CN101914556B - DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby - Google Patents
DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby Download PDFInfo
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Abstract
The invention discloses a DNA for high-level expression of N-acyl homoserine lactonase in yeasts and engineering bacteria constructed thereby. The DNA provided by the invention is the DNA expressed by the sequence 2 in a sequence list. On the basis of the traditional homoserine lactonase gene aiiaB546, the DNA sequence thereof is optimized to obtain the optimized gene aiiaB546 M by the method. The expression ability of the optimized gene in Pichia pastoris is obviously higher than that of the gene before optimization. An aiiaB546 M-containing recombinant expression plasmid is constructed; and recombination strains of the high-efficient homoserine lactonase can be obtained by introducing the recombinant expression plasmid into the Pichia pastoris. The expression ability of one of the recombination strains is particularly high. The DNA and the engineering bacteria have great value to the production of the homoserine lactonase.
Description
Technical field
The present invention relates in yeast, efficiently express the DNA of N-acyl homoserine lactones enzyme and the engineering bacteria of structure thereof.
Background technology
Because genetic code has degeneracy, a seed amino acid can have 1-6 the synonym that frequency of utilization is different.For specific species, the gene of high expression level often uses the specific synonym of part, and these codons are considered to the superior codon (optimal codon) of this species cance high-expression gene, and this phenomenon is called codon-bias (codon bias).The preferences of codon makes the foreign gene of DCRP often be difficult to express at the heterogeneity biological cell high-efficient.The foreign gene that in yeast, obtains to efficiently express often all is the coded gene of yeast preference codon; Statistical study through son that yeast genes is accessed to your password confirms have 25 to be that yeast is had a preference in 61 all codons, and therefore codon is carried out the preferences transformation can improve the expression amount of recombinant protein in this system in a large number.
N-acyl homoserine lactones enzyme is the proteolytic ferment of one type of specificity degraded N-acyl homoserine lactones class signaling molecule (AHLs); Generate acylhomoserine through hydrolysis AHLs; AHLs is lost activity; Thereby the quorum sensing mechanism of blocking-up pathogenic bacteria makes pathogenic bacteria lose pathogenecity, and it extensively is present in the multiple mikrobe.N-acyl homoserine lactones enzyme is as the toolenzyme of a kind of novel antibacterial strategy (quorum sensing cancellation strategy) and become the focus of research in recent years.
At present mainly is through making up transgenic plant, its mechanism of action in the quorum sensing cancellation of transgenic bacteria research, and there are the problem aspect Biosafety and the validity in transgenic plant or transgenic bacteria in practical application.
Summary of the invention
The purpose of this invention is to provide the DNA that in yeast, efficiently expresses N-acyl homoserine lactones enzyme and the engineering bacteria of structure thereof.
Dna molecular provided by the invention, for (a) as follows or (b):
(a) sequence 2 of sequence table is from the DNA shown in 5 ' terminal the 7th to 759 Nucleotide;
(b) DNA shown in the sequence 2 of sequence table.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said dna molecular all belong to protection scope of the present invention.
The MCS that said recombinant expression vector can be at the pPIC9 carrier inserts the recombinant plasmid that said dna molecular obtains, and is preferably at the EcoR of pPIC9 carrier I and Not I enzyme and cuts the recombinant plasmid that the said dna molecular of insertion obtains between the recognition site.
Said reorganization bacterium can be and in the host bacterium, imports the reorganization bacterium that said dna molecular obtains, and specifically can be and in the host bacterium, imports the reorganization bacterium that said recombinant expression vector obtains.Said host bacterium can be pichia spp (Pichia pastoris), specifically can be pichia spp (Pichia pastoris) GS115, preferred pichia pastoris phaff (Pichia pastoris) AiiA-B546M, CGMCC No.3975.
Pichia pastoris phaff (Pichia pastoris) AiiA-B546M; CGMCC No.3975 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 02nd, 2010 and (is called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.3975.
