CN106119224A - A kind of esterase EstP00714 and encoding gene thereof and application - Google Patents
A kind of esterase EstP00714 and encoding gene thereof and application Download PDFInfo
- Publication number
- CN106119224A CN106119224A CN201610605466.3A CN201610605466A CN106119224A CN 106119224 A CN106119224 A CN 106119224A CN 201610605466 A CN201610605466 A CN 201610605466A CN 106119224 A CN106119224 A CN 106119224A
- Authority
- CN
- China
- Prior art keywords
- estp00714
- esterase
- gene
- enzyme
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
- 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
-
- 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
- C12P7/62—Carboxylic acid esters
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of esterase EstP00714 and encoding gene thereof and application.The present invention clones from pseudomonas (Pseudonocardia antitumoralis) HUP007 and obtains an esterase gene EstP00714, total length is 1146bp, the aminoacid sequence of the esterase EstP00714 of its coding is as shown in SEQ ID NO.2, containing 381 aminoacid.The esterase EstP00714 of the present invention has good toleration to many kinds of metal ions, surfactant and organic solvent；This esterase Estp00714 can be used for preparing (S) methyl mandelate, utilizes esterase EstP00714 as catalyst, has obtained (S) methyl mandelate of substrate enantiomer excessive value more than 95%.The present invention has the advantage that reaction condition is gentle, pollution-free, enantiomeric excess value is high compared with traditional chemical resolution.The esterase EstP00714 of the present invention can be used for the fields such as biological medicine, cosmetics and fine chemistry industry, has the biggest using value.
Description
Technical field:
The invention belongs to biochemical industry and biological technical field, be specifically related to a kind of esterase EstP00714 and coding base thereof
Cause and application.
Background technology:
Esterase is widely present in animal, plant and microorganism, is a class catalyzing hydrolysis or the enzyme forming ester bond, effect
Substrate is typically the aliphatic chain esters less than ten carbon atoms.Esterase belongs to α/β and folds hydrolytic enzyme superfamily, and catalytic center is general
Being made up of serine, aspartic acid/glutamic acid and histidine, conserved sequence is pentapeptide (GXSXG) sequence near serine.
Esterase energy catalyzing hydrolysis, esterification, the multiple chemical reaction such as transesterification, be a kind of critically important industrial biocatalytic agent, by extensively
Apply to the fields such as fine chemistry industry, washing, medicine, food, papermaking, leather processing, weaving, waste water process and feed industry.From
From the point of view of catalysis characteristics, esterase has chemo-selective and the stereoisomerism selectivity of height, and reaction need not coenzyme, reaction bar
Part is gentle, by-product is few.Another distinguishing feature of esterase in production application is that it can be at outphasing system (i.e. oil-water interface)
Or organic facies acts on.In aqueous phase, the usual catalytic hydrolysis reaction of esterase, and in organic facies, it but can catalytic esterification and turning
Esterification.Prepare newtype drug intermediate by the bioconversion of microorganism esterase or remove the non-effective of medicine raceme
Composition, is a kind of important chiral technology, has boundless application prospect, and it can be that synthesis of chiral medicine provides new
Platform, for preparing the method that optical pure compound provides new in a large number.Enzyme process selectivity resolving racemic compound, has three-dimensional special
One property is high, and side reaction is few, and productivity is high, the advantage that product optical purity is good and reaction condition is gentle, so being a kind of by extensively
The method for splitting of accreditation.
Summary of the invention:
It is an object of the invention to provide a kind of alkaline-resisting esterase EstP00714 and encoding gene thereof and application.
The present invention develops from a pseudomonas (Pseudonocardia antitumoralis) HUP007 and obtains one
Plant esterase EstP00714 and encoding gene EstP00714 thereof, construct the recombinant expressed load containing esterase gene EstP00714
Body and genetic engineering bacterium, obtain esterase EstP00714 after culturing gene engineering bacteria, it can be applicable to be catalyzed ester-type hydrolysis reaction.
First purpose of the present invention is to provide a kind of esterase EstP00714, its aminoacid sequence such as SEQ ID NO.2 institute
Show.
Second object of the present invention is to provide a kind of esterase gene encoding described esterase EstP00714
EstP00714。
Preferably, the nucleotide sequence of described esterase gene EstP00714 is as shown in SEQ ID NO.1.
Present invention also offers a kind of recombinant expression carrier containing described esterase gene EstP00714.Described table
Reach carrier, preferably pET-28a (+) carrier.
Present invention also offers a kind of genetic engineering bacterium containing described esterase gene EstP00714.Described gene
Engineering bacteria, preferably e. coli bl21 (DE3).
