CN107236718A - A kind of low temperature esterase, encoding gene and its application from grand genome - Google Patents

A kind of low temperature esterase, encoding gene and its application from grand genome Download PDF

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CN107236718A
CN107236718A CN201710457737.XA CN201710457737A CN107236718A CN 107236718 A CN107236718 A CN 107236718A CN 201710457737 A CN201710457737 A CN 201710457737A CN 107236718 A CN107236718 A CN 107236718A
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est18
esterase
low
temperature alkali
recombinant
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CN107236718B (en
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吴菁
吴伟
李卓
梅江鹏
杨江科
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Wuhan Polytechnic University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01001—Carboxylesterase (3.1.1.1)

Abstract

The present invention relates to biotechnology and genetic engineering field, and in particular to a kind of low temperature esterase, encoding gene and its application from grand genome.Low temperature esterase is Est18, and its nucleotide sequence is as shown in SEQ ID NO.1；The amino acid sequence that it is encoded is as shown in SEQ ID NO.2.Est18 is low-temperature alkali esterase, is stablized relatively when below 40 DEG C of temperature, 20 DEG C of optimum temperature, maximum activity when optimal pH is 8.0, pH11.0>80%.

Description

A kind of low temperature esterase, encoding gene and its application from grand genome
Technical field
The present invention relates to biotechnology and bioengineering field, and in particular to a kind of low temperature from soil metagenome Esterase Est18 and its encoding gene and application.
Background technology
Esterase is widely present in animal, plant and microorganism, is the enzyme of a class catalyzing hydrolysis or formation ester bond, effect Substrate is typically the esters that aliphatic chain is less than ten carbon atoms.Esterase belongs to α/β and folds hydrolase superfamily, and catalytic center is general It is made up of serine, aspartic acid/glutamic acid and histidine, conserved sequence is pentapeptide (GXSXG) sequence near serine.Ester A variety of chemical reactions such as enzyme energy catalyzing hydrolysis, esterification, transesterification, are a kind of critically important industrial biocatalytic agent, are transported extensively For fields such as fine chemistry industry, washing, medicine, food, papermaking, leather processing, weaving, wastewater treatment and feed industries.From urging Change from the point of view of characteristic, esterase has the chemo-selective and alloisomerism selectivity of height, and reaction does not need coenzyme, reaction condition Gently, accessory substance is few.Another distinguishing feature of esterase in production application is that it can be in outphasing system（That is oil-water interface）Or Acted in organic phase.In aqueous phase, the usual catalytic hydrolysis reaction of esterase, and in organic phase, it but can catalytic esterification and transesterification Change reaction.
Grand genome, also known as first genome, refers to the summation of whole tiny organism inhereditary materials in habitat.Because grand gene Group technology is the environmental microorganism genomic dna cloning that will directly extract in different carriers and is transferred to suitable host, passes through Different triage techniqueses screens and obtains new gene or bioactivity class material, so the technology has actually bypassed microorganism training Support link, can preferably development and utilization Anticipated transient without scram resource, provided for industry, agricultural or each side broader Gene pool.At present, increasing patent enzyme comes from grand genomic library, and they are applicable to food, chemical industry, medicine Deng field.
The content of the invention
It is an object of the invention to provide a kind of new low-temperature alkali esterase Est18 from Mount Taishan soil metagenome, Encoding gene and preparation method thereof, the esterase can be used for the hydrolysis of the hydrolysis, especially short chain esters of ester bond.
The present invention utilizes functional screening from the soil metagenome library of Mount Taishan by specific substrate tributyrin Method (Function-driven Screening) therefrom screens one plant of bacterial strain TS-Est18 for showing esterase active, Using bioinformatic analysis method, analysis obtains an esterase geneest18.The full length gene is 957bp（From initiation codon Son arrives terminator codon）, encode 318 amino acid.By PCR method, clone is obtained into esterase geneest18Carried with expression Recombinant plasmid pEst18 is obtained after body pET-30b (+) connection, after e. coli bl21 (DE3) pLys induced expressions, is obtained Recombinant esterase Est18.Est18 is low-temperature alkali esterase, is stablized relatively when below 40 DEG C of temperature, 20 DEG C of optimum temperature, It is stable in the range of pH6-11, maximum activity when optimal pH is 8, pH11>80%.Recombinant bacterial strain comprising the gene is deposited in Positioned at the China typical culture collection center of Wuhan University, deposit number is CCTCC M 2017317, and strain classification is Escherichia coli。
First purpose of the present invention is to provide the esterase gene of the esterase Est18 described in coding a kind ofest18, it is described Esterase geneest18Nucleotide sequence as shown in SEQ ID NO.1.
