CN105176943A - Salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3 and gene and application thereof - Google Patents
Salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3 and gene and application thereof Download PDFInfo
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- CN105176943A CN105176943A CN201510668911.6A CN201510668911A CN105176943A CN 105176943 A CN105176943 A CN 105176943A CN 201510668911 A CN201510668911 A CN 201510668911A CN 105176943 A CN105176943 A CN 105176943A
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
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Classifications
-
- 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
- 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
- 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)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of genetic engineering, and provides a salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3, a genome coding gene estSL3 and a recombinant vector and a recombination strain comprising the gene, the esterase EstSL3 comes from halophilic and basophilic bacteria, the amino acid sequence of the esterase EstSL3 is shown as SEQ ID NO.1, and the nucleotide sequence of the genome coding gene estSL3 of the esterase is shown as SEQ ID NO.2. The esterase EstSL3 is adapted to pH9.0 and the reaction temperature of 30DEG C best, and the esterase EstSL3 still has about 70 percent of enzyme activity under 0DEG C, which indicates that the enzyme is a low-temperature esterase. Under the existence of 0.5 to 4.0M NaCl, more than 98 percent of enzyme activity remains, and moreover, under the concentration of 4M and 5M NaCl, the esterase EstSL3 is highly stable. The esterase disclosed by the invention has the characteristics of low temperature, alkalinity, salt tolerance, detergent tolerance and organic solvent tolerance, and can be applied in industrial fields such as fine chemical, pharmacy, detergents and food.
Description
Technical field
The present invention relates to genetically engineered field, particularly, the present invention relates to a kind of low-temperature alkali esterase EstSL3 of salt tolerant organic solvent-resistant and gene thereof and application.
Background technology
Esterase (EsteraseE.C.3.1.1.1) is the general name of the enzyme of a kind of catalysis ester linkage hydrolyzing and synthesis.During hydrolysis, catalysis ester bond produces alcohols and carboxylic acid, and the carboxyl of dehydrating condensation acid during synthesis and the hydroxyl of alcohol form ester and other spice products.Have greater catalytic specificity to substrate in the catalytic reaction process of esterase, its reaction conditions is gentle, by product is few and do not need coenzyme, has very high using value in industrial production and environmental improvement.
Produce esterase biological in occurring in nature distribution widely, from vegeto-animal tissue in the microorganism of varying environment all ubiquities.Microorganism esterase has that source is wide, kind is many, reaction conditions is gentle, good stability, substrate are single-minded, catalytic activity high, is the main source of industrial esterase.Be mainly derived from fungi from classification, comprise mould, monascus, aspergillus niger etc.; Secondly bacterium, comprises bacillus, Burkholderia etc.; The yeast of individual species and actinomyces also can produce some esterase.Some of them have industrial production be worth oneself used, as come from aspergillus niger (
aspergillus), head mold (
rhizopusarrhizus), mucormycosis (
mucorsp.), root Mucor (
rhizomucorsp.), Pseudomonas fluorescens (
pseudomanasfluoreseens), candiyeast (
candidathe esterase of microorganism such as sp.).
Microorganism esterase has important using value in food-processing, washing composition, chemical products, environmental protection etc.Such as, in food-processing, esterase can be applicable to the local flavor improving beer, white wine and other drinks, saccharomycetic esterase and Transacetylase is used in producing at beer technology, under both actings in conjunction, produce acid isobutyl ester pentyl ester, the local flavor of beer can be promoted.In fine chemical product, the conversion of its ester and building-up reactions can prepare the household chemicals with economic benefit, the synthesis of the bio-surfactant that esterase catalyzed sugar ester etc. are important.And chiral drug production aspect, main using acyl transfer and hydrolytic action synthetic drugs.
The microorganism in extreme environment source can produce some extreme enzymes, and these enzymes under extreme conditions have very high enzymic activity, therefore, it is possible to adapt to exacting terms in industrial production and have huge application prospect.What present research was more is Zimadzhunt L 340, basophilic enzyme, addicted to sour enzyme, addicted to cold enzyme, addicted to salt enzyme etc.Wherein under cryogenic there is high reactivity addicted to cold enzyme due to it and reaction process easily controls and is subject to increasing research.In addition, the enzyme that can adapt to multiple extreme condition also receives increasing concern.
