CN105176943B - The low-temperature alkali esterase EstSL3 and its gene of a kind of salt tolerant organic solvent-resistant and application - Google Patents
The low-temperature alkali esterase EstSL3 and its gene of a kind of salt tolerant organic solvent-resistant and application Download PDFInfo
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- CN105176943B CN105176943B CN201510668911.6A CN201510668911A CN105176943B CN 105176943 B CN105176943 B CN 105176943B CN 201510668911 A CN201510668911 A CN 201510668911A CN 105176943 B CN105176943 B CN 105176943B
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- estsl3
- esterase
- organic solvent
- resistant
- low
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- 239000003845 household chemical Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
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- 239000012474 protein marker Substances 0.000 description 1
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- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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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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- Enzymes And Modification Thereof (AREA)
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Abstract
The present invention relates to genetic engineering fields, and the present invention provides a kind of esterase EstSL3 from thermophilic salt basophilic bacterium, and amino acid sequence is as shown in SEQ ID NO.1, the genome encoding gene of above-mentioned esteraseestSL3, nucleotide sequence is as shown in SEQ ID NO.2, and the recombinant vector comprising the gene and recombinant bacterial strain.Esterase EstSL3 optimal pHs 9.0,30 DEG C of optimal reactive temperature also show that the enzyme is a low temperature esterase when 0 DEG C with 70% or so enzyme activity.The enzyme activity of 98% or more residue in the presence of 0.5-4.0 M NaCl, and it is highly stable under the concentration of 4 M and 5M NaCl.The esterase of the present invention has the characteristics that low temperature, alkalinity, salt tolerant, wash resistant agent and organic solvent-resistant, can be applied to the industrial circles such as fine chemistry industry, pharmacy, detergent and food.
Description
Technical field
The present invention relates to genetic engineering fields, in particular it relates to a kind of low-temperature alkali of salt tolerant organic solvent-resistant
Esterase EstSL3 and its gene and application.
Background technology
Esterase (Esterase E.C.3.1.1.1) is a kind of general name for the enzyme being catalyzed ester linkage hydrolyzing and synthesis.When hydrolysis
It is catalyzed ester bond and generates alcohols and carboxylic acid, the carboxyl of dehydrating condensation acid and the hydroxyl formation ester and other fragrance products of alcohol when synthesis.
There is greater catalytic specificity to substrate in the catalytic reaction process of esterase, reaction condition is mild, by-product is few and does not need
Coenzyme has very high application value in terms of industrial production and environmental improvement.
Production esterase biological is distributed widely in nature, from the tissue of animals and plants in the microorganism of varying environment
All generally existings.Microorganism esterase is with source is wide, type is more, reaction condition is mild, stability is good, substrate is single-minded, catalysis is lived
The features such as property is high, is the main source of industrial esterase.It is mainly derived from fungi from classification, including mould, monascus, black
Aspergillus etc.；Secondly bacterium, including bacillus, Burkholderia etc.；The saccharomycete of individual species and actinomyces
The esterase of some can be generated.Some of them have industrial production value oneself used, such as come from aspergillus niger
(Aspergillus), head mold (Rhizopus arrhizus), mucor (MucorSp.), root Mucor (Rhizomucorsp.)、
Pseudomonas fluorescens (Pseudomanas fluoreseens), Candida (Candida The esterase of microorganisms such as sp.).
Microorganism esterase has important application value in food processing, detergent, chemical products, environmental protection etc..Example
Such as, in food processing, esterase can be applied to improve the flavor of beer, white wine and other drinks, such as in beer technology production
Using the esterase and transacetylase of saccharomycete, under the collective effect of the two, acid isobutyl ester pentyl ester is generated, beer can be promoted
Flavor.In terms of fine chemical product, ester conversion and synthetic reaction can prepare the household chemicals with economic benefit, and esterase is urged
Change the synthesis of the important biosurfactants such as sugar ester.And in terms of chiral drug production, mainly shifts and hydrolyze using acyl group
Act on synthetic drug.
The microorganism in extreme environment source can generate some extreme enzymes, these enzymes have very high enzyme under extreme conditions
Activity, therefore can adapt to exacting terms in industrial production and there is huge application prospect.It is thermophilic to study now more
Hot enzyme, basophilic enzyme, acidophilus enzyme, thermophilic cold enzyme, thermophilic salt enzyme etc..Wherein thermophilic cold enzyme due to its under cryogenic have high activity and
Reaction process is easy to control and is more and more studied.Furthermore it is possible to adapt to the enzyme of a variety of extreme conditions also by more next
More concerns.
