CN103436506A - Alkaline thermal-stable esterase K91 Est8 and gene thereof - Google Patents
Alkaline thermal-stable esterase K91 Est8 and gene thereof Download PDFInfo
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Abstract
The invention provides an esterase K91 Est8 from Bacillussp.. The invention also provides an encoding gene K91 Est8 of the esterase, and a recombinant vector and a recombination strain thereof. The esterase provided by the invention has the properties of the optimum temperature at 50 DEG C, the optimum pH at 9.0, and the enzyme activity which is kept above 85% and 40% after heat preservation for 60 minutes at 37 DEG C and 50 DEG C, respectively; the esterase is still capable of keeping the activity above 40% after being treated at 37 DEG C for 60 minutes by using borax having the pH of 9.5 and a NaOH buffer solution; the esterase has excellent thermal stability and alkali resistance. Except ethanoic acid-4-nitrobenzene thiocyanate which is the optimum hydrolysis substrate for the esterase, the esterase is further capable of hydrolyzing alpha-naphthyl acetate, beta-naphthyl acetate, 4-nitrobenzene butyrate, 4-nitrophenyl hexanoate and 4-nitrophenyl caprylate; therefore, the esterase can be applied to the aspects of treating pesticide residue or pesticide pollution.
Description
Technical field
The present invention relates to gene engineering technology field, specifically esterase K91 Est8 and the gene thereof in a kind of heat-resistant bacillus source.
Background technology
Esterase (esterase, EC 3.1.1.1) is that a class can the catalysis ester linkage hydrolyzing and the enzyme of formation, be distributed widely in animal, plant and microorganism (Melloney J,
et al., Journal of Molecular Catalysis B:Enzymatic. 2005,32:261-270).The water miscible glyceride type that esterase can be hydrolyzed or the synthesizing acyl chain length is less than 10, and lipase generally to take the glyceryl ester that acyl chain length is more than or equal to 10 carbon atoms be substrate, but most of lipase also can be hydrolyzed the substrate of esterase.Most of esterase belongs to typical α/β lytic enzyme superfamily, and the conserved sequence that contains typical GxSxG, wherein at S(serine) site formed a serine-aspartate-histidine the catalysis triplet configuration (Kakugawa S,
et al., Appl Microbiol Biotechnol. 2007,74:585 – 591).In addition, also having in a class esterase sequence and contain conserved sequence, its conserved sequence that contains unique GDSL that is different from traditional GxSxG, is the esterase of a class new family.The esterase of GDSL family contains 5 sections conserved sequence districts (five consensus sequence blocks I-V), contain 4 more conservative catalytic site Ser, Gly, Asn and His in 5 sections conserved sequence districts, therefore can be divided into again SGNH subfamily esterase (John M
et al., The Journal of Biological Chemistry. 2013,288:2605-2613).Esterase K91 Est8 of the present invention belongs to SGNH subfamily esterase in GDSL esterase family.
Esterase is a kind of important industrial enzyme, in the processes such as, transesterify synthetic in catalysis ester hydrolysis, ester, significant application value is arranged, have good regioselectivity and stereoselectivity (Ramos Tombo,
et al., Agricultural and Biological Chemistry. 1987,51:1833-1838).At present from streptomyces (
streptomycessp.), Rhodopseudomonas (
pseudomonassp.), lactobacillus (
lactobacillussp.), micrococcus sp (
micrococcussp.) etc. be cloned into esterase gene in microorganism, be applied to, in the industry such as food, chemical industry, washing, leather, paper, be mainly used in hydrolysis reaction.In addition, esterase also is applied to the degraded of nitrophenyl phenolic toxic pollutants in the industrial and agricultural wastewaters such as pharmacy, dyestuff, sterilant and sterilant, biological technical field playing the part of extremely important role (Jaeger K.E,
et al., Trends in Biotechnology. 1998,16 (9): 396-403).