Third object of the present invention is to provide described esterase EstP00714 application in catalysis ester-type hydrolysis.
Preferably, described application be esterase EstP00714 catalysis split (±)-methyl mandelate obtains (S)-mandelic acid
Application in methyl ester.
Fourth object of the present invention is to provide described esterase EstP00714 at tolerance Mg2+、Li+、Na+、K+、Tween-
20, the application being catalyzed is carried out under TritonX-100, normal hexane, isobutyltrimethylmethane., heptane, DMSO and/or Decanol environment.
The esterase gene Estp00714 of the present invention is from pseudomonas (Pseudonocardia antitumoralis)
HUP007, is saved in Chinese Academy of Science Nanhai Ocean Research Institute's laboratory.The present invention utilizes bioinformatic analysis method, comparison
Obtain an esterase gene EstP00714.By the method for PCR, from above-mentioned pseudomonas (Pseudonocardia
Antitumoralis) in HUP007, clone obtains an esterase gene EstP00714, and total length is that 1146bp is (from start codon
To termination codon), the aminoacid sequence of the esterase EstP00714 of its coding is as shown in SEQ ID NO.2, containing 381 ammonia
Base acid.By esterase gene EstP00714 and expression vector pET-28a (+) be connected after convert e. coli bl21 (DE3), cultivate
And after abduction delivering, obtained recombinant expressed esterase EstP00714.The esterase EstP00714 of the present invention to various metals from
Son, surfactant and organic solvent have good toleration；This esterase Estp00714 can be used for preparing (S)-mandelic acid first
Ester, utilizes esterase EstP00714 as catalyst, has obtained (S)-mandelic acid first of substrate enantiomer excessive value more than 95%
Ester.The present invention has the advantage that reaction condition is gentle, pollution-free, enantiomeric excess value is high compared with traditional chemical resolution.This
The esterase EstP00714 of invention can be used for the fields such as biological medicine, cosmetics and fine chemistry industry, has the biggest application valency
Value.
Accompanying drawing illustrates:
Fig. 1 is the SDS-PAGE of esterase EstP00714.Wherein, M is albumen marker, and swimming lane 1 is induced for IPTG
Contain afterwards pET-28a (+) E.coli BL21 (DE3) the albumen supernatant of-EstP00714, swimming lane 2 contains after inducing for IPTG
PET-28a (+) E.coli BL21 (DE3) total protein of-EstP00714, swimming lane 3 is the esterase EstP00714 albumen of purification,
Swimming lane 4 for before IPTG induction containing pET-28a (+) E.coli BL21 (DE3) total protein of-EstP00714.
Fig. 2 is the esterase EstP00714 specificity to the p-nitrophenyl phenolic ester of different side chain lengths.
Fig. 3 is the pH impact on esterase EstP00714 activity.
Fig. 4 is the different pH stability influences to esterase EstP00714.
Fig. 5 is the temperature impact on esterase EstP00714 activity.
Fig. 6 is the different temperatures impact on esterase EstP00714 stability.
Fig. 7 is concentration of substrate to be split esterase EstP00714 (±) impact of-methyl mandelate.
Fig. 8 is the response time to be split esterase EstP00714 (±) impact of-methyl mandelate.
Fig. 9 be (±)-methyl mandelate gas chromatogram；Wherein, S represents (S)-methyl mandelate, and R represents (R)-almond
Acid methyl ester.
Figure 10 be esterase EstP00714 to (±) the selective hydrolysis gas chromatogram of-methyl mandelate；Wherein, S represents
(S)-methyl mandelate, R represents (R)-methyl mandelate.
Detailed description of the invention:
Following example are to further illustrate the present invention rather than limitation of the present invention.
The esterase gene EstP00714 of the present invention derives from the pseudomonas (Pseudonocardia of gene order-checking
Antitumoralis) HUP007, this bacterium is saved in Chinese Academy of Science Nanhai Ocean Research Institute's laboratory.
Embodiment 1: esterase gene EstP00714 open reading frame border determines and design of primers
Extract the genomic DNA of pseudomonas (Pseudonocardia antitumoralis) HUP007, through full genome
After group order-checking, utilize bioinformatics means that genome is annotated, analyze esterase gene therein, it is determined that esterase gene
The open reading frame of EstP00714, its gene order as shown in SEQ ID NO.1, total length be 1146bp (from start codon to
Termination codon), the aminoacid sequence of esterase EstP00714 of its coding as shown in SEQ ID NO.2, totally 381 aminoacid.
