CN101402947A - Novel gene of esterase and recombinant expression system - Google Patents
Novel gene of esterase and recombinant expression system Download PDFInfo
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- CN101402947A CN101402947A CNA2008102269426A CN200810226942A CN101402947A CN 101402947 A CN101402947 A CN 101402947A CN A2008102269426 A CNA2008102269426 A CN A2008102269426A CN 200810226942 A CN200810226942 A CN 200810226942A CN 101402947 A CN101402947 A CN 101402947A
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Abstract
The invention provides a new esterase gene named Est_p1 and a recombination expression system thereof. The gene is cloned from the metagenomic library of the marginal mud 100 meters under the South China Sea. The gene is an 891bp long amino acid numbered 296, which successfully constructs a recombination expression system of a colon bacillus named pET28a-Est_p1/ BL21. The molecular weight of a target protein is 33.5 kD. With pNP-butyric ester as catalyzing substrates, the Est_p1 is proved to be a mildly alkaline medium temperature esterase. The Est_p1 has strong catalytic activity for short esters and is applicable to esterification and transesterification industries.
Description
Technical field
The present invention relates to biological technical field, relate to particularly can not culturing micro-organisms gene clone and expression.
Background technology
Enormous amount can not cultured microorganism in the resources advantage that is richly stored with, grand Study on Genome provides reliable guidance for excavating this resources advantage.95% marine microorganism can't or be difficult to be realized artificial culture under the current experiments condition, directly extract DNA from environment, can overlay environment on theorizing in the grand genomic libraries of genetic information of all microorganisms.The gene fragment that obtains in the extreme environment of ocean depth more than 100 meters with these gene heterogenous expressions, might obtain having the new gene of using value.By making up the grand genomic library of ocean bed mud, preserve the gene diversity of marine microorganism, be beneficial to the research and development utilization of dissimilar novel genes.
Lipase and esterase are important industrial enzyme preparation kinds, can catalysis separate reactions such as fat, transesterify, ester is synthetic, are widely used in industry such as grease processing, food, medicine, daily use chemicals.The esterase of different sources has different catalysis characteristics and catalysis activity with lipase.The large-scale production that wherein is used for the lipase with transesterificationization or esterification function of organic synthesis and esterase is significant for synthetic fine chemicals of enzyme catalysis and chipal compounds.
Current energy dilemma enjoys people's attention; the tradition fossil energy can not satisfy human wants; by the technology of lipase biocatalysis biodiesel synthesis, have advantages such as extraction is simple, gentle, the pure consumption of reaction conditions is little, glycerine easily reclaims and no waste produces, pollution-free.
The present invention intends screening strong stress resistance from the grand genomic library of ocean bed mud, and novel esterases that character is good and lipase gene are for the conversion and the preparation technology of biofuel provides alternative resource.
The contriver has successfully made up 0～100 meter grand genomic library of dark bed mud in China South Sea in the work in early stage, this storage capacity DNA reaches 194MB, cut, connect and transform through enzyme, obtain 118,000 recons that contain exogenous genetic fragment altogether, its external source fragment length is between 1.0-8.0Kb.Set up the screening model of esterase and lipase, for next step screening provides technical guarantee.
Summary of the invention
The purpose of this invention is to provide a kind of new esterase gene, be used for the bio-transformation of lipid degradation and other oil compounds.
The present invention obtains a kind of new esterase gene est_p1 by functional screening from the grand genomic library of South Sea bed mud, its nucleotide sequence is shown in SEQ ID No.1.The est_p1 size is 891 bp, based composition: 226A (25.4%), 232C (26.0%), 247G (27.7%), 186T (20.9%), 0 other (0.0%); The proteins encoded size is 296aa, and its aminoacid sequence is shown in sequence table SEQ ID No.2.This gene order is carried out homology search in GenBank, find one similarly property be up to 59% dietary protein origin in strain esterase/lipase of culturing bacterium (its number of registration at GenBank is ABQ11268) not.The structure prediction result shows, Est_p1 has the folding conserved domain (COG0596) of complete α/β hydrolysis, this structural domain has comprised the catalysis triplet that Serine, L-glutamic acid and Histidine are formed, constitute the catalytic center of esterase/lipase, belong to the super family of esterase/lipase (cl09107).In sum, Est_p1 should be newcomer of the super family of esterase/lipase (cl09107).