Described reorganization bacterium can be used for producing N-acyl homoserine lactones enzyme.
The present invention also protects a kind of method of the N-of production acyl homoserine lactones enzyme, is to cultivate said reorganization bacterium, obtains N-acyl homoserine lactones enzyme.
Said method specifically can be: (4-5 * g) cultivate said reorganization bacterium obtains N-acyl homoserine lactones enzyme for 30 ℃, 250-280rpm; Adopt methanol induction in the culturing process.The concentration of said methyl alcohol in said nutrient solution is preferably 0.5% (volumn concentration).The substrate of said N-acyl homoserine lactones enzyme specifically can be N-3-oxygen-decoyl homoserinelactone (3-oxo-C8-HSL).
The present invention is on the basis of existing N-acyl homoserine lactones enzyme gene aiiaB546; Its dna sequence dna is optimized; Be transformed into the coded gene of yeast preference codon; Gene aiiaB546M after having obtained optimizing, gene before the ability to express of this optimization back gene in pichia spp is significantly higher than and optimizes.The present invention has made up the recombinant expression plasmid that contains aiiaB546M, and this recombinant expression plasmid is imported pichia spp, can obtain the reorganization bacterium of efficient homoserinelactone enzyme.Wherein the ability to express of strain reorganization bacterium is particularly efficient.The present invention can reduce N-acyl homoserine lactones enzyme production cost, and security and validity that improve to use have great value for the production of homoserinelactone enzyme.
Description of drawings
Fig. 1 is gene before optimizing and the comparison of optimizing the back gene.
Fig. 2 is the electrophorogram of aiiaB546M/pUC57; 1 is 1kb plus ladder Marker; 2 is plasmid aiiaB546M/pUC57.
Fig. 3 cuts the electrophorogram of back plasmid for enzyme; 1 for being pPIC9 carrier double digestion fragment; 2 is two bar segment behind the plasmid aiiaB546M/pUC57 double digestion, and goal gene aiiaB546M is 753bp; 3 is 1kb plusladder Marker.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment like no specified otherwise, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Plasmid or bacterial strain used among the embodiment are following:
Intestinal bacteria (Escherichia coli) Trans1: buy from Beijing full Shi Jin biotech firm.
Pichia spp (Pichia pastoris) GS115: buy from Beijing full Shi Jin biotech firm.
PPIC9 carrier: buy from Invitrogen company.
KYC55 indicator (Agrobacterium tumefaciens KYC55): Institute of Feeds,China Academy of Agriculture Sciences guarantees to provide to the public; Reference: Zhu J, et al.Applied and Environmental Microbiology.69:6949-6953.
PUC57 carrier: buy from Nanjing Genscript Biotechnology Co., Ltd..
Substratum and solution used among the embodiment are following:
LB substratum: 1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0.
YPD substratum: 1% yeast extract, 2% peptone, 2% glucose.
MD solid medium: YNB 13.4g/L, glucose 20g/L, vitamin H 4 * 10
-4G/L, agar powder 20g/L.
BMGY substratum: yeast extract 10g/L, peptone 20g/L, yeast nitrogen (YNB) 13.4g/L, vitamin H 4 * 10
-4G/L, glycerine 10mL.
The BMMY substratum: replace glycerine with 0.5% methyl alcohol (volumn concentration), all the other compositions are identical with BMGY.
The glucone basal culture medium: glucose 0.75g, agar powder 3g, ultrapure water 138g, sterilization cooling back adds ATMM salts solution 15mL, X-gal solution 200 μ L.
Solution I: 50mmol/L glucose, 25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA;
Solution II: 0.2mol/L NaOH, 1%SDS (at present joining existing usefulness);
Solution III: 3mol/L Potassium ethanoate, 5mol/L acetic acid (pH4.8);
TAE (50 *): 242g Tris alkali, the 57.1mL glacial acetic acid, 100mL 0.5mol/L EDTA (pH8.0) is settled to 1000mL with sterilized water.