Yet natural strain enzyme-producing is low, be difficult to a large amount of production, and production cost is high, having limited it applies, and the esterase be cloned into from microorganism is also still less, be difficult to meet the industry such as food, chemical industry fast-developing, and the demand of continuity of environment, sound development.In addition, general esterase is due to poor stability, and easily inactivation, also limited its extensive utilization.The esterase in thermophilic and thermoduric bacteria source, due to good stability in high temperature and organic solvent, not only can extend the duration of service of esterase, can also enlarge its range of application, has become the focus that biological technology application is paid close attention to.Therefore, the esterase that exploitation has the greater catalytic vigor under hot environment is applied to industrial circle, and good prospect and value are arranged, and also is conducive to study the catalyst mechanism of esterase.
Summary of the invention
The purpose of this invention is to provide the thermally-stabilised esterase K91 of a kind of alkalescence Est8.
A further object of the present invention is to provide the gene of the above-mentioned esterase of coding.
Another object of the present invention is to provide the recombinant vectors that comprises said gene.
Another object of the present invention is to provide the recombinant bacterial strain that comprises said gene.
The present invention is cloned into esterase gene from heat-resistant bacillus K91 genome
k91 Est8, belong to the SGNH-serine lytic enzyme subfamily in GDSL esterase family.
k91 Est8with expression vector pEASY
tMafter-E2 connects, be transformed into the middle abduction delivering of e. coli bl21 (DE3) and its zymologic property has been carried out to preliminary study, for further studying esterase K91 Est8, the degraded of agricultural chemicals having been established to theoretical basis.
Esterase K91 Est8 of the present invention can derive from genus bacillus (
bacillussp.), as
bacillussp. ATCC 31072, and the aminoacid sequence of K91 Est8 is as shown in SEQ ID NO.1.
Esterase K91 Est8 of the present invention contains 217 amino acid altogether, and theoretical molecular is 24.52kDa, and 6 HIS that comprise its C end are histidine-tagged, and the total molecular weight size is about 25.35kDa.With acetic acid-4-nitro phenyl ester (pNPC
2) be substrate: 50 ℃ of optimum temperutures; Optimal pH 9.0; Be incubated 1h under 37 ℃ and 50 ℃, enzyme is lived and is remained on respectively more than 85% and 40%; Process 1h in the scope of pH7.5-pH9.5 after, still can keep the activity more than 40%; There is good alkali tolerance and thermostability.
The invention provides the gene of the above-mentioned esterase of coding
k91 Est8, this gene order is as shown in SEQ ID NO.2.
The present invention has cloned the thermostability esterase gene by PCR method
k91 Est8, its total length 654bp, initiation codon is ATG, termination codon is TGA.Through BLAST comparison, this esterase gene
k91 Est8in the aminoacid sequence and GeneBank of coding
clostridium thermocellumthe esterase (YP_001039529.1) in ATCC27405 source has the highest consistence, is 61%; With
acetivibrio cellulolyticusthe esterase (WP_010243524.1) in source has 55% consistence; With
streptomycessp. the esterase (YP_007863706.1) in PAMC26508 source has 44% consistence.Illustrate that alkaline thermally-stabilised esterase K91 Est8 is a kind of new esterase.
The present invention also provides and has comprised above-mentioned thermostability esterase gene
k91 Est8recombinant vectors, be preferably pEASY
tM-E2-
k91 Est8.By esterase gene of the present invention and pEASY
tM-E2 carries out the T-A connection, and its nucleotide sequence is connected with expression regulation sequence, obtains expression of recombinant e. coli plasmid pEASY
tM-E2-
k91 Est8.
The present invention also provides and has comprised above-mentioned esterase gene
k91 Est8recombinant bacterial strain, preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably recombinant bacterial strain BL21 (DE3)
/ K91 Est8.
The present invention prepares the method for esterase K91 Est8 and carries out according to the following steps:
1) with above-mentioned recombinant vectors transformed host cell, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce Recombinant esterase to express;
3) reclaim the also expressed esterase K91 Est8 of purifying.