Est_p1 is cloned into expression vector pET28a and transformed into escherichia coli BL21 (DE3), obtains the pET28a-Est_p1/BL21 recombinant expression system.Shake-flask culture and abduction delivering Est_p1, purified back obtains Est_p1, and SDS-PAGE shows that its apparent molecular weight is 33.5kD, conforms to predictor; Show that by esterase activity mensuration Est_p1 can interrupt the ester bond of compound pNP-butyric ester specifically; Show that by optimal reaction pH mensuration its optimal reaction pH value is pH8.57, meta-alkalescence to Est_p1; By being measured, the optimal reactive temperature of Est_p1 shows that its optimal reactive temperature is about 40 ℃.The kinetic constant of enzyme is measured and is shown that the suitableeest substrate of Est_p1 is the pNP-butyric ester, is better than the long-chain ester class for the hydrolysis vigor of the shorter ester class of acyl group carbochain.
Should be appreciated that under the prerequisite that does not influence the Est_p1 protein-active those skilled in the art can carry out various replacements, interpolation and/or lack the aminoacid sequence that one or several amino acid obtains to have same function the aminoacid sequence shown in the SEQ ID NO:2.
In addition, consider the degeneracy of codon, for example can under the condition that does not change aminoacid sequence, or not influence at its non-coding region under the condition of est_p1 expression, can make amendment the above-mentioned proteic gene order of encoding in its coding region.Therefore, the present invention also comprises replacement, the interpolation that the nucleotide sequence shown in the SEQ ID No.1 is carried out and/or lacks one or more Nucleotide, and the above-mentioned proteic nucleotide sequence of encoding.
And then the present invention also provides a kind of cloning vector or expression vector that contains said gene, contains the host cell of described carrier.
Further, the present invention also provides the method for a kind of Est_p1 of preparation, and it is the host cell that contains above-mentioned expression vector by cultivation, separates obtaining Est_p1 from culture.
The present invention also provides described Est_p1 to separate the application in synthetic of ester, transesterify or ester in catalysis.Preferred pH value in reaction is pH8.57, and preferable reaction temperature is 40 ℃.
The present invention angles out novel esterase gene not only to increase the newcomer for esterase family from the grand genomic library of China's ocean bed mud, the albumen of finding this genes encoding has good zymologic property, the synthetic ester product of enzyme process can be applied to, the choice who replenishes and replace the new production process of part chemosynthesis product will be expected to become.The production of this enzyme will show its important economy and social value biodiesel manufacture (fat is separated in catalysis, reactions such as transesterify) in the technologies such as paper waste deinking regeneration.
Description of drawings
What Fig. 1 showed is the typical curve of pNP;
That Fig. 2 shows is the SDS-PAGE figure of each purification phase of expression product Est_p1, wherein, and M: low molecular weight protein (LMWP) Marker; CL: thick zyme extract; FL:NTA-Agarose post effluent liquid; EL:Est_p1 albumen is in 50mM imidazoles eluant solution;
What Fig. 3 showed is that pH is to the active influence of Est_p1;
What Fig. 4 showed is that temperature is to the active influence of esterase Est_p1;
That Fig. 5 shows is the double reciprocal curve figure of Est_p1 under pNP-butyric ester and pNP-decylate substrate condition, wherein Fig. 5 A measures the Est_p1 kinetic constant mensuration that substrate is the pNP-butyric ester, and Fig. 5 B measures the Est_p1 kinetic constant mensuration that substrate is the pNP-decylate.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The acquisition of embodiment 1 est_p1 gene
1. library primary dcreening operation: the dilution of the bacterial strain in the library is coated on the LB flat board that contains 1% tributyrin, 37 ℃ of cultivations, it is standby to choose the bacterium colony that produces transparent circle after 1～2 day;
2. sieve again in the library: picking is got bacterial strain put again and be connected on the LB flat board that contains 1% tributyrin, 37 ℃ of cultivations are determined after 1～2 day and are picked out the bacterium colony that produces transparent circle.
3. the bacterial strain of picking generation transparent circle carries out liquid culture, extracts plasmid by the TIANGEN plasmid extraction kit, send company's order-checking;
4. sequencing result: it is 3650bp that this plasmid foreign DNA inserts segment size.Obtain 4 sections complete ORF sequences by ORF Finder prediction.Wherein, the gene est_p1 of coding esterase or lipase is positioned at and inserts pulsating 728～1618bp.The est_p1 size is 891bp, and its nucleotide sequence proteins encoded size shown in sequence table SEQ ID No.1 is 296aa.This gene order is carried out homology search in GenBank, find one similarly property be up to 59% dietary protein origin in strain esterase/lipase of culturing bacterium (its number of registration at GenBank is ABQ11268) not.The structure prediction result shows, Est_p1 has the folding conserved domain (COG0596) of complete α/β hydrolysis, this structural domain has comprised the catalysis triplet that Serine, L-glutamic acid and Histidine are formed, constitute the catalytic center of esterase/lipase, belong to the super family of esterase/lipase (cl09107).In sum, Est_p1 should be newcomer of the super family of esterase/lipase (cl09107).
