CN101603037A - A kind of thermostable carboxylesterase gene, proteins encoded and application thereof - Google Patents
A kind of thermostable carboxylesterase gene, proteins encoded and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 claims description 2
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- PHYFQTYBJUILEZ-UHFFFAOYSA-N Trioleoylglycerol Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCCCCCCCC)COC(=O)CCCCCCCC=CCCCCCCCC PHYFQTYBJUILEZ-UHFFFAOYSA-N 0.000 description 1
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- KZRPHCQLJZXMJV-UHFFFAOYSA-N sodium 4-(pyridin-2-yldiazenyl)benzene-1,3-diol Chemical compound [Na+].OC1=CC(O)=CC=C1N=NC1=CC=CC=N1 KZRPHCQLJZXMJV-UHFFFAOYSA-N 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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- 229940117972 triolein Drugs 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of thermostable carboxylesterase gene, proteins encoded and application thereof, described esterase has SEQ ID NO:1, shown aminoacid sequence.The present invention compared with prior art, the esterase of the thermostability that the esterase that contains EstW obtains for the streptomycete from known mesophilic bacteria strain at present can be widely used in the production of non-steroidal anti-inflammatory drug or forulic acid.
Description
Technical field
The present invention relates to a kind of Procaine esterase gene, proteins encoded and application thereof, particularly have Procaine esterase gene, proteins encoded and the application thereof of thermostability.
Background technology
Esterase (esterases, EC 3.1.1.X) is that a big class can the catalysis ester linkage hydrolyzing and the enzyme of formation, is distributed widely in animal, plant and microorganism.Esterase mainly comprise Procaine esterase (carboxylesterases, carboxylester hydrolases, EC 3.1.1.1) and lipase (lipase, triacylglycerol hydrolases, EC3.1.1.3).Procaine esterase can hydrolysis or the synthesizing acyl chain length less than 10 glycerolipid; with tributyrin (tributyryglycerol) is the standard substrate; and lipase is substrate with acyl chain length more than or equal to the glyceryl ester of 10 carbon atoms generally; triolein is its standard substrate, but the substrate that most of lipase also can hydrolysising carboxy acid ester's enzyme.Esterase belongs to typical α/β lytic enzyme superfamily, has by Ser, catalytic center three combinations (catalytic triad) that His and Asp constitute, and wherein Ser is usually located at and has in the conservative Gly-Xaa-Ser-Xaa-Gly pentapeptide structure.Three amino acid primary structures that constitute catalytic center three combinations fall far short, but space structure is arranged on the close position, and nucleophilic amino acid Ser residue links to each other with His by hydrogen bond, and the Asp residue also links to each other with His by the hydrogen bond that is formed by carboxyl.
Esterase occupies significant proportion as a kind of important industrial enzymes on global zymin market.Because the character of the expression amount of esterase gene and reorganization esterase is subjected to the influence of many factors: (1) esterase is the virose material of a class pair cell, and regular meeting causes toxicity to cause the cracking of cell to host cell when heterologous host is expressed; (2) esterase protein often needs molecular chaperones to help it to be folded into correct conformation in folding process; (3) esterase often needs the existence of inductor in culturing process; (4) non-translational region of esterase gene, the glycosylation of the existence of esterase propetide and signal peptide, the difference of codon and different expression system expression reorganization esterases etc. all can influence the heterogenous expression of esterase gene.Intestinal bacteria particularly, it is as the low prokaryotic organism of waiting, lack perfect posttranslational modification system, esterase expression therein often exists with the inclusion body form, few activated protein that obtains, the thermostability esterase mainly comes from the thermophilic or super thermophilic environment simultaneously, does not appear in the newspapers as yet from the thermostability esterase in the mesophilic bacteria strain.
Summary of the invention
The 1st technical problem to be solved by this invention provides a kind of Procaine esterase gene of thermostability.
It is said gene encoded protein matter that the 2nd technical problem to be solved by this invention provides.
The 3rd the above-mentioned proteinic application of technical problem to be solved by this invention.
The present invention obtains the new gene estW of thermostable carboxylesterase from muta lead mycillin, aminoacid sequence shown in SEQID NO:1 of its coding, above-mentioned this gene coded protein of amino acid primary sequence analysis revealed of being derived out by gene are the newcomer of true lipases subfamily in I.7; EstW is connected with the pET28b plasmid, makes up recombinant expression plasmid pET28b-estW; This recombinant plasmid transformed competent escherichia coli cell makes up genetic engineering bacterium Rosetta (DE3) pLysS/pET28b-estW that is used for the estW expression; Above-mentioned engineering bacteria has obtained to express preferably under 25 ℃ of low temperature and 0.5mM IPTG condition; Procaine esterase EstW is carried out purifying and detect zymologic property showing this enzyme optimal reactive temperature and pH are respectively 50 ℃, pH 8.0.EstW has high thermostability, and enzyme work is 51.34% after 95 ℃ of following incubation 12.5h, begins to reach the transformation period subsequently.
