CN102061290B - Method for realizing over expression of thermostable laccase gene through location transformation - Google Patents
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Abstract
The invention discloses a method for realizing over expression of the thermostable laccase gene through location transformation. Thermostable laccase produced from thermus thermophilus bacteria has great application potential in the industrial processes of pulp bleaching, textile decoloring, biomass hydrolysate detoxifying and the like, but the thermostable laccase gene has low expression level and can not become an enzyme source. In the invention, a carrier in a pHsh series is firstly applied to carrying out cloning, in-situ mutagenesis and induced expression on the laccase gene. By carrying out case analysis and multiple explorative site-directed mutagenesis on a special sequence of the thermostable laccase gene, the invention has the effects of reducing rare codons in the gene, lowering the GC base content and weakening formation of a nucleic acid secondary structure and the like, thereby enhancing the expression level of the gene in escherichia coli by 317 times compared with a natural gene.
Description
Technical field
Make the gene of utmost point thermotolerance laccase realize the method for overexpression by location transformation, relate to the fields such as molecular biology, genetically engineered and zymetology.Thereby be specifically related to the gene of a coding utmost point thermotolerance laccase is carried out the method that efficiently expresses of the location transformation realization laccase gene of base sequence.
Technical background
Laccase (Laccase, EC1.10.3.2) is a kind of polyphenoloxidase, belongs to blue many copper oxydase family, and the catalytic center of zymoprotein generally comprises 4 cupric ions, therefore also is referred to as many copper oxydase.Laccase has unique substrate catalyst mechanism, and in catalytic reaction process, the electronics of laccase in can the abstraction reaction substrate is oxidized to active radical form with substrate, simultaneously, and the O around the electronics of catching is sent to by cupric ion
2, finally produce H
2O.
Since laccase can the various different types of structure of oxidation phenols and non-phenolic compound, all have broad application prospects in fields such as pulping and paper-making, food, textile printing and dyeing, Pollutant Treatment and synthetic chemistries.In paper industry, traditional paper pulp delignification/bleaching method adopts chloride chemical SYNTHETIC OPTICAL WHITNER, and medicament causes obvious destruction to paper cellulose, affects paper pulp yield and paper strength, causes simultaneously serious environmental pollution.Laccase has the macromolecular ability of xylogen degradation, utilizes laccase to remove residual lignin in the paper pulp, has the atopic height, and environment amenable advantage can reduce the consumption of chemical bleaching agent, the more clean and effective that bleaching process is become.In foodstuffs industry, laccase is mainly used in beverage processing, makes the polyphenol substance oxidative degradation in the beverage, effectively improves degradation problem under the rear color and luster intensification of beverage storage, muddiness and the mouthfeel; Can also be used for the analysis of food ingredient, as measuring the content of Ascorbic Acid in Foods.Laccase has good decolorizing effect, therefore is used to the fabric decolouring in textile printing and dyeing industry; At field of Environment Protection, laccase is used for chlorophenols compound and the aniline substituent of degrading pesticide and waste water; In the Application and Development of wood fibre, laccase is used for the detoxification of biomass hydrolysate.
Pay close attention to today of environmental protection and " low-carbon (LC) " people, the Application and Development of laccase enjoys attention.There have been a variety of laccases successfully to be purified or clone, but have at present two problems: the commercialization laccase that (1) has been developed all derives from fungal laccase, and the fungal growth cycle is long, and production of enzyme is low, even employing genetic engineering means, laccase output also are difficult to reach industrial level; (2) the laccase ubiquity poor stability of having developed is generally 15 minutes to 1 hour work-ing life, and this greatly reduces application of enzymes efficient, has further improved the trace utilization cost of enzyme.
Have a liking for enzyme that high temperature microbe produces have thermus thermophilus (
Thermus thermophilus) be a kind of extreme thermophilic bacterium, the transformation period of the laccase that produces under 80 ℃ reaches 14 hours, work-ing life, the laccase more microbe-derived than normal temperature grown more than ten times, and at high temperature accelerated reaction, improve catalytic efficiency (K. Miyazaki, Extremophiles, 2005,9:415-425).But because extreme thermophilic bacterium growth conditions is harsh, the output of enzyme is very low, by natural bacterium producing multi enzyme preparation fermentation, is difficult to obtain the rational enzyme product of production cost.Existing investigator attempts this laccase gene is carried out heterogenous expression, but the expression level of this nature gene is very low, is difficult to detect laccase activity (K. Miyazaki, Extremophiles, 2005,9:415-425), also do not realize so far the report of overexpression about this gene.