The esterase gene EstP00714 sequence obtained according to analysis, design total length amplimer is as follows: forward primer: 5 '-
CATGAATTCGTGCAGATCCAGGGTCAC-3 ' (underscore is EcoR I restriction enzyme site)；Downstream primer: 5 '-
CACCTCGAGTTAAAGGCAACTGCCAAG-3 ' (underscore is Xho I restriction enzyme site).
Embodiment 2: the clone of esterase gene EstP00714 and vector construction
2.1PCR amplification
Primer (the forward primer: 5 '-CAT that embodiment 1 is designedGAATTCGTGCAGATCCAGGGTCAC-3′；Downstream is drawn
Thing: 5 '-CACCTCGAGTTAAAGGCAACTGCCAAG-3 ') deliver to the synthesis of Shanghai biological engineering company limited, the primer of synthesis
TE buffer solution is used to become final concentration of 10 μMs, with the pseudomonas (Pseudonocardia antitumoralis) extracted
The STb gene of HUP007, as DNA profiling, sets up reaction system as shown in table 1:
Table 1 PCR reaction system
Following PCR amplification program is used to expand EstP00714:95 DEG C of degeneration 5min of esterase gene；95 DEG C of degeneration 30s, 55
DEG C annealing 30s, 72 DEG C extend 2min, carry out 32 circulations；72 DEG C extend 10min, are cooled to 18 DEG C.
By PCR primer in 0.8% agarose gel, electrophoresis 20min under 120V voltage, it is placed in gel imaging system sight
Examine, reclaim the band of about 1146bp.The method that PCR primer reclaims test kit according to glue reclaims, and uses 30 μ L sterilized water
Eluting, obtains the PCR primer that purification reclaims.
2.2 enzyme action
The PCR primer reclaimed by purification uses following enzyme action system to carry out double digestion, enzyme action time 1.5h.Enzyme action system
For: EcoR I 2 μ L, Xho I 2 μ L, DNA < 0.3 μ g, the distilled water of sterilizing adds to 40 μ L.Test kit is reclaimed according to glue after enzyme action
Method reclaim through the PCR primer of double digestion.
Plasmid pET-28a (+) double digestion: picking contain plasmid pET-28a (+) bacillus coli DH 5 alpha list bacterium colony, mistake
Night cultivates.Use plasmid extraction kit to extract plasmid, carry out double digestion, enzyme with EcoR I and Xho I by following enzyme action system
Cut time 1.5h.Enzyme action system is: EcoR I 2 μ L, Xho I 2 μ L, and < 0.3 μ g, the distilled water of sterilizing adds to 40 μ to plasmid DNA
L.After enzyme action in 0.8% agarose gel electrophoresis, reclaim kit method according to glue and reclaim through the linear pET-of double digestion
28a (+) carrier.
The restricted enzyme that above-mentioned double digestion uses is the quick restriction endonuclease that Thermo company produces.Purification after enzyme action
Reclaim and use nucleic acid purification to reclaim test kit (Magen, Hipure Gel Pure DNA Micro Kit), plasmid extraction reagent
Box is the Plasmid Miniprep Kit of Shanghai Jierui Biology Engineering Co., Ltd, and operational approach presses its operation instructions.
2.3 connect
By through the PCR primer of double digestion and double digestion pET-28a (+) carrier is attached by following system: double enzymes
The PCR primer 5 μ L cut, the pET-28a of double digestion (+) carrier 1 μ L, T4 ligase (5U/ μ L) 0.5 μ L, connect buffer (5 ×)
1 μ L, supplies 5 μ L with deionized water, and connecting temperature is 20 DEG C, Connection Time 20min.Thus obtain connecting product.
2.4 convert and screening
Take 5 μ L connect products add in 50 μ L escherichia coli DH5a competent cells, ice bath 20～30min, after in 42 DEG C
Water-bath heat shock 90s, adds 500 μ L LB fluid mediums after ice bath 2min, under 37 DEG C of 200rpm rotating speeds, hatch cultivation 1h.Take
A certain amount of bacterium solution coats the LB flat board containing 50 μ L/mL kanamycin, cultivates picking individual colonies after 20h.Single bacterium colony is in 5mL
Extracting plasmid in LB culture medium after incubated overnight, carry out double digestion checking, what endonuclease bamhi was identical with gene size is the positive
Clone.