ATMM salts solution: KH
2PO
40.079mol/L, NaOH 0.044mol/L, (NH
4)
2SO
40.015mol/L, MgSO
47H
2O 0.6mmol/L, CaCl
20.06mmol/L, FeSO
47H
2O 0.027mmol/L, MnSO
4H
2O0.007mmol/L.
PBS damping fluid: NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO4 4.3mmol/L, KH
2PO
41.4mmol/L, pH7.3,121 ℃ of high pressure moist heat sterilization 20min.
X-gal is available from Sigma company among the embodiment, and DNA reclaims test kit available from TakaRa company, and various restriction enzymes and Taq enzyme are all available from TakaRa company, and ligase enzyme is available from Invitrogen company.
Enzyme activity determination method among the embodiment is following:
Squared paper: the length of side is to arrange 22 rectangles on the square of paper of 18cm, and each rectangular length is 15 lattices, and wide is 2 lattices, and each lattice is length of side 4mm.
Preparation feedback liquid: 20 μ L solution to be measured+180 μ L PBS damping fluid (pH8.0)+1 μ L 1mg/ml3-oxo-C8-HSL; 30 ℃ of water-baths, reaction 0.5h.
Preparation agar strip: the glucone basal culture medium is poured in the massive plate that diameter is 18cm, after solidifying, with aseptic pocket knife substratum is cut into 22 rectangle slices of interval 4mm according to the lines of squared paper; Use diameter to be pressed into the circle ring as the aseptic punch tool of 6mm at the preceding 8mm place of each slice, every afterwards separated 4mm inserts the KYC55 indicator with aseptic toothpick, reaction solution 10 μ L is joined in the circle ring of punching again; After waiting to infiltrate substratum; Seal and put into 30 ℃ of incubators with sealing film, cultivate observations behind the 24h, counting becomes blue point; Be converted into distance, calculate enzyme according to the typical curve that has made and live.With the PBS damping fluid that does not add enzyme liquid is CK.
Typical curve preparation: with absolute ethyl alcohol N-acyl homoserine lactones class signaling molecule (AHLs) is diluted to the concentration gradient of series, and equal-volume adds in the application of sample circle of above-mentioned agar strip, each extent of dilution set three parallel, flat board places 30 ℃ to cultivate 24h.Count the point that becomes blue, be converted into distance, promptly measure the colour developing radius, the relation of amount of analytical signal molecular substance (nmol/L) and radius (cm) makes up typical curve.
U=6.52* (1.4163
Rck-1.4163
Rs) * 50/30/10
6(solution to be measured is 20 μ l);
E=2.718281828459045, R (apart from mm).Rck: the diffusion length that does not add the PBS damping fluid of enzyme liquid.Rs: the diffusion length of reaction solution sample.More than two formula identical, second formula is the simplification to first formula.
The definition of enzyme unit alive (U): at 30 ℃, under the pH8.0 condition, the PM degraded 1nmol substrate N-3-oxygen-needed enzyme amount of decoyl homoserinelactone (3-oxo-C8-HSL).
The optimization of embodiment 1, N-acyl homoserine lactones enzyme gene codon
One, the optimization of homoserinelactone enzyme gene codon
N-acyl homoserine lactones enzyme gene aiiaB546 (shown in the sequence 1 of sequence table) is carried out codon optimized, the gene aiiaB546M after the optimization is shown in the sequence 2 of sequence table.Fig. 1 is seen in the comparison of gene aiiaB546M after gene aiiaB546 and the optimization.
Two, the structure of recombinant plasmid aiiaB546/pUC57
1, the aiiaB546 gene shown in the sequence 1 of artificial synthesized sequence table.
2,, reclaim enzyme and cut product with restriction enzyme EcoR I and Not I double digestion step 1 synthetic gene.
3,, reclaim carrier framework with restriction enzyme EcoR I and Not I double digestion pUC57 carrier.