Esterase of the present invention is in the application of the production of preparation non-steroidal anti-inflammatory drug or forulic acid.
The present invention compared with prior art, the esterase of the thermostability that the esterase that contains EstW obtains for the streptomycete from known mesophilic bacteria strain at present can be widely used in the production of non-steroidal anti-inflammatory drug or forulic acid.
Description of drawings
Fig. 1 is the pcr amplification figure of estW gene.
In Fig. 1: M1 is Marker III, and 1 is the pcr amplification product of estW gene.
Fig. 2 is the pcr amplification figure that Nde I/Xho I double digestion is identified recombinant plasmid pET28b-estW.
In Fig. 2: M2 is 1kb DNA Marker; 2 is that pET28b-estW is through Nde I/Xho I double digestion product.
Fig. 3 SDS-PAGE analyzes the table result schematic diagram of EstW.
In Fig. 3: M3 is protein Marker; 31 for carrying the crude enzyme liquid of pET28b plasmid; 32 for carry the pET28b-estW plasmid without IPTG inductive crude enzyme liquid; 33 for having the 0.5mM IPTG inductive crude enzyme liquid of pET28b-estW plasmid; 34 is the purifying protein of EstW
Fig. 4 is the enzyme work of EstW and the graph of a relation of temperature.
Fig. 5 is the enzyme work of EstW and the relation of pH value.
In Fig. 5, ■ is Na-citrate damping fluid (pH 5.5-7.0); ● be Tris-HCl (7.0-9.0) damping fluid; ▲ be KH
2PO
4-NaOH (8.5-9.5) damping fluid.
Fig. 6 is under 95 ℃ of conditions, the remnant enzyme activity of EstW and time relation.
Embodiment
Below in conjunction with embodiment the present invention is done detailed explanation.
Embodiment 1:
1, Procaine esterase gene estW obtains
Cultivating muta lead mycillin TK64 and extract genome, is template with its genomic dna, with estW-F:ATACGTC
CATATGAGTTTCCTCAATCCCCGCAT and estW-R:TAA
CTCGAGCGATTATAAAGCTTCCCG (underscore is respectively NdeI and XhoI restriction enzyme site) is a primer, at 98 ℃, and 10s, 55 ℃, 10s, 72 ℃, 1min 10s carries out pcr amplification under 35 round-robin conditions, and product carries out agarose gel electrophoresis (Fig. 1), subsequently, amplified production and carrier pET28b (+) behind NdeI, Xho I double digestion, connect transformed into escherichia coli DH5 α, kalamycin resistance screening positive clone respectively, obtain expression plasmid pET28b-estW, double digestion is identified (Fig. 2); PET28b-estW checks order with expression plasmid, and aminoacid sequence is shown in SEQ ID NO:1.
2, expressing engineering bacteria makes up
Expression plasmid pET28b-estW is converted into intestinal bacteria Rosetta (DE3) competent cell according to prior art, and kantlex and paraxin screening positive clone make up recombinant bacterial strain Rosetta (DE3)/pET28b-estW.
3, the expression of EstW and purifying
Picking recombinant bacterial strain Rosetta (DE3)/pET28b-estW in the test tube that contains 5ml LB liquid nutrient medium, 37 ℃, 225rpm, the enlarged culturing of spending the night; With culture with in the tap web bottle that is inoculated in the LB liquid nutrient medium at 1: 100,37 ℃, 225rpm, shaking culture is to OD
600During for 0.4-0.6; Adding 0.5mM isopropylthiogalactoside (isopropyl-β-D-thiogalactopyranoside, IPTG), 25 ℃ of abduction delivering 10h; 4 ℃, 5,000rpm, centrifugal 5min collects thalline, ultrasonic disruption.Use Co
2+The ion affinity column (
Purification Kit) purifying.Protein behind the purifying is identified (Fig. 3) through polyacrylamide gel electrophoresis (SDS-PAGE), as shown in Figure 3: the very high band of one specificity is arranged at molecular weight 33kDa place, illustrate that the purity of protein behind the purifying is higher, SDS-PAGE shows that the size of EstW is about 33kDa, and is consistent with the EstW molecular weight size of calculating by aminoacid sequence (32.4kDa) that has 6 His-tag.