Wherein, preferred described host cell is Bacillus coli cells, preferably by expression of recombinant e. coli plasmid transformation escherichia coli cell BL21 (DE3), obtains recombinant bacterial strain BL21 (DE3)
/ K91 Est8.
The invention provides a new esterase gene, the esterase of its coding is with pNPC
2for substrate: 50 ℃ of optimum temperutures; Optimal pH 9.0; Be incubated 1h under 37 ℃ and 50 ℃, enzyme is lived and is remained on respectively more than 85% and 40%; Process 1h in the scope of pH7.5-pH9.5 after, still can keep the activity more than 40%; There is good alkali tolerance and thermostability.In addition, esterase K91 Est8 also can be hydrolyzed α-naphthaleneaceticacidester, β-naphthyl acetate, 4-oil of mirbane butyric ester (pNPC
4), caproic acid 4-nitro phenyl ester (pNPC
6) and 4-nitrophenyl octanoate (pNPC
8) etc.; Can be applicable to pesticide residue or pesticidal contamination improvement aspect.The esterase that has the greater catalytic vigor under hot environment is applied to industrial circle, and good prospect and value are arranged.
The accompanying drawing explanation
Fig. 1: the SDS-PAGE at the Recombinant esterase K91 of expression in escherichia coli Est8 analyzes, wherein, and M: low molecular weight protein Marker; 1:BL21 (DE3)
/ K91 Est8induce the total protein of rear expression; 2: the Recombinant esterase K91 Est8 before purifying; 3: the Recombinant esterase K91 Est8 that contains 6 HIS labels after purifying.
Fig. 2: the optimum temperuture of Recombinant esterase K91 Est8.
Fig. 3: the thermostability of Recombinant esterase K91 Est8.
Fig. 4: the optimal pH of Recombinant esterase K91 Est8.
Fig. 5: the pH stability of Recombinant esterase K91 Est8.
Fig. 6: 1mM metal ion and the part chemical reagent impact on Recombinant esterase K91 Est8 activity.
Fig. 7: 10mM metal ion and the part chemical reagent impact on Recombinant esterase K91 Est8 activity.
Fig. 8: the impact that 1% and 5% final concentration organic solvent is lived on enzyme.
Fig. 9: Recombinant esterase K91 Est8 is with pNPC
2kinetics during for substrate.
Embodiment
Experiment material and reagent
1, bacterial strain and carrier: genus bacillus (
bacillussp.) with document report bacterial classification character, as
bacillussp. ATCC 31072; Intestinal bacteria
escherichia colibL21 (DE3) is purchased from Novagen company; Expression vector pEASY
tM-E2 is purchased from biological limited (TransGen) company of full formula gold.
2, enzyme and other biochemical reagents: restriction enzyme, archaeal dna polymerase and dNTP are purchased from TaKaRa company; The T4 ligase enzyme is purchased from Promega company; Firm blue B, α-naphthaleneaceticacidester, β-naphthyl acetate, 4-oil of mirbane butyric ester (pNPC
4), caproic acid 4-nitro phenyl ester (pNPC
6) and 4-nitrophenyl octanoate (pNPC
8) purchased from Sigma company; Other reagent is all purchased from traditional Chinese medicines group.
3, substratum:
LB substratum: Tryptones 1%, yeast powder 0.5%, sodium-chlor 1%, pH nature (being about 7.0).Solid medium adds 2.0%(w/v on this basis) agar.
Illustrate: do not make the experimental methods of molecular biology illustrated in following examples, all with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book, carry out, or carry out according to test kit and product description.
Embodiment 1: esterase gene
k91 Est8the clone
Adopt a day root bacterial genomes to extract test kit (DP302) and extract genus bacillus K91 genomic dna.