Most of esterase obtained at present derives from conventional environment, and for optimum temperature at 40 DEG C or so, optimal pH is neutrality
Meta-alkalescence cannot be applied in many industry.Salt alkali lake is the alkaline environment of a natural stabilization, and pH has between 9-11
When can reach 12 or so, usually there is medium-altitude salt to exist therefore simultaneously.There is a large amount of thermophilic saline and alkaline microorganism in salt alkali lake.
And obtain the alkaline enzyme with industrial value such as alkali protease, alkaline fiber from the thermophilic saline and alkaline microorganism that these are detached
Plain enzyme, alkali starch enzyme etc..
The present invention has detached the thermophilic salt basophilic bacterium of bacterial strain that one plant can produce low-temperature alkali salt tolerant esterase from salt alkali lake
（Alkalibacteriumsp. SL3）, the low temperature Saline alkali tolerance is obtained using the method for molecular biology and genetic engineering
The encoding gene of esterase, and the gene is recombinantly expressed, recombinase has low temperature（30 DEG C of optimum temperature, 0 DEG C also has
70% or so enzyme activity）, alkalinity（Optimal pH 9.0）.The enzyme also tolerance and stability with good salt（At 5M NaCl
Manage the enzyme activity of 2h also surplus 88%）.In addition, the enzyme has preferably some metal ions, common organic solvent and detergent
Resistance.Esterase EstSL3 has the characteristics that low temperature, alkalinity, salt tolerant, wash resistant agent and organic solvent-resistant, can be applied to refine
The industrial circles such as work, pharmacy, detergent and food.
Invention content
The low-temperature alkali esterase EstSL3 and its gene of a kind of salt tolerant organic solvent-resistant of the present invention and application, to be solved first
Certainly the technical issues of is to overcome the deficiencies of the prior art and provide a kind of good properties, be suitable for, in fine chemistry industry, pharmacy, washing
Wash the new esterase of agent and Applications in Food Industry.The present invention obtains a kind of esterase of low-temperature alkali salt tolerant organic solvent-resistant
EstSL3 has very high enzyme activity under low-temperature alkali and high salt conditions, and the enzyme is to some common organic solvents, chemistry
Reagent and detergent have preferable resistance.These properties meet food industry, and fine chemistry industry and pharmaceuticals industry are wanted
It asks, reduces energy consumption, increase efficiency of pcr product, optimize production process.
To achieve the above object, the present invention adopts the following technical scheme that：
The present invention provides a kind of low-temperature alkali salt tolerant organic solvent-resistant esterase, amino acid sequence such as SEQ ID NO. 1
It is shown.
The present invention provides encode above-mentioned low-temperature alkali salt tolerant organic solvent-resistant esteraseestSL3Gene.Specifically, the base
The gene order of cause is as shown in SEQ ID NO. 2.
The present invention also provides include above-mentioned low-temperature alkali salt tolerant organic solvent-resistant esteraseestSL3Recombinant vector, preferably
ForpET28a-estSL3。
It is excellent the present invention also provides the recombinant bacterial strain of the esterase EstSL3 comprising above-mentioned low-temperature alkali salt tolerant organic solvent-resistant
It is Escherichia coli, saccharomycete, bacillus and filamentous fungi to select the bacterial strain.
The present invention also provides a kind of method preparing low-temperature alkali salt tolerant organic solvent-resistant esterase EstSL3, including it is following
Step：
1）Host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
2）Cultivate recombinant bacterial strain, induction Recombinant esterase expression；And
3）It recycles and purifies expressed esterase EstSL3.
The present invention also provides the applications of above-mentioned low-temperature alkali salt tolerant organic solvent-resistant esterase EstSL3.
The present invention first the technical problem to be solved is that overcome the deficiencies of the prior art and provide a kind of good properties,
It is suitable in fine chemistry industry, pharmacy, the new esterase of detergent and Applications in Food Industry.It is resistance to that the present invention obtains a kind of low-temperature alkali
The esterase EstSL3 of salt organic solvent-resistant has very high enzyme activity under low-temperature alkali and high salt conditions, and the enzyme is to some
Common organic solvent, chemical reagent and detergent have preferable resistance.These properties meet food industry, fine chemistry industry
And the requirement of pharmaceuticals industry, energy consumption is reduced, efficiency of pcr product is increased, optimizes production process.
The esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant of the present invention is available from big cloth Soviet Union salt alkali lake bed mud point
From thermophilic salt basophilic bacterium（Alkalibacteriumsp.）, amino acid sequence is as shown in SEQ ID NO. 1：
MKINKNDTLLFIGDSITDVGRDRMDGEDLGKGFPLMVASHLQSRYPAKRLTVLNRGIGGDSLKDLKRRWEDDCLITN
PDIVTLLIGVNDTWRNQNDGVELTDEELDEFESDYRFLLKSLHQRTDARVILMESFVLPYPKRRVGWRNDLDKRIQI
VRKMARDYQTELIPLDGLLNAAGIRDGFSYYTGDDGVHPTVAGHGLIANSWLKAVDE。
The esterase EstSL3 of the present invention contains 211 amino acid, signal peptide of the N-terminal without prediction in total, and theoretical molecular weight is
24.04 kDa, theoretical isoelectric point are 5.28.
The apparent optimal pH that the optimal pH of the esterase EstSL3 of the present invention is 9.0, EstSL3 is 9.0, when pH is less than 6.0
Enzymatic activity drastically declines, and in pH5.0, is substantially not detectable enzymatic activity.The esterase is very steady between pH 6.0-10.0
Fixed, for remaining enzymatic activity 80% or more, this illustrates that this enzyme is stablized with preferable pH after 60min is handled within the scope of this pH
Property.
The optimum temperature of the esterase EstSL3 of the present invention is 30 DEG C, the enzyme activity of residue 70% or so at 0 DEG C.At 50 DEG C
Lower stabilization, handle 60 min after remain to keep 90 % or more activity.Unstable at 60 DEG C, half-life period is less than 5min.
The esterase EstSL3 of the present invention also tolerance and stability with good salt.Exist in 0.5-4.0 M NaCl
In the case of residue 98% or more enzyme activity.60 min, the enzyme of 88% or more residue are handled under the concentration of 4 M and 5M NaCl
It is living.Show that the esterase has extraordinary tolerance and stability to salt.
Li+, Na+, K+, Mg2+, Ca2+, EDTA and β-mercaptoethanol are in the case of low concentration and high concentration
Enzyme activity can be promoted；Co2+, Zn2+, Mn2+And Cu2+There is partial inhibition to enzyme；Ag+, Fe2+, Hg2+And Pb2++To enzyme activity
Power has strong inhibition.
The Tween 80 and Triton X-100 of low concentration have facilitation to EstSL3, wherein 1% Tween 80 promotes
Effect is the most apparent, up to 131% or so；And there is partial inhibition to EstSL3 under high concentration.20 Hes of Tween of low concentration
SDS has partial inhibition to enzyme；And there is strong inhibiting effect under high concentration to enzyme.
10% and 20% ethyl alcohol, propyl alcohol, n-hexane influence enzyme activity little, and enzyme activity remains to be maintained at 90% or more.Methanol,
Glycerine, acetonitrile then have slight inhibiting effect to enzyme activity.Butanol, isobutanol, acetone, the chloroform of low concentration influence not enzyme activity
Greatly, and when a concentration of 20% with obvious inhibiting effect, enzyme activity is 50% or less.
The present invention also provides the genes for the esterase EstSL3 for encoding above-mentioned low-temperature alkali salt tolerant organic solvent-resistantestSL3,
The gene order is as shown in SEQ ID NO. 2（Its accession number in NCBI is KT225466）：
ATGAAAATCAACAAGAATGATACGTTGCTGTTCATCGGTGACAGCATAACAGATGTAGGCCGTGACCGTATGGATGG
CGAAGATTTAGGTAAGGGATTCCCATTGATGGTCGCTTCGCACCTTCAGTCACGCTATCCGGCGAAAAGGCTGACGG
TTTTGAATCGGGGCATTGGCGGGGACAGTCTGAAAGATCTTAAAAGGCGATGGGAAGATGATTGCCTGATCACTAAT
CCTGACATCGTCACATTACTTATAGGTGTAAATGACACCTGGCGTAATCAGAATGACGGAGTAGAACTTACAGATGA
GGAACTGGATGAGTTTGAATCAGACTACCGCTTTCTTCTGAAATCGCTTCACCAGAGAACGGATGCTCGAGTCATTT
TAATGGAGTCTTTTGTACTGCCTTATCCGAAAAGAAGAGTGGGCTGGAGGAATGACCTGGATAAACGAATCCAGATC
GTCAGGAAGATGGCCAGAGATTATCAGACTGAACTCATTCCGCTGGATGGACTTTTGAATGCCGCTGGAATAAGGGA
CGGGTTCAGCTATTACACAGGAGATGACGGCGTCCATCCGACTGTAGCCGGTCACGGACTAATCGCGAACAGCTGGC
TGAAAGCTGTCGATGAATAA。
The present invention has cloned this esterase gene by the method separation of PCRestSL3, DNA complete sequence analysis result tables
It is bright, esterase EstSL3 structural genesestSL3Overall length 636 bp, initiation codon ATG, terminator codon TAA, GC
47.2 % of content, the polypeptide (EstSL3) of coding 211 amino acid composition, signal peptide sequence of the N-terminal without prediction are intermediate
For the catalyst structure domain of a SGNH family.Comparison result in GenBank shows it and comes fromAlkalibacteriumSp. the highest consistency for paroling esterase for not doing function of AK22 (WP_034300718) is
69%, show that EstSL3 is a new esterase.