4, sequential analysis
Utilize BioEdit to analyze the estW gene order, this full length gene 870bp, initiator codon is GTG, and terminator codon is TGA, and GC content is 68.39%, has embodied the high GC content feature of streptomyces gene group.Utilize Clustal X1.8 and Megalign 6.1. to analyze different families Procaine esterase/lipase sequence, show that this Procaine esterase belongs to I.7 family member of true lipases subfamily.The EstW primary structure has Gly137-Phe138-Ser139-Glu140-Gly141 pentapeptide conserved regions, meet the conservative Gly-Xaa-Ser-Xaa-Gly pentapeptide structure of esterase, tertiary structure contains Ser139-Asp235-His270 catalysis three subunits, and Ser139 links to each other by hydrogen bond with Asp235, His270 as nucleophilic group.
Embodiment 2:
The EstW optimum temperuture detects:
The accurate system 1ml of enzyme biopsy mark of EstW, 50mM Tris-HCL (pH 8.0), 1% acetonitrile, the p-NP capronate of 0.5mM, the pure enzyme liquid of 5 μ l, the Cary 300 UV spectrophotometers that use can be temperature automatically controlled, the detection wavelength is 410nm.Temperature of reaction (10 ℃-60 ℃) is set, and test is independent to be repeated 3 times, averages, and the result shows that the optimum temperuture of this enzyme is 50 ℃ (Fig. 4).
Embodiment 3:
The EstW optimal pH detects
The mensuration system of employing standard, temperature of reaction are 50 ℃, use corresponding damping fluid, pH5.0 to 7.04,50mM sodium citrate buffer in corresponding pH scope; PH 7.0-9.0,50mM TrisHCl buffer; PH 8.5-9.0,50mM K
2HPO
4-KOH buffer.The detection wavelength is OD
348, test is independent to be repeated 3 times, and the result shows that the optimal pH of this enzyme is 8.0 (Fig. 5).
Embodiment 6:
The EstW heat stability test
With the pure enzyme of EstW differing temps incubation certain hour between 20 ℃ of-95 ℃ of scopes, adopt the standard detection system, 50 ℃ are detected remnant enzyme activity, determine thermostability, are made as 100% with untreated pure enzymic activity.The result shows, this enzyme is placed 173h and 13.7h respectively under 50 ℃ and 90 ℃ of conditions, and enzymic activity does not have considerable change.95 ℃ of following EstW place 6.7h, and enzyme is lived still more than 90%, and 12.5h reaches the transformation period (Fig. 6) later on gradually.EstW is at present known from the highest esterase of thermostability in the streptomycete, also is the true lipasessubfamily the highest enzyme of thermostability in the family I.7 under this enzyme.This enzyme even more many esterases from thermophilic environment reach all high from the Procaine esterase thermostability in Archaeoglobus fulgidus and the thermophilic environment macro genomic library as the lipase from Thermophilic Bacillus sp. and Thermoanaerobacter tengcongensis.
Sequence table
＜110〉Anhui Normal University
＜120〉a kind of thermostable carboxylesterase gene, proteins encoded and application thereof
<130>1
<160>1
<170>PatentIn?version?3.3
<210>1
<211>289
<212>PRT
＜213〉muta lead mycillin (streptomyces lividands)
<400>1
Val?Ser?Phe?Leu?Asn?Pro?Arg?Ile?Pro?Gly?Val?Asn?Ser?Asp?Asp?Val
1 5 10 15
Val?Thr?Asp?Ile?Thr?Glu?Ala?Pro?His?Arg?Leu?Gly?Ala?Val?Val?Gln
20 25 30
Ala?Gly?Gly?Ala?Gln?Val?Ile?Ala?Thr?Val?Asp?Val?Gln?Ala?Gly?Glu
35 40 45
Asp?Asp?Trp?Asn?Thr?Pro?Pro?Ser?Glu?Glu?His?Pro?Arg?Pro?Val?Val
50 55 60
Leu?Val?His?Gly?Thr?Phe?Gly?Asn?Arg?Gly?Tyr?Thr?Trp?Asn?Thr?Ala
65 70 75 80
Val?Pro?Leu?Leu?Arg?Arg?His?Gly?His?Arg?Val?Phe?Arg?Leu?Asp?Tyr
85 90 95
Val?Pro?Leu?Leu?Arg?Arg?His?Gly?His?Arg?Val?Phe?Arg?Leu?Asp?Tyr
85 90 95
Gly?Gln?His?Gly?Asn?Pro?Leu?Ile?Phe?Gly?Leu?Gly?Asp?Ile?Lys?His
100 105 110
Ser?Ala?Arg?Gln?Leu?Ala?Asp?Phe?Val?Asp?Glu?Val?Leu?Arg?Arg?Thr
115 120 125
Gly?Ala?Gln?Gln?Val?Asp?Leu?Val?Gly?Phe?Ser?Gln?Gly?Gly?Met?Met