The esterase great majority obtained at present derive from conventional environment, and optimum temperuture is at about 40 DEG C, and optimal pH is neutral meta-alkalescence, cannot apply in a lot of industry.Salt alkali lake is a natural stable alkaline environment, and pH, between 9-11, reaches about 12 sometimes, usually has medium-altitude salt simultaneously and exists therefore.Exist a large amount of addicted to saline and alkaline microorganism in salt alkali lake.And from these be separated addicted to obtain saline and alkaline microorganism there is industrial value alkaline enzyme as Sumizyme MP, alkali cellulose enzyme, alkali starch enzyme etc.
The present invention be separated from salt alkali lake bacterial strain that a strain can produce low-temperature alkali salt tolerant esterase addicted to salt basophilic bacterium (
alkalibacteriumsp.SL3), utilize molecular biology and engineered method to obtain the encoding gene of this low temperature Saline alkali tolerance esterase, and carry out this gene recombinant expressed, recombinase has low temperature (optimum temperuture 30 DEG C, 0 DEG C also have about 70% enzyme live), alkalescence (optimal pH 9.0).This enzyme also has tolerance and the stability (5MNaCl process 2h also remains the enzyme work of 88%) of good salt.In addition, this enzyme to some metal ions, common are machine solvent and washing composition has good resistance.Esterase EstSL3 has the feature of low temperature, alkalescence, salt tolerant, wash resistant agent and organic solvent-resistant, can be applicable to the industrial circles such as fine chemistry industry, pharmacy, washing composition and food.
Summary of the invention
The low-temperature alkali esterase EstSL3 of a kind of salt tolerant organic solvent-resistant of the present invention and gene thereof and application, first technical problem to be solved overcomes the deficiencies in the prior art, there is provided a kind of good properties, be suitable in fine chemistry industry, pharmacy, the new esterase of washing composition and Applications in Food Industry.The present invention obtains a kind of esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant, has very high enzyme and live under low-temperature alkali and high salt condition, and this enzyme common are machine solvent, chemical reagent and washing composition to some and has good resistance.These character meet foodstuffs industry, fine chemistry industry and pharmaceutical industry requirement, reduce energy consumption, increase efficiency of pcr product, optimization production process.
For achieving the above object, the present invention adopts following technical scheme:
The invention provides a kind of low-temperature alkali salt tolerant organic solvent-resistant esterase, its aminoacid sequence is as shown in SEQIDNO.1.
The invention provides the above-mentioned low-temperature alkali salt tolerant organic solvent-resistant esterase of coding
estSL3gene.Particularly, the gene order of this gene is as shown in SEQIDNO.2.
Present invention also offers and comprise above-mentioned low-temperature alkali salt tolerant organic solvent-resistant esterase
estSL3recombinant vectors, be preferably
pET28a-estSL3.
Present invention also offers the recombinant bacterial strain of the esterase EstSL3 comprising above-mentioned low-temperature alkali salt tolerant organic solvent-resistant, preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus and filamentous fungus.
Present invention also offers a kind of method preparing low-temperature alkali salt tolerant organic solvent-resistant esterase EstSL3, comprise the following steps:
1) with above-mentioned recombinant vectors transformed host cell, recombinant bacterial strain is obtained;
2) cultivate recombinant bacterial strain, induction Recombinant esterase is expressed; And
3) the esterase EstSL3 also expressed by purifying is reclaimed.
Present invention also offers the application of above-mentioned low-temperature alkali salt tolerant organic solvent-resistant esterase EstSL3.
The present invention's technical problem first to be solved overcomes the deficiencies in the prior art, provide a kind of good properties, be suitable in fine chemistry industry, pharmacy, the new esterase of washing composition and Applications in Food Industry.The present invention obtains a kind of esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant, has very high enzyme and live under low-temperature alkali and high salt condition, and this enzyme common are machine solvent, chemical reagent and washing composition to some and has good resistance.These character meet foodstuffs industry, fine chemistry industry and pharmaceutical industry requirement, reduce energy consumption, increase efficiency of pcr product, optimization production process.
The esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant of the present invention can obtain that arrogant cloth revives that saline and alkaline lakebed mud is separated addicted to salt basophilic bacterium (
alkalibacteriumsp.), its aminoacid sequence is as shown in SEQIDNO.1:
MKINKNDTLLFIGDSITDVGRDRMDGEDLGKGFPLMVASHLQSRYPAKRLTVLNRGIGGDSLKDLKRRWEDDCLITNPDIVTLLIGVNDTWRNQNDGVELTDEELDEFESDYRFLLKSLHQRTDARVILMESFVLPYPKRRVGWRNDLDKRIQIVRKMARDYQTELIPLDGLLNAAGIRDGFSYYTGDDGVHPTVAGHGLIANSWLKAVDE。
Esterase EstSL3 of the present invention is altogether containing 211 amino acid, and N end is without the signal peptide of prediction, and theoretical molecular is 24.04kDa, and theoretical iso-electric point is 5.28.