5, according to sequencing result, the design primer carries out PCR, obtains the est_p1 complete genome.The PCR primer:
5’-CATGCCATGGCAAACATTATTGCG
5’-ACGCGTCGACGATAAAAATTTTTTGGGT
The PCR reaction system:
dNTPs(2mM) 1μl
KOD enzyme (1U/ μ l) 0.2 μ l
10 * PCR reaction buffer, 2 μ l
MgSO
4(25mM) 0.5μl
Upstream primer 1 μ l
Downstream primer 1 μ l
ddH
2O 13.3μl
The PCR reaction conditions:
94 ℃, pre-sex change 2min; 94 ℃, sex change 15s; 51 ℃, annealing 30s; 68 ℃, extend 1min; Totally 30 circulations; 68 ℃, extend 10min again.
With Marker IV is dna molecular amount mark, and the methyl bromjophenol blue is a dyestuff, detects the PCR product with 1% agarose gel electrophoresis.
The structure of embodiment 2 Est_p1 expression systems and the enzyme activity thereof are investigated
1, the structure of Est_p1 expression system
Design is introduced NcoI and XhoI restriction enzyme site respectively at 5 ' end and the 3 ' end of Est_p1, pays the living worker in Shanghai and synthesizes, and is used for construction of expression vector after order-checking is correct.With NcoI and XhoI synthetic est_p1 gene is carried out double digestion; NcoI and SalI carry out double digestion to pET28a (Novagen) carrier, with dna ligase above-mentioned segment is connected then, to connect product at last is transformed in the e. coli bl21 (DE3), on LB (containing kan 50 μ g/ml) flat board, cultivate, the preliminary screening positive colony obtains the pET28a-Est_p1/BL21 recombinant expression system after enzyme is cut checking.
2, the abduction delivering of target protein and purifying;
This example adopts Ni-agarose affinitive layer purification (pET expression system) target protein, and concrete steps are as follows:
1) inducing culture
Expression strain is inoculated into is equipped with in the 5ml LB test tube of (containing kan 50 μ g/ml), 37 ℃, rotating speed 250rpm, be cultured to OD600 ≈ 0.5.With 1% (V/V) inoculum size bacterium liquid is transferred in the same substratum of 500ml, when OD600 is between 0.5～1.0 the time, adding IPTG is that 0.5mM induced 12～15 hours to final concentration.
2) albumen is slightly carried
Will be through inductive culture 10000g, 4 ℃, centrifugal 10 minutes collecting cells, with lysis buffer washing 2 times.Be dissolved in the 30ml lysis buffer, behind the ultrasonic disruption cell (240 volts, 3 seconds supersonic wave wall breakings, 7 seconds intermittently, 60 circulations), centrifugal (15000g, 4 ℃, 10 minutes) gets supernatant.
3) protein purification
I. add 1ml Ni-agarose in the pillar, wash with 10 times of volume distilled waters, and with an amount of lysis buffer balance pillar.
Ii. Ni-agarose is taken out, mix at 1: 4 with volume ratio with protein crude extract, rotating speed 200rpm, ice bath was in conjunction with 1 hour.
Iii. Ni-agarose after the combination and protein crude extract are added in the pillar again, with the lysis buffer balance pillar of 50 times of volumes.
Iv. use the unconjugated albumen of cleaning buffer solution flush away of 50 times of volumes, protein content is zero in effluent liquid.
V. use the elution buffer wash-out of 5 times of column volumes.Use the 1.5ml centrifuge tube, every pipe is collected 10.
Vi. carrying out cylinder after wash-out finishes preserves.0.3M acetate, 10% glycerine, distilled water with 10 times of column volumes washs 30% alcohol immersion, 4 ℃ of preservations successively.
Vi. the electrophoretic analysis target protein with the albumen dialysis that meets the requirements, carries out zymologic property and measures.
Lysis buffer:
50mM NaH
2PO
4, 300mM NaCl, 10mM imidazoles, transfer pH to 8.0 with NaOH.
Cleaning buffer solution:
50mM NaH
2PO
4, 300mM NaCl, 20mM imidazoles, transfer pH to 8.0 with NaOH.
Elution buffer:
50mM NaH
2PO
4, 300mM NaCl, 50mM imidazoles, transfer pH to 8.0 with NaOH.
Dialysis buffer liquid:
50mM NaH
2PO
4, 300mM NaCl, transfer pH to 8.0 with NaOH.
3, esterase enzyme activity determination
Esterase can interrupt the ester bond of compound pNP-fatty acid ester specifically, discharge the lipid acid and the pNP of the amount of same substance, and there is special absorption peak at pNP 405nm place under alkaline environment, calculates the vigor of lipase/esterase by the growing amount of measuring product pNP in the certain hour.
Reaction system is:
Buffer 940μl
PNP-fatty acid ester 10 μ l
With damping fluid, substrate solution (pNP-butyric ester, 4-Nitrophenyl butyrate is available from Sigma) and dehydrated alcohol in 40 ℃ of water-baths, be incubated 10min, add the suitably enzyme liquid (0.128mg/ml) of dilution, reaction 3min, measure the light absorption value of 405nm, reference standard curve calculation enzyme is lived.Enzyme unit alive (unit) is defined as, and under above-mentioned reaction conditions, the enzyme amount that per minute discharges 1 μ mol pNP is an enzyme unit alive.。
Enzyme activity (μ mol/minml)=(14.312 ÷, 3 ÷ 16.731) * 100=28.514
Than enzyme (μ mol/minmg)=28.514 ÷ 0.128=222.765 alive
4, zymologic property analysis
1) SDS-PAGE method determining molecular weight
The SDS-PAGE method is measured the molecular weight subunit of enzyme, and the relative mobility Rf mapping of standard protein in SDS-PAGE according to the known molecular amount gets its molecular weight.The result as shown in Figure 2, the SDS-PAGE apparent molecular weight of Est_p1 is about 33.5kDa.And the SDS-PAGE electrophoresis shows that it is pure to reach electrophoresis through the Est_p1 behind the Ni-agarose affinitive layer purification.
2) the optimal reaction pH value of enzyme
Prepare the damping fluid of various 50mM, pH is respectively 3.12,4.55,5.11,5.53,6.15,6.45 citrate buffer solution, pH is respectively 6.33,6.62,6.94,7.33,7.45,7.83,8.09 MOPS damping fluid, pH is respectively 6.9,7.16,7.55,8.05,8.57,8.93 Tri-HCl damping fluid, pH is respectively 8.94,9.42,9.95 CHES damping fluid, and pH is respectively 9.73,9.9,10.41,10.88 CAPS damping fluid.Enzyme liquid is joined respectively in the damping fluid of above-mentioned pH, measure enzyme activity by standard method, soprano alive locatees 100% with enzyme, with relative vigor and corresponding pH value mapping.The result shows (Fig. 3), and the optimum pH of Est_p1 is about pH8.57, meta-alkalescence.
3) optimal reactive temperature of enzyme
The Tri-HCl damping fluid of substrate solution and pH8.57,50mmol/L is incubated 10min respectively in the water-bath of 0 ℃, 5 ℃, 9 ℃, 15 ℃, 18 ℃, 24.5 ℃, 30.5 ℃, 35 ℃, 37 ℃, 40 ℃, 42 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ differing temps, add enzyme liquid then and measure enzyme activity according to standard method, obtain the enzyme activity that enzyme liquid has under the differential responses temperature, soprano alive is decided to be 100% with enzyme, with relative vigor temperature is mapped., the result shows (Fig. 4), the optimal reactive temperature of enzyme is about 40 ℃.
4) kinetic constant of enzyme is measured
The substrate that adopts is that pNP-butyric ester (4-Nitrophenyl butyrate) and pNP-decylate (4-Nitrophenyl stearate) are available from Sigma company, prepare the substrate of a series of different concns, make that the concentration of substrate is respectively 0.1mM, 0.05mM, 0.025mM, 0.0125mM, 0.00625mM, 0.00313mM in the reaction system.With the Tri-HCl damping fluid of the substrate, dehydrated alcohol and the pH8.57 that prepare at 40 ℃ of following preheating 10min, add the suitably enzyme liquid of dilution (dilution back 0.0128mg/ml) of 10 μ l respectively, according to concentration of substrate and the initial velocity of reaction v (the same enzyme activity determination of calculation formula) that records, with 1/[S] 1/v is made the Lineweaver-Burk double reciprocal plot, try to achieve Km, K
Cat, K
Cat/ Km.The result shows (seeing Fig. 4 and table 2), and the preferable substrate of Est_p1 is the pNP-butyric ester, and not as the short-chain ester class, therefore, Est_p1 is an esterase for the hydrolysis vigor of the longer ester class of acyl group carbochain.
Calculation formula:
V＝V
max[S]/(Km+[S]) 1/V＝(Km/V
max)·(1/[S])+1/V
max
K
Cat=V
Max/ [E] [E] is zymoprotein concentration
The enzyme kinetics constant of the Est_p1 of the different substrates of table 2
＜110〉China Agricultural University
＜120〉novel gene of esterase and recombinant expression system thereof
<130>KHP08112923.6
<160>5
<170>PatentIn?version?3.5
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<211>891
<212>DNA
＜213〉the unknown
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<221>CDS
<222>(1)..(891)
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atg?gca?aac?att?att?gcg?aat?ggc?atc?cag?atc?gaa?tac?gat?acc?ttt 48
Met?Ala?Asn?Ile?Ile?Ala?Asn?Gly?Ile?Gln?Ile?Glu?Tyr?Asp?Thr?Phe
1 5 10 15
gga?aat?ccc?gat?gag?cct?cca?ctt?ctt?ttg?att?atg?gga?ctg?gct?tgc 96
Gly?Asn?Pro?Asp?Glu?Pro?Pro?Leu?Leu?Leu?Ile?Met?Gly?Leu?Ala?Cys
20 25 30
cag?cta?att?cac?tgg?gat?gag?gac?ttg?tgc?gag?caa?ctg?gcg?cgg?cga 144
Gln?Leu?Ile?His?Trp?Asp?Glu?Asp?Leu?Cys?Glu?Gln?Leu?Ala?Arg?Arg
35 40 45
ggc?cat?tac?gtg?atc?cga?ttt?gat?aat?cgc?gat?acg?ggc?ctt?tca?acc 192
Gly?His?Tyr?Val?Ile?Arg?Phe?Asp?Asn?Arg?Asp?Thr?Gly?Leu?Ser?Thr
50 55 60
aaa?ttc?gca?gaa?gcc?ggc?ata?ccc?gat?atc?ggg?cag?att?atc?gaa?gcc 240
Lys?Phe?Ala?Glu?Ala?Gly?Ile?Pro?Asp?Ile?Gly?Gln?Ile?Ile?Glu?Ala
65 70 75 80
cgt?atg?aag?ggt?gag?gag?ttc?agg?cct?ccc?tac?acg?ctc?gag?gac?atg 288
Arg?Met?Lys?Gly?Glu?Glu?Phe?Arg?Pro?Pro?Tyr?Thr?Leu?Glu?Asp?Met
85 90 95
gcc?gac?gat?gcg?gtc?ggg?ctg?ctg?gat?gct?tta?gaa?atc?gaa?aag?gcc 336
Ala?Asp?Asp?Ala?Val?Gly?Leu?Leu?Asp?Ala?Leu?Glu?Ile?Glu?Lys?Ala
100 105 110
cat?gtc?tgc?ggt?atg?tca?atg?gga?ggg?atg?atc?gcc?cag?acc?atc?ggg 384
His?Val?Cys?Gly?Met?Ser?Met?Gly?Gly?Met?Ile?Ala?Gln?Thr?Ile?Gly
115 120 125
ctg?aat?cat?cag?cag?cgg?gtg?tta?agc?ctc?att?tcg?ata?tac?agc?cat 432
Leu?Asn?His?Gln?Gln?Arg?Val?Leu?Ser?Leu?Ile?Ser?Ile?Tyr?Ser?His
130 135 140
acg?ggc?aac?cct?gaa?ctt?ccg?ccg?ccg?aca?ccc?gaa?gca?ttg?gaa?tac 480
Thr?Gly?Asn?Pro?Glu?Leu?Pro?Pro?Pro?Thr?Pro?Glu?Ala?Leu?Glu?Tyr
145 150 155 160
ctg?gtc?acg?ccg?cca?cct?atg?gaa?cgt?gaa?gcc?aac?atc?acc?tat?acc 528
Leu?Val?Thr?Pro?Pro?Pro?Met?Glu?Arg?Glu?Ala?Asn?Ile?Thr?Tyr?Thr
165 170 175
ctc?gat?gtg?tgg?cgc?acg?ttt?tca?ggg?aag?ggc?ttt?ccg?ctg?gat?gag 576
Leu?Asp?Val?Trp?Arg?Thr?Phe?Ser?Gly?Lys?Gly?Phe?Pro?Leu?Asp?Glu
180 185 190
gat?tgg?aac?aga?aaa?atc?gcc?gcc?aaa?gcc?tat?gac?cgc?gcc?ttt?tac 624
Asp?Trp?Asn?Arg?Lys?Ile?Ala?Ala?Lys?Ala?Tyr?Asp?Arg?Ala?Phe?Tyr
195 200 205
ccg?gaa?ggc?gtg?gcc?cgt?cag?atg?gcg?gcc?gtc?tta?acc?caa?aaa?aat 672
Pro?Glu?Gly?Val?Ala?Arg?Gln?Met?Ala?Ala?Val?Leu?Thr?Gln?Lys?Asn
210 215 220
cga?aaa?ttc?gaa?ctg?ggt?tcg?gtc?acc?atg?ccc?aca?ctg?gtg?att?cac 720
Arg?Lys?Phe?Glu?Leu?Gly?Ser?Val?Thr?Met?Pro?Thr?Leu?Val?Ile?His
225 230 235 240
gga?gcc?gat?gac?ccg?ctg?gtg?ccg?gtt?gaa?ggc?ggc?aaa?gat?acg?gct 768
Gly?Ala?Asp?Asp?Pro?Leu?Val?Pro?Val?Glu?Gly?Gly?Lys?Asp?Thr?Ala
245 250 255
gaa?gca?ata?ccg?ggg?gct?gag?ctg?ata?atc?att?gac?gga?atg?gga?cac 816
Glu?Ala?Ile?Pro?Gly?Ala?Glu?Leu?Ile?Ile?Ile?Asp?Gly?Met?Gly?His
260 265 270
gat?ctg?ccg?cat?ggt?gga?gcc?tgg?ccg?cag?ata?gtc?gat?gcg?atc?gta 864
Asp?Leu?Pro?His?Gly?Gly?Ala?Trp?Pro?Gln?Ile?Val?Asp?Ala?Ile?Val
275 280 285
aat?cac?acc?caa?aaa?att?ttt?atc?tga 891
Asn?His?Thr?Gln?Lys?Ile?Phe?Ile
290 295
<210>2
<211>296
<212>PRT
＜213〉the unknown
<400>2
Met?Ala?Asn?Ile?Ile?Ala?Ash?Gly?Ile?Gln?Ile?Glu?Tyr?Asp?Thr?Phe
1 5 10 15
Gly?Asn?Pro?Asp?Glu?Pro?Pro?Leu?Leu?Leu?Ile?Met?Gly?Leu?Ala?Cys
20 25 30
Gln?Leu?Ile?His?Trp?Asp?Glu?Asp?Leu?Cys?Glu?Gln?Leu?Ala?Arg?Arg
35 40 45
Gly?His?Tyr?Val?Ile?Arg?Phe?Asp?Asn?Arg?Asp?Thr?Gly?Leu?Ser?Thr
50 55 60
Lys?Phe?Ala?Glu?Ala?GlyIle?Pro?AspIle?Gly?GlnIle?Ile?Glu?Ala
65 70 75 80
Arg?Met?Lys?Gly?Glu?Glu?Phe?Arg?Pro?Pro?Tyr?Thr?Leu?Glu?Asp?Met
85 90 95
Ala?Asp?Asp?Ala?Val?Gly?Leu?Leu?Asp?Ala?Leu?Glu?Ile?Glu?Lys?Ala
100 105 110
His?Val?Cys?Gly?Met?Ser?Met?Gly?Gly?Met?Ile?Ala?Gln?Thr?Ile?Gly
115 120 125
Leu?Asn?His?Gln?Gln?Arg?Val?Leu?Ser?Leu?Ile?Ser?Ile?Tyr?Ser?His
130 135 140
Thr?Gly?Asn?Pro?Glu?Leu?Pro?Pro?Pro?Thr?Pro?Glu?Ala?Leu?Glu?Tyr
145 150 155 160
Leu?Val?Thr?Pro?Pro?Pro?Met?Glu?Arg?Glu?Ala?Asn?Ile?Thr?Tyr?Thr
165 170 175
Leu?Asp?Val?Trp?Arg?Thr?Phe?Ser?Gly?Lys?Gly?Phe?Pro?Leu?Asp?Glu
180 185 190
Asp?Trp?Asn?Arg?Lys?Ile?Ala?Ala?Lys?Ala?Tyr?Asp?Arg?Ala?Phe?Tyr
195 200 205
Pro?Glu?Gly?Val?Ala?Arg?Gln?Met?Ala?Ala?Val?Leu?Thr?Gln?Lys?Asn
210 215 220
Arg?Lys?Phe?Glu?Leu?Gly?Ser?Val?Thr?Met?Pro?Thr?Leu?Val?Ile?His