130 135 140
Pro?Arg?Tyr?Tyr?Leu?Asn?Ala?Leu?Gly?Gly?Gly?Pro?Lys?Val?His?Asn
145 150 155 160
Phe?Val?Gly?Ile?Ser?Pro?Ser?Asn?His?Gly?Val?Thr?Ala?Gln?Gly?Leu
165 170 175
Met?Asn?Leu?Ala?Arg?Gln?Ile?Pro?Gly?Ala?Val?Glu?Leu?Leu?Glu?Gln
180 185 190
Gly?Ala?Val?Gly?Glu?Val?Val?Pro?Ala?Trp?Pro?Gln?Leu?Gln?His?Asp
195 200 205
His?Leu?Phe?Gln?Arg?Glu?Leu?Ala?Asp?Leu?Gly?Glu?Thr?Thr?Glu?Gly
210 215 220
Val?Arg?His?Thr?Val?Ile?Ala?Thr?Gln?Tyr?Asp?Asp?Val?Val?Thr?Pro
225 230 235 240
Tyr?Thr?Ser?Cys?Ala?Leu?Ala?Lys?Thr?Glu?Gly?Cys?Tyr?Val?Asn?Asn
245 250 255
Ile?Val?Leu?Gln?Asp?Ile?Asp?Pro?Asp?Asp?His?Thr?Pro?His?Val?Ser
260 265 270
Met?Pro?Tyr?Asn?Ala?Thr?Val?Leu?Asn?Glu?Val?Leu?Lys?Ala?Leu?Ala
275 280 285
Asp
Claims (3)
1, a kind of thermostable carboxylesterase is characterized in that: it has SEQ ID NO:1, shown aminoacid sequence.
2, a kind of gene of the described thermostable carboxylesterase of claim 1 of encoding.
3, the described thermostable carboxylesterase of claim 1 is in the application of the production of preparation non-steroidal anti-inflammatory drug or forulic acid.
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| CN2009101442130A CN101603037B (en) | 2009-07-23 | 2009-07-23 | Thermostable carboxylesterase gene, coding protein and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886063A (en) * | 2010-06-23 | 2010-11-17 | 中国科学院微生物研究所 | Esterase with racemic phenyl glycidyl ester split activity, encoding gene and application thereof |
| CN102465118A (en) * | 2010-11-10 | 2012-05-23 | 中国科学院微生物研究所 | Alkaline esterase and encoding gene and application thereof |
| CN102559719A (en) * | 2012-01-20 | 2012-07-11 | 上海海洋大学 | Carboxylesterase gene and protein coded by same |
| CN102559718A (en) * | 2011-12-08 | 2012-07-11 | 上海交通大学 | Construction of thermophilic carboxylesterase gene engineering strain and application of carboxylesterase of strain |
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2009
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| CN101886063A (en) * | 2010-06-23 | 2010-11-17 | 中国科学院微生物研究所 | Esterase with racemic phenyl glycidyl ester split activity, encoding gene and application thereof |
| CN102465118A (en) * | 2010-11-10 | 2012-05-23 | 中国科学院微生物研究所 | Alkaline esterase and encoding gene and application thereof |
| CN102465118B (en) * | 2010-11-10 | 2013-07-10 | 中国科学院微生物研究所 | Alkaline esterase and encoding gene and application thereof |
| CN102559718B (en) * | 2011-12-08 | 2013-09-11 | 上海交通大学 | Construction of thermophilic carboxylesterase gene engineering strain and application of carboxylesterase of strain |
| CN102559718A (en) * | 2011-12-08 | 2012-07-11 | 上海交通大学 | Construction of thermophilic carboxylesterase gene engineering strain and application of carboxylesterase of strain |
| CN102559719A (en) * | 2012-01-20 | 2012-07-11 | 上海海洋大学 | Carboxylesterase gene and protein coded by same |
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| CN109554383A (en) * | 2018-05-29 | 2019-04-02 | 安徽师范大学 | A kind of heat-resisting esterase encoding gene, carrier, engineering bacteria and its purposes for encoding albumen |
| CN109554383B (en) * | 2018-05-29 | 2022-05-06 | 安徽师范大学 | Heat-resistant esterase encoding gene, vector, engineering bacterium and application of encoding protein thereof |
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| CN112779186B (en) * | 2021-01-19 | 2022-06-17 | 自然资源部第二海洋研究所 | Intermediate-temperature bacterium for producing ester hydrolase simultaneously resisting high salinity, organic solvent and detergent and application thereof |
| CN112813131A (en) * | 2021-02-18 | 2021-05-18 | 江南大学 | Carboxylesterase and application thereof in producing cyclohexenecarboxylic acid by kinetic resolution of cyclohexenecarboxylate |
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