CN105543190A - Esterase BSE00077 and encoding gene and application thereof - Google Patents
Esterase BSE00077 and encoding gene and application thereof Download PDFInfo
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- CN105543190A CN105543190A CN201610021561.9A CN201610021561A CN105543190A CN 105543190 A CN105543190 A CN 105543190A CN 201610021561 A CN201610021561 A CN 201610021561A CN 105543190 A CN105543190 A CN 105543190A
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- 108090000623 proteins and genes Proteins 0.000 title abstract description 13
- 238000003259 recombinant expression Methods 0.000 claims abstract description 7
- WAPNOHKVXSQRPX-SSDOTTSWSA-N (R)-1-phenylethanol Chemical compound C[C@@H](O)C1=CC=CC=C1 WAPNOHKVXSQRPX-SSDOTTSWSA-N 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108010088545 esterase B Proteins 0.000 claims description 88
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- 241000894006 Bacteria Species 0.000 claims description 13
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- 238000010353 genetic engineering Methods 0.000 claims description 6
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- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
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- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- HWGNBUXHKFFFIH-UHFFFAOYSA-I pentasodium;[oxido(phosphonatooxy)phosphoryl] phosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O HWGNBUXHKFFFIH-UHFFFAOYSA-I 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- 229910052708 sodium Inorganic materials 0.000 description 1
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- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- HFQQZARZPUDIFP-UHFFFAOYSA-M sodium;2-dodecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HFQQZARZPUDIFP-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical group OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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- 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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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses esterase BSE00077 and an encoding gene and application thereof. A new esterase gene bse00077 is developed from bacillus sp. SCSIO 15121, the nucleotide sequence of the esterase gene bse00077 is shown as SEQ ID NO.1, the total length is 741 bp, and the amino acid sequence of the esterase BSE00077 encoded by the new esterase gene bse00077 is shown as SEQ ID NO.2 and contains 246 amino acids. After the cloned esterase gene bse00077 is heterologously expressed in escherichia coli BL21(DE3), esterase BSE00077 obtained after recombinant expression is obtained. The esterase serves as a catalyst and can catalytically prepare (R)-1-phenethyl alcohol through an ester hydrolysis reaction at normal temperature, good stability is achieved, and good tolerance is achieved for part of a surface active agent and an organic solvent. The esterase BSE00077 can be used for the fields of preparation of a chiral intermediate, detergent, biological medicine, cosmetics, fine chemical engineering and the like.
Description
Technical field
The invention belongs to biochemical industry and biological technical field, be specifically related to a kind of esterase B SE00077 and encoding gene thereof and application.
Background technology
Esterase (EC3.1.1.1) is extensively present in animal, plant and microorganism, is a class catalytic hydrolysis or forms the enzyme of ester bond, and the substrate of effect normally aliphatic chain is less than the ester class of ten carbon atoms.Esterase belongs to α/β and folds lytic enzyme superfamily, and catalytic center is made up of Serine, aspartic acid/L-glutamic acid and Histidine, and conserved sequence is pentapeptide (GXSXG) sequence near Serine.Esterase energy catalytic hydrolysis, esterification, the number of chemical such as transesterification reaction, be a kind of very important industrial biocatalytic agent, be widely used in fields such as fine chemistry industry, washing, medicine, food, papermaking, leather processing, weaving, wastewater treatment and fodder industries.From catalysis characteristics, esterase has chemo-selective and the stereoisomerism selectivity of height, and reaction does not need, and coenzyme, reaction conditions are gentle, by product is few.Another distinguishing feature of esterase in production application is that it can act in outphasing system (i.e. oil-water interface) or organic phase.In aqueous phase, the usual catalytic hydrolysis reaction of esterase, and in organic phase, it but can catalytic esterification and transesterification.Prepare newtype drug intermediate by the bio-transformation of microorganism esterase or remove the non-active ingredients of medicine raceme, it is a kind of important chiral technology, there is boundless application prospect, it can provide new platform for synthesis of chiral medicine, for preparing the method that optical pure compound provides new in a large number.Enzyme process selectivity resolving racemic compound, have stereospecificity high, side reaction is few, and productive rate is high, the advantage that product optical purity is good and reaction conditions is gentle, so be a kind of by the method for splitting extensively approved.
