceed to the Pichia pastoris GS115/pPIC9K of empty plasmid pPIC9K as comparison, determine positive
Transformant.
G, on the basis of determining positive transformant, first with the high heredity of resistant panel screening containing variable concentrations Geneticin
The transformant of chloramphenicol resistance, then measures the enzyme activity of the novel laccase enzyme of the transformant of these high geneticin resistant respectively, with
Superior strain GS115/pPIC9K-Lac to novel laccase enzyme.
Embodiment 4：The mensure of laccase activity
The laccase of separate sources (plant, animal, funguses, antibacterial) is different from the affinity of substrate reactions, enzyme activity determination side
In method, reaction substrate used, reaction condition and enzyme-activity unit definition have a significant impact to the measurement result of laccase activity.Right
Phenylenediamine, guaiacol, o-tolidine, catechol, laccol, syringaldazine, 2,6- syringol, 2,2 '-connection
Double (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) di-ammonium salts of nitrogen can be used as the substrate of laccase activity mensure.Wherein, adjacent ditolyl
Aqueous amine dissolubility is poor；During with guaiacol for substrate, reaction is highly stable, but the response time is longer, the laccase activity that records
It is worth low, less employing；When laccase activity is measured for substrate with syringaldazine, although laccase activity value is higher, work as
Substrate and enzyme liquid consumption react unstable when higher, if taking the method reducing substrate and enzyme liquid consumption, can be largely
Reaction is overcome to be difficult the defect terminating.With ABTS for substrate measure laccase enzyme activity when, have be quick on the draw, good stability, weight
Existing good the features such as.
The method measuring laccase activity has HPLC method, oximetry, spectrophotography, level spectrometry etc..With ABTS as substrate
Measuring laccase activity is one of method most commonly seen abroad at present.It has following pluses and minuses：(1) ABTS is soluble in water,
Under room temperature place 6 months still more stable.(2) its reaction only has a step, that is, from ABTS to its radical cation.The sun of ABTS
Ion radical is light blue green in aqueous, and more stable, generally in several hours or be stable within these few days.
(3) in the presence of laccase, the molar absorption coefficient of ABTS colored solutions is higher, illustrate with its for substrate measure sensitivity relatively
High.(4) up to the present, also not it has been reported that ABTS has carcinogenesis, mutagenesises or strong toxicity.(5) with
ABTS carries out the quantitative analyses of laccase for substrate, measures the change of 3min internal absorbance generally under 420nm.Entered with this substrate
Row quantitative analyses have one disadvantage in that, that is, the absorbance of ABTS radical cation solution is easily subject to unreacted ABTS in solution
The impact of concentration.This generates a problem：Molar absorption coefficient 36000L/ (mol cm) under 420nm is in pure ABTS
Try to achieve under conditions of radical cation, and in practical measurement reaction, ABTS is excessive, and this results in people and underestimates
Enzyme activity.This impact can not be ignored, because during reaction terminating, the ABTS of 1mmol/L at least also to be had in solution.Although using ABTS
There are the problems referred to above for substrate, but it is still one of optimal substrate that laccase activity measures.
1st, laccase is measured for substrate with ABTS (double (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) di-ammonium salts of 2,2 '-azino)
Enzyme activity
(1) principle of laccase activity is measured with ABTS for substrate
Laccase decomposes ABTS and is changed into ABTS radical cation, however at 420nm ABTS radical cation extinction
Coefficient is far longer than substrate A BTS.So, with the continuous reaction of laccase and substrate A BTS, the concentration of ABTS free radical gradually increases
Greatly, its absorbance is also gradually increased.
The definition of enzyme activity：Under certain conditions, the enzyme amount required for ABTS of 1 μm of ol of oxidation per minute is defined as one
Enzyme-activity unit.
(2) method and the step of laccase activity is measured with ABTS for substrate
A, take the Cu containing 4mmol/L of 200 μ L2+0.1mol/L citrate-phosphate disodium hydrogen buffer (pH=2,
2.5th, 3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8) or 200 μ L containing 4mmol/LCu2+0.05mol/L glycine-
Sodium hydrate buffer solution (pH=8.5,9,9.5,10,10.5,11), in 96 orifice plates, 20,25,30,35,40,45,50,55,
60th, 65,70,75,80,85 or 90 DEG C of insulation 1min.
B, the enzyme liquid of the dilution suitable multiple of addition 10 μ L, add the substrate (ABTS of 50mmol/L) of 30 μ L, mix,
The initial OD value of record reaction and the reaction OD value of 3 minutes.
2nd, laccase activity is measured for substrate with 2,6- syringol (2,6-dimethoxyphenol, DMP)
(1) principle of laccase activity is measured with DMP for substrate
DMP can be oxidized to 3,5 by laccase, 3 ', 5 '-tetramethoxy diphenyl quinone, and this product has absorption maximum at 468nm
Peak, its molar absorption coefficient 49.6mM-1cm-1, the concentration of this product and the linear pass of its absorbance in the range of finite concentration
System, by surveying the change of absorbance come the generating rate of measure and calculation product.