2.5 gene nucleotide series measure
The positive colony of screening is delivered to Shanghai Mei Ji biological medicine company limited check order, sequencing result and esterase base
Because the nucleotide sequence (as shown in SEQ ID NO.1) of EstP00714 is compared, further confirm that it is by esterase gene
EstP00714 be inserted into pET-28a (+) in plasmid, confirm to obtain with esterase gene EstP00714's after result is completely correct
PET-28a (+) plasmid (named pET-28a (+)-EstP00714), can be used for carrying out next step test.
Embodiment 3: the esterase EstP00714 high efficient expression in E.coli BL21 (DE3)
Prepared by 3.1 e. coli bl21s (DE3) competent cell
1. a small amount of e. coli bl21 (DE3) strain is accessed in 5mL LB test tube liquid, 200rpm, 37 DEG C of incubated overnight；
2. by the inoculum concentration of 1% volume ratio, e. coli bl21 (DE3) bacterium solution in test tube is inoculated into 200mL LB to shake
In Ping, 200rpm, 20 DEG C of incubated overnight, obtain stock culture；
3. cultured shaking flask is rapidly cooled to 0 DEG C in frozen water, by the centrifuge tube of stock culture subpackage to ice pre-cooling
(50mL), ice puts several minutes；
4.4 DEG C, 4000rpm is centrifuged 10min and reclaims cell, abandons supernatant；
The CaCl of the most ice-cold 10mL 0.1M2Re-suspended cell, 4 DEG C, it is thin that 4000rpm is centrifuged 10～15min recovery
Born of the same parents；
6. repeat 5, with the CaCl of 10mL 0.1M2Re-suspended cell, more than ice bath 30min；
7.4 DEG C, 4000rpm is centrifuged 10min and reclaims cell；
8. the cell 5mL that every 50mL stock culture obtains is containing 7.5%DMSO+CaCl2Come resuspended, be sub-packed in 1.5mL from
Heart pipe, 50～100 μ L often manage.-80 DEG C of preservations.Thus obtain e. coli bl21 (DE3) competent cell.
3.2 convert
The pET-28a that obtains in Example 2 (+)-EstP00714 plasmid 0.5～1 μ L and 50 μ L e. coli bl21s
(DE3) competent cell mixing, ice bath 30min, in 42 DEG C of water-bath heat shock 90s, add 500 μ L LB liquid after ice bath 2min
Culture medium, 37 DEG C of 200rpm cultivate 1h.It is coated with the kanamycin LB flat board of 50 μ L/mL after culture is centrifugal, chooses after cultivating 20 h
Menu bacterium.Thus obtain containing pET-28a (+) e. coli bl21 (DE3) of-EstP00714.
Embodiment 4: the expression of esterase EstP00714 and purification
4.1 it is protein induced
Containing pET-28a (+) e. coli bl21 (DE3) of-EstP00714 cultivates to OD in LB culture medium600For
About 0.85, add IPTG to concentration 0.2mM, cultivate 16h for 22 DEG C.300mL bacterium solution 4000rpm, 4 DEG C of centrifugal 20min, collect bacterium
Body, with 30mL (50mM, pH 7.4) the resuspended thalline of Tris-HCl buffer, ultrasonic 400w, super 4s, stops 6s, broken 15min and divides
Clock, centrifugal, collect supernatant.
The purification of 4.2 esterase EstP00714 and SDS-PAGE electrophoresis
With the supernatant collected in nickel ion affinity chromatograph column purification 4.1, specific embodiments is as follows: use the imidazoles of 25mM
5 column volumes of eluting, 40mM imidazoles eluting 20～30 column volumes, finally use 3.5mL 100mM imidazoles eluting, collects last
3mL eluent.Carrying out desalination with desalting column SephadexG25, concrete operation method is carried out with reference to the workbook of GE company.
The esterase of purification being carried out PAGE gel electrophoresis, obtains the esterase EstP00714 (see Fig. 1) of purification, the albumen of purification is big
Little about 41.2kD, coincidence theory is expected.
4.3 esterase EstP00714 determinations of activity
Esterase EstP00714 vitality test uses p-nitrophenyl phenolic ester, and concrete grammar is as follows: 1. with acetontrile 5mM's
P-nitrophenyl phenolic ester；2. in 0.2mL reaction system, add 187.5 μ L Tris-HCl buffer (50mM, pH 8.0), 10 μ L
P-nitrophenyl phenolic ester, 2.5 μ L esterase EstP00714 pure enzyme liquid (0.0645 μ g/ μ L)；3., at 20 DEG C, after reaction 4min, 50 are added
μ L normal propyl alcohol terminates reaction, measures absorbance in 405nm.