Analyze esterase gene according to genus bacillus K91 genome sequencing and gene annotation
k91 Est8and primer CarF and CarR are expressed in design:
Upstream primer CarF:5 '-GCAAATCATATTTATCTTGC-3 '
Downstream primer CarR:5 '-CCTTTCTTTG ATGATCGATT C-3 '
Upstream primer T7:5 '-TAATACGACTCACTATAGGG-3 '
Downstream primer T7 ter:5 '-TGCTAGTTATTGCTCAGCGG-3 '
T7 and T7 ter are that PCR detects the sub-primer of positive colony.
The heat-resistant bacillus K91 genomic dna of take carries out pcr amplification as template, and the PCR reaction parameter is: 95 ℃ of denaturation 5 min; Then 95 ℃ of sex change 30 sec; 57 ℃ of annealing (0.5 ℃ of each cycle down), 30 sec; 72 ℃ are extended 1 min; 30 rear 95 ℃ of sex change 30 sec of circulation; 43 ℃ of annealing 30sec; 72 ℃ are extended 1 min; 7 rear 72 ℃ of insulation 10min of circulation.Get 4 μ L PCR products and 1 μ L expression vector pEASY
tM-E2 carries out the T-A connection, and 25 ℃ connect 10min, connects product and all proceeds in the Trans-I competent cell, 37 ℃ of incubator incubated overnight.The single bacterium colony of several strains of picking reformer plate, (this upstream region of gene primer T7ter) carries out positive colony that colony PCR amplification screening forward connects to use primer.Bacterium colony PCR is verified to correct clone's adopts T7 and the order-checking of T7 ter primer, is completed by Beijing Hua Da gene sequencing.Plasmid pEASY is extracted in the positive colony inoculation of checking order correct
tM-E2-
k91 Est8, get 0.01uL pEASY
tM-E2-
k91 Est8plasmid Transformation is expressed in competent cell to 100 uL BL21 (DE3), coats containing Amp
rthe LB solid plate on, 37 ℃ of incubator incubated overnight, reformer plate, the single bacterium colony of several strains of picking, carry out colony PCR amplification screening positive clone, thereby obtain recombinant escherichia coli strain BL21 (DE3)
/ K91 Est8.
Embodiment 2: the preparation of Recombinant esterase K91 Est8
Get and contain recombinant plasmid pEASY
tM-E2-
k91 Est8bL21 (DE3) bacterial strain and only contain pEASY
tMthe empty plasmid of-E2
bL21 (DE3)bacterial strain, the inoculum size with 0.1% is inoculated in LB(containing 100 ug/mL Amp
r) in nutrient solution, 37 ℃ of quick oscillation 16 h.Then the bacterium liquid of this activation is inoculated into to fresh LB(containing 100ug/mL Amp with 1% inoculum size
r) in nutrient solution, quick oscillation is cultivated approximately 2 – 3 h(OD
600reach 0.4-0.7) after, add the IPTG of final concentration 0.7 mM to be induced, continue approximately 20 h of shaking culture in 20 ℃.Centrifugal 10 min of 12000 rpm, collect thalline.With appropriate pH 7.0 citric acids-Na
2hPO
4after damping fluid suspension thalline, ultrasonic disruption thalline under the low temperature water-bath., draw supernatant and purify target protein with Nickel-NTA Agarose after centrifugal 10 min of 12,000 rpm with crude enzyme liquid concentrated in upper eye lid.SDS-PAGE result (Fig. 1) shows, Recombinant esterase has obtained expression in intestinal bacteria, after Nickel-NTA Agarose purifying, is single band.