The present invention also provides include above-mentioned esterase geneestSL3Recombinant vector, preferablypET28a- estSL3。
By the esterase gene of the present inventionestSL3It is inserted between suitable restriction enzyme cleavage sites of the expression vector, makes its nucleotide sequence
It is operable to be linked to the expression control sequence.As the most preferred embodiment of the present invention, preferably by esterase base
CauseestSL3It is inserted on plasmid pET28aNcoIWith HindIIIBetween restriction enzyme site, recombinant expression plasmid is obtainedpET28a- estSL3。
The present invention also provides include above-mentioned esterase geneestSL3Recombinant bacterial strain, the preferably described bacterial strain be large intestine bar
Bacterium, saccharomycete, bacillus and filamentous fungi, preferably recombinant bacterial strain BL21 (DE3)/estSL3。
The advantage of the invention is that：The present invention provides a kind of good properties, be suitable in food, fine chemistry industry and system
The new esterase applied in the industry such as medicine.It is usually carried out in organic solvent in fine chemistry industry and pharmacy procedure.Institute of the present invention
The esterase optimal reactive temperature of offer is 30 DEG C, and the thermal stability of 50 DEG C or more enzymes is very poor, is controlled convenient for enzymatic reaction.In addition,
The enzyme has good tolerance to common organic solvent, can meet the requirement industrially to a variety of different solvents.The esterase
Also there is good pH tolerances, it is highly stable in pH6.0~10.0.In addition, the enzyme also has good salt tolerance and steady
It is qualitative.Therefore the low-temperature alkali salt tolerant wash resistant agent of the present invention and the esterase of organic solvent-resistant can be in fine chemistry industry, pharmaceuticals industries
Middle application can reduce production temperature, reduce energy consumption；Nonspecific reaction is reduced, purpose product yield is increased.Since the enzyme exists
There is very high enzyme activity and the good tolerability to detergent, therefore the enzyme can also be applied to washers under cryogenic conditions
Industry.In addition, the enzyme can be also used for food industry, improve the flavor etc. of drinks and liquid food.
Description of the drawings
Fig. 1：In the SDS-PAGE analyses of the Recombinant esterase EstSL3 of expression in escherichia coli.Wherein, M:Low molecule
Measure protein Marker；1:What is do not induced contains esterase gene carrier pET-estSL3E. coli culture supernatant liquid；2:
Induction contains esterase gene carrier pET-EstSL3E. coli culture supernatant concentrated liquor；3:Pass through ni-sepharose purification
Reach electrophoretically pure EstSL3 albumen.
Fig. 2：The optimal pH of Recombinant esterase EstSL3.
Fig. 3：The pH stability of Recombinant esterase EstSL3.
Fig. 4：The optimum temperature of Recombinant esterase EstSL3.
Fig. 5：The thermal stability of Recombinant esterase EstSL3.
Fig. 6：The salt tolerance of Recombinant esterase EstSL3.
Fig. 7：The salt-stable of Recombinant esterase EstSL3.
Specific implementation mode
Test material and reagent
1, bacterial strain and carrier：I-T1 and BL21 (DE3) of Escherichia coli (Escherichia coli) Trans are purchased from Beijing
TransGen companies, carrier pET-28a (+) and pMD19-T carriers are purchased from Novagen companies of the U.S. and Japan TaKaRa respectively
Company.
2, enzyme and other biochemical reagents：Restriction enzyme, T4 DNA ligases, archaeal dna polymerase number and dNTPs purchases
In Japanese TaKaRa companies.Genome extracts kit is purchased from Beijing Tiangen companies, and purifying and plasmid extraction kit are purchased from
OMEGA companies of the U.S..P-nitrophenol（pNP）, p-nitrophenyl esters（pNP-esters）It is purchased from Sigma Co., USA；Albumen
Peptone (Tryptone), yeast extract (Yeast Extract) are Britain's OXOID Products, remaining reagent is domestic analysis
It is pure.