The optimal pH of esterase EstSL3 of the present invention is the apparent optimal pH of 9.0, EstSL3 is 9.0, sharply declines, when pH5.0, substantially can't detect enzymic activity when pH is less than 6.0 enzymic activitys.This esterase is all very stable between pH6.0-10.0, and process 60min within the scope of this pH after, residual enzyme is active more than 80%, and this illustrates that this enzyme has good pH stability.
The optimum temperuture of esterase EstSL3 of the present invention is 30 DEG C, and the enzyme remaining about 70% 0 DEG C time is lived.Stable at 50 DEG C, still can keep the activity of more than 90% after process 60min.Unstable at 60 DEG C, the transformation period is less than 5min.
Esterase EstSL3 of the present invention also has tolerance and the stability of good salt.When 0.5 – 4.0MNaCl exists, the enzyme of residue more than 98% is lived.Under the concentration of 4M and 5MNaCl, process 60min, the enzyme of residue more than 88% is lived.Show that this esterase has extraordinary tolerance and stability to salt.
Li
+, Na
+, K
+, Mg
2+, Ca
2+, EDTA and β-mercaptoethanol all can promote that when lower concentration and high density enzyme is lived; Co
2+, Zn
2+, Mn
2+and Cu
2+partial inhibition is had to enzyme; Ag
+, Fe
2+, Hg
2+and Pb
2++strongly inhibited is had to enzyme activity.
Tween80 and TritonX-100 of lower concentration has promoter action to EstSL3, and wherein 1%Tween80 promoter action is the most obvious, can reach about 131%; And have partial inhibition to EstSL3 under high density.Tween20 and SDS of lower concentration has partial inhibition to enzyme; And have strong restraining effect to enzyme under high density.
The ethanol of 10% and 20%, propyl alcohol, normal hexane are lived on enzyme affect little, and enzyme work still can remain on more than 90%.Methyl alcohol, glycerine, acetonitrile are then lived to enzyme and are had slight restraining effect.The butanols of lower concentration, isopropylcarbinol, acetone, chloroform are little on enzyme impact of living, and when concentration is 20% have obvious restraining effect, enzyme work is less than 50%.
Present invention also offers the gene of the esterase EstSL3 of above-mentioned low-temperature alkali salt tolerant organic solvent-resistant of encoding
estSL3, this gene order (its accession number be KT225466) in NCBI as shown in SEQIDNO.2:
ATGAAAATCAACAAGAATGATACGTTGCTGTTCATCGGTGACAGCATAACAGATGTAGGCCGTGACCGTATGGATGGCGAAGATTTAGGTAAGGGATTCCCATTGATGGTCGCTTCGCACCTTCAGTCACGCTATCCGGCGAAAAGGCTGACGGTTTTGAATCGGGGCATTGGCGGGGACAGTCTGAAAGATCTTAAAAGGCGATGGGAAGATGATTGCCTGATCACTAATCCTGACATCGTCACATTACTTATAGGTGTAAATGACACCTGGCGTAATCAGAATGACGGAGTAGAACTTACAGATGAGGAACTGGATGAGTTTGAATCAGACTACCGCTTTCTTCTGAAATCGCTTCACCAGAGAACGGATGCTCGAGTCATTTTAATGGAGTCTTTTGTACTGCCTTATCCGAAAAGAAGAGTGGGCTGGAGGAATGACCTGGATAAACGAATCCAGATCGTCAGGAAGATGGCCAGAGATTATCAGACTGAACTCATTCCGCTGGATGGACTTTTGAATGCCGCTGGAATAAGGGACGGGTTCAGCTATTACACAGGAGATGACGGCGTCCATCCGACTGTAGCCGGTCACGGACTAATCGCGAACAGCTGGCTGAAAGCTGTCGATGAATAA。
The present invention passes through this esterase gene of the method separating clone of PCR
estSL3, DNA complete sequence analysis result shows, esterase EstSL3 structure gene
estSL3total length 636bp, initiator codon is ATG, and terminator codon is TAA, GC content 47.2%, and the polypeptide (EstSL3) of 211 amino acid compositions of encoding, N end is without the signal peptide sequence of prediction, and centre is the catalyst structure domain of a SGNH family.Comparison result in GenBank shows it and comes from
alkalibacteriumthe highest consistence of paroling esterase not doing function of sp.AK22 (WP_034300718) is 69%, shows that EstSL3 is a new esterase.