4, the enzyme of step 2 is cut product and be connected, obtain connecting product with the carrier framework of step 3.
5, will connect product and check order, sequencing result shows, the recombinant plasmid aiiaB546/pUC57 that has obtained (has inserted the DNA shown in the sequence 1 of sequence table between the EcoR of pUC57 carrier I and Not I restriction enzyme site.
Three, the structure of recombinant plasmid aiiaB546M/pUC57
1, the aiiaB546M gene shown in the sequence 2 of artificial synthesized sequence table.
2,, reclaim enzyme and cut product with restriction enzyme EcoR I and Not I double digestion step 1 synthetic gene.
3,, reclaim carrier framework with restriction enzyme EcoR I and Not I double digestion pUC57 carrier.
4, the enzyme of step 2 is cut product and be connected, obtain connecting product with the carrier framework of step 3.
5, will connect product and check order, sequencing result shows, the recombinant plasmid aiiaB546M/pUC57 that has obtained (has inserted the DNA shown in the sequence 2 of sequence table between the EcoR of pUC57 carrier I and Not I restriction enzyme site.
The structure of embodiment 2, recombinant expression vector
One, the structure of pPIC9-aiiaB546M
1, extracts recombinant plasmid aiiaB546M/pUC57 and plasmid pPIC9 respectively
Adopt the little extraction reagent kit of plasmid of sky, Beijing root biotech company to extract plasmid, 1.2% agarose gel electrophoresis detects (electrophoretic buffer 1 * TAE, voltage 1-5V/cm, about 30min of time).The electrophorogram of recombinant plasmid aiiaB546M/pUC57 is seen Fig. 2.
2,, reclaim carrier framework with restriction enzyme EcoR I and Not I double digestion plasmid pPIC9.1.0% agarose gel electrophoresis detects, and electrophorogram is seen Fig. 3.
3,, reclaim the dna fragmentation (aiiaB546M) about 750bp with restriction enzyme EcoR I and Not I double digestion recombinant plasmid aiiaB546M/pUC57.1.0% agarose gel electrophoresis detects, and electrophorogram is seen Fig. 3.
4, the dna fragmentation that carrier framework that step 2 is reclaimed and step 3 reclaim spends the night for 4 ℃ with the T4DNA ligase enzyme and is connected, and obtains connecting product.
5, connect the evaluation of product
(1) preparation of intestinal bacteria (E.coli) Trans 1 competent cell
1. get the intestinal bacteria Trans 1 bacterium liquid of 1/100 volume, be inoculated in the 500mL LB nutrient solution, 37 ℃ of shaking culture are crossed liquid, make its OD
600Be about 0.5-0.7;
2. with nutrient solution ice bath 20min, later step remains on 0 ℃ as far as possible, all precoolings before adding cell of all containers.
3. with step nutrient solution 4000g, 4 ℃ of centrifugal 15min 2., abandon supernatant;
4. use 10% (10g/100ml) the aqueous glycerin solution re-suspended cell (deposition) of 500mL precooling; 4000g, 4 ℃ of centrifugal 15min abandon supernatant; With 10% (10g/100ml) aqueous glycerin solution re-suspended cell of 250mL precooling, 4000g, 4 ℃ of centrifugal 15min abandon supernatant; With 10% (10g/100ml) aqueous glycerin solution re-suspended cell of 20mL precooling, transfer in the aseptic centrifuge tube of 30mL, 4000g, 4 ℃ of centrifugal 15min abandon supernatant; 10% (10g/100ml) aqueous glycerin solution re-suspended cell with the 1-2mL precooling; Cell is pressed every pipe 40 μ L packing, liquid nitrogen flash freezer ,-70 ℃ of preservations, subsequent use (competent cell).