Second object of the present invention is to provide a kind of esterase Est18, and its amino acid sequence is as shown in SEQ ID NO.2.
The present invention also provides a kind of containing described esterase geneest18Recombinant expression carrier.Described expression vector, It is preferred that pET-30b (+) carrier.
The present invention also provides a kind of containing described esterase geneest18 genetic engineering bacterium.Described genetic engineering bacterium, It is preferred that Escherichia coliEscherichia coli BL21 (DE3) pLys。
Third object of the present invention is to provide a kind of method for preparing low-temperature alkali esterase Est18, comprises the following steps：
1) host cell is converted with above-mentioned recombinant vectorEscherichia coliBL21 (DE3) pLys., obtain recombinant bacterial strain；
2) recombinant bacterial strain, induction Recombinant esterase expression are cultivated；
3) reclaim and purify expressed esterase Est18.
Low-temperature alkali esterase Est18 of the present invention comes from Mount Taishan soil metagenome（Host Strains are unknown）, its amino acid Sequence is as shown in SEQ ID NO. 2：
MSTLDPKLFSDKAIAPETRAVNDAIIAAMTGMPEWWDVGAAKTRAARARGEGIFPAAAKSERARWIEIDGPAG KVPLRVIAPENPRGVYLHIHGGGHVLGAADQQDRLLERISNETRLTAISVEYRLAPENPYPAGPDDCEAAALWVNEH AADFGGHKIAIGGESAGAHLSALTILRLRDKHGLTPFNAANLVFGVFDLGMTPSARAFGDERLVLRTRDIEKFGEAF LPGTDDEQKRAPEFSPLYANLAGLCPALFTIGTRDALLDDSLFMHARWVAAGNVGELDIYPGGCHGFIAFPYPQAFA SIARQATFLNAALG
The present invention also provides above-mentioned low-temperature alkali esterase Est18 answering in catalysis ester-type hydrolysis, esterification or transesterification With.It is preferred that short-chain aliphatic ester be the p-nitrophenyl phenolic ester with C2-C8 short carbon chains, such as paranitrophenylacetic acid ester, to nitre Base benzenebutanoic acid ester, p-nitrophenyl caprylate etc., wherein substrate is p-nitrophenyl butyrate（C4）When catalytic activity highest.
Esterase Est18 is a kind of low-temperature alkali esterase, and altogether containing 318 amino acid, the enzyme activity between 20-45 DEG C reaches More than 80%, enzymatic activity drastically declines when temperature is more than 50 DEG C.Optimal pH is 8.0, is incubated in pH 11 buffer solution 20 min still preserve more than 80% enzyme activity, there is high enzyme activity, preferably 7.0 ~ 9.0 between pH value 7-11.1 mM Cu2+、Fe2+And Zn2+The strong inhibitory action of enzyme activity generation to Est18, and Ca2+, Mg2+, Ni2+And Co2+, with And metal-chelator EDTA has humidification to enzyme activity.
Present invention screening from the soil metagenome library of Mount Taishan obtains new esterase gene, finds the egg of the gene code White stability and high efficiency, fast deactivation at 50 DEG C at 20 DEG C（It is easy to enzymatic reaction control）, while shown in alkaline environment compared with High stability and activity.Therefore the low-temperature alkali esterase of the present invention can be applied in the industry such as food, chemical industry, washing, pharmacy, with Reduction production temperature reduces energy consumption.
Brief description of the drawings
Fig. 1：E. coliThe Recombinant esterase Est18 of middle expression SDS-PAGE analyses.Swimming lane M is protein Marker, swimming lane A are the soluble component before esterase Est18 is induced, and swimming lane B is solvable after esterase Est18 is induced Property component, swimming lane C be the albumen of esterase Est18 after purification.
Fig. 2：Recombinant esterase Est18 optimal reactions pH.
Fig. 3：Recombinant esterase Est18 optimal reactive temperatures.
Fig. 4：Recombinant esterase Est18 heat endurance.
Fig. 5：Bivalent cation is to esterase Est18 activity influences.
Fig. 6：Organic solvent, denaturant and chela and agent are to Est18 activity influences.
Embodiment
Embodiment 1：Esterase geneest18Acquisition
Screen and purify from the soil metagenome library of Mount Taishan by substrate of tributyrin and obtain showing esterase active Bacterial strain TS-Est18.TS-Est18 DNA is extracted, the sequence information of Insert Fragment is obtained through subclone sequencing.Utilize Bioinformatics method is annotated to gene information, analyzes and determine the ORFs of wherein esterase gene（Open Reading Frame, ORF）, the ORF is named asest18, its gene order is as shown in SEQ ID NO.1, and total length is 957bp（ATG is originated, and TAG is terminated）, its esterase Est18 encoded amino acid sequence is as shown in SEQ ID NO.2, totally 318 Amino acid.
Embodiment 2：Esterase geneest18Clone, the structure of recombinant vector and recombinant bacterial strain
The esterase gene that the present invention is obtainedest18It is cloned on expression vector, builds recombinant expression carrier.Obtained according to analysis Esterase gene sequence, utilize primer5 Software for Design expand esterase full genome sense primer Forwards（5’- CCAAGCTTTCCGGTTTTTGACGAAGCGC,BamHI）With anti-sense primer Reverse（5’- CGGGATCCGAGCGCGATCCGCAAACTAG,HindIII）.Esterase gene is expanded using following PCR amplification programsest18：94 DEG C min of pre-degeneration 3；94 DEG C of denaturation 1min, renaturation temperature 56 DEG C of 1 min, 72 DEG C of 1 min of extension, carry out 25 circulations；Follow 72 DEG C of extensions 10 min, 4 DEG C of 10 min after ring terminates, the length of amplification purpose fragment is about 1 kb.PCR primer after purification ThroughBamHI andHinDIII double digestions, rubber tapping reclaim after withBamHI andHinPlasmid pET-30b (+) connection of dIII double digestions, Bacillus coli DH 5 alpha is converted, positive colony is obtained by kalamycin resistance and tributyrin screening active ingredients.Extracting is positive The plasmid of clone obtains recombinant vector pEst18, through double digestion and sequencing identification Insert Fragment with SEQ ID NO.1.Include restructuring Carrier pEst18 Escherichia coli Escherichia coli DH5 α bacterial strains are deposited in the Chinese Typical Representative culture positioned at Wuhan University Thing collection, deposit number is CCTCC M 2017317, and preservation date is on June 8th, 2017.
Embodiment 3：Express the structure of esterase Est18 genetic engineering bacterium
It is prepared by 3.1 e. coli bl21s (DE3) pLys competent cells
1. by a small amount of e. coli bl21 (DE3) pLys strains access 3 ml LB test tube liquid in, 200 rpm, 37 DEG C overnight Culture；
2. the bacterium solution in test tube is inoculated into 200ml LB shaking flasks by the inoculum concentration of 1% volume ratio, 200 rpm, 20 DEG C overnight Culture；
3. cultured shaking flask is rapidly cooled into 4 DEG C in frozen water, dispense to the centrifuge tube (50 ml) of ice precooling, ice bath number Minute；
4. 4 DEG C, 4000 rpm centrifugation 10 min recovery cells, abandon supernatant；
5. the ice-cold M of 10 ml 0.1 CaCl2Cell is resuspended, 4 DEG C of 4000 rpm centrifuges 10~15 min and reclaim thin Born of the same parents；
6. 5 are repeated, with the M of 10 ml 0.1 CaCl2Cell, more than ice bath 30min is resuspended；
7. 4 DEG C, 4000 rpm centrifuge 10 min and reclaim cell；
8. every 50 ml stock cultures, 1 ml 0.1M CaCl2After resuspension, after the glycerine for adding 1 ml 50% is mixed, point Loaded on 1.5 ml centrifuge tubes, 50~100 μ l are often managed.- 80 DEG C of preservations.Thus e. coli bl21 (DE3) pLys impressions are obtained State cell.
3.2 conversion
The μ l of pEst18 plasmids 0.5~1 obtained in Example 2 and 50 μ l e. coli bl21s (DE3) pLys competence Mixing with cells, the min of ice bath 30, in adding 500 μ l LB fluid nutrient mediums after 42 DEG C of s of water-bath heat shock 90, the min of ice bath 2, 37 DEG C of 200 rpm cultivates 45 min.The LB flat boards of the kanamycins containing 50 μ g/ml are coated on after culture centrifugation, thus Obtain e. coli bl21 (DE3) pLys containing pEst18.
Embodiment 4：Esterase Est18 expression and purifying
4.1 protein induced expression
E. coli bl21 (DE3) pLys containing pEst18 37 DEG C of cultures in 200 ml LB culture mediums are to OD600 During 0.4-0.6, plus IPTG to the mM of final concentration 0.2,20 DEG C of overnight incubations.Every rpm of 50 ml bacterium solutions 4000,4 DEG C of centrifugations 10 Min, collects thalline, and 2 ml (20 mM, pH 7.4) PBS（KH2PO4-K2HPO4）Thalline, ultrasonic disruption is resuspended 15 min minutes, supernatant was collected in centrifugation.
4.2 esterase Est18 purifying and SDS-PAGE electrophoresis
Purify the supernatant collected in 4.1 with His affinity columns, specific embodiment is as follows：
The Ni of 1.His affinity columns2+With reference to Binding Buffer（20 mM PBSs, 5 mM imidazoles and 500 MM NaCl are simultaneously filtered with 0.22 μm of filter）Balance.Add（0.5 times of column volume）0.1 M NiSO4Solution carries out Ni2+'s It is corresponding to combine, after having filled, add dH2O（Filtered with 0.22 μm of filter）The uncombined Ni of washing2+, then with 5 times of cylinders Long-pending Binding Buffer are balanced.
2. the purifying of esterase Est18 albumen.Loading after balance, sample is the supernatant collected in 4.1（Salinity in supernatant It is identical with Binding Buffer with imidazole concentration）10 ml, are washed away uncombined with the Binding Buffer of 5 times of column volumes（It is miscellaneous Albumen）Albumen.Then elute, elution volume is 20 ml（In this 20 ml, Elution Buffer（20 mM PBS are buffered Liquid, 500 mM imidazoles and 500 mM NaCl are simultaneously filtered with 0.22 μm of filter）Carried out with Binding Buffer in the step Mixing, Elution Buffer ratios gradually increase, and imidazole concentration is at the uniform velocity increased to 500 mM from 5 mM）；Finally again with 5 times Volume Elution Buffer are eluted completely, collect protein peak (1 ml/tube).
3. examine the esterase active for the protein eluate collected.Drawn in the LB culture dishes bottom containing tributyrin Then each lattice are entered line label by lattice, and the eluent of collection is respectively taken into 10 μ l, then add corresponding small lattice according to sequence number In sub, it is placed in 37 DEG C of incubators and just puts 3 h of culture or so result can be observed（Eluent containing esterase Est18 albumen Have hydrolysis circle）.
4. pair eluent collected carries out desalination and concentration.Use super filter tube（Merck Mi Libo Merck Millipore）Enter Row desalination and concentration, concrete operation method are carried out with reference to the operation manual of Merck Mi Libo Merck Millipore companies.
5. esterase Est18 SDS-PAGE electrophoresis.The esterase of purifying is subjected to PAGE gel electrophoresis（Fig. 1）, obtain The esterase Est18 of purifying, the albumen size about 37kDa of purifying, meets theory expectation.
4.3 esterase Est18 determinations of activity
Esterase Est18 vitality tests use p-nitrophenyl phenolic ester, and specific method is as follows：
1 uses dimethyl sulfoxide (DMSO)（DMSO）Prepare 200 mM p-nitrophenyl phenolic ester；
2 add the mM Tris-HCl Buffer of 980 μ l 50 in 1 ml reaction systems（100 mM NaCl, 50 mM Tris- HCl, 0.3 %（V/V）Triton X-100, pH 8.0）, 10 μ l p-nitrophenyl phenolic esters, the 10 pure enzyme liquids of μ l（After 100 times of dilution The μ g/ μ l of concentration 0.012276）；
3 detect the content of free p-nitrophenol using spectrophotometer under 400 nm absorbances, determine under this condition The extinction coefficient of content of p-nitrophenol is 16,642 M-1cm-1（That is the M of ε=16,642-1cm-1）.The definition of unit of enzyme activity is At the standard conditions（25 DEG C, 101.325 kPa）Enzyme amount needed for 1 μm of ol p-nitrophenol of generation per minute.
Embodiment 5：Esterase Est18 substrate specificity analysis
According to 4.3 condition determination, compare the activity of the p-nitrophenyl phenolic ester of esterase Est18 hydrolysis different length acyl groups.Substrate Using paranitrophenylacetic acid ester（C2）, p-nitrophenyl butyrate（C4）, p-nitrophenyl caprylate（C8）.As a result show, Est18 pairs The shorter p-nitrophenyl phenolic ester of acyl group carbochain has catalytic activity, and wherein substrate is p-nitrophenol butyrate（C4）Shi Cuihua lives Property highest.
Embodiment 6：Esterase Est18 optimum reaction conditionses analysis
Esterase Est18 optimal reaction pH is determined in the range of 4.0 ~ 11.0.The different cushioning liquid of preparation, wherein HAc- NaAc pH of buffer is 4.0-6.0, K2HPO3- KH2PO3PH of buffer is 6.0-8.0, H3BO3- Na2B4O7Buffering Liquid pH is 7.4-9.0, Na2CO3- NaHCO3PH of buffer is 9.0-11.0.These different pH buffer concentration It is 50 mM.Buffer solution (Tris-HCl buffer) described in condition determination in 4.3 is distinguished with the cushioning liquid prepared It is replaced, determines Recombinant esterase Est18 enzyme activity, substrate is p-nitrophenol butyrate.PH lives to Recombinant esterase Est18 The influence result of property is shown in Fig. 3.In 50mM H3BO3- Na2B4O7During pH of buffer 8.0, esterase Est18 enzymatic activity highest, pH Value has higher enzymatic activity between 7.0-11.0.When pH is less than 7.0, the rapid reduction of its activity（Fig. 2）
Esterase Est18 optimal reactive temperature is determined in the range of 20 ~ 70 DEG C.Delayed using 50 mM described in condition determination in 4.3 Fliud flushing (Tris-HCl buffer) pH8.0 is as buffer solution, and p-nitrophenol butyrate is as substrate, according to anti-in 4.3 System is answered, enzyme activity is determined at different temperatures.As a result show, Est18 is cold-adapted enzyme, temperature more low reaction speed is higher, in 20- Enzyme activity between 45 DEG C is up to more than 80%, and enzymatic activity drastically declines when temperature is more than 50 DEG C（Fig. 3）.
Embodiment 7：Esterase Est18 zymetology stability analyses
Esterase Est18 heat endurance is determined in the range of 20 ~ 50 DEG C.Enzyme liquid after purification is incubated 20 respectively, 30,40, At 45 and 50 DEG C, separated in time takes out the detection method as shown in 4.3 and determines enzymatic activity, as a result sees Fig. 4.Esterase Est18 with The rise for the treatment of temperature, esterase residual enzyme activity is gradually reduced, enzymatic activity is drastically reduced when temperature is higher than 50 DEG C, at 50 DEG C Handle after 5min, residual enzyme activity is lost substantially（Fig. 4）.
The measure of bivalent cation esterase Est18 activity influences.1 mM Fe is separately added into reaction system2+、Zn2+、Cu2+、 Mn2+、Ca2+、Mg2+、Ni2+And Co2+, 20 min are incubated at room temperature, by 4.3 assay methods（Made with p-nitrophenol butyrate For substrate）Relative enzyme activity is determined, to be not added with the enzyme activity in the reaction system of any ion as 100%.As a result show, it is and right Photograph ratio, the Fe of low concentration2+、Zn2+And Cu2+There is different degrees of suppression to the vigor of esterase catalyzed p-nitrophenol butyrate Effect.And Mn2+To the influence very little of esterase Est18 enzyme activities, low concentration Ca2+、Mg2+、Ni2+And Co2+To esterase Est18 enzyme activity Power has enhancing effect（Fig. 5）.
The measure to Est18 activity influences such as organic solvent and denaturant.15% is separately added into reaction system（v/v） With 30%（v/v）Organic solvent（Methanol, ethanol, isopropanol, acetone and dimethyl sulfoxide (DMSO) DMSO）, 1% denaturant（Dodecyl sulphur Sour sodium SDS, polysorbas20）And chelating agent（10% EDTA）It is incubated after 20 min, by 4.3 assay methods（With p-nitrophenol butyric acid Ester is used as substrate）Relative enzyme activity is determined, using the enzyme activity in primitive reaction system as 100%.As a result show, survey organic solvent There is different degrees of suppression to Est18 activity, and with the increase of organic solvent concentration, inhibition level enhancing.Denaturant pair The suppression of enzyme activity is that strongly, the SDS of addition 1% is incubated after 20 min only remaining 0.14% activity.But Est18 In chelating agent EDTA（122%）In the presence of enzymatic activity have larger increase（Fig. 6）.
SEQUENCE LISTING
<110>Wuhan Polytechnic University
<120>A kind of low temperature esterase, encoding gene and its application from grand genome
<160> 2
<210> SEQ ID NO.1
<211> 957
<212> DNA
<213> environmental DNA
<400> SEQ ID NO.1
atgagcacgc tcgatccgaa actgttcagc gataaagcga ttgccccgga aacgcgcgcg 60
gtgaacgatg ccattatcgc cgcgatgacc ggcatgcctg aatggtggga tgtcggcgcg 120
gccaagacgc gcgcggcgcg cgccaggggc gaaggcattt tcccagcggc ggcgaaatcc 180
gaacgcgcgc gctggatcga gatagatggg cccgccggca aggtgccgtt gcgcgtcatc 240
gcgccggaaa atccgcgcgg cgtatatctg cacattcacg gcggcggcca cgtgttgggc 300
gccgcggatc aacaggatcg gttgctcgag cgcatctcaa acgaaaccag gctgacggcg 360
atcagcgttg agtatcgctt ggcgccggaa aatccctatc ccgctgggcc cgacgattgt 420
gaagcggcgg cgctttgggt gaacgagcat gcggcggatt ttggcggcca caagattgcc 480
atcggcggcg aaagcgcggg cgcgcacctg tcagcgctga ctattctgcg cttgcgcgac 540
aagcatgggc tgacgccctt caatgcggcg aaccttgtgt tcggcgtttt cgatttggga 600
atgacgccaa gtgcgcgcgc gtttggcgat gagcggctgg tcttgcgcac gcgcgacatc 660
gagaagttcg gcgaggcgtt cttgcccggt acggacgacg agcagaagcg cgcgccggaa 720
ttctcgccgc tctacgcgaa cctcgccggc ttgtgccccg cgctcttcac catcggcacg 780
cgcgatgcgc tcctggacga cagtctcttc atgcacgcgc gctgggtggc ggcgggaaat 840
gtcggcgaac tcgacatcta tccgggcggc tgtcatggct tcatcgcctt cccgtacccg 900
caggcgttcg cgtcgatcgc gcgccaagcc acgtttttga acgcggccct gggttag 957
<210> SEQ ID NO.2
<211> 318
<212> PRT
<213> environmental DNA
<400> SEQ ID NO.2
Met Ser Thr Leu Asp Pro Lys Leu Phe Ser Asp Lys Ala Ile Ala Pro
1 5 10 15
Glu Thr Arg Ala Val Asn Asp Ala Ile Ile Ala Ala Met Thr Gly Met
20 25 30
Pro Glu Trp Trp Asp Val Gly Ala Ala Lys Thr Arg Ala Ala Arg Ala
35 40 45
Arg Gly Glu Gly Ile Phe Pro Ala Ala Ala Lys Ser Glu Arg Ala Arg
50 55 60
Trp Ile Glu Ile Asp Gly Pro Ala Gly Lys Val Pro Leu Arg Val Ile
65 70 75 80
Ala Pro Glu Asn Pro Arg Gly Val Tyr Leu His Ile His Gly Gly Gly
85 90 95
His Val Leu Gly Ala Ala Asp Gln Gln Asp Arg Leu Leu Glu Arg Ile
100 105 110
Ser Asn Glu Thr Arg Leu Thr Ala Ile Ser Val Glu Tyr Arg Leu Ala
115 120 125
Pro Glu Asn Pro Tyr Pro Ala Gly Pro Asp Asp Cys Glu Ala Ala Ala
130 135 140
Leu Trp Val Asn Glu His Ala Ala Asp Phe Gly Gly His Lys Ile Ala
145 150 155 160
Ile Gly Gly Glu Ser Ala Gly Ala His Leu Ser Ala Leu Thr Ile Leu
165 170 175
Arg Leu Arg Asp Lys His Gly Leu Thr Pro Phe Asn Ala Ala Asn Leu
180 185 190
Val Phe Gly Val Phe Asp Leu Gly Met Thr Pro Ser Ala Arg Ala Phe
195 200 205
Gly Asp Glu Arg Leu Val Leu Arg Thr Arg Asp Ile Glu Lys Phe Gly
210 215 220
Glu Ala Phe Leu Pro Gly Thr Asp Asp Glu Gln Lys Arg Ala Pro Glu
225 230 235 240
Phe Ser Pro Leu Tyr Ala Asn Leu Ala Gly Leu Cys Pro Ala Leu Phe
245 250 255
Thr Ile Gly Thr Arg Asp Ala Leu Leu Asp Asp Ser Leu Phe Met His
260 265 270
Ala Arg Trp Val Ala Ala Gly Asn Val Gly Glu Leu Asp Ile Tyr Pro
275 280 285
Gly Gly Cys His Gly Phe Ile Ala Phe Pro Tyr Pro Gln Ala Phe Ala
290 295 300
Ser Ile Ala Arg Gln Ala Thr Phe Leu Asn Ala Ala Leu Gly
305 310 315

Claims (9)

1. a kind of low-temperature alkali esterase Est18, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of low-temperature alkali esterase geneest18, the low-temperature alkali esterase Est18 described in claim 1 is encoded, its feature exists In corresponding nucleotide sequence is as shown in SEQ ID NO.1.
3. carry the low-temperature alkali esterase gene described in claim 2est18Recombinant vector, it is characterised in that described table It is pET-30b (+) up to carrier.
4. comprising low-temperature alkali esterase gene described in Claims 2 or 3est18Recombinant bacterial strain.
5. recombinant bacterial strain according to claim 4, it is characterised in that the bacterial strain is bacterium, yeast or zooblast.
6. a kind of ETEC for including Est18 low-temperature alkali esterase encoding genes（Escherichia coli）, it is protected It is CCTCC M 2017317 to hide numbering.
7. a kind of method for preparing low-temperature alkali esterase Est18, it is characterised in that comprise the following steps：
Host cell is converted with recombinant vector described in claim 3, recombinant bacterial strain is obtained；
Cultivate recombinant bacterial strain, induction Recombinant esterase expression；
Reclaim and purify expressed esterase Est18.
8. low-temperature alkali esterase Est18 as claimed in claim 1 answering in catalysis ester-type hydrolysis, esterification or transesterification With.
9. application according to claim 8, it is characterised in that described esters are short chain esters.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2883874A1 (en) * 2005-04-04 2006-10-06 Substipharm Lab Preparation of perhydroindole compounds comprises enantiomeric resolution of an ester by enzymatic hydrolysis in presence of protease, isolation ester/acid, saponification or hydrolysis of ester to an acid, reduction of acid
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
CN103436506A (en) * 2013-09-18 2013-12-11 云南师范大学 Alkaline thermal-stable esterase K91 Est8 and gene thereof
CN104894081A (en) * 2015-04-15 2015-09-09 云南师范大学 Alkaline thermal-stability SGNH family esterase EstD1 and gene thereof
CN105176943A (en) * 2015-10-13 2015-12-23 福州大学 Salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3 and gene and application thereof
CN105368802A (en) * 2015-12-09 2016-03-02 广东轻工职业技术学院 Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2883874A1 (en) * 2005-04-04 2006-10-06 Substipharm Lab Preparation of perhydroindole compounds comprises enantiomeric resolution of an ester by enzymatic hydrolysis in presence of protease, isolation ester/acid, saponification or hydrolysis of ester to an acid, reduction of acid
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
CN103436506A (en) * 2013-09-18 2013-12-11 云南师范大学 Alkaline thermal-stable esterase K91 Est8 and gene thereof
CN104894081A (en) * 2015-04-15 2015-09-09 云南师范大学 Alkaline thermal-stability SGNH family esterase EstD1 and gene thereof
CN105176943A (en) * 2015-10-13 2015-12-23 福州大学 Salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3 and gene and application thereof
CN105368802A (en) * 2015-12-09 2016-03-02 广东轻工职业技术学院 Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase

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