The definition of enzyme activity：Under certain conditions, the enzyme amount required for DMP of 1 μm of ol of oxidation per minute is defined as one
Enzyme-activity unit.
(2) method and the step of laccase activity is measured with DMP for substrate
A, take the Cu containing 4mmol/L of 200 μ L2+0.1mol/L citrate-phosphate disodium hydrogen buffer (pH=2,
2.5th, 3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8) or 200 μ L containing 4mmol/LCu2+0.05mol/L glycine-
Sodium hydrate buffer solution (pH=8.5,9,9.5,10,10.5,11), in 96 orifice plates, 20,25,30,35,40,45,50,55,
60th, 65,70,75,80,85 or 90 DEG C of insulation 1min.
B, the enzyme liquid of the dilution suitable multiple of addition 10 μ L, add the substrate (DMP of 8mmol/L) of 30 μ L, mix, note
The initial OD value of record reaction and the reaction OD value of 3 minutes.
3rd, enzyme activity calculates
Enzyme activity
In formula：△ OD represents from reaction start to finish, the variable quantity of absorbance.
V1Represent the cumulative volume of reaction system.
△ t represented from reaction start to finish, time used.
V2Represent in reaction system, the volume of enzyme liquid.
When ε represents with ABTS for substrate, its product molar absorption coefficient 36mM at 420nm-1cm-1.
When ε represents with DMP for substrate, its product molar absorption coefficient 49.6mM at 468nm-1cm-1.
D represents internal diameter or the light path thickness (cm) of extinction cup.
Measure the zymologic property of novel laccase enzyme by said method, this novel laccase enzyme zymologic property is as follows：
During with ABTS for substrate mensure novel laccase enzyme zymologic property, optimum temperature is 35 DEG C, and the most suitable action pH is 4；
During with DMP for substrate mensure novel laccase enzyme zymologic property, optimum temperature is 70 DEG C, and the most suitable action pH is 8；
Measure pH stability and the heat stability of this novel laccase enzyme with DMP for substrate, result shows：This novel bacterial laccase
Have good stability in the range of pH5～9, be embodied in and be incubated 35h in pH5, pH6 and pH9, measure residual at pH8,70 DEG C
Remaining enzyme activity, more than 60%, is incubated 35h in pH7 and pH8, measures remnant enzyme activity at pH8,70 DEG C more than 85%；
Good in 30～60 DEG C of heat stability, it is embodied in and is incubated 5h at 30～40 DEG C, measure remaining at pH8,70 DEG C
Enzyme activity is more than 95%, is incubated 4h at 50～60 DEG C, and measuring remnant enzyme activity at pH8,70 DEG C is more than 50%；With report
Laccase from Klebsiella pneumonia is compared, the pH stability of the claimed novel laccase enzyme of this patent and temperature stability
More preferably.
Embodiment 5：Expression in Bacillus subtillis recombinant bacterial strain for the novel laccase enzyme and preparation
By Bacillus subtillis recombinant bacterial strain WB600/pBSA43-Lac be inoculated in 5mL LB fluid medium (containing card that
Mycin, 50 μ g/mL) in, 37 DEG C, 220r/min overnight incubation, transfer in 50mL fresh LB according to 2% inoculum concentration,
Continue with 37 DEG C, 220r/min cultivates 48h, you can prepare high stability novel laccase enzyme crude enzyme liquid, surveyed for substrate with ABTS
Determine novel laccase enzyme crude enzyme liquid enzyme activity (pH4,35 DEG C under the conditions of), after Bacillus subtillis expression novel laccase enzyme recombinant bacterial strain fermentation
Laccase activity can reach 16.8U/mL, measures novel laccase enzyme crude enzyme liquid enzyme activity (pH8,70 DEG C under the conditions of) with DMP for substrate, withered
After careless bacilluss expression novel laccase enzyme recombinant bacterial strain fermentation, laccase activity can arrive 19.7U/mL；Then salt fractionation method is adopted to sink
Shallow lake novel laccase enzyme, collects protein precipitation, after dissolving, desalination of dialysing, then after ion-exchange chromatography, gel chromatography, freezing is dry
The pure enzyme enzyme powder of dry prepared novel laccase enzyme.
Embodiment 7：Expression and preparation that novel laccase enzyme dissociates in expression recombinant bacterial strain in Pichia sp.