Foreign gene Expression in Escherichia coli level is subject to impact and the restriction of many factors, and the relation between each factor is intricate, and therefore, most of nature genes have expression level very low or produce the phenomenon of inclusion body.The research that improves the exogenous gene expression level must be for the particular sequence of different target gene, attempt secondary structure, the initial sum termination element of test translation, selection expressive host, the correction codon preference of different expression vectors and expression regulation mode, analysis and change mRNA, and optimization of fermentation conditions etc.This laboratory pair
T. thermophilusLaccase gene launch research, the new and effective expression vector pHsh that utilization has independent intellectual property right makes up recombinant expression plasmid, use site-directed mutagenesis technique, laccase gene in the plasmid is carried out various design and rational and the transformation of sequence location, through repeatedly testing and transforming, realize utmost point thermotolerance laccase efficiently expressing in intestinal bacteria, successfully created the recombinant production technology of utmost point thermotolerance laccase.
Summary of the invention
1. goal of the invention:
Laccase is the large biological catalyst of the wide demand of a kind of purposes, in order to solve the key issues such as the poor stability that yields poorly that exist in the laccase use, we carry out multiple Design ﹠ reform to the gene of a coding utmost point thermotolerance laccase, increase substantially this gene Expression in Escherichia coli level, to reach industrial demand.
2
.Technical scheme:
The present invention is cloned into the laccase gene in the thermus thermophilus karyomit(e) and is carried out bioinformatic analysis, original position site-directed mutagenesis and abduction delivering test among the bacillus coli gene expression vector pHsh.Genetic analysis is the result show, codon usage frequency has been compared very big-difference with intestinal bacteria in the nature gene sequence; GC content surpasses 70%; The mRNA that transcribes generation forms complicated secondary structure in the Escherichia coli Growth temperature range.All of these factors taken together all is the potential feature that hinders the gene effective expression.Therefore, main technical schemes of the present invention all centers on the analysis of gene order, the design in transformation site, the large-scale location of DNA enforcement is transformed with the GC content of adjusting codon preference, reduction gene, the free energy that slackens the mRNA secondary structure, and the DNA fusion etc.
The said method that makes utmost point thermotolerance laccase gene realization overexpression by locating transformation may further comprise the steps:
(1) to the formation that mainly is conceived to reduce colibacillary rare codon, reduction GC base contents, reduction nucleic acid secondary structure of the location transformation of utmost point thermotolerance laccase gene;
(2) except 5 ' end at the mature peptide gene merges 7 ~ 12 codons, all the other codons are only done the synonym conversion to keep the natural character of enzyme;
(3) there were significant differences for the dna sequence dna of the improved gene in location and nature gene;
(4) carrier that uses pHsh series to laccase gene clone, original position mutagenesis and abduction delivering.
The invention discloses the utmost point thermotolerance laccase gene that transform a location, its base sequence is shown in SEQ ID NO:5.
The main operational steps of gene positioning reconstruction of the present invention comprises:
(1) numbers according to laccase gene TTC1370(NCB database: AAS81712) design pair of primers, from the genome of thermus thermophilus, amplify the gene (called after of natural laccase mature peptide
Lcs-0), sequence and is cloned into it among the pHsh and is produced recombinant plasmid pHsh-shown in SEQ ID NO:1
Lcs-0.
(2) at plasmid pHsh-
LcsCodon preference to the 1st to the 27th amino-acid residue of mature peptide in-0 is analyzed and the mutagenesis of setting up an office (Fig. 1).After suddenling change successfully, with the unnamed gene that produces be
Lcs-1, sequence and will contain the plasmid pHsh-of this gene shown in SEQ ID NO:2
Lcs-1 is transformed into and carries out abduction delivering in the intestinal bacteria, the variation of checking gene expression dose.
(3) at plasmid pHsh-
LcsIn-1, right
LcsThe codon (numbering 90 ~ 174 among Fig. 1) of the 30th to the 58th amino-acid residue of-1 mature peptide is optimized design and mutagenesis.After suddenling change successfully, with the unnamed gene that produces be
Lcs-2, sequence is shown in SEQ ID NO:3, and with plasmid pHsh-
Lcs-2 are transformed into and carry out abduction delivering in the intestinal bacteria, the variation of checking gene expression dose.