Summary of the invention
The object of this invention is to provide a kind of new esterase B SE00077 and encoding gene thereof and application.
The present invention obtains a kind of esterase B SE00077 and encoding gene esterase gene bse00077 thereof from a strain from exploitation genus bacillus (Bacillussp.) SCSIO15121 of ocean, construct the recombinant expression vector containing esterase gene bse00077 and genetic engineering bacterium, obtain esterase B SE00077 after culturing gene engineering bacteria, it can be applicable to the reaction of catalysis ester-type hydrolysis.
First object of the present invention is to provide a kind of esterase B SE00077, and its aminoacid sequence is as shown in SEQIDNO.2.
Second object of the present invention is to provide the esterase gene bse00077 of a kind of described esterase B SE00077 that encodes.
Preferably, the nucleotide sequence of described esterase gene bse00077 is as shown in SEQIDNO.1.
The present invention also provides a kind of recombinant expression vector containing described esterase gene bse00077.Described recombinant expression vector, preferred pET-28a (+) carrier.
The present invention also provides a kind of genetic engineering bacterium containing described esterase gene bse00077.Described genetic engineering bacterium, preferred e. coli bl21 (DE3).
3rd object of the present invention is to provide the application of described esterase B SE00077 in catalysis ester-type hydrolysis.
Preferably, the described esterase B SE00077 that is applied as is hydrolyzed the application generated in (R)-1-phenylethyl alcohol at catalysis styroyl acetate.
4th object of the present invention is to provide the application that described esterase B SE00077 carries out catalysis under tolerance normal hexane and/or normal heptane environment.
(its source is genus bacillus (Bacillussp.) SCSIO15121 that esterase gene bse00077 of the present invention originates from deep-sea: 89 ° of 29.22 ' E, 10 ° of 00.12 ' N ,-3400m, pH7.8,2 DEG C), be kept at Chinese Academy of Science Nanhai Ocean Research Institute.The present invention utilizes bioinformatic analysis method, from genus bacillus (Bacillussp.) SCSIO15121 of gene order-checking, clone obtains esterase gene bse00077, total length is 741bp (from initiator codon to terminator codon), and the esterase B SE00077 of its coding contains 246 amino acid.Transformation of E. coli BL21 (DE3) after being connected with expression vector pET-28a (+) by esterase gene bse00077, cultivates and after abduction delivering, obtains Recombinant esterase BSE00077.Using the esterase B SE00077 of purifying as the reaction of catalyst ester-type hydrolysis, there is good stability, and to part surface promoting agent and organic solvent, there is good tolerance.The characteristic conforms detergent additives of esterase B SE00077, the industrial demand of greenization, can be used for the fields such as washing composition, biological medicine, makeup and fine chemistry industry.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of esterase B SE00077.Wherein, M is albumen marker, swimming lane 1 is e. coli bl21 (DE3) crude protein containing pET-28 α (+)-bse00077 before IPTG induction, swimming lane 2 is e. coli bl21 (DE3) crude protein containing pET-28 α (+)-bse00077 after IPTG induction, and swimming lane 3 is the esterase B SE00077 albumen of purifying.
Fig. 2 is the specificity of esterase B SE00077 to the p-nitrophenyl phenolic ester of different side chain lengths acyl group.
Fig. 3 is the impact of pH on esterase B SE00077 activity.
Fig. 4 is the stability influence of different pH to esterase B SE00077.
Fig. 5 is the impact of temperature on esterase B SE00077 activity.
Fig. 6 is the impact of differing temps on esterase B SE00077 stability.
Fig. 7 is the GC figure that esterase B SE00077 splits styroyl acetate.