3, culture medium：
（1）LB culture mediums（g/l）：Yeast powder 5.0, peptone 10.0, NaCl 10.0, extremely with the sodium carbonate tune pH of 1M
9.6。
（2）LB solid mediums（g/l）：Yeast powder 5.0, peptone 10.0, NaCl 10.0, agar 15.0, with 1 M
Sodium carbonate tune pH to 9.6.
Explanation：Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiment《Molecular Cloning: A Laboratory
Guide》（The third edition）J. specific method listed in one book of Pehanorm Brooker carries out, or according to kit and product description
It carries out.
Embodiment 1：Esterase geneEstSL3Clone
The extraction of thermophilic salt basophilic bacterium genomic DNA：It is carried using the bacterial genomes extracts kit (DP302) of Tiangeng company
Genomic DNA, concrete operations is taken to be carried out fully according to the specification of the kit.
When we clone xylanase gene from the bacterium, 3 ' ends of the esterase gene, sequence alignment point are obtained
Analysis shows that the similitude of the gene is also relatively low, and does not all do property, therefore the esterase gene obtained according to sequencing
The nucleotide sequence at 3 ' ends, designs the TAIL-PCR specific primers of upstream three：Design direction is the zone of ignorance for needing to expand
Direction, for the Position Design of sp2 in the inside of sp1, sp3 is located at the inside of sp2.The distance between each two primer is not advised strictly
It is fixed（For convenience of electrophoresis recognition result, sp2 and general 100 bp of spacing of sp3）, general 22~30 nt of primer length, annealing temperature
At 62 DEG C.And they are respectively designated as estSL3-uSP1, estSL3-uSP2, estSL3-uSP3 is shown in Table 1.According to TAIL-
Program pair 5 ' in PCR holds flanking sequence to expand.
Primer needed for 1 esterase EstSL3 TAIL-PCR amplifications of table and overall length amplification
The flanking sequence of known sequence is obtained by TAIL-PCR, amplification send handsome company to survey after obtaining product recycling
Sequence.The upstream and downstream flanking sequence of the segment is obtained by sequence assembly, complete sequence is total to long 1.17kb, finds one and completely opens
Reading frame（ORF）.Esterase geneestSL3(GenBank accession no. KT225466) is compiled by 636 base compositions
211 amino acid of code and a terminator codon, the comparison result in GenBank show it and derive fromAlkalibacteriumSp. the imaginary esterase gene complete sequence similitude of AK22 (WP_034300718) is 69%, Er Qiewei
Did functional study.estSL3Coding albumen estimated molecular weight is 24.04kDa, isoelectric point 5.28.It is predicted, EstSL3 without
Signal peptide sequence, centre are the catalyst structure domain of a SGNH family.
The preparation of 2 Recombinant esterase of embodiment.
To be introduced respectively in gene 5 ' and 3 ' endsNcoIWith HindIIIRestriction enzyme siteestSL3-m-F andestSL3-m-
R is primer pair（It is shown in Table one）, thermophilic salt basophilic bacterium genomic DNA is template, carries out PCR amplifications.PCR response parameters are：94
DEG C 5 min of pre-degeneration；94 DEG C of 30 sec of denaturation, 51 DEG C of 30 sec of annealing, 72 DEG C of 1 min of extension, 30 cycles, 72 DEG C keep the temperature
5 min.By expression vector pET28a (+) carry out double digestion (NcoI+ HindIII), while the gene that esterase will be encodedestSL3Double digestion (NcoI+ HindIII), genetic fragment and the expression vector pET28a (+) of the encoding mature esterase cut connect
It connects, acquisition contains esterase geneestSL3Recombinant plasmid pET28a-estSL3And e. coli bl21 (DE3) is converted, it obtains
Recombinant escherichia coli strain BL21/estSL3。
It takes containing recombinant plasmid pET28a-estSL3BL21 bacterial strains, being inoculated in 5 mL LB, (the ammonia benzyl of 100 μ g/mL is green
Mycin) in culture solution, 37 DEG C of overnight incubations.Cultured bacterium solution is inoculated in 20 mL LB (added with the ammonia of 100 μ g/mL by 1%
Parasiticin), the derivant of 0.5 mmol/L of final concentration is added in 37 DEG C of about 2 ~ 3 h (OD600 reaches 0.5) of shaken cultivation afterwards
IPTG, 30 DEG C of 180 12 h of rpm shake cultures.12000 rpm centrifuge 5 min, collect thalline and carry out ultrasonic disruption.It utilizes
The vigor of paranitrophenol colorimetric method for determining esterase enzyme activity determination esterase.SDS-PAGE results (Fig. 1) show Recombinant esterase in large intestine
It is expressed in bacillus.For expressed esterase after purifying, the content of protein reaches 95% of total protein or more.