The Pichia sp. being incubated on YPD solid plate is dissociated and expresses novel laccase enzyme recombinant bacterium GS115/pPIC9K-Lac
It is seeded in YPD fluid medium, 30 DEG C, 220r/min cultivates 24h.Fresh BMGY culture medium is transferred to 2% inoculum concentration
In, continue with 30 DEG C, 220r/min cultivates 24h, then 10min collects thalline is centrifuged with 6000rpm/min, thalline is transferred to
In BMMY culture medium.Again with 30 DEG C, 220r/min cultivates, and adds a methanol so as to final concentration is maintained at every 12h
0.5%V/V, can get the crude enzyme liquid of novel laccase enzyme, measures novel laccase enzyme crude enzyme liquid enzyme activity with ABTS for substrate after culture 120h
Power (pH4,35 DEG C under the conditions of), the Pichia sp. laccase activity after expression novel laccase enzyme recombinant bacterial strain fermentation that dissociates can reach 15.3U/
ML, measures novel laccase enzyme crude enzyme liquid enzyme activity (pH8,70 DEG C under the conditions of) with DMP for substrate, and Pichia sp. dissociates and expresses new paint
After the fermentation of enzyme recombinant bacterial strain, laccase activity can reach 18.6U/mL；Then adopt salt fractionation method to precipitate novel laccase enzyme, collect egg
White matter precipitates, after dissolving, desalination of dialysing, then after ion-exchange chromatography, gel chromatography, lyophilization is obtained the pure enzyme of novel laccase enzyme
Enzyme powder.
Embodiment 8：The desolventing technology to dyestuff for the novel laccase enzyme and the calculating of percent of decolourization
Using novel laccase enzyme enzyme liquid, desolventing technology is carried out to anthraquinone and azo dye, need to consider in catalystic converter system：
The factor such as reaction temperature, pH value in reaction, kind of dyes and concentration, mediator species and concentration, enzyme liquid consumption, bleaching time.Originally grind
Used in studying carefully, dyestuff includes：Reactive Brilliant Blue X-BR, reactive brilliant bule K-GR, Reactive Brilliant Blue KN-R, reactive dark blue M-2GE, the Congo
Red, cotton is blue, malachite green oxalate, azophloxine, bromophenol blue, acid mordant black PV.
(1) novel laccase enzyme is to dye decolored method and step
The dye decolored reaction system processing using 300 μ L of Laccase Catalyzed：
A, take the Cu containing 4mmol/L of 275 μ L2+Citrate-phosphate disodium hydrogen buffer (pH=4) of 0.1mol/L or
Person Cu containing 4mmol/L2+0.05mol/L Glycine-NaOH buffer (pH=8), in 96 orifice plates, 70 DEG C insulation
1min.
B, the concentration of addition 15 μ L are the above-mentioned dyestuff of 2000mg/L.
C, add 10 μ L dilution suitable multiple laccase enzyme liquid (corresponding enzyme activity be 0.05U/mL) (or identical
Under experiment condition, the enzyme liquid of 100 DEG C of inactivation 15min adding equivalent is as blank), survey its absorbance A0.
D, under the conditions of 70 DEG C decolour 60min, measure its absorbance be A1.
Each of the above desolventing technology is all repeated 3 times, results averaged.
(2) calculating of percent of decolourization：
After obtain two plants of recombinant bacterium fermentations, using the novel laccase enzyme enzyme powder of preparation, according to above-mentioned decolorization process, to upper
State dyestuff and carry out desolventing technology, 60min percent of decolourization has all reached more than 85%.
Claims (7)
1. a kind of novel bacterial laccase from Klebsiella pneumonia is it is characterised in that the aminoacid of described bacterial laccase
SEQ ID No in sequence such as sequence table：Shown in 2.
2. a kind of novel bacterial laccase from Klebsiella pneumonia according to claim 1 is it is characterised in that compile
The gene of the described bacterial laccase of code is Lac, its base sequence such as sequence table SEQ ID No：Shown in 1.
3. a kind of novel bacterial laccase from Klebsiella pneumonia according to claim 1 is it is characterised in that institute
The preparation method stating bacterial laccase comprises the steps：
(1) gene described in claim 2 is carried out enzyme action, be connected with expression vector and obtained new recombinant vector；
(2) recombinant vector is transformed in host cell, obtains recombinant bacterial strain, afterwards recombinant bacterial strain ferments, obtain high stable
Property bacterial laccase.
4. the cloning vehicle containing the novel laccase enzyme gene described in claim 2, expression vector and host cell.
5. the cloning vehicle containing novel laccase enzyme gene according to claim 4, expression vector and host cell, its
It is characterised by, described cloning vehicle is pBSA43 carrier, described expression vector is pBSA43, described host cell is withered
Careless bacilluss WB600.
6. the cloning vehicle of novel laccase enzyme gene according to claim 4, expression vector and host cell, its feature
It is, described cloning vehicle is pPIC9K carrier, described expression vector is pPIC9K, described host cell is to finish red ferment
Female GS115.
7. a kind of novel bacterial laccase from Klebsiella pneumonia according to claim 1 is in azo and Anthraquinones
The application of dye decolored aspect.
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