(4) take turns on the basis of mutagenesis front 2, (from 834 to 909 bp) carry out site-directed mutagenesis to the section that is rich in the GC base in the nature gene, and the unnamed gene that success suddenlys change is
Lcs-3, sequence is shown in SEQ ID NO:4 (Fig. 2), and with plasmid pHsh-
Lcs-3 are transformed into and carry out abduction delivering in the intestinal bacteria, the variation of checking gene expression dose.
(5) at plasmid pHsh-
LcsBase (numbering 171 ~ 252 among Fig. 1) to the 57th to the 84th amino-acid residue of coding laccase mature peptide in-3 is carried out site-directed mutagenesis, and the unnamed gene that success suddenlys change is
Lcs-4, sequence is shown in SEQ ID NO:5, and with plasmid pHsh-
Lcs-4 are transformed into and carry out abduction delivering in the intestinal bacteria, the variation of checking gene expression dose.
(6) for the ease of the restructuring separation and purification of enzyme,
Lcs5 ' one of end fusion of-4 genes is histidine-tagged, and removal remains in initiator codon multiple clone site original series before, generation plasmid pHsh-
Lcs-H.
3. the obtained beneficial effect of the present invention:
The present invention carries out comprehensive bioinformatic analysis by the nature gene to utmost point thermotolerance laccase, and carry out multistage, many wheels/time the location transform and recombinant expressed test, under the prerequisite that does not change aminoacid sequence and zymologic property, produce a conservative property with nature gene and be 90% utmost point thermotolerance laccase gene, the expression level of gene is brought up to every milligram of albumen 3.17 units from every milligram of albumen 0.01 unit, make utmost point thermotolerance laccase output increased 317 times (Fig. 3).
Description of drawings
Fig. 1. behind front 4 site-directed mutagenesises of taking turns 252 bases of 5 ' of utmost point thermotolerance laccase gene end from
Lcs-0 arrives
Lcs-3 variation
Fig. 2. cut down the potentiality that are positioned at the GC island between 834 ~ 909 in the nature gene and form secondary structure by site-directed mutagenesis.
Fig. 3. the effect that gene positioning reconstruction is received improving utmost point thermotolerance laccase output.Swimming lane M, the protein molecule quality standard; Swimming lane 1 to 7, bacterium coli solubility albumen contains respectively expression plasmid pHsh, pHsh-in the cell
Lcs-0, pHsh-
Lcs-1, pHsh-
Lcs-2, pHsh-
Lcs-3, pHsh-
Lcs-4, pHsh-
Lcs-H; Swimming lane 8, purified utmost point thermotolerance laccase.
Embodiment
Employed term unless other explanation is arranged, generally has the implication that those of ordinary skills understand usually in the present invention.Below method in conjunction with specific embodiments, and comparable data is described the present invention in further detail.Should be understood that embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
1. the clone of thermus thermophilus laccase gene
The present invention adopt the method for K. Miyazaki report carry out the extraction of cultural method and the genomic dna of thermus thermophilus (Extremophiles, 2005,9:415-125).The genetic manipulations such as the separation of the extraction of plasmid DNA, pcr amplification, DNA, purifying, enzyme are cut, connection, colibacillary conversion are by " (Sambrook and Russell is carried out in the standard method on the molecular cloning handbook third edition, 2001, CSHL Press, Cold Spring Harbor, New York); Test is purchased (TaKaRa Biotech, Dalian) with toolenzymes such as archaeal dna polymerase, restriction enzyme, T4 dna ligases from precious biotechnology company limited; Primer is synthetic by biosynthesizing center, Chinese Academy of Sciences Shanghai; The mensuration of dna sequence dna is finished by the large Gene science limited-liability company of Beijing China.
2. utmost point thermotolerance laccase gene and through the mutant gene Expression in Escherichia coli behind the site-directed mutagenesis
PHsh is the heat-inducible type expression vector (U.S. Patent number US 7,807,460 B2) of an autonomous invention, research paper (Wu, et al., Biotechnol Lett that its characteristic and use-pattern are delivered referring to Wu etc., 2010,32:795 – 801).Use recombinant plasmid pHsh-
Lcs-0 and mutant transform
E.coliDH10B, and 30 ℃ of cultivations, picking 1 ~ a plurality of single colony inoculations contain the LB liquid nutrient medium of 0.1 mg/ml penbritin, are cultured to OD in 30 ℃ of concussions
600Reach needed value, test tube is moved into 42 ℃ of shaking bath inducible gene expressions, continue to cultivate 2-6 hour, centrifugal collecting cell after the activity of monitoring objective enzyme, activity reach is steadily used the ultrasonic disruption cell, centrifugal removal cell relic, the activity of laccase in the mensuration supernatant liquor.
3. the purifying of enzyme assay and enzyme
Enzyme activity determination reaction system cumulative volume 1000 mL.The reaction substrate ABTS(2 of 10 mmol/L, 2 '-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), the aqueous solution 50 mL, 0.1 mol/L CuSO
4Sodium acetate buffer 920 mL of solution 10 mL and 20 mmol/L pH 4.5,85 ℃ of lower insulations add enzyme liquid 10 mL that suitably dilute and start reaction after 10 minutes, react the sodium azide solution 10 mL termination reactions that add rapidly 0.1 mol/L after 2 minutes, reaction tubes is put to cool off in the ice-water bath and is surveyed measured reaction liquid light absorption value A under 420 nm after 5 minutes
420ABTS free radical by the laccase oxidation makes A
420Numerical value improves, reaction product optical extinction coefficient e
420Be 3.6 * 10
4M
-1Cm
-1
The activity unit of enzyme (U) is defined as under this reaction conditions, and oxidation 1 mmol ABTS generates the required enzyme amount of corresponding ABTS free radical in 1 min.Use the Bradford reagent of U.S. Bole (Bio-Rad) company to measure protein concn, use according to the method that product description is recommended.Adopt U.S. Navagen company that test kit BugBuster Ni-NTA His-Bind Purification Kit and operational guidance are provided, by will the recombinate purifying (Fig. 3) of laccase of nickel ion affinity chromatograph.
4. genetic analysis and mutational site design
Adopt DNA analysis software (DNA2.0-Gene Designer) that the laccase original gene is carried out the codon preference analysis, seek the non-Preference of intestinal bacteria or the rare codon that exist in the gene.Through DNA on-line analysis software mfold(http: //frontend.bioinfo.rpi.edu/applications/mfold) analyze and find that there is a zone of being rich in the GC base (GC island) that is easy to form complicated secondary structure in laccase gene sequence middle part, the existence in this zone causes the gene sequencing signal interruption, and affects the stability of recombinant expression plasmid in escherichia coli host.The mutational site is designed main GC content take adjustment codon preference, reduction gene, is slackened the mRNA secondary structure as purpose (Fig. 1, Fig. 2).
5. gene site-directed mutagenesis method
Transform the location that the present invention adopts the gene site-directed mutagenesis test kit TaKaRa MutanBEST Kit of precious biotechnology company limited (TaKaRa) to implement utmost point thermotolerance laccase.The basic experiment method is undertaken by the operational guidance that the test kit supplier provides.Synthetic a pair of adjacent reverse primer, the 5 ' end of the base that will make a variation design beyond 15 bases of distance 3 ' end; Carry out inverse PCR to contain the gene expression plasmid of intending mutator gene as template, the linear PCR product that obtains carries out from cyclisation with the T4 dna ligase after DNA purifying and phosphokinase processing again; Transform intestinal bacteria and filter out transformant.Concrete operations are referring to the product description of TaKaRa company.
Embodiment 1: the clone of utmost point thermotolerance laccase gene
(1) according to utmost point thermotolerance laccase gene TTC1370 design pair of primers: 5 '-TTCTGCAGAT GGCCCAAGGC CCTTCCTTC-3 ' (SEQ ID NO:6) and 5 '-CCCGCATGCC TAACCCACCT CGAGGAC-3 ' (SEQ ID NO:7).
(2) take the thermus thermophilus genomic dna as template, with above-mentioned primer laccase gene is carried out pcr amplification.
(3) use
PstI and
SphI processes the PCR product, and with it and warp
PstI and
SphThe heat shock expression vector pHsh of I double digestion connects.
(4) transform importing by electricity
E.coliBehind the DH10B, be applied on the LB flat board that contains 0.1 mg/ml penbritin, in 30 ℃ of cultivations.
(5) picking list bacterium colony from the LB flat board is inoculated into respectively in the LB liquid nutrient medium that 4 ml contain 0.1 mg/ml penbritin, after 30 ℃ of overnight incubation, extracts plasmid by standard method.
(6) plasmid is addressed to the large Gene science limited-liability company of China and carries out sequencing, and the dna sequence dna plasmid consistent with TTC1370 named and be pHsh-
Lcs-0, and as parent material and the experiment contrast of follow-up study.
Embodiment 2: the site-directed mutagenesis of utmost point thermotolerance laccase gene
(1) analyzes the laccase nature gene with data analysis software DNA2.0-Gene Designer
LcsCodon in-0 is analyzed the frequency of utilization of these codons in intestinal bacteria, finds that intestinal bacteria rare codon and non-optimal codon exist
LcsDistribution situation in-0.
(2) design and synthesize a pair of reverse long primer the rare and non-optimal codon in the 1st to the 27th codon is replaced, the sequence of primer is: 5 ’ – ACGAACAACT TTCGGTTCCG GAAAGCTCGG ACCCTGCATGG GTATATCTCC TTCTTG –, 3 ' (SEQ ID NO:8), 5 ’ – AGCCAGGGTG GTCTGCTGAG CCTGAAACTG AGCGCAACCC CGACCCCGCT TGCCCTGG –, 3 ' (SEQ ID NO:9).
(3) with pHsh-
Lcs-0 is template, carries out pcr amplification with above-mentioned primer.The PCR condition setting is: 95 ℃, and 5 min; Time out adds the Pyrobest polysaccharase, adds the sealing of 40 mL paraffin oils; 35 circulations (94 ℃, 30 s; 60 ℃, 60 s; 72 ℃, 4 min); 72 ℃, 10 min; Reaction stops, 4 ℃ of insulations.
(4) separate PCR product and template by agarose gel electrophoresis, the dna fragmentation in the pcr amplification band is reclaimed in rubber tapping.After processing the PCR product and make it phosphorylation with T4 DNA kinases, add the T4 dna ligase and make it recirculation.
(5) transform importing by electricity
E.coliBehind the DH10B, be applied on the LB flat board that contains 0.1 mg/ml penbritin, in 30 ℃ of cultivations.Picking list bacterium colony from the LB flat board is inoculated into respectively in the LB liquid nutrient medium that 4 ml contain 0.1 mg/ml penbritin, after 30 ℃ of overnight incubation, extracts plasmid by standard method.
(6) plasmid is addressed to the large Gene science limited-liability company of China and carries out sequencing, and the plasmid that dna sequence dna meets the site-directed mutagenesis design is named and is pHsh-
Lcs-1, and as parent material and the experiment contrast of follow-up study.
Embodiment 3: the next round site-directed mutagenesis of utmost point thermotolerance laccase gene
The method of using is substantially the same manner as Example 2, and difference is the target base section of mutagenesis and the sequence of inverse PCR primer: 5 ’ – GCTCGGACCC TGCGGCATTG CATGTGCCAT ACCACCCATA TCCATCATGC CCATGGCC –, 3 ' (SEQ ID NO:10), 5 ’ – CGTCCGGAAA CACTGCTGTA TCTGATTGCA CCGAAAAATC CGAAGCCCTT ACCCCTGCC –, 3 ' (SEQ ID NO:11).In order to obtain the cumulative of Mutagenic Effect, this mutagenesis of taking turns can take the mutant of previous round as template, produce plasmid pHsh-
Lcs-2.
Embodiment 4: the next round site-directed mutagenesis of utmost point thermotolerance laccase gene
(1) in the experiment, the gene sequencing signal interrupts through a zone (GC island) of being rich in the GC base of the laccase gene of being everlasting, with DNA on-line analysis software mfold(http: //frontend.bioinfo.rpi.edu/applications/mfold) to analyze and find that the GC island causes DNA to form complicated secondary structure, the mutant of the present invention's design can reduce this structure (Fig. 2).
(2)-(6) substantially the same manner as Example 2, difference is the target base section of mutagenesis and the sequence of inverse PCR primer: 5 '-ATATACCCAT GCAGGGTCCG TCTTTCCCGG AACCGAAAGT TGTTCG –, 3 ' (SEQ ID NO:12), 5 '-CGAACAACTT TCGGTTCCGG GAAAGACGGA CCCTGCATGGG TATAT –, 3 ' (SEQ ID NO:13).In order to obtain the cumulative of Mutagenic Effect, this mutagenesis of taking turns can be take the mutant of previous round as template.
Embodiment 5: the next round site-directed mutagenesis of utmost point thermotolerance laccase gene
The method of using is substantially the same manner as Example 2, and difference is the target base section of mutagenesis and the sequence of inverse PCR primer: 5 ’ – TCGGTTCCGG CAGACGGTTT TCCAGGGTCA GACGAACGGT GTCACGCGGA CGAACACGC –, 3 ' (SEQ ID NO:14), 5 ’ – CCAACCTGCA CTGGCACGGT CTGCCGATCT CTCCGAAAGT TGACGACCCC TTCCTGGAG –, 3 ' (SEQ ID NO:15).In order to obtain the cumulative of Mutagenic Effect, this mutagenesis of taking turns can be take the mutant of previous round as template.