Fig. 8 is the impact that concentration of substrate splits esterase B SE00077.
Fig. 9 is that esterase B SE00077 splits the change curve of styroyl acetate reaction with the reaction times.
Embodiment
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: esterase gene bse00077 open reading frame border is determined and design of primers
Extract the genomic dna of genus bacillus (Bacillussp.) SCSIO15121, after genome sequencing, information biology means are utilized to carry out gene annotation to genome, analyze esterase gene wherein, determine the open reading frame of wherein esterase gene bse00077, its gene order is as shown in SEQIDNO.1, total length is 741bp (from initiator codon to terminator codon), the aminoacid sequence of esterase B SE00077 of its coding as shown in SEQIDNO.2, totally 246 amino acid.According to analyzing the esterase gene bse00077 sequence obtained, design total length amplimer is as follows: upstream primer: 5 '-TGCTAGC
cATATGaAAGTTGTGACACCAAAACC-3 ' (underscore is NdeI restriction enzyme site); Downstream primer: 5 '-AGG
aAGCTTtTACCAATCGAGCTTCTCTAAAA-3 ' (underscore is HindIII restriction enzyme site).
Embodiment 2: the clone of esterase gene bse00077 and vector construction
2.1PCR amplification
Primer (the upstream primer: 5 '-TGCTAGC that embodiment 1 is designed
cATATGaAAGTTGTGACAC-CAAAACC-3 '; Downstream primer: 5 '-AGG
aAGCTTtTACCAATCGAGCTTCTCTAAAA-3 ') deliver to the synthesis of Shanghai biotechnology company limited, the primer of synthesis uses TE buffer solution to become final concentration to be 10 μMs, using genus bacillus (Bacillussp.) the SCSIO15121 STb gene extracted as DNA profiling, set up reaction system as shown in table 1:
Table 1PCR reaction system
Use following pcr amplification program amplification esterase gene bse00077:94 DEG C of sex change 5min; 94 DEG C of sex change 1min, 62 DEG C of annealing 30s, 72 DEG C extend 1.5min, carry out 30 circulations; 72 DEG C extend 10min, are cooled to 18 DEG C.
By PCR primer in 0.8% sepharose, electrophoresis 20min under 120V voltage, is placed in gel imaging system and observes, and reclaims the band of about 750bp.The method that PCR primer reclaims test kit according to glue reclaims, and uses 20 μ L sterilized water wash-outs, obtains the PCR primer that purifying reclaims.
2.2 enzymes are cut
PCR primer uses following system to carry out enzyme and cuts, and enzyme cuts time 1.5h.The enzyme system of cutting is: NdeI1 μ L, HindIII1 μ L, DNA<0.3 μ g, and the distilled water of sterilizing adds to 50 μ L.Enzyme reclaims kit method according to glue after cutting and reclaims the PCR primer obtained through double digestion.
The double digestion of plasmid pET-28a (+): picking contains the bacillus coli DH 5 alpha list bacterium colony of this plasmid, incubated overnight.Use plasmid extraction kit to extract plasmid, with NdeI and HindIII by following system double digestion, enzyme cuts time 1.5h.The enzyme system of cutting is: NdeI1 μ L, HindIII1 μ L, DNA<0.3 μ g, and the distilled water of sterilizing adds to 50 μ L.Enzyme cut after in 0.8% sepharose electrophoresis, according to glue reclaim kit method reclaim linear pET-28a (+) carrier obtained through double digestion.
The quick restriction endonuclease that the restriction enzyme that above-mentioned double digestion uses is produced for Thermo company, enzyme cut after purifying reclaim and use nucleic acid purification to reclaim test kit (Magen, HipureGelPureDNAMicroKit), plasmid extraction kit is the Plasmid Miniprep Kit of Shanghai Jierui Biology Engineering Co., Ltd, and working method is by its working instructions.