Present invention also offers and comprise above-mentioned esterase gene
estSL3recombinant vectors, be preferably
pET28a-estSL3.By esterase gene of the present invention
estSL3be inserted between the suitable restriction enzyme site of expression vector, make that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably esterase gene
estSL3be inserted on plasmid pET28a
ncoIwith
hindIIIbetween restriction enzyme site, obtain recombinant expression plasmid
pET28a-estSL3.
Present invention also offers and comprise above-mentioned esterase gene
estSL3recombinant bacterial strain, preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus and filamentous fungus, be preferably recombinant bacterial strain BL21 (DE3)/
estSL3.
The invention has the advantages that: the invention provides a kind of good properties, be suitable for the new esterase applied in the industry such as food, fine chemistry industry and pharmacy.Usually carry out in organic solvent in fine chemistry industry and pharmacy procedure.Esterase optimal reactive temperature provided by the present invention is 30 DEG C, and the thermostability of more than 50 DEG C enzymes is very poor, is convenient to enzymatic reaction and controls.In addition, this enzyme has good tolerance to the machine solvent of common are, and can meet the industrial requirement to multiple different solvents.This esterase also has good pH tolerance, highly stable in pH6.0 ~ 10.0.In addition, this enzyme also has good salt tolerance and stability.Therefore the esterase of low-temperature alkali salt tolerant wash resistant agent of the present invention and organic solvent-resistant can be applied in fine chemistry industry, pharmaceutical industry, can reduce production temperature, reduces energy consumption; Reduce nonspecific reaction, increase object efficiency of pcr product.Because this enzyme has very high enzyme work and the well tolerable property to washing composition under cryogenic, therefore this enzyme can also be applied to detergent industry.In addition, this enzyme can also be used for foodstuffs industry, improves the local flavor etc. of drinks and liquid foodstuff.
Accompanying drawing explanation
Fig. 1: analyze at the SDS-PAGE of the Recombinant esterase EstSL3 of expression in escherichia coli.Wherein, M: low molecular weight protein Marker; 1: that does not induce contains esterase gene carrier pET-
estSL3e. coli culture supernatant liquid; 2: induction containing esterase gene carrier pET-
estSL3e. coli culture supernatant concentrated liquor; 3: reach electrophoretically pure EstSL3 albumen by ni-sepharose purification.
Fig. 2: the optimal pH of Recombinant esterase EstSL3.
The pH stability of Fig. 3: Recombinant esterase EstSL3.
Fig. 4: the optimum temperuture of Recombinant esterase EstSL3.
Fig. 5: the thermostability of Recombinant esterase EstSL3.
The salt tolerance of Fig. 6: Recombinant esterase EstSL3.
Fig. 7: the salt-stable of Recombinant esterase EstSL3.
Embodiment
Test materials and reagent
1, bacterial strain and carrier: intestinal bacteria (Escherichiacoli) Trans I-T1 and BL21 (DE3) is purchased from Beijing TransGen company, carrier pET-28a (+) and pMD19-T carrier are purchased from Novagen company of the U.S. and Japanese TaKaRa company respectively.
2, enzyme and other biochemical reagents: restriction enzyme, T4DNA ligase enzyme, archaeal dna polymerase number and dNTPs are purchased from Japanese TaKaRa company.Genome extracts test kit and is purchased from Beijing Tiangen company, and purifying and plasmid extraction kit are purchased from OMEGA company of the U.S..P-NP (pNP), p-nitrophenyl ester class (pNP-esters) are purchased from Sigma Co., USA; Peptone (Tryptone), yeast extract (YeastExtract) for Britain OXOID Products, all the other reagent be domestic analytical pure.
3, substratum:
(1) LB substratum (g/l): yeast powder 5.0, peptone 10.0, NaCl10.0, adjusts pH to 9.6 with the sodium carbonate of 1M.
(2) LB solid medium (g/l): yeast powder 5.0, peptone 10.0, NaCl10.0, agar 15.0, adjusts pH to 9.6 with the sodium carbonate of 1M.
Illustrate: in following examples, do not make the experimental methods of molecular biology illustrated, all carry out with reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book, or carry out according to test kit and product description.
embodiment 1: esterase gene
estSL3clone
Extraction addicted to salt basophilic bacterium genomic dna: adopt the bacterial genomes of Tian Gen company to extract test kit (DP302) and extract genomic dna, concrete operations are carried out according to the specification sheets of this test kit completely.