Embodiment 3: the property testing of the Recombinant esterase K91 Est8 of purifying
1, the activation analysis of Recombinant esterase K91 Est8
The Recombinant esterase K91 Est8 employing para-nitrophenol method of purifying (
p-nitrophenol) carry out determination of activity: get 4 1.5mL centrifuge tubes, number respectively 1,2,3,4, wherein 1,2, No. 3 test tube is experimental group (3 repetition), is for No. 4 control group.To the 50mM Tris-HCL damping fluid and the 30 μ L 10mM substrate pNPC that add respectively 420 μ L pH8.0 in four centrifuge tubes
2, after 37 ℃ of preheating 5min, every 10s, respectively to the enzyme liquid that adds 50 μ L dilution suitable multiple in 1,2 and No. 3 pipe, No. 4 control tube add and wait water gaging, and 37 ℃ add 50 μ L 0.1M Na every 10s after reacting 5min in 1,2,3 experiment tubes
2cO
3termination reaction, No. 4 control tube are inserted into 4 centrifuge tubes on ice after adding equally the equal-volume stop buffer immediately, and microplate reader 405nm reads the OD value.1 enzyme unit alive (U/mL) is defined as under certain condition, and per minute decomposes substrate pNPC
2generate the needed enzyme amount of 1 μ moL p-NP.With pNPC
4, pNPC
6, pNPC
8, α-naphthaleneaceticacidester and β-naphthyl acetate be that substrate measuring method and enzyme activity calculate same pNPC
2.
2, the mensuration of the optimum temperuture of Recombinant esterase K91 Est8 and thermostability
The optimum temperuture of enzyme is measured: Recombinant esterase K91 Est8, under the Tris-HCL of 50mM pH9.0 buffer conditions, is carried out respectively to enzymatic reaction under 20 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 95 ℃.The mensuration of temperature stability: by Recombinant esterase K91 Est8 under the Tris-HCL of 50mM pH9.0 buffer conditions, carry out enzymatic reaction be incubated respectively 5min, 10 min, 20 min, 30 min and 60 min at 37 ℃, 50 ℃ and 60 ℃ of three temperature after, with untreated enzyme liquid in contrast.With pNPC
2for substrate, react 5 min, measure the zymologic property of Recombinant esterase K91 Est8.Result shows: 50 ℃ of the optimum temperutures of K91 Est8, but this enzyme also has the activity (Fig. 2) more than 80% in the time of 37 ℃; Under 37 ℃, after insulation 1h, enzymic activity remains on more than 85%, 50 ℃ and 60 ℃ of insulation 1h, and enzyme is lived and is still remained on (Fig. 3) more than 35%.
3, the mensuration of the optimal pH of Recombinant esterase K91 Est8 and pH stability
The mensuration of the optimal pH of enzyme: by esterase K91 Est8 at 37 ℃, respectively at the different pH6.5,7.0,7.5 of 1/15 mol/L Sodium phosphate dibasic-potassium phosphate buffer; The different pH7.5,8.5,8.8,9.0,9.3,9.5 of 50mM Tris-HCL damping fluid; Carry out enzymatic reaction under the different pH9.5,10.0,11.0 of 50mM borax-NaOH damping fluid, 12.0 conditions.The mensuration of pH stability: by Recombinant esterase K91 pH2.0,4.0 for Est8,0.1M citric acid-Na of 5.0
2hPO
4damping fluid; PH6.5,7.0,1/15 mol/L Sodium phosphate dibasic-potassium phosphate buffer of 7.5; PH7.5,8.5,8.8,9.0,9.3,9.5 50mM Tris-HCL damping fluid; Carry out enzymatic reaction after tolerance 60 min respectively under 37 ℃ after pH9.5,10.0,11.0,12.0 50mM borax-NaOH damping fluid dilution suitable multiple, with untreated enzyme liquid in contrast.With pNPC
2for substrate, react 5 min, measure the zymologic property of the Recombinant esterase K91 Est8 of purifying.Result shows: the optimal pH of K91 Est8 is 9.0(Fig. 4); Through pH7.5,8.5,8.8,9.0,9.3,9.5 50mM Tris-HCL damping fluid; Still keep the activity (Fig. 5) more than 40% after processing 60min under pH9.5 borax-37 ℃ of NaOH damping fluids condition, illustrate that Recombinant esterase K91 Est8 has good alkali tolerance.