2.3 connect
Be connected through the PCR primer of double digestion by following system with linear pET-28a (+) carrier of double digestion: double digestion PCR primer 5 μ L, linear pET-28a (+) the carrier 0.5 μ L of double digestion, T4 ligase enzyme (5U/ μ L) 0.5 μ L, connect damping fluid (5 ×) 2 μ L, supply 10 μ L with deionized water; Connecting temperature is 25 DEG C, 20min.Obtain thus connecting product.
2.4 transform and screening
Get 10 μ L connect products add in 50 μ L bacillus coli DH 5 alpha competent cells, ice bath 20 ~ 30min, after in 42 DEG C of water-bath heat shock 90s, add 500 μ LLB liquid nutrient mediums after ice bath 2min, under 37 DEG C of 200rpm rotating speeds, hatch cultivate 30min.Get the LB that a certain amount of bacterium liquid coats containing 50 μ g/mL kantlex dull and stereotyped, cultivate picking individual colonies after 20h.Single bacterium colony extracts plasmid after incubated overnight in 5mLLB substratum, and carry out double digestion checking, what endonuclease bamhi was identical with gene size is positive colony.
2.5 gene nucleotide series measure
The positive colony of screening is delivered to Shanghai Mei Ji biological medicine company limited to check order, sequencing result and esterase gene sequence are compared, be confirmed to be further and esterase gene bse00077 (its nucleotide sequence is as shown in SEQIDNO.1) is inserted in pET-28a (+) plasmid, the entirely true rear confirmation of result obtains pET-28a (+) plasmid (called after pET-28a (+)-bse00077) with esterase gene bse00077, can be used for carrying out next step test.
The high expression of embodiment 3: esterase B SE00077 in e. coli bl21 (DE3)
Prepared by 3.1 e. coli bl21s (DE3) competent cell
1. by a small amount of e. coli bl21 (DE3) bacterial classification access 5mLLB test tube liquid, 200rpm, 37 DEG C of incubated overnight;
2. be inoculated in 200mLLB shaking flask by the inoculum size of 1% volume ratio by the bacterium liquid in test tube, 200rpm, 25 DEG C of incubated overnight, obtain stock culture;
3. cultured shaking flask is cooled to rapidly 0 DEG C in frozen water, stock culture is divided the centrifuge tube being filled to ice precooling (50mL), ice puts several minutes;
4.4 DEG C, the centrifugal 15min of 4000rpm reclaims cell, abandons supernatant;
5. the CaCl of ice-cold 10mL0.1M
2re-suspended cell, 4 DEG C, the centrifugal 10 ~ 15min of 4000rpm reclaims cell;
6. repeat 5, with the CaCl of 10mL0.1M
2re-suspended cell, more than ice bath 1h;
7.4 DEG C, the centrifugal 10min of 4000rpm reclaims cell;
8. cell 2 ~ 3mL that every 50mL stock culture obtains contains 15%DMSO+CaCl
2come resuspended, be sub-packed in 1.5mL centrifuge tube, 50 ~ 100 μ L often manage.-80 DEG C of preservations.Obtain e. coli bl21 (DE3) competent cell thus.
3.2 transform
PET-28a (+)-bse00077 plasmid 0.5 ~ 1 μ L obtained in Example 2 mixes with 50 μ L e. coli bl21 (DE3) competent cells, ice bath 30min, in 42 DEG C of water-bath heat shock 90s, add 500 μ LLB liquid nutrient mediums after ice bath 2min, 37 DEG C of 200rpm cultivate 1h.The kantlex LB of the centrifugal rear coating 50 μ g/mL of culture is dull and stereotyped, selects single bacterium after overnight incubation 20h.Obtain the e. coli bl21 (DE3) containing pET-28 α (+)-bse00077 thus.
The expression and purification of embodiment 4: esterase B SE00077
4.1 protein induced
E. coli bl21 (DE3) containing pET-28a (+)-bse00077 is cultured to OD in LB substratum
600be about 0.85, add IPTG to final concentration 0.2mM, cultivate 18h for 22 DEG C.300mL bacterium liquid 4000rpm, 4 DEG C of centrifugal 20min, collect thalline, and with the resuspended thalline of 30mL (50mM, pH7.4) PBS damping fluid, ultrasonic 400w, super 5s, stops 5s, broken 10min minute, centrifugal, collects supernatant.