225 230 235 240
Gly?Ala?Asp?Asp?Pro?Leu?Val?Pro?Val?Glu?Gly?Gly?Lys?Asp?Thr?Ala
245 250 255
Glu?Ala?Ile?Pro?Gly?Ala?Glu?Leu?Ile?Ile?Ile?Asp?Gly?Met?Gly?His
260 265 270
Asp?Leu?Pro?His?Gly?Gly?Ala?Trp?Pro?Gln?Ile?Val?Asp?Ala?Ile?Val
275 280 285
Asn?His?Thr?Gln?Lys?Ile?Phe?Ile
290 295
<210>3
<211>304
<212>PRT
＜213〉the unknown
<400>3
Met?Ala?Asn?Ile?Ile?Ala?Asn?Gly?Ile?Gln?Ile?Glu?Tyr?Asp?Thr?Phe
1 5 10 15
Gly?Asn?Pro?Asp?Glu?Pro?Pro?Leu?Leu?Leu?Ile?Met?Gly?Leu?Ala?Cys
20 25 30
Gln?Leu?Ile?His?Trp?Asp?Glu?Asp?Leu?Cys?Glu?Gln?Leu?Ala?Arg?Arg
35 40 45
Gly?His?Tyr?Val?Ile?Arg?Phe?Asp?Asn?Arg?Asp?Thr?Gly?Leu?Ser?Thr
50 55 60
Lys?Phe?Ala?Glu?Ala?Gly?Ile?Pro?Asp?Ile?Gly?Gln?Ile?Ile?Glu?Ala
65 70 75 80
Arg?Met?Lys?Gly?Glu?Glu?Phe?Arg?Pro?Pro?Tyr?Thr?Leu?Glu?Asp?Met
85 90 95
Ala?Asp?Asp?Ala?Val?Gly?Leu?Leu?Asp?Ala?Leu?Glu?Ile?Glu?Lys?Ala
100 105 110
His?Val?Cys?Gly?Met?Ser?Met?Gly?Gly?Met?Ile?Ala?Gln?Thr?Ile?Gly
115 120 125
Leu?Asn?His?Gln?Gln?Arg?Val?Leu?Ser?Leu?Ile?Ser?Ile?Tyr?Ser?His
130 135 140
Thr?Gly?Asn?Pro?Glu?Leu?Pro?Pro?Pro?Thr?Pro?Glu?Ala?Leu?Glu?Tyr
145 150 155 160
Leu?Val?Thr?Pro?Pro?Pro?Met?Glu?Arg?Glu?Ala?Asn?Ile?Thr?Tyr?Thr
165 170 175
Leu?Asp?Val?Trp?Arg?Thr?Phe?Ser?Gly?Lys?Gly?Phe?Pro?Leu?Asp?Glu
180 185 190
Asp?Trp?Asn?Arg?Lys?Ile?Ala?Ala?Lys?Ala?Tyr?Asp?Arg?Ala?Phe?Tyr
195 200 205
Pro?Glu?Gly?Val?Ala?Arg?Gln?Met?Ala?Ala?Val?Leu?Thr?Gln?Lys?Asn
210 215 220
Arg?Lys?Phe?Glu?Leu?Gly?Ser?Val?Thr?Met?Pro?Thr?Leu?Val?Ile?His
225 230 235 240
Gly?Ala?Asp?Asp?Pro?Leu?Val?Pro?Val?Glu?Gly?Gly?Lys?Asp?Thr?Ala
245 250 255
Glu?Ala?Ile?Pro?Gly?Ala?Glu?Leu?Ile?Ile?Ile?Asp?Gly?Met?Gly?His
260 265 270
Asp?Leu?Pro?His?Gly?Gly?Ala?Trp?Pro?Gln?Ile?Val?Asp?Ala?Ile?Val
275 280 285
Asn?His?Thr?Gln?Lys?Ile?Phe?Ile?Val?Glu?His?His?His?His?His?His
290 295 300
<210>4
<211>24
<212>DNA
＜213〉artificial sequence
<400>4
CATGCCATGG?CAAACATTAT?TGCG 24
<210>5
<211>28
<212>DNA
＜213〉artificial sequence
<400>5
ACGCGTCGAC?GATAAAAATT?TTTTGGGT 28
Claims (10)
1, a kind of esterase, its aminoacid sequence is: the aminoacid sequence shown in the SEQ ID No.2, or this sequence through disappearance, add and/or replace one or several amino acid and have the aminoacid sequence of same function.
2, the gene of the described esterase of coding claim 1.
3, gene as claimed in claim 2, its nucleotides sequence is classified as: the nucleotide sequence shown in the SEQ ID No.1, or this sequence through disappearance, add and/or replace one or several Nucleotide, and the nucleotide sequence of coding same protein.
4, contain claim 2 or 3 described expression carrier.
5, expression vector as claimed in claim 4, it is derived from pET28a.
6, the host cell that contains claim 4 or 5 described expression vectors.
7, host as claimed in claim 6, it is intestinal bacteria.
8, the described esterase of claim 1 is separated the application in synthetic of ester, transesterify or ester in catalysis.
9, a kind of method for preparing the described esterase of claim 1, it is by cultivating claim 6 or 7 described host cells, separate obtaining described esterase from culture.
10, method as claimed in claim 9 is characterized in that, this method comprises the steps:
1) activation: the weighting profit requires single bacterium colony of 7 described hosts to contain in the LB substratum of kan 50 μ g/ml in 5ml, is cultured to OD600 ≈ 0.5 in 37 ℃, 250rpm;
2) cultivate: with 1% inoculum size bacterium liquid is transferred to 500ml and contains in the LB substratum of kan 50 μ g/ml, being cultured to OD600 in 37 ℃, 250rpm is between 0.5～1.0;
3) induce: in bacterium liquid, add IPTG to final concentration be 0.5mM, in 30 ℃, 250rpm inducing culture 12～15 hours.
4) collecting cell: 4 ℃, centrifugal 10 minutes collecting cells of 10000g; Abandon supernatant, precipitation is with 1 * PBS damping fluid washing 2 times, and cell is resuspended in 30～40ml PBS damping fluid;
5) obtain thick zyme extract: under the condition of ice bath, the ultrasonic disruption cell; Behind the cell wall breaking, with centrifugal 10 minutes of 4 ℃, 15000g, collecting supernatant was thick zyme extract.
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|---|---|---|---|
| CN2008102269426A CN101402947B (en) | 2008-11-20 | 2008-11-20 | Novel gene of esterase and recombinant expression system |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102269426A CN101402947B (en) | 2008-11-20 | 2008-11-20 | Novel gene of esterase and recombinant expression system |
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| Publication Number | Publication Date |
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| CN101402947A true CN101402947A (en) | 2009-04-08 |
| CN101402947B CN101402947B (en) | 2010-11-10 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286441A (en) * | 2011-07-24 | 2011-12-21 | 国家海洋局第二海洋研究所 | Low-temperature esterase and coding gene and use thereof |
| CN102715485A (en) * | 2012-06-20 | 2012-10-10 | 中国农业大学 | Method for preparing milky essence from esterase Est_p1 |
| CN105255917A (en) * | 2015-11-09 | 2016-01-20 | 中国科学院海洋研究所 | Moderate-temperature alkaline lipase as well as preparation method and application thereof |
| CN113234742A (en) * | 2021-05-18 | 2021-08-10 | 中国农业科学院农产品加工研究所 | Preparation of novel high-activity ferulic acid esterase and application of novel high-activity ferulic acid esterase in agricultural wastes |
-
2008
- 2008-11-20 CN CN2008102269426A patent/CN101402947B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286441A (en) * | 2011-07-24 | 2011-12-21 | 国家海洋局第二海洋研究所 | Low-temperature esterase and coding gene and use thereof |
| CN102286441B (en) * | 2011-07-24 | 2012-12-12 | 国家海洋局第二海洋研究所 | Low-temperature esterase and coding gene and use thereof |
| CN102715485A (en) * | 2012-06-20 | 2012-10-10 | 中国农业大学 | Method for preparing milky essence from esterase Est_p1 |
| CN105255917A (en) * | 2015-11-09 | 2016-01-20 | 中国科学院海洋研究所 | Moderate-temperature alkaline lipase as well as preparation method and application thereof |
| CN113234742A (en) * | 2021-05-18 | 2021-08-10 | 中国农业科学院农产品加工研究所 | Preparation of novel high-activity ferulic acid esterase and application of novel high-activity ferulic acid esterase in agricultural wastes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101402947B (en) | 2010-11-10 |
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