Time we clone xylanase gene from this bacterium, obtain 3 ' end of this esterase gene, sequence alignment analysis shows that the similarity of this gene is also lower, and all do not do character, therefore according to the 3 ' nucleotide sequence held of the esterase gene obtained that checks order, design three, upstream TAIL-PCR Auele Specific Primer: design direction is the zone of ignorance direction needing amplification, and the Position Design of sp2 is in the inner side of sp1, and sp3 is positioned at the inner side of sp2.Distance between every two primers does not have strict regulation (for convenience of electrophoresis recognition result, the general spacing 100bp of sp2 and sp3), and the general 22 ~ 30nt of primer length, annealing temperature is at 62 DEG C.And by they difference called after estSL3-uSP1, estSL3-uSP2, estSL3-uSP3 are in table 1.According to the program in TAIL-PCR, 5 ' end flanking sequence is increased.
Primer needed for table 1 esterase EstSL3TAIL-PCR increases and total length increases
Obtained the flanking sequence of known sequence by TAIL-PCR, amplification obtains sending handsome company to check order after product reclaims.Obtained the upstream and downstream flanking sequence of this fragment by sequence assembly, complete sequence is long 1.17kb altogether, finds the open reading frame (ORF) that complete.Esterase gene
estSL3(GenBankaccessionno.KT225466) by 636 based compositions, encode 211 amino acid and a terminator codon, the comparison result in GenBank shows it and derives from
alkalibacteriumthe imaginary esterase gene complete sequence similarity of sp.AK22 (WP_034300718) is 69%, and does not do functional study.
estSL3proteins encoded estimated molecular weight is 24.04kDa, and iso-electric point is 5.28.Through prediction, EstSL3 no signal peptide sequence, centre is the catalyst structure domain of a SGNH family.
the preparation of embodiment 2 Recombinant esterase.
To introduce respectively at gene 5 ' and 3 ' end
ncoIwith
hindIIIrestriction enzyme site
estSL3-m-f and
estSL3-m-r is primer pair (see table one), is template, carries out pcr amplification addicted to salt basophilic bacterium genomic dna.PCR reaction parameter is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30sec, 51 DEG C of annealing 30sec, 72 DEG C extend 1min, 30 circulations, 72 DEG C of insulation 5min.Expression vector pET28a (+) is carried out double digestion (
ncoI+
hindIII), simultaneously by the gene of coding esterase
estSL3double digestion (
ncoI+
hindIII), the gene fragment of the encoding mature esterase cut is connected with expression vector pET28a (+), obtains containing esterase gene
estSL3recombinant plasmid pET28a-
estSL3and transformation of E. coli BL21 (DE3), obtain recombinant escherichia coli strain BL21/
estSL3.
Get containing recombinant plasmid pET28a-
estSL3bL21 bacterial strain, be inoculated in 5mLLB (penbritins of 100 μ g/mL) nutrient solution, 37 DEG C of overnight incubation.Cultured bacterium liquid is inoculated in 20mLLB (being added with the penbritin of 100 μ g/mL) by 1%, add the inductor IPTG of final concentration 0.5mmol/L after 37 DEG C of shaking culture about 2 ~ 3h (OD600 reaches 0.5), 12h is cultivated in 30 DEG C of 180rpm concussions.The centrifugal 5min of 12000rpm, collects thalline and carries out ultrasonic disruption.Utilize the vigor of p-nitrophenol colorimetric method for determining esterase enzyme activity determination esterase.SDS-PAGE result (Fig. 1) shows, Recombinant esterase obtains expression in intestinal bacteria.Expressed esterase is after purifying, and the content of its protein reaches more than 95% of total protein.
the property testing of embodiment 3 Recombinant esterase EstSL3
1, the activation analysis of Recombinant esterase
The determination of activity of Recombinant esterase takes colorimetric method for determining esterase enzyme to live: concrete grammar is as follows: at pH9.0, under 30 DEG C of conditions, the reaction system of 1mL comprises the suitable dilution enzyme liquid of 100 μ L, 20 μ L substrates (10mM), add 880 μ L50mmol/LpH9.0 phosphoric acid buffers, immediately at OD after reaction 10min
404place measures its light absorption value.1 Ge Meihuo unit (U) is defined as per minute under given conditions and discharges enzyme amount needed for 1 μm of ol p-NP.