Embodiment 6: inverse PCR adds histidine-tagged
Step (2)-(6) among method and the embodiment 2 are basic identical, and difference is the sequence of inverse PCR primer: 5 ’ – CACCACCACG CCCAAGGC CCTTCCTTC – 3 ' (SEQ ID NO:16), 5 '-GTGGTGGTGC ATGGGTATAT CTCCTTCTT – 3 ' (SEQ ID NO:17).In order finally to add the distance between histidine-tagged and adjustment SD sequence and the initiator codon, carry out on the basis of former all sudden changes of mutagenesis that this is taken turns, product is pHsh-
Lcs-H.
The detection of the expression level of embodiment 7 utmost point thermotolerance laccase genes
(1) uses respectively recombinant expressed various Plasmid Transformation
E.coliDH10B, coating is contained in penbritin 0.1 mg/ml LB culture medium flat plate, obtains recombinant bacterium after 30 ℃ of cultivations.
(2) picking 1 ~ a plurality of single colony inoculations contain the LB liquid nutrient medium of 0.1 mg/ml penbritin, are cultured to OD in 30 ℃ of concussions
600=0.6 ~ 0.8.
(3) test tube is moved into 42 ℃ of shaking bath inducible gene expressions, continue to cultivate 6 hours, centrifugal collecting cell is used the ultrasonic disruption cell, and centrifugal removal cell relic obtains crude enzyme liquid.
(4) measure the activity of laccase in the supernatant liquor, and pass through sds gel electrophoresis observing protein band.
Embodiment 8: produce enzyme with the improved utmost point thermotolerance laccase gene Expression in Escherichia coli in location
(1) uses recombinant expression plasmid pHsh-
Lcs-H transforms
E.coliBL21, coating is contained in penbritin 0.1 mg/ml LB culture medium flat plate, obtains recombinant bacterium after 30 ℃ of cultivations
E.coli(pHsh-
Lcs-H).
(2) picking recombinant bacterium list bacterium colony enters to contain in the TB substratum test tube of penbritin 0.1 mg/ml 30 ℃ of shaking tables and cultivates, and is about 2.0 to the concentration OD600 of recombinant bacterium.
The bottled 25ml TB of (3) 100 ml triangles substratum adds penbritin to 0.1 mg/ml, with the bacterium liquid in the 2% inoculum size access test tube; Triangular flask is put 30 ℃ of shaking table shaking culture, grow to cell density (OD
600) when reaching 13 left and right sides shaking flask being moved into 42 ℃ of shaking baths continuation cultivations, heat-inducible is expressed centrifugal receipts bacterium after 2 hours.
(4) the This-HCl damping fluid re-suspended cell of usefulness pH 8.0, the centrifugal cell conditioned medium liquid that obtains after the high pressure fragmentation, the mensuration of the laccase activity of recombinating, the result shows that restructuring laccase output is 4000 U ∕ L in the supernatant cell extract, obtains the utmost point thermotolerance laccase of the overexpression in intestinal bacteria.
(5) provide sample buffer diluting cells extracting solution among the test kit BugBuster Ni-NTA His-Bind Purification Kit to 1x concentration with Navagen company, allow sample flow cross Ni
2+Affinity column (5 mL), with after the sample buffer balance of 1x, the elutriant with 1x washes laccase again, detects the purity (Fig. 3) of enzyme after the dialysis.
Claims (1)
1. a utmost point thermotolerance laccase gene of transforming through the location is characterized in that its sequence is shown in SEQ ID NO.5.
2. the overexpression method of a utmost point thermotolerance laccase gene is that the described utmost point thermotolerance of claim 1 laccase gene is cloned into the abduction delivering that carries out laccase gene among the expression vector pHsh.
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| Huawei Wu 等.pHsh vectors, a novel expression system of Escherichia coli for the large-scale production of recombinant enzymes.《Biotechnol Lett》.2010,第32卷(第6期),第795-801页. * |
| HuaweiWu等.pHshvectors a novel expression system of Escherichia coli for the large-scale production of recombinant enzymes.《Biotechnol Lett》.2010 |
| Kentaro Miyazaki.A hyperthermophilic laccase from Thermus thermophilus HB27.《Extremophiles》.2005,第9卷第415-525页. * |
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