The purifying of 4.2 esterase B SE00077 and SDS-PAGE electrophoresis
With the supernatant collected in nickel ion affinity chromatograph column purification 4.1, specific embodiments is as follows: imidazoles wash-out 5 column volumes using 20mM, 40mM imidazoles wash-out 20 ~ 30 column volumes, finally use 3.5mL300mM imidazoles wash-out, collect last 2.5mL elutriant.Carry out desalination with desalting column SephadexG25, concrete operation method carries out with reference to the operational manual of GE company.The expression product of purifying is carried out SDS-PAGE gel electrophoresis, and obtain the esterase B SE00077 (Fig. 1) of purifying, the albumen size of purifying is about 30.2kD, and coincidence theory is expected.
4.3 esterase B SE00077 determinations of activity
Esterase B SE00077 vitality test adopts p-nitrophenyl phenolic ester, and concrete grammar is as follows: the p-NP ester solution 1. preparing 200mM with DMSO; 2. in 0.5mL reaction system, 495 μ LPBSbuffer (50mM, pH8.0) are added, 2 μ L p-NP ester solutions, the pure enzyme liquid of 3 μ L esterase B SE00077 (0.0645 μ g/ μ L); 3., at 38 DEG C, after reaction 2min, add 0.5mL n-propyl alcohol termination reaction, measure absorbancy in 405nm.
Enzyme is lived, and unit definition: 1min is interior is hydrolyzed p-nitrophenyl phenolic ester, and the enzyme amount discharged needed for 1 μM of p-NP is defined as a Ge Meihuo unit.
The zymologic property of embodiment 5: esterase B SE00077
The p-nitrophenyl phenolic ester of the different side chain lengths of 5.1 hydrolysis
According to the condition determination of 4.3, compare the p-nitrophenyl phenolic ester C that esterase B SE00077 is hydrolyzed different lengths acyl group
2-C
12, result is as Fig. 2.Illustrate that esterase B SE00077 is to long-chain p-nitrophenyl phenolic ester poor specificity, and better for the action effect of the p-nitrophenyl phenolic ester of short chain, and best substrate is C
4, i.e. p-NP butyric ester.
5.2 optimal pHs and pH stability
Prepare different buffered soln, these buffered soln have different pH, as shown in table 2, and its concentration is 50mM.
The buffer system of the different pH of table 2
Replaced respectively according to the buffered soln in table 2 by damping fluid (PBS damping fluid) in 4.3 described in condition determination, measure the enzyme activity of Recombinant esterase BSE00077 in the buffered soln of different pH, substrate is p-NP butyric ester.The impact of pH on restructuring esterase B SE00077 activity the results are shown in Figure 3.Time in the PBS buffered soln of pH8.0, the enzymic activity of esterase B SE00077 is the highest, has higher enzymic activity in the buffered soln between pH7.0-8.5; When pH lower than 7 or higher than 9 time, it is active reduces rapidly.The stability of esterase B SE00077 in the buffered soln of 4 DEG C of different pH is shown in Fig. 4.PH is 8.0, and when 9.0, enzymic activity is more stable, and after process 12h, residual activity is respectively 67%, 71%.And under alkali condition crossed by peracid, enzyme forfeiture alive is then comparatively obvious.
5.2 optimum temperutures and temperature stability
Use the PBSpH8.0 of 50mM as damping fluid, p-NP butyric ester, as substrate, according to the reaction system in 4.3, measures enzyme at different temperatures and lives.The optimum temperuture recording esterase B SE00077 catalytic hydrolysis p-NP butyric ester is 38 DEG C (Fig. 5).Enzyme activity between 25-42 DEG C reaches more than 74.50%, and when temperature is greater than 42 DEG C, enzymic activity sharply declines.By esterase B SE00077 pre-treatment under differing temps (25-50 DEG C), separated in time taking-up is pressed measuring method survey enzyme in 4.3 and is lived, and the results are shown in Figure 6.Esterase B SE00077 is when lower than 40 DEG C, and enzyme is lived and kept higher, and after process 12h, remnant enzyme activity still keeps more than 50%; After 40 DEG C of process 12h, remnant enzyme activity is 46.10%; Along with the rising for the treatment of temp, esterase residual enzyme activity declines gradually, and when temperature sharply reduces higher than enzyme when 45 DEG C is alive, after 50 DEG C of process 1h, residual enzyme activity is lost substantially (Fig. 6), illustrates that esterase B SE00077 is better lower than stability when 40 DEG C.
5.3 esterase B SE00077 are hydrolyzed p-nitrophenyl phenolic ester kinetic parameter
Under optimum temperuture 38 DEG C and optimal pH 8.0 condition, change concentration of substrate with different p-nitrophenyl phenolic ester for substrate, the method according to 4.3 measures the change of the speed of reaction of enzyme, adopt Lineweaver-Burk double-reciprocal plot, obtain Km and Vmax of reaction.Measurement result is in table 3.
Table 3 esterase B SE00077 is hydrolyzed the kinetic parameter of p-nitrophenyl phenolic ester
Can be found out by the result in table 3, the Km value that esterase B SE00077 is hydrolyzed p-NP butyric ester is minimum, and Kcat is maximum, illustrate to the avidity of butyric ester and hydrolysis efficiency the highest.
5.4 metal ions, tensio-active agent are on the impact of esterase B SE00077 activity
According to the metal ion in table 4 or kinds of surfactants and corresponding final concentration, metal ion or tensio-active agent process esterase B SE00077 is added in reaction system PBS (pH8.0) buffered soln, reaction conditions is 38 DEG C, hatch 1h, be 100% in contrast with the enzyme work do not added in the reaction system of any ion or tensio-active agent, then measure relative enzyme by 4.3 measuring methods (using p-NP butyric ester as substrate) and live.Metal ion on the impact of esterase B SE00077 enzymic activity in table 4.Compared with the control, Ca
2+activation is had, the Ca of 10mM to the vigor of esterase B SE00077 catalysis p-NP butyric ester
2+under mass action, the vigor of enzyme is up to 153.11%, and the Ca of lower concentration
2+more obvious than the activation of high density.The Mg of lower concentration
2+, K
+on esterase B SE00077 enzyme live without impact, but increase concentration can reduce enzyme live.The Cu of 10mM
2+, Ni
2+, Zn
2+, Mn
2+esterase B SE00077 enzyme is lived and has restraining effect.Tensio-active agent on the impact of esterase B SE00077 enzymic activity in table 4.Under 0.1% and 0.5% two mass concentration, TPP and Tween-80 lives impact not quite to enzyme, and the restraining effect that the Tween-20 of 0.1% concentration lives to enzyme is larger than above-mentioned two kinds of Surfactant Effects, and strengthens along with concentration increases restraining effect.SDS, SDBS, TritonX-100 live to esterase B SE00077 enzyme and have strong rejection capability under 0.1% concentration.
Table 4 metal ion and tensio-active agent are on the impact of esterase B SE00077 vigor
A represents and does not detect
5.5 organic solvents are on the impact of esterase B SE00077 activity
According to the kind of organic solvent shown in table 5 and final concentration, organic solvent process esterase B SE00077 enzyme liquid is added in reaction system PBS (pH8.0) buffered soln, reaction conditions is 38 DEG C, hatch 1h, be 100% in contrast with esterase B SE00077 activity in the reaction system not adding organic solvent, measure the work of relative enzyme according to the measuring method (using p-NP butyric ester as substrate) of 4.3 again, result is as shown in table 5.Under the concentration of 10%, normal heptane has activation to esterase B SE00077, and normal hexane, octane, toluene, dimethylbenzene, dimethyl sulfoxide (DMSO) have no significant effect, and ethanol, acetone, n-propyl alcohol and trichloromethane have restraining effect to esterase B SE00077.Under 20% concentration, normal hexane and normal heptane are lived without impact on enzyme, but other solvents make enzymic activity reduce.Under high density (50%), esterase B SE00077 still keeps higher enzyme to live at normal hexane, normal heptane, toluene and dimethylbenzene, and being up to is 86.25%.These results suggest that esterase B SE00077 can organic solvent-resistant, especially better to the tolerance of normal hexane and normal heptane.