The property of 3 Recombinant esterase EstSL3 of embodiment measures
1, the activity analysis of Recombinant esterase
Colorimetric method for determining esterase enzyme activity is taken in the determination of activity of Recombinant esterase：The specific method is as follows：In pH9.0,30 DEG C of items
Under part, the reaction system of 1 mL includes 100 μ L dilution enzyme solutions appropriate, 20 μ L substrates（10 mM）, 880 μ L 50 are added
9.0 phosphate buffers of mmol/L pH, react 10 min after immediately in OD404Place measures its light absorption value.1 enzyme-activity unit (U)
It is defined as the enzyme amount per minute released needed for 1 μm of ol p-nitrophenol under given conditions.
2, the measurement of the optimal pH of Recombinant esterase EstSL3 and pH stability
Optimal pH measures：By purified Recombinant esterase EstSL3 at 37 DEG C and the buffering of 0.1M pH5.0-9.0
Enzymatic reaction is carried out in liquid.The pH Stability Determinations of enzyme：Enzyme solution is placed in the buffer solution of 0.1M pH 4.0-11.0,37
1h is handled at DEG C, enzymatic reaction is then carried out at pH9.0 and 30 DEG C, as a contrast with untreated enzyme solution.Buffer solution is：
0.1M McIlvaine buffer （pH4.0–7.0）, 0.1 M Tris-HCl buffer（pH7.0–10.0）And 0.1M
glycine-NaOH（pH10.0–11.0）.Using pNP-C2 as substrate, 10min is reacted, the vigor of EstSL3 is measured.As a result table
It is bright：The optimal pH of EstSL3 is 9.0,60% or more (Fig. 2) of residue maximum enzyme activity when between pH 7.5 to 9.0；This ester
Enzyme is very stable between pH 6.0-10.0, remaining enzymatic activity is handled after 60min within the scope of this pH 80% or more,
This illustrates that this enzyme has preferable pH stability（Fig. 3）.
3, the optimum temperature and thermal stability determination of Recombinant esterase EstSL3
The measurement of the optimum temperature of enzyme：In the buffer solution of pH 9.0, enzymatic reaction is carried out at 0-70 DEG C.The heat of enzyme
Stability Determination：The enzyme solution of same enzyme amount is placed in 50 DEG C, 55 DEG C and 60 DEG C, after handling 0-60min, in pH 9.0 and 30
Enzymatic reaction is carried out at DEG C, as a contrast with untreated enzyme solution.Using pNP-C2 as substrate, 10min is reacted.Enzyme reaction most thermophilic
Degree measurement result (Fig. 4) shows that optimum temperature is 30 DEG C.The esterase, also with 70% or so enzyme activity, shows the enzyme at 0 DEG C
It is a cold-adapted enzyme.The thermal stability of enzyme is experiments have shown that (Fig. 5), recombinase stability at 50 DEG C are very good.It is protected at 55 DEG C
60 min of temperature, remaining enzymatic activity are 21.3%, and 60 DEG C of 60 min enzyme activity almost all of processing are lost.
4, Recombinant esterase EstSL3V maxAnd KmMeasurement
With the substrate pNP-C2 of different final concentrations (0.02,0.05,0.1,0.15,0.2 and 0.3 mmol) for substrate, most
Enzymatic activity is measured under the conditions of suitable, calculates corresponding reaction speed, K is acquired using the double counting backward techniques of Michaelis-Menten equationmValue and Vmax.As a result
Show：The enzymeV maxFor 769.23 ± 6.55 μm of ol min–1mg–1, KmFor 0.15 ± 0.008 mg mL–1。
5, influence of the different metal ions chemical reagent to EstSL3 enzyme activity measures
The metal ion of 1 and 5 mM is added in enzymatic reaction system, studies its influence to enzymatic activity.At 30 DEG C and
Under the conditions of pH 9.0, enzymatic activity is measured by substrate of pNP-C2.As a result（Table 2）Show：Li+, Na+, K+, Mg2+, Ca2+, EDTA
It can promote enzyme activity in the case of low concentration and high concentration with β-mercaptoethanol；Co2+, Zn2+, Mn2+With
Cu2+There is partial inhibition to enzyme；Ag+, Fe2+, Hg2+And Pb2++There is strong inhibition to enzyme activity.
Two metal ion of table and chemical reagent are on the active influences of Recombinant esterase EstSL3
6, the influence of different detergent and organic solvent to EstSL3 enzyme activity measures
3 kinds of different three concentration (a concentration of 1%, 5 %L and 10% of final volume) are separately added into enzymatic reaction system
Polysorbas20, Tween 80, Triton X-100 and SDS, to study its influence to enzymatic activity.In the item of optimal pH and optimum temperature
Enzymatic activity is measured under part, not add the enzymatic reaction of any reagent as control group under similarity condition.Divide in enzymatic reaction system
Not Jia Ru 11 kinds of different two concentration (final volume a concentration of 10% and 20%) organic solvent, including：Methanol, ethyl alcohol, propyl alcohol,
Butanol, isobutanol, isoamyl alcohol, acetone, n-hexane, glycerine, chloroform, acetonitrile.To study its influence to enzymatic activity.In optimal pH
With enzymatic activity is measured under conditions of optimum temperature, using under similarity condition not added with the enzymatic reaction of solvent as control group.
The result shows that（Table three）, the Tween 80 and Triton X-100 of low concentration have facilitation to EstSL3, wherein
1% Tween, 80 facilitations are the most apparent, up to 131% or so；And there is partial inhibition to EstSL3 under high concentration.It is low
The Tween 20 and SDS of concentration have partial inhibition to enzyme；And there is strong inhibiting effect under high concentration to enzyme.10% and 20%
Ethyl alcohol, propyl alcohol, n-hexane enzyme activity is influenced little, enzyme activity remains to be maintained at 90% or more.Methanol, glycerine, acetonitrile are then to enzyme activity
With slight inhibiting effect.Butanol, isobutanol, acetone, the chloroform of low concentration influence less enzyme activity, and work as a concentration of 20%
With obvious inhibiting effect, enzyme activity is 50% or less.Wherein isoamyl alcohol is the most apparent, and enzyme activity is only 7.3% or so.
Three detergent of table and organic solvent are on the active influences of Recombinant esterase EstSL3
7, various concentration NaCl is to the activity of Recombinant esterase EstSL3 and the measurement of stabilization
Activity influences of the NaCl to Recombinant esterase EstSL3：Various concentration is added in enzymatic reaction system（0.5-4M）'s
NaCl studies its influence to enzymatic activity.Under the conditions of 30 DEG C and 9.0 pH, enzymatic activity is measured by substrate of pNP-C2.NaCl
Influence to the stability of Recombinant esterase EstSL3：Enzyme solution is respectively placed in the buffer solution of the pH 9.0 containing 4M and 5 M,
2h is handled at 37 DEG C, then carries out enzymatic reaction at pH 9.0 and 30 DEG C, as a contrast with untreated enzyme solution.As a result
Show：In the presence of 0.5-4.0 M NaCl, which is capable of the enzyme activity of 98% or more residue（Fig. 6）, show that the enzyme has
Good salt tolerance.120 min, the enzyme activity of 88% or more residue are handled under the concentration of 4 M and 5M NaCl（Fig. 7）, show
The enzyme has extraordinary salt-stable.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>The low-temperature alkali esterase EstSL3 and its gene of a kind of salt tolerant organic solvent-resistant and application
<130> 7
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 211
<212> PRT
<213>Esterase EstSL3 amino acid sequences
<400> 1
Met Lys Ile Asn Lys Asn Asp Thr Leu Leu Phe Ile Gly Asp Ser Ile
1 5 10 15
Thr Asp Val Gly Arg Asp Arg Met Asp Gly Glu Asp Leu Gly Lys Gly
20 25 30
Phe Pro Leu Met Val Ala Ser His Leu Gln Ser Arg Tyr Pro Ala Lys
35 40 45
Arg Leu Thr Val Leu Asn Arg Gly Ile Gly Gly Asp Ser Leu Lys Asp
50 55 60
Leu Lys Arg Arg Trp Glu Asp Asp Cys Leu Ile Thr Asn Pro Asp Ile
65 70 75 80
Val Thr Leu Leu Ile Gly Val Asn Asp Thr Trp Arg Asn Gln Asn Asp
85 90 95
Gly Val Glu Leu Thr Asp Glu Glu Leu Asp Glu Phe Glu Ser Asp Tyr
100 105 110
Arg Phe Leu Leu Lys Ser Leu His Gln Arg Thr Asp Ala Arg Val Ile
115 120 125
Leu Met Glu Ser Phe Val Leu Pro Tyr Pro Lys Arg Arg Val Gly Trp
130 135 140
Arg Asn Asp Leu Asp Lys Arg Ile Gln Ile Val Arg Lys Met Ala Arg
145 150 155 160
Asp Tyr Gln Thr Glu Leu Ile Pro Leu Asp Gly Leu Leu Asn Ala Ala
165 170 175
Gly Ile Arg Asp Gly Phe Ser Tyr Tyr Thr Gly Asp Asp Gly Val His
180 185 190
Pro Thr Val Ala Gly His Gly Leu Ile Ala Asn Ser Trp Leu Lys Ala
195 200 205
Val Asp Glu
210
<210> 2
<211> 636
<212> DNA
<213>Gene estSL3
<400> 2
atgaaaatca acaagaatga tacgttgctg ttcatcggtg acagcataac agatgtaggc 60
cgtgaccgta tggatggcga agatttaggt aagggattcc cattgatggt cgcttcgcac 120
cttcagtcac gctatccggc gaaaaggctg acggttttga atcggggcat tggcggggac 180
agtctgaaag atcttaaaag gcgatgggaa gatgattgcc tgatcactaa tcctgacatc 240
gtcacattac ttataggtgt aaatgacacc tggcgtaatc agaatgacgg agtagaactt 300
acagatgagg aactggatga gtttgaatca gactaccgct ttcttctgaa atcgcttcac 360
cagagaacgg atgctcgagt cattttaatg gagtcttttg tactgcctta tccgaaaaga 420
agagtgggct ggaggaatga cctggataaa cgaatccaga tcgtcaggaa gatggccaga 480
gattatcaga ctgaactcat tccgctggat ggacttttga atgccgctgg aataagggac 540
gggttcagct attacacagg agatgacggc gtccatccga ctgtagccgg tcacggacta 600
atcgcgaaca gctggctgaa agctgtcgat gaataa 636
<210> 3
<211> 29
<212> DNA
<213>Artificial sequence
<400> 3
cagcccactc ttcttttcgg ataaggcag 29
<210> 4
<211> 27
<212> DNA
<213>Artificial sequence
<400> 4
gactcgagca tccgttctct ggtgaag 27
<210> 5
<211> 31
<212> DNA
<213>Artificial sequence
<400> 5
cctcatctgt aagttctact ccgtcattct g 31
<210> 6
<211> 35
<212> DNA
<213>Artificial sequence
<400> 6
gcgccatggg catgaaaatc aacaagaatg atacg 35
<210> 7
<211> 29
<212> DNA
<213>Artificial sequence
<400> 7
ccgaagcttt tcatcgacag ctttcagcc 29
Claims (10)
1. a kind of esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant, which is characterized in that its amino acid sequence such as SEQ ID
NO. shown in 1.
2. a kind of esterase gene of low-temperature alkali salt tolerant organic solvent-resistantestSL3, which is characterized in that described in coding claim 1
Low-temperature alkali salt tolerant organic solvent-resistant esterase EstSL3.
3. the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant according to claim 2estSL3, which is characterized in that its
Base sequence is as shown in SEQ ID NO. 2.
4. including the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant described in Claims 2 or 3estSL3Recombinant vector.
5. including the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant described in Claims 2 or 3estSL3Recombinant vectorpET28a-estSL3。
6. including the esterase gene of low-temperature alkali salt tolerant organic solvent-resistant described in Claims 2 or 3estSL3Recombinant bacterial strain.
7. recombinant bacterial strain according to claim 6, which is characterized in that the bacterial strain be Escherichia coli, saccharomycete, bacillus and
Filamentous fungi.
8. a kind of method for the esterase EstSL3 preparing low-temperature alkali salt tolerant organic solvent-resistant, which is characterized in that including following step
Suddenly：
1）The recombinant vector described in claim 4 converts host cell, obtains recombinant bacterial strain；
2）Cultivate recombinant bacterial strain, induction Recombinant esterase expression；
3）It recycles and purifies expressed esterase EstSL3.
9. esterase EstSL3 the answering in fine chemistry industry and pharmacy of low-temperature alkali salt tolerant organic solvent-resistant as described in claim 1
With.
10. the esterase EstSL3 of low-temperature alkali salt tolerant organic solvent-resistant according to claim 1 is for producing specificization
Learn product or prodrug；Application in detergent；Improve the application in the flavor of drinks and liquid food on food.
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| CN102286441A (en) * | 2011-07-24 | 2011-12-21 | 国家***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***第二海洋研究所 | Abyssal deposit-derived esterase EST22 as well as coding gene and application thereof |
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| CN102286441A (en) * | 2011-07-24 | 2011-12-21 | 国家***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***第二海洋研究所 | Abyssal deposit-derived esterase EST22 as well as coding gene and application thereof |
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