Enzyme is lived, and unit definition: 1min is interior hydrolyzes p-nitrophenyl phenolic ester, and the enzyme amount needed for discharging 1 μM of paranitrophenol is defined as
One enzyme unit alive.
Embodiment 5: the zymologic property of esterase EstP00714
The p-nitrophenyl phenolic ester of the 5.1 different side chain lengths of hydrolysis
According to the condition determination of 4.3, compare the p-nitrophenyl phenolic ester C of esterase EstP00714 hydrolysis different length acyl group2-
C12, result such as Fig. 2.Illustrate esterase EstP00714 to long-chain p-nitrophenyl phenolic ester poor specificity, and for short chain to nitre
The action effect of base phenol ester is preferable, and optimal substrate is C4, i.e. paranitrophenol butyrate.
5.2 optimum pHs and pH stability
Preparing different buffer solution, these buffer solution have different pH, and as shown in table 2, its concentration is 50mM.
The buffer system of the different pH of table 2
By the buffer (pH 8.0Tris-HCl buffer) described in condition determination in 4.3 according to the buffer solution in table 2
Being replaced respectively, measure the enzyme activity of esterase EstP00714 in the buffer solution of different pH, substrate is paranitrophenol butanoic acid
Ester.The result that affects of esterase EstP00714 activity is shown in Fig. 3 by pH.In the Tris-HCl buffer solution of pH9.0, esterase
The enzymatic activity of EstP00714 is the highest, has higher enzymatic activity when pH value is between 8.0-10.0.When pH is less than 7, its activity is fast
Prompt drop is low.Esterase EstP00714 stability in the buffer of 20 DEG C of different pH is shown in Fig. 4.PH is enzymatic activity when 8.0-12.0
More stable, after processing 5h, residual activity is more than 60%.And under the conditions of peracid, enzyme forfeiture alive is the most obvious.
5.3 optimum temperatures and temperature stability
Using the Tris-HCl pH8.0 of 50mM as buffer, paranitrophenol butyrate is as substrate, in 4.3
Reaction system, under different temperatures (4～65 DEG C) measure enzyme live.Record esterase EstP00714 catalyzing hydrolysis paranitrophenol
The optimum temperature of butyrate is 40 DEG C (Fig. 5).Enzyme activity between 30-55 DEG C reaches more than 80%, and when temperature is more than 55 DEG C, enzyme is lived
Impatient acute decline.By esterase EstP00714 pretreatment under different temperatures (30-50 DEG C), separated in time takes out to be pressed in 4.3
Assay method survey enzyme live, result is shown in Fig. 6.Esterase EstP00714 is when less than 40 DEG C, and enzyme is lived and kept higher, residual after processing 1h
Remaining enzyme is lived and is still kept more than 70%.Along with the rising for the treatment of temperature, esterase EstP00714 residual enzyme activity is gradually reduced, and works as temperature
When degree is higher than 50 DEG C, enzyme is lived and is drastically reduced, and after processing 45min at 50 DEG C, residual enzyme activity loses (Fig. 6) substantially, and esterase is described
EstP00714 stability when less than 40 DEG C is preferable.
The impact on esterase EstP00714 activity of 5.4 metal ions
According to the species of metal ion in table 3 and corresponding final concentration, buffer in reaction system Tris-HCl (pH 9.0)
Solution adds metal ion treatment esterase EstP00714, lives as 100% with the enzyme being not added with in the reaction system of any ion
As comparison, hatching 3h at 20 DEG C, the assay method (using paranitrophenol butyrate as substrate) by 4.3 measures relative enzyme
Live.Metal ion is shown in Table 3 to the impact of esterase EstP00714 enzymatic activity.Compared with the control, the Ba of 2mM2+、Fe3+、Al3+、Cu2+、
Mn2+、Mg2+、Ca2+The vigor of esterase EstP00714 catalysis paranitrophenol butyrate there is activation, the Mg of 2mM2+Concentration
The vigor of the lower enzyme of effect reaches 127.96%.The Li of low concentration+、Ni2+、Na+、K+Impact of living esterase EstP00714 enzyme is less, and
Co2+And Zn2+The vigor of esterase EstP00714 there is obvious inhibitory action.With the increase of concentration of metal ions, inhibitory action
Strengthen.The Cu of 10mM2+、Fe3+、Zn2+The suppression living esterase EstP00714 enzyme is stronger.