2, the optimal pH of Recombinant esterase EstSL3 and the mensuration of pH stability
Optimal pH measures: Recombinant esterase EstSL3 good for purifying is carried out enzymatic reaction at 37 DEG C and in the damping fluid of 0.1MpH5.0 – 9.0.The pH Stability Determination of enzyme: damping fluid enzyme liquid being placed in 0.1MpH4.0 – 11.0, processes 1h at 37 DEG C, then at pH9.0 and 30 DEG C, carries out enzymatic reaction, with untreated enzyme liquid in contrast.Damping fluid is: 0.1MMcIlvainebuffer(pH4.0 – 7.0), 0.1MTris-HClbuffer(pH7.0 – 10.0) and 0.1Mglycine-NaOH(pH10.0 – 11.0).Take pNP-C2 as substrate, reaction 10min, measures the vigor of EstSL3.Result shows: the optimal pH of EstSL3 is 9.0, remains more than 60% (Fig. 2) of maximum enzyme activity time between pH7.5 is to 9.0; This esterase is all very stable between pH6.0-10.0, and process 60min within the scope of this pH after, residual enzyme is active more than 80%, and this illustrates that this enzyme has good pH stability (Fig. 3).
3, the optimum temperuture of Recombinant esterase EstSL3 and thermal stability determination
The mensuration of the optimum temperuture of enzyme: in the damping fluid of pH9.0, carries out enzymatic reaction at 0 – 70 DEG C.The thermal stability determination of enzyme: the enzyme liquid of same enzyme amount is placed in 50 DEG C, 55 DEG C and 60 DEG C, after processing 0 – 60min, carries out enzymatic reaction, with untreated enzyme liquid in contrast at pH9.0 and 30 DEG C.Take pNP-C2 as substrate, reaction 10min.Enzyme reaction optimum temperuture measurement result (Fig. 4) shows, its optimum temperuture is 30 DEG C.This esterase also has the enzyme work of about 70% at 0 DEG C, shows that this enzyme is a cold-adapted enzyme.The thermostability test of enzyme shows (Fig. 5), and recombinase stability 50 DEG C time is very good.Be incubated 60min at 55 DEG C, residual enzyme activity is 21.3%, 60 DEG C of process 60min enzymes almost total losses alive.
4, Recombinant esterase EstSL3
v maxand K
mmensuration
Be substrate with the substrate pNP-C2 of different final concentration (0.02,0.05,0.1,0.15,0.2 and 0.3mmol), under optimum condition, measure enzymic activity, calculate corresponding speed of response, utilize the two counting backward technique of Michaelis-Menton equation to try to achieve K
mvalue and V
max.Result shows: this enzyme
v maxbe 769.23 ± 6.55 μm of olmin
– 1mg
– 1, K
mbe 0.15 ± 0.008mgmL
– 1.
5, different metal ion chemistry reagent measures the impact that EstSL3 enzyme is lived
In enzymatic reaction system, add the metal ion of 1 and 5mM, study its impact on enzymic activity.Under 30 DEG C and pH9.0 condition, be that substrate measures enzymic activity with pNP-C2.Result (table 2) shows: Li
+, Na
+, K
+, Mg
2+, Ca
2+, EDTA and β-mercaptoethanol all can promote that when lower concentration and high density enzyme is lived; Co
2+, Zn
2+, Mn
2+and Cu
2+partial inhibition is had to enzyme; Ag
+, Fe
2+, Hg
2+and Pb
2++strongly inhibited is had to enzyme activity.
Table two metal ion and chemical reagent are on the impact of Recombinant esterase EstSL3 activity
6, different washing composition and organic solvent measure the impact that EstSL3 enzyme is lived
Polysorbas20, tween 80, TritonX-100 and SDS of 3 kinds of different three concentration (final volume concentration be 1%, 5%L and 10%) is added respectively, to study its impact on enzymic activity in enzymatic reaction system.Enzymic activity is measured, not add the enzymatic reaction of any reagent under similarity condition for control group under the condition of optimal pH and optimum temperuture.In enzymatic reaction system, add the organic solvent of 11 kinds of different two concentration (final volume concentration is 10% and 20%) respectively, comprising: methyl alcohol, ethanol, propyl alcohol, butanols, isopropylcarbinol, primary isoamyl alcohol, acetone, normal hexane, glycerine, chloroform, acetonitrile.To study its impact on enzymic activity.Enzymic activity is measured, not add the enzymatic reaction of organic solvent under similarity condition for control group under the condition of optimal pH and optimum temperuture.