4, different metal ion and the chemical reagent impact on Recombinant esterase K91 Est8 activity
The metal ion and the chemical reagent that add final concentration 1 mM and 10 mM in enzymatic reaction system, study its impact on enzymic activity.With pNPC
2for substrate, at 50 ℃, under the pH9.0 condition, react 5 min, measure the enzyme activity of the Recombinant esterase K91 Est8 of purifying.Result (Fig. 6) shows, the metal ion Co of final concentration 1 mM
2+, K
1+, Na
1+, Al
3+, Li
2+, Ba
2+, Zn
2+, Ca
2+, Cu
2+, final concentration 1 mM chemical reagent EDTA, CTAB, urea, final concentration 1% the Tween-80 of organic solvent Tween-80, TritonX-100, ethanol, cyclohexane, n-hexane, isopropanol and final concentration 5% enzyme is had to stronger activation; Fe
3+enzyme is had to slight promoter action; The Ni of 1 mM
2+, Mn
2+, Fe
2+, Ag
+, DTT, SDS, the K of final concentration 10 mM
1+, Na
1+, Li
2+, Mg
2+, Ni
2+, SDS, urea, DMSO, the methanol of the DMSO of final concentration 1%, methanol and final concentration 5%, ethanol, cyclohexane, isopropanol have certain restraining effect to enzyme; The Hg of 10 mM
2+, CTAB, DTT and final concentration 5% TritonX-100, n-propanol enzyme is had to stronger restraining effect (Fig. 7); The Mg of 1mM
2+, Hg
2+, urea, the EDTA of 10 mM, 1% n-propanol, 5% n-hexane, n-propanol on enzyme almost without impact (Fig. 8).
5, the mensuration of the kinetic parameter of Recombinant esterase K91 Est8
At 50 ℃, under the pH9.0 condition, with pNPC
2for substrate, termination reaction measure enzymic activity in the 1-10 of enzymatic reaction min, calculate the ratio in enzymic activity and reaction times successively, if this ratio keeps stable within a certain period of time, this time is the first order reaction time.PNPC with 0.1mM-1.2mM
2for substrate ([S]), at 50 ℃, pH9.0 and first order reaction are measured enzyme and are lived under the time, calculate corresponding speed of response (ν), as Fig. 9, according to the Lineweaver-Burk method, measure
kmwith
vmax.After measured, at 50 ℃, under the pH9.0 condition, Recombinant esterase K91 Est8 is to acetic acid-4-nitro phenyl ester (pNPC
2)
kmwith
vmaxbe respectively 0.1882mM and 117.647U/mg.
6, Recombinant esterase K91 Est8 is to substrate pNPC2, pNPC4, pNPC6, pNPC8 hydrolysis
At 50 ℃, under the pH9.0 condition, the pNPC of Recombinant esterase K91 Est8 to 10mM
2, pNPC
4, pNPC
6, pNPC
8the ratio vigor be respectively 45.382U/mg, 1.317U/mg, 0.131U/mg, 0.033U/mg.
Sequence table
SEQ ID NO.1
<110 > Yunnan Normal University
<120 > a kind of alkalescence thermally-stabilised esterase K91 Est8 and gene thereof
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 217
<212> PRT
<213>genus bacillus (
bacillussp.)
<400> 1
Met Ala Asn His Ile Tyr Leu Ala Gly Asp Ser Thr Val Gln Thr Tyr
1 5 10 15
Gly Asp Ser Thr Asn Gln Gly Gly Trp Gly Gln Phe Leu Gly Ser His
20 25 30
Leu Pro Glu His Ile Gln Val Ile Asn Arg Ala Ile Gly Gly Arg Ser
35 40 45
Ser Lys Thr Phe Val Glu Glu Gly Arg Leu Gln Ala Ile Leu Asp Val
50 55 60
Ile Glu Pro Asp Asp Trp Leu Phe Val Gln Met Gly His Asn Asp Ala
65 70 75 80
Ser Lys Asn Lys Pro Glu Arg Tyr Thr Glu Pro Tyr Thr Thr Tyr Lys
85 90 95
Gln Tyr Leu Lys Gln Tyr Ile Ala Gly Ala Arg Glu Lys Gly Ala His
100 105 110
Pro Leu Leu Ile Thr Pro Val Ala Arg Phe His Tyr Glu Asn Gly Val
115 120 125
Phe Leu Asn Asp Phe Pro Asp Tyr Cys Ile Ala Met Lys Gln Thr Ala
130 135 140
Glu Glu Glu Asn Val Gln Leu Ile Asp Leu Met Glu Lys Ser Leu Ala
145 150 155 160
Phe Phe Thr Glu Lys Gly Glu Glu Lys Val Tyr Thr Tyr Phe Met Ile
165 170 175
Ser Glu Gly Ile Asn Asp Tyr Thr His Phe Thr Lys Lys Gly Ala Asn
180 185 190
Glu Met Ala Lys Leu Val Ala Lys Gly Ile Lys Glu Leu Gly Leu Pro
195 200 205
Leu Thr Glu Ser Ile Ile Lys Glu Arg
210 215
SEQ ID NO.2
<210> 2
<211> 654
<212> DNA
<213>genus bacillus (
bacillussp.)
<400> 2
atggcaaatc atatttatct tgccggcgat tcgactgttc aaacgtatgg agacagcaca 60
aatcaagggg gctgggggca gtttctcggc tcgcatctgc cggagcatat tcaagtgatc 120
aacagagcga tcgggggaag aagctcgaaa acatttgtgg aagagggcag gcttcaggca 180
atcctcgatg tgattgagcc ggatgattgg ctgttcgtgc agatgggcca taatgacgcg 240
tcaaaaaata agccggagcg ctacaccgag ccctatacta cttataaaca atatttaaag 300
cagtatatcg caggcgcgcg ggaaaaaggc gcccatccgc ttctcattac ccccgtagcc 360
cgctttcatt acgaaaacgg cgtgtttttg aacgattttc ctgattactg cattgccatg 420
aagcagacgg ctgaagagga gaatgtccag ctcattgatc tgatggagaa aagtctcgct 480
ttctttactg agaagggcga ggaaaaagtg tacacctatt ttatgatttc agaagggatt 540
aatgattaca cgcattttac aaaaaaaggc gcaaatgaaa tggcgaaact tgtggcaaaa 600
ggcataaagg agctcggcct gccattgaca gaatcgatca tcaaagaaag gtga 654
Claims (4)
1. the thermally-stabilised esterase K91 of an alkalescence Est8, it is characterized in that: it has the aminoacid sequence shown in SEQ ID NO.1.
One kind the coding esterase gene claimed in claim 1
k91 Est8, it is characterized in that its nucleotide sequence is as shown in SEQ ID NO.2.
3. one kind comprises the described esterase gene of claim 2
k91 Est8recombinant vectors.
4. one kind comprises the described esterase gene of claim 2
k91 Est8recombinant bacterial strain.
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| 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 |
| CN109628552A (en) * | 2018-12-27 | 2019-04-16 | 北京森根比亚生物工程技术有限公司 | A kind of detection method of carboxylesterase activities |
| CN113637656A (en) * | 2021-08-05 | 2021-11-12 | 云南师范大学 | GDSL family deacetylation esterase mutant Est8-G45R and application thereof |
| CN113637653A (en) * | 2021-08-05 | 2021-11-12 | 云南师范大学 | Esterase mutant Est8-XL with improved activity and application thereof |
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| CN109628552A (en) * | 2018-12-27 | 2019-04-16 | 北京森根比亚生物工程技术有限公司 | A kind of detection method of carboxylesterase activities |
| CN113637656A (en) * | 2021-08-05 | 2021-11-12 | 云南师范大学 | GDSL family deacetylation esterase mutant Est8-G45R and application thereof |
| CN113637653A (en) * | 2021-08-05 | 2021-11-12 | 云南师范大学 | Esterase mutant Est8-XL with improved activity and application thereof |
| CN113637653B (en) * | 2021-08-05 | 2023-05-23 | 云南师范大学 | Esterase mutant Est8-XL with improved activity and application thereof |
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