The impact on esterase EstP00714 activity of table 3 metal ion
The impact on esterase EstP00714 activity of 5.5 surfactants
According to kind and the corresponding final concentration of the surfactant in table 4, at reaction system Tris-HCl (pH 9.0)
Buffer solution adds surfactant and processes esterase EstP00714, with the enzyme being not added with in the reaction system of surfactant
Work is 100% conduct comparison, hatches 3h, the assay method (using paranitrophenol butyrate as substrate) by 4.3 at 20 DEG C
Measure relative enzyme to live.Surfactant is shown in Table 4 to the impact of esterase EstP00714 enzymatic activity.TritonX-100 is 0.1% He
Impact of under 0.5% two mass concentration living enzyme is little, Tween-20 and S10 of 0.1% concentration is alive on enzyme, and impact is little, and
Strengthen along with concentration increases inhibitory action.Esterase EstP00714 enzyme is lived under 0.1% concentration and is had by force by SDS, SDBS, CTAB
Inhibition.
The impact on esterase EstP00714 activity of table 4 surfactant
The impact on esterase EstP00714 activity of 5.6 organic solvents
According to organic solvent kind shown in table 5 and corresponding final concentration, delay in reaction system Tris-HCl (pH 9.0)
Adding organic solvent in dissolved liquid and process esterase EstP00714 enzyme liquid, treatment conditions are 20 DEG C, hatch 3h, organic not add
In the reaction system of solvent, esterase active is 100% conduct comparison, and the assay method according still further to 4.3 is (with paranitrophenol butanoic acid
Ester is as substrate) measure the work of relative enzyme, result is as shown in table 5.Under the concentration of 10%, normal hexane and isobutyltrimethylmethane. are to esterase
EstP00714 has activation, heptane and DMSO and has no significant effect, and esterase EstP00714 is had not by other organic solvents
Inhibitory action with degree.Under high concentration (50%), esterase EstP00714 keeps higher in normal hexane, heptane, Decanol
Enzyme live, residual enzyme activity is more than 50%.These results suggest that this esterase EstP00714 is resistant to organic solvent.
The impact on esterase EstP00714 activity of table 5 organic solvent
Embodiment 6: esterase EstP00714 fractionation (±)-methyl mandelate
6.1pH esterase EstP00714 is split (±) impact of-methyl mandelate
The final concentration of 60 μ g/mL pure enzymes of esterase EstP00714 are added in different pH reaction systems, 30mM (±)-almond
Acid methyl ester substrate, reacts 2h at 40 DEG C, detects with gas chromatography chiral post, according to calculated by peak area substrate enantiomer excessive value
(e.e.s) and conversion ratio (C), the results are shown in Table 6.As can be seen from the table, when pH 8.5, e.e.sReach 99%, but conversion ratio
Also reaching 99%, therefore the yield of (S)-methyl mandelate is the lowest, considers, and the pH after optimization is 7.0.
Formula 1:Formula 2:
In formula: ASAnd ARRepresent the peak area of (S)-methyl mandelate and (R)-methyl mandelate, A respectively0With A table respectively
The peak area of methyl mandelate after showing before reacting and reacting.
Table 6 pH esterase EstP00714 is split (±) impact of-methyl mandelate
6.2 organic solvents esterase EstP00714 is split (±) impact of-methyl mandelate
Final concentration of 60 μ g/mL esterase EstP00714 are added in reaction system Tris-HCl (pH 7.0) buffer solution
Pure enzyme, 30mM (±)-methyl mandelate substrate, the organic solvent in 10% (v/v) table 7, with do not add organic solvent for comparison,
React 2h at 40 DEG C, detect with gas chromatography chiral post, according to calculated by peak area substrate enantiomer excessive value (e.e.s) and turn
Rate (C), the results are shown in Table 7.
Table 7 organic solvent esterase EstP00714 is split (±) impact of-methyl mandelate
The existence of most organic solvents reduces the stereo selectivity of esterase EstP00714 as can be seen from Table 7
And conversion ratio.The stereo selectivity of esterase EstP00714 is affected relatively small by ethanol.
6.3 reaction temperatures esterase EstP00714 is split (±) impact of-methyl mandelate
The final concentration 60 μ g/mL pure enzyme of esterase EstP00714 is added in reaction system Tris-HCl (pH 7.0) buffer,
30mM (±)-methyl mandelate substrate, reacting 2h at different temperatures, Chiral gas chromatography measures esterase EstP00714 and splits
The stereo selectivity of (±)-methyl mandelate, the results are shown in Table 8.
As can be seen from Table 8, e.e.sIncrease with the rising of temperature with conversion ratio, between 45-50 DEG C, e.e.sBase
This no change, maintains 95%, for obtaining (S)-methyl mandelate maximum yield, selects at identical e.e.sLower conversion ratio is minimum
Temperature be optimum temperature, i.e. 40 DEG C.