Result shows (table three), Tween80 and TritonX-100 of lower concentration has promoter action to EstSL3, and wherein 1%Tween80 promoter action is the most obvious, can reach about 131%; And have partial inhibition to EstSL3 under high density.Tween20 and SDS of lower concentration has partial inhibition to enzyme; And have strong restraining effect to enzyme under high density.The ethanol of 10% and 20%, propyl alcohol, normal hexane are lived on enzyme affect little, and enzyme work still can remain on more than 90%.Methyl alcohol, glycerine, acetonitrile are then lived to enzyme and are had slight restraining effect.The butanols of lower concentration, isopropylcarbinol, acetone, chloroform are little on enzyme impact of living, and when concentration is 20% have obvious restraining effect, enzyme work is less than 50%.Wherein primary isoamyl alcohol is the most obvious, and enzyme is lived and is only about 7.3%.
Table three washing composition and organic solvent are on the impact of Recombinant esterase EstSL3 activity
7, different concns NaCl is to the activity of Recombinant esterase EstSL3 and stable mensuration
NaCl is to the activity influence of Recombinant esterase EstSL3: the NaCl adding different concns (0.5-4M) in enzymatic reaction system, studies its impact on enzymic activity.Under 30 DEG C and pH9.0 condition, be that substrate measures enzymic activity with pNP-C2.NaCl is on the impact of the stability of Recombinant esterase EstSL3: damping fluid enzyme liquid being placed in respectively the pH9.0 containing 4M and 5M, at 37 DEG C, process 2h, then at pH9.0 and 30 DEG C, carries out enzymatic reaction, with untreated enzyme liquid in contrast.Result shows: deposit in case at 0.5 – 4.0MNaCl, and this enzyme can remain the enzyme (Fig. 6) alive of more than 98%, shows that this enzyme has good salt tolerance.Under the concentration of 4M and 5MNaCl, process 120min, the enzyme of residue more than 88% is lived (Fig. 7), shows that this enzyme has extraordinary salt-stable.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
SEQUENCELISTING
<110> University of Fuzhou
The low-temperature alkali esterase EstSL3 of a <120> salt tolerant organic solvent-resistant and gene thereof and application
<130>7
<160>7
<170>PatentInversion3.3
<210>1
<211>211
<212>PRT
<213> esterase EstSL3 aminoacid sequence
<400>1
MetLysIleAsnLysAsnAspThrLeuLeuPheIleGlyAspSerIle
151015
ThrAspValGlyArgAspArgMetAspGlyGluAspLeuGlyLysGly
202530
PheProLeuMetValAlaSerHisLeuGlnSerArgTyrProAlaLys
354045
ArgLeuThrValLeuAsnArgGlyIleGlyGlyAspSerLeuLysAsp
505560
LeuLysArgArgTrpGluAspAspCysLeuIleThrAsnProAspIle
65707580
ValThrLeuLeuIleGlyValAsnAspThrTrpArgAsnGlnAsnAsp
859095
GlyValGluLeuThrAspGluGluLeuAspGluPheGluSerAspTyr
100105110
ArgPheLeuLeuLysSerLeuHisGlnArgThrAspAlaArgValIle
115120125
LeuMetGluSerPheValLeuProTyrProLysArgArgValGlyTrp
130135140
ArgAsnAspLeuAspLysArgIleGlnIleValArgLysMetAlaArg
145150155160
AspTyrGlnThrGluLeuIleProLeuAspGlyLeuLeuAsnAlaAla
165170175
GlyIleArgAspGlyPheSerTyrTyrThrGlyAspAspGlyValHis
180185190
ProThrValAlaGlyHisGlyLeuIleAlaAsnSerTrpLeuLysAla
195200205
ValAspGlu
210
<210>2
<211>636
<212>DNA
<213> gene estSL3
<400>2
atgaaaatcaacaagaatgatacgttgctgttcatcggtgacagcataacagatgtaggc60
cgtgaccgtatggatggcgaagatttaggtaagggattcccattgatggtcgcttcgcac120
cttcagtcacgctatccggcgaaaaggctgacggttttgaatcggggcattggcggggac180
agtctgaaagatcttaaaaggcgatgggaagatgattgcctgatcactaatcctgacatc240
gtcacattacttataggtgtaaatgacacctggcgtaatcagaatgacggagtagaactt300
acagatgaggaactggatgagtttgaatcagactaccgctttcttctgaaatcgcttcac360
cagagaacggatgctcgagtcattttaatggagtcttttgtactgccttatccgaaaaga420
agagtgggctggaggaatgacctggataaacgaatccagatcgtcaggaagatggccaga480
gattatcagactgaactcattccgctggatggacttttgaatgccgctggaataagggac540
gggttcagctattacacaggagatgacggcgtccatccgactgtagccggtcacggacta600
atcgcgaacagctggctgaaagctgtcgatgaataa636
<210>3
<211>29
<212>DNA
<213> artificial sequence
<400>3
cagcccactcttcttttcggataaggcag29
<210>4
<211>27
<212>DNA
<213> artificial sequence
<400>4
gactcgagcatccgttctctggtgaag27
<210>5
<211>31
<212>DNA
<213> artificial sequence
<400>5
cctcatctgtaagttctactccgtcattctg31
<210>6
<211>35
<212>DNA
<213> artificial sequence
<400>6
gcgccatgggcatgaaaatcaacaagaatgatacg35
<210>7
<211>29
<212>DNA
<213> artificial sequence
<400>7
ccgaagcttttcatcgacagctttcagcc29
Claims (10)
1. an esterase EstSL3 for low-temperature alkali salt tolerant organic solvent-resistant, is characterized in that, its aminoacid sequence is as shown in SEQIDNO.1.