(2) evaluation of connection product
Adopt heat shock to transform competent cell: 40 μ L competent cells to be connected with 10 μ L to produce to mix ice bath 30min with connection product step of converting (1) preparation of step 4; 42 ℃ of water-bath 60sec place 2min on ice; Add 500 μ L LB liquid nutrient mediums, 37 ℃ of shaking tables are cultivated 1h; It is dull and stereotyped to coat the solid LB that contains 100 μ g/mL penbritins, and 37 ℃ of incubators are cultivated 16h.
40 mono-clonal bacterium colonies of picking carry out the PCR checking; Choose the PCR positive colony and send Bo Maide company order-checking, obtain recombinant plasmid pPIC9-aiiaB546M (between the EcoR of skeleton carrier pPIC9 I and Not I restriction enzyme site, having inserted the DNA shown in the sequence 2 of sequence table).
Two, the structure of pPIC9-aiiaB546
Replace recombinant plasmid aiiaB546M/pUC57 with recombinant plasmid aiiaB546/pUC57; Other same step 1 obtains recombinant plasmid pPIC9-aiiaB546 (between the EcoR of skeleton carrier pPIC9 I and Not I restriction enzyme site, having inserted the DNA shown in the sequence 1 of sequence table)
The preparation of embodiment 3, reorganization bacterium
One, changes the preparation and the screening of aiiaB546M gene recombination bacterium
1, a large amount of extractions of DNA
1. get the intestinal bacteria Trans1 that contains recombinant plasmid pPIC9-aiiaB546M of the process sequence verification that obtains among the embodiment 2, be inoculated in the 50mL LB substratum that contains 100 μ g/mL penbritins, 37 ℃ of shaken overnight are cultivated;
2. get incubated overnight liquid 50mL, 10,000rpm, 4 ℃ of centrifugal 5min abandon supernatant, are inverted centrifuge tube on thieving paper, inhale and go excess liquid;
3. deposition is resuspended in the 2mL solution I, thermal agitation fully suspends; Add the solution II of the fresh configuration of 4mL, put upside down mixing, place 4min on ice; Add the 3mL solution III, put upside down mixing, place 5min on ice, 13,000rpm, 4 ℃ of centrifugal 10min;
4. shift out supernatant, filter with lens wiping paper; Add the Virahol of 0.6-1 times of volume in the supernatant, room temperature is placed 10min; 13,000rpm, 4 ℃ of centrifugal 10min; Abandon supernatant, 70% ethanol is washed deposition, and ethanol is removed in centrifugal slightly hypsokinesis, will precipitate vacuum-drying 10min;
5. in dried centrifuge tube, every pipe adds 500 μ L TE, breaks up deposition, moves in the Eppendorf pipe, adds 10 μ L RNase (20 μ g/mL), 37 ℃ of insulation 30min; Every pipe adds the saturated phenol of 500 μ L Tris, mixing, 12, the centrifugal 10min of 000rpm; Draw the upper strata water in new Eppendorf pipe, add 500 μ L phenol: chloroform mixed solution, mixing, 12, the centrifugal 10min of 000rpm; The careful upper strata water of drawing adds 500 μ L chloroform mixings in new Eppendorf pipe, and 12, the centrifugal 10min of 000rpm; Get supernatant, add the equal-volume Virahol, place 15min on ice, 14, the centrifugal 15min of 000rpm; Abandon supernatant, deposition adds 500 μ L, 70% ethanol and washes centrifugal 3min.
6. discard ethanol, deposition (pPIC9-aiiaB546M plasmid) dry 10min in vacuum drier; Add 50 μ L TE liquid in the centrifuge tube ,-20 ℃ of preservations are subsequent use.
2, the linearizing of plasmid
Get 50 μ L pPIC9-aiiaB546M plasmids, cut 2-3h, obtain enzyme and cut product with a restriction enzyme BglII37 ℃ enzyme.Cut the sodium acetate (pH5.2) that product adds 1/10 volume and the absolute ethyl alcohol deposition of 2-3 times of volume, 13, the centrifugal 15min of 000rpm to 100 μ L enzymes; With 70% ethanol rinsing; Vacuum-drying adds 10 μ L sterilized waters in the centrifuge tube, place-20 ℃ (linearization plasmids) for use.
3, the preparation of pichia spp GS115 competent cell
1. the single bacterium colony of the pichia spp GS115 on the picking YPD flat board is in 5mL YPD liquid nutrient medium, and 30 ℃, 250rpm shaking table spend the night;
2. the fresh GS115 bacterium liquid that shaking table is spent the night is forwarded in the 100mL YPD liquid nutrient medium by 1/1000 inoculum size, and 30 ℃, 250rpm shaking table spend the night, when OD600 is about 1.3-1.5, and the failure of oscillations;
3. the bacterium liquid with precooling is transferred in the centrifuge tube of ice precooling 4 ℃, the centrifugal 5min of 1500g; Precipitate resuspended with the sterilized water of 200mL precooling, 4 ℃, the centrifugal 5min of 1500g; Precipitate resuspended with the sterilized water of 100mL precooling, 4 ℃, the centrifugal 5min of 1500g; Precipitate resuspended with the 1mol/L sorbitol aqueous solution of 20mL precooling, 4 ℃, the centrifugal 5min of 1500g; Precipitate resuspended with the 1mol/L sorbitol aqueous solution of 1mL precooling, by every pipe 80 μ L packing, liquid nitrogen flash freezer ,-70 ℃ of preservations, subsequent use (competent cell).
4, the preparation of reorganization bacterium
1. the competent cell with 80 μ L steps 3 mixes with the linearization plasmid of 10 μ L steps 2, and it is gone in the electricity conversion cup;
2. the conversion cup ice bath 5min of mixed solution will be housed; Adjust the parameter (placing the PIC shelves) of gene introducing apparatus, shock by electricity once, voltage 2000V, the time is about 5ms;
3. immediately toward transforming the 1mol/L sorbitol aqueous solution that adds 700 μ L precoolings in the cup, mixing, and it is gone in the centrifuge tube of the bacterium of going out;
4. mixed solution is applied on the MD plate, places 30 ℃ of incubators to cultivate 2-3 days the MD plate, till growing bacterium colony, being changes aiiaB546M gene recombination bacterium (transformant).
5, the screening of efficient expression strain
1. with the toothpick of the bacterium of going out picking list bacterium colony from the long MD plate that transformant arranged, according to the numbering point to the MD flat board, each dull and stereotyped 100 single bacterium colony of generic point of going up;
2. be inoculated in the 3mL BMGY substratum by the mono-clonal on the numbering picking MD flat board, 30 ℃, 250-280rpm shaking table were cultivated 2-3 days;
3. shaking table is cultivated 2-3 days the centrifugal 5min of nutrient solution 3250rpm, got deposition (as far as possible supernatant being eliminated), in deposition, add 1mL BMMY substratum again, again at 30 ℃, 250-280rpm inducing culture;
4. behind the inducing culture 48h, that thalline is centrifugal, get supernatant and live as solution detection enzyme to be measured, filter out enzyme high bacterial strain alive.If three groups of parallel laboratory tests filter out more stable high expression level bacterial strain.
Screening obtains the reorganization bacterium of a plant height efficient expression homoserinelactone enzyme, with its called after pichia pastoris phaff (Pichiapastoris) AiiA-B546M.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 02nd, 2010, and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.3975.
The enzyme work of the solution to be measured that pichia pastoris phaff AiiA-B546M obtains is 36.1 ± 0.9U/mL, and the enzyme work that contrasts the solution to be measured that bacterium obtains has improved 6.92%.
Two, change the preparation of aiiaB546 gene recombination bacterium
Get the intestinal bacteria Trans1 that contains recombinant plasmid pPIC9-aiiaB546 of the process sequence verification that obtains among the embodiment 2, replace step 11 1. in intestinal bacteria, other same step 1 obtains changeing aiiaB546 gene recombination bacterium (contrast bacterium).
Contrast bacterium mono-clonal on the picking MD flat board is inoculated in the 3mL BMGY substratum, and 30 ℃, 250-280rpm shaking table were cultivated 2-3 days; Shaking table is cultivated 2-3 days the centrifugal 5min of nutrient solution 3250rpm, get deposition (as far as possible supernatant being eliminated), in deposition, add 1mL BMMY substratum again, again at 30 ℃, 250-280rpm inducing culture; Behind the inducing culture 48h, thalline is centrifugal, get supernatant and live as solution detection enzyme to be measured; If three groups of parallel laboratory tests.
The enzyme work of the solution to be measured that the contrast bacterium obtains is 35.6 ± 1.1U/mL.
The enzyme activity determination of embodiment 4, reorganization bacterium
Respectively pichia pastoris phaff AiiA-B546M is cultivated and enzyme activity determination with the contrast bacterium as follows:
1, with inoculation in the 500mL triangular flask that 150mL BMGY substratum is housed, 30 ℃, 250-280rpm shaking table were cultivated 2-3 days;
2, get nutrient solution, the centrifugal 5min of 3250rpm gets deposition (as far as possible supernatant being eliminated);
3, in deposition, add 80mL BMMY substratum, 30 ℃, 250-280rpm inducing culture; Be the volatilization loss of compensation methyl alcohol, every separated 12h adds methanol solution, makes the methanol concentration in the bacterium liquid remain on 0.5% (volumn concentration); Every separated 12h takes a sample once, until 48h;
4, the bacterium liquid of sampling is centrifugal, supernatant carries out enzyme activity determination as solution to be measured; If three groups of parallel laboratory tests.
The enzyme work that pichia pastoris phaff AiiA-B546M obtains solution to be measured is 36.2 ± 1.0U/mL.The enzyme work of the solution to be measured that the contrast bacterium obtains is 33.1 ± 0.7U/mL.Be after codon is transformed, to have improved 9.53%, significance (P＜0.05) than the enzymic activity of transforming preceding bacterial strain.
Claims (10)
1.DNA molecule, for (a) as follows or (b):
(a) sequence 2 of sequence table is from the DNA shown in 5 ' terminal the 7th to 759 Nucleotide;
(b) DNA shown in the sequence 2 of sequence table.
2. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain the said dna molecular of claim 1.
3. recombinant expression vector as claimed in claim 2 is characterized in that: said recombinant expression vector inserts the recombinant plasmid that the said dna molecular of claim 1 obtains for the MCS at the pPIC9 carrier.
4. recombinant expression vector as claimed in claim 3 is characterized in that: said recombinant expression vector is for cutting the recombinant plasmid that the said dna molecular of insertion claim 1 obtains between the recognition site at the EcoR of pPIC9 carrier I and Not I enzyme.
5. reorganization bacterium as claimed in claim 2 is characterized in that: said reorganization bacterium is in the host bacterium, to import the reorganization bacterium that the said dna molecular of claim 1 obtains; Said host bacterium is pichia spp (Pichia pastoris).
6. reorganization bacterium as claimed in claim 5 is characterized in that: said pichia spp (Pichia pastoris) is pichia spp (Pichia pastoris) GS115.
7. reorganization bacterium as claimed in claim 5 is characterized in that: the said dna molecular of claim 1 imports said host bacterium through claim 3 or 4 said recombinant expression vectors.
8. reorganization bacterium as claimed in claim 7 is characterized in that: said reorganization bacterium is pichia pastoris phaff (Pichia pastoris) AiiA-B546M, and its deposit number is CGMCC No.3975.
9. claim 2,5,6,7 or 8 application of described reorganization bacterium in producing N-acyl homoserine lactones enzyme.
10. a method of producing N-acyl homoserine lactones enzyme is to cultivate claim 2,5,6,7 or 8 described reorganization bacterium, obtains N-acyl homoserine lactones enzyme.
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