Table 8 temperature esterase EstP00714 is split (±) impact of-methyl mandelate
6.4 surfactants esterase EstP00714 is split (±) impact of-methyl mandelate
With optimal conditions (40 DEG C, pH7.0 Tris-HCl buffer), reaction system adds final concentration 60 μ g/mL
The pure enzyme of esterase EstP00714,30mM (±)-methyl mandelate substrate, the surfactant in 0.01% (w/v) table 9
(Tween-20, Tween-80, TritonX-100, sodium tripolyphosphate), with do not add surfactant for comparison, reaction 2h after, sample
Product are used for gas chromatographic detection, the results are shown in Table 9.As can be seen from Table 9, compared with the control, the feelings existed at surfactant
Under condition, e.e.sHaving reduction in various degree, in the presence of sodium tripolyphosphate, conversion ratio improves, but e.e.sSubstantially reduce.
TritonX-100 esterase EstP00714 is split (±) impact of-methyl mandelate is relatively small.
Table 9 surfactant esterase EstP00714 is split (±) impact of-methyl mandelate
6.5 concentration of substrate esterase EstP00714 is split (±) impact of-methyl mandelate
With optimal conditions (40 DEG C, pH7.0 Tris-HCl buffer), reaction system adds final concentration of 60 μ g/
The pure enzyme of esterase EstP00714 of mL, 20-90mM (±)-methyl mandelate substrate, after reaction 2h, sample detects for GC, result
See Fig. 7.It can be seen from figure 7 that along with the increase of concentration of substrate, e.e.sCan decline rapidly with conversion ratio.Illustrate higher
Under concentration of substrate, the conversion ratio of reaction declines.When concentration of substrate is 20mM, e.e.sReach more than 99%, but conversion ratio also reaches
To more than 99%, (S)-methyl mandelate productivity is the lowest, and therefore selecting 30mM is the suitableeest concentration of substrate.
6.6 response time esterase EstP00714 is split (±) impact of-methyl mandelate
With optimal conditions (40 DEG C, pH7.0 Tris-HCl buffer), reaction system adds final concentration of 60 μ g/
The pure enzyme of esterase EstP00714 of mL, 30mM (±)-methyl mandelate substrate, take out 500 μ L every different time, use ethyl acetate
Extraction, anhydrous sodium sulfate removes water, and gas chromatography chiral post detects, and result is shown in Fig. 8.As can be seen from Figure 8, along with the response time
Prolongation, e.e.sGradually rise with C.After reaction 2h, e.e.sInconspicuous with conversion ratio change, therefore esterase EstP00714 catalysis
The optimum time of (±)-methyl mandelate is 2h, the e.e. under optimum reaction conditionssBeing 95%, conversion ratio is 86%.The suitableeest bar
Under part, before and after reaction, gas chromatogram is shown in Fig. 9 and Figure 10.
Claims (10)
1. an esterase EstP00714, it is characterised in that its aminoacid sequence is as shown in SEQ ID NO.2.
2. the esterase gene EstP00714 of the esterase EstP00714 that a kind encodes described in claim 1.
Esterase gene EstP00714 the most according to claim 2, it is characterised in that described esterase gene EstP00714
Nucleotide sequence as shown in SEQ ID NO.1.
4. the recombinant expression carrier containing the esterase gene EstP00714 described in Claims 2 or 3.
Recombinant expression carrier the most according to claim 4, it is characterised in that described expression vector be pET-28a (+) carry
Body.
6. the genetic engineering bacterium containing the esterase gene EstP00714 described in Claims 2 or 3.
Genetic engineering bacterium the most according to claim 6, it is characterised in that described genetic engineering bacterium is e. coli bl21
(DE3)。
8. the application in catalysis ester-type hydrolysis of the esterase EstP00714 described in claim 1.
Application the most according to claim 8, it is characterised in that described application is that esterase EstP00714 splits in catalysis
(±)-methyl mandelate obtains the application in (S)-methyl mandelate.