2. the esterase gene of a low-temperature alkali salt tolerant organic solvent-resistant
estSL3, it is characterized in that, the esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant according to claim 1 of encoding.
3. the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant according to claim 2
estSL3, it is characterized in that, its base sequence is as shown in SEQIDNO.2.
4. comprise the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant described in Claims 2 or 3
estSL3recombinant vectors.
5. comprise the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant described in Claims 2 or 3
estSL3recombinant vectors
pET28a-estSL3.
6. comprise the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant described in Claims 2 or 3
estSL3recombinant bacterial strain.
7. recombinant bacterial strain according to claim 6, is characterized in that, described bacterial strain is intestinal bacteria, yeast, genus bacillus and filamentous fungus.
8. prepare a method of the esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant, it is characterized in that, comprise the following steps:
1) with recombinant vectors transformed host cell described in claim 4, recombinant bacterial strain is obtained;
2) cultivate recombinant bacterial strain, induction Recombinant esterase is expressed;
3) the esterase EstSL3 also expressed by purifying is reclaimed.
9. the application of esterase EstSL3 in fine chemistry industry and pharmacy of low-temperature alkali salt tolerant organic solvent-resistant as claimed in claim 1.
10. the esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant according to claim 9 is for the production of specific chemical products or prodrug; Application in washing composition; Food improves the application in the local flavor of drinks and liquid foodstuff.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107236718A (en) * | 2017-06-16 | 2017-10-10 | 武汉轻工大学 | A kind of low temperature esterase, encoding gene and its application from grand genome |
| CN109355323A (en) * | 2018-11-23 | 2019-02-19 | 福州大学 | A kind of method that low temperature compound lipases improve glycerol ester type omega-fatty acid content in transesterification |
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| 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 |
| CN104561059A (en) * | 2015-01-19 | 2015-04-29 | 山东大学 | Ocean cold-adapted esterase as well as coding gene E40 and application thereof |
| CN104762276A (en) * | 2014-01-08 | 2015-07-08 | 中国科学院微生物研究所 | Esterase protein and encoding gene thereof as well as application of both esterase protein and encoding gene thereof |
| CN104962534A (en) * | 2015-07-31 | 2015-10-07 | 国家海洋局第二海洋研究所 | Abyssal deposit-derived esterase EST22 as well as coding gene and application thereof |
-
2015
- 2015-10-13 CN CN201510668911.6A patent/CN105176943B/en not_active Expired - Fee Related
Patent Citations (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 |
| CN104762276A (en) * | 2014-01-08 | 2015-07-08 | 中国科学院微生物研究所 | Esterase protein and encoding gene thereof as well as application of both esterase protein and encoding gene thereof |
| CN104561059A (en) * | 2015-01-19 | 2015-04-29 | 山东大学 | Ocean cold-adapted esterase as well as coding gene E40 and application thereof |
| CN104962534A (en) * | 2015-07-31 | 2015-10-07 | 国家海洋局第二海洋研究所 | Abyssal deposit-derived esterase EST22 as well as coding gene and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107236718A (en) * | 2017-06-16 | 2017-10-10 | 武汉轻工大学 | A kind of low temperature esterase, encoding gene and its application from grand genome |
| CN107236718B (en) * | 2017-06-16 | 2021-04-13 | 武汉轻工大学 | Low-temperature esterase from metagenome, coding gene and application thereof |
| CN109355323A (en) * | 2018-11-23 | 2019-02-19 | 福州大学 | A kind of method that low temperature compound lipases improve glycerol ester type omega-fatty acid content in transesterification |
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