Table 5 organic solvent is on the impact of esterase B SE00077 activity
A represents and can't detect activity, and b represents and do not detect
Embodiment 6: esterase B SE00077 splits styroyl acetate
6.1 organic solvents split the impact of styroyl acetate to esterase B SE00077
In reaction system PBS (pH8.0) buffered soln, add final concentration is the pure enzyme of 0.26mg/mL esterase B SE00077,50mM styroyl acetate substrate, organic solvent in 10% (v/v) table 6, not add organic solvent for contrast, 3h is reacted at 38 DEG C, detect, according to calculated by peak area substrate enantiomer excessive value (ee with gas chromatography chiral post
s), product enantiomeric excess value (ee
p) and transformation efficiency (C), the results are shown in Table 6.
Formula 1:
formula 2:
formula 3:
in formula: A
sand A
rrepresent the peak area of S-styroyl acetate and R-styroyl acetate respectively, B
sand B
rrepresent product (S)-1 phenylethyl alcohol and (R)-1-phenylethyl alcohol peak area respectively, A
0with the peak area reacting rear styroyl acetate before representing reaction respectively with A.
Table 6 organic solvent splits the impact of styroyl acetate to esterase B SE00077
The stereoselectivity impact of organic solvent on esterase B SE00077 is little as can be seen from Table 6, but can improve transformation efficiency to a certain extent.The stereoselectivity taking toluene as solvent is the highest, is that the stereoselectivity of solvent is taken second place with alkanes, but transformation efficiency in toluene is minimum, and the transformation efficiency of alkanes is better.In lower concentration normal heptane, the activity of esterase B SE00077 is activated, and therefore follow-up experiment adopts normal heptane to be solvent.Fig. 7 is the GC figure of the esterase B SE00077 fractionation styroyl acetate adding 10% (v/v) normal heptane group in reaction system, under the katalysis of esterase B SE00077, the styroyl acetate overwhelming majority be converted into (R)-1-phenylethyl alcohol.
6.2 temperature of reaction and pH split the impact of styroyl acetate to esterase B SE00077
In reaction system PBS (pH8.0) buffered soln, add final concentration is the pure enzyme of 0.26mg/mL esterase B SE00077,50mM styroyl acetate substrate, and 10% (v/v) normal heptane is solvent, and the reaction times is 6h.Measure the stereoselectivity that esterase B SE00077 splits styroyl acetate at different temperatures, the results are shown in Table 7.Ee
sreduce afterwards along with temperature raises first to increase with C, between 25-40 DEG C, ee
preduce not obvious.When temperature is higher than 40 DEG C of ee
s, ee
psignificantly reduce with C, this is relevant with the activity of temperatures involved enzyme.
In the reaction system of different pH, add final concentration is the pure enzyme of 0.26mg/mL esterase B SE00077,50mM styroyl acetate substrate, and 10% (v/v) normal heptane is solvent, 30 DEG C of reaction 6h.Measure the stereoselectivity that esterase B SE00077 splits styroyl acetate, the results are shown in Table 7.When pH6.0, ee
pthe highest, but transformation efficiency is minimum, only has 9.2%.In neutral and alkaline conditions, ee
preduce gradually.Consider, the condition after optimization is 30 DEG C, pH7.0PBS damping fluid.
Table 7 temperature of reaction and pH split the impact of styroyl acetate to esterase
A represents PBS damping fluid, and b represents Tris/HCl damping fluid
6.3 tensio-active agents split the impact of styroyl acetate to esterase B SE00077
(30 DEG C with optimal conditions, pH7.0PBS damping fluid), adding final concentration in reaction system is the pure enzyme of 0.26mg/mL, 50mM styroyl acetate substrate, tensio-active agent (tripoly phosphate sodium STPP (TPP), Tween-20, Tween-80) in 0.1% (w/v) table 8, after reaction 6h, sample is used for GC and detects, and the results are shown in Table 8.As can be seen from Table 8, compared with the control, three kinds of tensio-active agents have no significant effect esterase B SE00077 fractionation styroyl acetate.
Table 8 tensio-active agent splits the impact of styroyl acetate to esterase
The impact that 6.4 concentration of substrate split esterase B SE00077
(30 DEG C with optimal conditions, pH7.0PBS damping fluid), adding final concentration in reaction system is the pure enzyme of 0.26mg/mL esterase B SE00077,10-100mM styroyl acetate substrate, 10% (v/v) normal heptane is solvent, after reaction 6h, sample is used for GC and detects, and the results are shown in Figure 8.As can be seen from Figure 8, along with concentration of substrate increases, product enantiomeric excess value ee
pslow increase, but substrate enantiomer excessive value ee
scan decline rapidly with transformation efficiency C.The reaction of the substrate meeting inhibitory enzyme of higher concentration is described, transformation efficiency is declined.
6.5 reaction times split the impact of styroyl acetate to esterase B SE00077
(30 DEG C with optimal conditions, pH7.0PBS damping fluid), adding final concentration in reaction system is the pure enzyme of 0.26mg/mL esterase B SE00077,50mM styroyl acetate substrate, and 10% (v/v) normal heptane is solvent, 500 μ L are taken out at interval of 1h, be extracted with ethyl acetate, anhydrous sodium sulphate dewaters, and adds dodecane as interior mark, detect, according to calculated by peak area substrate enantiomer excessive value (ee with gas chromatography chiral post
s), product enantiomeric excess value (ee
p), transformation efficiency (C) and selectivity (E).Differential responses time esterase B SE00077 split styroyl acetate the results are shown in Table 9 and Fig. 9.
Formula 4:
Can find out from table 9, Fig. 9, along with the prolongation of time, ee
sraise gradually with C, through 3h reaction, transformation efficiency reaches the highest substantially.This may be the carrying out along with reaction, and the acetic acid of generation reduces the pH value in reaction system, thus affects the activity of enzyme, and transformation efficiency is no longer increased.After 3h, the selectivity of esterase B SE00077 reduces gradually.
Table 9 differential responses time esterase B SE00077 splits the change of styroyl acetate
Claims (10)
1. an esterase B SE00077, is characterized in that, its aminoacid sequence is as shown in SEQIDNO.2.
2. the esterase gene bse00077 of a coding esterase B SE00077 according to claim 1.
3. esterase gene bse00077 according to claim 2, is characterized in that, the nucleotide sequence of described esterase gene bse00077 is as shown in SEQIDNO.1.
4. the recombinant expression vector containing esterase gene bse00077 according to claim 2.
5. recombinant expression vector according to claim 4, is characterized in that, described recombinant expression vector is pET-28a (+) carrier.
6. the genetic engineering bacterium containing esterase gene bse00077 according to claim 2.
7. genetic engineering bacterium according to claim 6, is characterized in that, described genetic engineering bacterium is e. coli bl21 (DE3).
8. the application of esterase B SE00077 according to claim 1 in catalysis ester-type hydrolysis.
9. application according to claim 8, is characterized in that, described is applied as the application of esterase B SE00077 in catalysis styroyl acetate hydrolysis generation (R)-1-phenylethyl alcohol.
10. esterase B SE00077 according to claim 1 carries out the application of catalysis under tolerance normal hexane and/or normal heptane environment.
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