10. the esterase EstP00714 described in claim 1 is at tolerance Mg2+、Li+、Na+、K+、Tween-20、TritonX-100、
The application being catalyzed is carried out under normal hexane, isobutyltrimethylmethane., heptane, DMSO and/or Decanol environment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610605466.3A CN106119224B (en) | 2016-07-27 | 2016-07-27 | Esterase EstP00714 and coding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610605466.3A CN106119224B (en) | 2016-07-27 | 2016-07-27 | Esterase EstP00714 and coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106119224A true CN106119224A (en) | 2016-11-16 |
| CN106119224B CN106119224B (en) | 2019-12-10 |
Family
ID=57254247
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610605466.3A Active CN106119224B (en) | 2016-07-27 | 2016-07-27 | Esterase EstP00714 and coding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106119224B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330154A (en) * | 2018-01-26 | 2018-07-27 | 中国科学院南海海洋研究所 | Applications of the esterase EstP00714 in catalysis resolution of racemic bergamio obtains (S)-linalool |
| CN109652470A (en) * | 2018-12-29 | 2019-04-19 | 浙江工业大学 | A kind of application of lipase in fractionation (R, S)-methyl mandelate |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560912A (en) * | 2015-01-06 | 2015-04-29 | 浙江大学 | Esterase as well as encoding genes and application thereof |
| CN105543191A (en) * | 2016-02-24 | 2016-05-04 | 中国科学院南海海洋研究所 | Esterase PHE21 and encoding gene and application thereof |
-
2016
- 2016-07-27 CN CN201610605466.3A patent/CN106119224B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560912A (en) * | 2015-01-06 | 2015-04-29 | 浙江大学 | Esterase as well as encoding genes and application thereof |
| CN105543191A (en) * | 2016-02-24 | 2016-05-04 | 中国科学院南海海洋研究所 | Esterase PHE21 and encoding gene and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 无: "Multispecies:hypothetical protein[Pseudomonas]", 《NCBI:WP_050704223.1》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330154A (en) * | 2018-01-26 | 2018-07-27 | 中国科学院南海海洋研究所 | Applications of the esterase EstP00714 in catalysis resolution of racemic bergamio obtains (S)-linalool |
| CN108330154B (en) * | 2018-01-26 | 2020-08-18 | 中国科学院南海海洋研究所 | Application of esterase EstP00714 in catalytic resolution of racemic linalyl acetate to obtain (S) -linalool |
| CN109652470A (en) * | 2018-12-29 | 2019-04-19 | 浙江工业大学 | A kind of application of lipase in fractionation (R, S)-methyl mandelate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106119224B (en) | 2019-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104774825B (en) | Nitrilase mutants and its application | |
| CN105543191B (en) | A kind of esterase PHE21 and its encoding gene and application | |
| CN108285895A (en) | A kind of esterase EstC11 and its encoding gene and application | |
| CN104673810A (en) | Malic dehydrogenase gene MIMDH1 and recombinant expression vector thereof | |
| CN105543190B (en) | Esterase BSE00077 and coding gene and application thereof | |
| CN106244569A (en) | A kind of esterase EstC10 and encoding gene thereof and application | |
| CN104293744B (en) | Talaromyces thermophilus derived lipase mutant and application thereof | |
| CN105802935B (en) | A kind of esterase PHE14 and its encoding gene and application | |
| CN104673809B (en) | A kind of malate dehydrogenase gene and its recombinant expression carrier | |
| CN106591258B (en) | A kind of lipase gene, carrier, engineering bacteria and its application | |
| CN106119224A (en) | A kind of esterase EstP00714 and encoding gene thereof and application | |
| CN104745547A (en) | Epoxide hydrolase mutant, engineering bacteria and application of epoxide hydrolase mutant | |
| CN103981160A (en) | Thermomyces lanuginosus lipase mutant, coding genes, and applications of thermomyces lanuginosus lipase mutant | |
| CN104673814B (en) | A kind of L threonine aldolases for coming from enterobacter cloacae and its application | |
| CN106754818B (en) | Heat-resistant esterase mutant and preparation method and application thereof | |
| CN105543192B (en) | A kind of esterase B SE01701 and its encoding gene and application | |
| CN105238769B (en) | A kind of lipase LIPASE7 and its encoding gene and application | |
| CN112175919A (en) | Lactone hydrolase mutant and application thereof | |
| Su et al. | Functional expression of Serratia marcescens H30 lipase in Escherichia coli for efficient kinetic resolution of racemic alcohols in organic solvents | |
| KR101180239B1 (en) | Improved halohydrin epoxidase | |
| CN106701629B (en) | Pseudomonas fluorescens, pseudomonas fluorescens lipase LIPASEBJ10 and application thereof | |
| CN105132394B (en) | A kind of lipase LIPASE6 and its encoding gene and application | |
| CN105296446B (en) | A kind of lipase L-1 and its encoding gene and application | |
| CN106085985B (en) | A kind of esterase WDEst9 and its encoding gene and application | |
| CN104560912B (en) | Esterase and its encoding gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |