CN106119224B - Esterase EstP00714 and coding gene and application thereof - Google Patents
Esterase EstP00714 and coding gene and application thereof Download PDFInfo
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- CN106119224B CN106119224B CN201610605466.3A CN201610605466A CN106119224B CN 106119224 B CN106119224 B CN 106119224B CN 201610605466 A CN201610605466 A CN 201610605466A CN 106119224 B CN106119224 B CN 106119224B
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- ITATYELQCJRCCK-QMMMGPOBSA-N methyl (2s)-2-hydroxy-2-phenylacetate Chemical compound COC(=O)[C@@H](O)C1=CC=CC=C1 ITATYELQCJRCCK-QMMMGPOBSA-N 0.000 claims abstract description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- ITATYELQCJRCCK-UHFFFAOYSA-N Mandelic Acid, Methyl Ester Chemical compound COC(=O)C(O)C1=CC=CC=C1 ITATYELQCJRCCK-UHFFFAOYSA-N 0.000 claims description 22
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 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)
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- 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
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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
- C12P7/62—Carboxylic acid esters
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Abstract
The invention discloses esterase EstP00714 and a coding gene and application thereof. The invention clones an esterase gene EstP00714 from Pseudomonas antipruralis (HUP 007), the total length is 1146bp, the amino acid sequence of the encoded esterase EstP00714 is shown in SEQ ID NO.2, and the esterase gene EstP00714 contains 381 amino acids. The esterase EstP00714 has good tolerance to various metal ions, surfactants and organic solvents; the esterase EstP00714 can be used for preparing (S) -methyl mandelate, and the esterase EstP00714 is used as a catalyst to obtain the (S) -methyl mandelate with the substrate enantiomer excess value of more than 95%. Compared with the traditional chemical resolution, the method has the advantages of mild reaction conditions, no pollution and high enantiomeric excess value. The esterase EstP00714 can be used in the fields of biological medicine, cosmetics, fine chemical engineering and the like, and has great application value.
Description
The technical field is as follows:
the invention belongs to the fields of biochemical engineering and biotechnology, and particularly relates to esterase EstP00714 and a coding gene and application thereof.
Background art:
Esterases are widely found in animals, plants and microorganisms, and are enzymes that catalyze hydrolysis or the formation of ester bonds, and the substrates for the action are usually esters with less than ten carbon atoms in the aliphatic chain. Esterases belong to the alpha/beta sheet hydrolase superfamily, catalytic centers are generally composed of serine, aspartate/glutamate, and histidine, and conserved sequences are pentapeptide (GXSXG) sequences near serine. Esterase can catalyze various chemical reactions such as hydrolysis, esterification, transesterification and the like, is an important industrial biocatalyst, and is widely applied to the fields of fine chemical engineering, washing, medicines, food, papermaking, leather processing, textile, wastewater treatment, feed industry and the like. From the catalytic property, the esterase has high chemoselectivity and stereoselectivity, and the reaction does not need coenzyme, has mild reaction condition and less byproducts. Another significant feature of esterases in production applications is their ability to function in heterogeneous systems (i.e., oil-water interfaces) or in the organic phase. In the aqueous phase, esterases generally catalyze hydrolysis reactions, while in the organic phase, esterases catalyze esterification and transesterification reactions. The method for preparing the novel drug intermediate or removing the non-effective components of the drug racemate by the biotransformation of microbial esterase is an important chiral technology, has very wide application prospect, can provide a new platform for synthesizing chiral drugs, and provides a new method for preparing optical pure compounds in large quantities. The enzymatic method for selectively resolving the racemate compound has the advantages of high stereospecificity, less side reaction, high yield, good optical purity of the product and mild reaction conditions, so the method is a widely accepted resolving method.
the invention content is as follows:
The invention aims to provide an alkali-resistant esterase EstP00714 and a coding gene and application thereof.
The esterase EstP00714 and a coding gene EstP00714 thereof are developed from a strain of pseudomonas (Pseudomonas antiaturtium) HUP007, a recombinant expression vector containing the esterase gene EstP00714 and a genetic engineering bacterium are constructed, and the esterase EstP00714 is obtained after the genetic engineering bacterium is cultured and can be applied to catalyzing ester hydrolysis reaction.
The first purpose of the invention is to provide esterase EstP00714, the amino acid sequence of which is shown as SEQ ID NO. 2.
The second purpose of the invention is to provide an esterase gene EstP00714 for coding the esterase EstP 00714.
Preferably, the nucleotide sequence of the esterase gene EstP00714 is shown as SEQ ID No. 1.
The invention also provides a recombinant expression vector containing the esterase gene EstP 00714. The expression vector is preferably a pET-28a (+) vector.
The invention also provides a genetic engineering bacterium containing the esterase gene EstP 00714. The genetic engineering bacteria is preferably Escherichia coli BL21(DE 3).
The third purpose of the invention is to provide the application of the esterase EstP00714 in catalyzing ester hydrolysis.
Preferably, the application is the application of esterase EstP00714 in catalytic resolution of (+/-) -methyl mandelate to obtain (S) -methyl mandelate.
The fourth purpose of the invention is to provide the esterase EstP00714 with Mg tolerance2+、Li+、Na+、K+Tween-20, TritonX-100, n-hexane, isooctane, heptane, DMSO and/or n-decanol.
The esterase gene Estp00714 is derived from pseudomonas (Pseudomonas antipruralis) HUP007 and is stored in a south China sea institute laboratory. The invention utilizes a bioinformatics analysis method to obtain an esterase gene EstP00714 by comparison. An esterase gene EstP00714 is cloned from the Pseudomonas (Pseudomonas antaitus) HUP007 by a PCR method, the total length is 1146bp (from an initiation codon to a stop codon), and the amino acid sequence of the encoded esterase EstP00714 is shown as SEQ ID NO.2 and contains 381 amino acids. The esterase gene EstP00714 is connected with an expression vector pET-28a (+) and then transformed into Escherichia coli BL21(DE3), and after culture and induced expression, the recombinant esterase EstP00714 is obtained. The esterase EstP00714 has good tolerance to various metal ions, surfactants and organic solvents; the esterase EstP00714 can be used for preparing (S) -methyl mandelate, and the esterase EstP00714 is used as a catalyst to obtain the (S) -methyl mandelate with the substrate enantiomer excess value of more than 95%. Compared with the traditional chemical resolution, the method has the advantages of mild reaction conditions, no pollution and high enantiomeric excess value. The esterase EstP00714 can be used in the fields of biological medicine, cosmetics, fine chemical engineering and the like, and has great application value.
Description of the drawings:
FIG. 1 is an SDS-PAGE electrophoresis of esterase EstP 00714. Wherein, M is protein marker, lane 1 is E.coli BL21(DE3) protein supernatant containing pET-28a (+) -EstP00714 after IPTG induction, lane 2 is E.coli BL21(DE3) total protein containing pET-28a (+) -EstP00714 after IPTG induction, lane 3 is purified esterase EstP00714 protein, lane 4 is E.coli BL21(DE3) total protein containing pET-28a (+) -EstP00714 before IPTG induction.
FIG. 2 shows the specificity of esterase EstP00714 for p-nitrophenol esters of different side chain lengths.
FIG. 3 is the effect of pH on esterase EstP00714 activity.
FIG. 4 shows the stability effect of different pH on esterase EstP 00714.
FIG. 5 is the effect of temperature on the activity of esterase EstP 00714.
FIG. 6 shows the effect of different temperatures on the stability of esterase EstP 00714.
FIG. 7 shows the effect of substrate concentration on the resolution of (. + -.) -methyl mandelate by esterase EstP 00714.
FIG. 8 shows the effect of reaction time on the resolution of (+ -) -mandelic acid methyl ester by esterase EstP 00714.
FIG. 9 is a gas chromatogram of (. + -.) -methyl mandelate; wherein S represents (S) -methyl mandelate and R represents (R) -methyl mandelate.
FIG. 10 is a gas chromatogram of the selective hydrolysis of the methyl (. + -.) -mandelate by esterase EstP 00714; wherein S represents (S) -methyl mandelate and R represents (R) -methyl mandelate.
The specific implementation mode is as follows:
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The esterase gene EstP00714 is derived from genome sequenced pseudomonas (Pseudomonas antitumoralis) HUP007, and the bacterium is stored in a south China sea ocean institute laboratory.
Example 1: esterase gene EstP00714 open reading frame boundary determination and primer design
Extracting genome DNA of pseudomonas (Pseudomonas antitumualis) HUP007, carrying out whole genome sequencing, annotating the genome by using a bioinformatics method, analyzing an esterase gene in the genome, and determining the esterase gene EstP00714, its gene sequence is shown in SEQ ID NO.1, the total length is 1146bp (from start codon to stop codon), its coded esterase EstP00714 amino acid sequence is shown in SEQ ID NO.2, total 381 amino acids. According to the sequence of esterase gene EstP00714 obtained by analysis, a full-length amplification primer is designed as follows: an upstream primer: 5' -CATGAATTCGTGCAGATCCAGGGTCAC-3' (the EcoR I cleavage site is underlined); a downstream primer: 5' -CACCTCGAGTTAAAGGCAACTGCCAAG-3' (Xho I cleavage site underlined).
Example 2: cloning and vector construction of esterase gene EstP00714
2.1PCR amplification
The primer designed in example 1 (upstream primer: 5' -CAT)GAATTCGTGCAGATCCAGGGTCAC-3'; a downstream primer: 5' -CACCTCGAGTTAAAGGCAACTGCCAAG-3') was synthesized by Shanghai bioengineering, and the synthesized primers were dissolved in TE buffer solution to a final concentration of 10. mu.M, and the total DNA of extracted Pseudomonas (Pseudomonas antipruralis) HUP007 was used as a DNA template to establish a reaction system as shown in Table 1:
TABLE 1PCR reaction System
The esterase gene EstP00714 was amplified using the following PCR amplification procedure: denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and performing 32 cycles; extension at 72 ℃ for 10min and cooling to 18 ℃.
The PCR product was electrophoresed in 0.8% agarose gel at 120V for 20min, and observed in a gel imaging system, and a band of about 1146bp was recovered. And (3) recovering the PCR product according to the method of the gel recovery kit, and eluting by using 30 mu L of sterile water to obtain the purified and recovered PCR product.
2.2 enzyme digestion
And carrying out double enzyme digestion on the purified and recovered PCR product by using the following enzyme digestion system, wherein the enzyme digestion time is 1.5 h. The enzyme cutting system is as follows: EcoR I2. mu.L, Xho I2. mu.L, DNA < 0.3. mu.g, sterile double distilled water to 40. mu.L. And recovering the PCR product subjected to double enzyme digestion according to the method of the gel recovery kit after enzyme digestion.
Double digestion of plasmid pET-28a (+): a single colony of E.coli DH 5. alpha. containing plasmid pET-28a (+) was picked and cultured overnight. Plasmid was extracted using a plasmid extraction kit, and double digestion was carried out with EcoR I and Xho I according to the following digestion system for 1.5 h. The enzyme cutting system is as follows: EcoR I2. mu.L, Xho I2. mu.L, plasmid DNA < 0.3. mu.g, sterile double distilled water to 40. mu.L. After enzyme digestion, the mixture is electrophoresed in 0.8% agarose gel, and a linear pET-28a (+) vector subjected to double enzyme digestion is recovered according to a gel recovery kit method.
The restriction enzyme used in the above double digestion is a rapid endonuclease produced by Thermo. The purification and recovery after enzyme digestion uses a nucleic acid purification and recovery Kit (magenta, Hipure Gel Pure DNA Micro Kit), the plasmid extraction Kit is a plasmid miniprep Kit of Shanghai Czeri bioengineering, Inc., and the operation method is according to the use instruction.
2.3 connection
Connecting the PCR product subjected to double enzyme digestion and a pET-28a (+) vector subjected to double enzyme digestion according to the following system: mu.L of the double digested PCR product, 1. mu.L of the double digested pET-28a (+) vector, 0.5. mu.L of T4 ligase (5U/. mu.L), 1. mu.L of ligation buffer (5X), and 5. mu.L of deionized water for 20min at 20 ℃ for ligation. Thus, a ligation product was obtained.
2.4 transformation and selection
and adding 5 mu L of the ligation product into 50 mu L of escherichia coli DH5a competent cells, carrying out ice bath for 20-30 min, carrying out heat shock in a water bath kettle at 42 ℃ for 90s, carrying out ice bath for 2min, adding 500 mu L of LB liquid culture medium, and carrying out incubation culture at 37 ℃ and 200rpm for 1 h. A certain amount of bacterial liquid is taken and coated on an LB plate containing 50 mu L/mL kanamycin, and after 20 hours of culture, a single colony is selected. After single colony is cultured in 5mLLB culture medium overnight, plasmid is extracted, double enzyme digestion verification is carried out, and positive clone is obtained when enzyme digestion fragments and genes have the same size.
2.5 determination of the nucleotide sequence of the Gene
And (3) sending the screened positive clones to Shanghai Meiji biological medicine Limited company for sequencing, comparing a sequencing result with a nucleotide sequence (shown as SEQ ID NO. 1) of an esterase gene EstP00714, further confirming that the esterase gene EstP00714 is inserted into a pET-28a (+) plasmid, and confirming that the pET-28a (+) plasmid (named as pET-28a (+) -EstP00714) with the esterase gene EstP00714 is obtained after the result is completely correct, wherein the screened positive clones can be used for carrying out next-step experiments.
example 3: efficient expression of esterase EstP00714 in E.coli BL21(DE3)
3.1 preparation of competent cells of E.coli BL21(DE3)
1. Inoculating a small amount of Escherichia coli BL21(DE3) strain into 5mL LB tube liquid, and culturing at 37 ℃ overnight at 200 rpm;
2. Inoculating Escherichia coli BL21(DE3) bacterial liquid in a test tube into a 200mL LB shake flask according to the inoculation amount of 1% volume ratio, and culturing at 200rpm and 20 ℃ overnight to obtain a primary culture;
3. Rapidly cooling the cultured shake flask to 0 ℃ in ice water, subpackaging the original culture into a centrifuge tube (50mL) precooled by ice, and standing the ice for a plurality of minutes;
Centrifuging at 4.4 deg.C and 4000rpm for 10min to recover cells, and discarding supernatant;
5. 10mL of 0.1M CaCl precooled with ice2Resuspending the cells, centrifuging at 4 ℃ and 4000rpm for 10-15 min, and recovering the cells;
6. Repeat 5 with 10mL of 0.1M CaCl2Resuspending the cells, and ice-cooling for more than 30 min;
centrifuging at 7.4 deg.C and 4000rpm for 10min to recover cells;
8.5 mL of cells obtained from 50mL of stock culture contained 7.5% DMSO + CaCl2and (4) resuspending, and subpackaging in a 1.5mL centrifuge tube and 50-100 mu L of each tube. Storage at-80 ℃. Coli BL21(DE3) competent cells were thus obtained.
3.2 transformation
0.5-1 μ L of the pET-28a (+) -EstP00714 plasmid obtained in example 2 was mixed with 50 μ L of competent cells of Escherichia coli BL21(DE3), and the mixture was subjected to ice bath for 30min, heat shock in a water bath at 42 ℃ for 90s, ice bath for 2min, then 500 μ L of LB liquid medium was added, and the mixture was cultured at 37 ℃ and 200rpm for 1 h. After centrifugation, the culture was plated on 50. mu.L/mL kanamycin LB plates, and after 20 hours of culture, the individual bacteria were selected. Thus, Escherichia coli BL21(DE3) containing pET-28a (+) -EstP00714 was obtained.
Example 4: expression and purification of esterase EstP00714
4.1 protein Induction
Escherichia coli BL21(DE3) containing pET-28a (+) -EstP00714 was cultured in LB medium to OD600About 0.85, IPTG was added to a concentration of 0.2mM, and the mixture was cultured at 22 ℃ for 16 hours. 300mL of bacterial liquid at 4000rpm and 4 ℃ for 20min, collecting thalli, resuspending the thalli by using 30mL (50mM, pH 7.4) of Tris-HCl buffer solution, carrying out ultrasonic treatment for 400w for 4s, stopping for 6s, crushing for 15min, centrifuging, and collecting supernatant.
4.2 purification of esterase EstP00714 and SDS-PAGE electrophoresis
The supernatant collected in 4.1 was purified by a nickel ion affinity column, and the specific embodiment was as follows: eluting 5 column volumes with 25mM imidazole, eluting 20-30 column volumes with 40mM imidazole, and finally eluting with 3.5mL 100mM imidazole, and collecting the final 3mL eluate. Desalting was performed by SephadexG25 desalting column, and the specific operation was carried out according to the manual of GE corporation. The purified esterase was subjected to SDS-PAGE gel electrophoresis to obtain purified esterase EstP00714 (see FIG. 1), and the purified protein was about 41.2kD in size, which was in line with theoretical expectations.
4.3 esterase EstP00714 Activity assay
The esterase EstP00714 activity is measured by adopting p-nitrophenol ester, and the specific method is as follows: preparing 5mM p-nitrophenol ester by using acetonitrile; ② 187.5. mu.L Tris-HCl buffer (50mM, pH 8.0), 10. mu.L p-nitrophenol ester, 2.5. mu.L esterase EstP00714 pure enzyme solution (0.0645. mu.g/. mu.L) are added into the 0.2mL reaction system; ③ reacting for 4min at 20 ℃, adding 50 mu L of n-propanol to terminate the reaction, and measuring the absorbance at 405 nm.
Definition of enzyme activity unit: the enzyme amount required for hydrolyzing p-nitrophenol ester within 1min and releasing 1. mu.M of p-nitrophenol is defined as one enzyme activity unit.
Example 5: enzymatic Properties of esterase EstP00714
5.1 hydrolysis of p-nitrophenol esters of different side chain lengths
comparison of hydrolysis of p-nitrophenol esters C of different acyl lengths by the esterase EstP00714 according to the assay conditions of 4.32-C12the results are shown in FIG. 2. Indicating that esterase EstP00714 is a pairThe long-chain p-nitrophenol ester has poor specificity, the effect on the short-chain p-nitrophenol ester is better, and the optimal substrate is C4I.e., p-nitrophenol butyrate.
5.2 optimum pH and pH stability
Different buffer solutions were prepared, having different pH values, as shown in Table 2, at concentrations of 50mM each.
TABLE 2 buffer systems of different pH
And (3) respectively replacing the buffer solution (pH 8.0Tris-HCl buffer solution) in the determination condition in 4.3 according to the buffer solution in the table 2, and determining the enzyme activity of esterase EstP00714 in the buffer solution with different pH values, wherein the substrate is p-nitrophenol butyrate. The results of the effect of pH on esterase EstP00714 activity are shown in FIG. 3. In Tris-HCl buffer solution with pH9.0, esterase EstP00714 has the highest enzyme activity, and has higher enzyme activity when the pH value is between 8.0 and 10.0. When the pH is below 7, the activity decreases rapidly. The stability of esterase EstP00714 in buffers at different pH values at 20 ℃ is shown in FIG. 4. The enzyme activity is stable when the pH value is 8.0-12.0, and the residual activity is more than 60 percent after the treatment for 5 hours. Under the condition of peracid, the enzyme activity is obviously lost.
5.3 optimum temperature and temperature stability
50mM Tris-HCl pH8.0 is used as buffer solution, p-nitrophenol butyrate is used as substrate, and enzyme activity is measured at different temperatures (4-65 ℃) according to a reaction system in 4.3. The optimum temperature for esterase EstP00714 to catalyze the hydrolysis of p-nitrophenol butyrate was found to be 40 deg.C (FIG. 5). The enzyme activity reaches more than 80% at 30-55 deg.C, and the enzyme activity decreases sharply when the temperature is higher than 55 deg.C. The esterase EstP00714 is pretreated at different temperatures (30-50 ℃), taken out at intervals and subjected to enzyme activity measurement according to the measurement method in 4.3, and the result is shown in figure 6. When the esterase EstP00714 is lower than 40 ℃, the enzyme activity is kept high, and the residual enzyme activity is still kept above 70% after 1 hour of treatment. Along with the increase of the treatment temperature, the residual enzyme activity of the esterase EstP00714 is gradually reduced, the enzyme activity is rapidly reduced when the temperature is higher than 50 ℃, and the residual enzyme activity is basically lost after the treatment at 50 ℃ for 45min (figure 6), which shows that the esterase EstP00714 has better stability when the temperature is lower than 40 ℃.
5.4 Effect of Metal ions on esterase EstP00714 Activity
According to the metal ion species and the corresponding final concentration in the table 3, the metal ion treatment esterase EstP00714 is added into a Tris-HCl (pH 9.0) buffer solution of a reaction system, the enzyme activity in the reaction system without any ion is taken as 100 percent as a reference, incubation is carried out for 3h at 20 ℃, and the relative enzyme activity is measured according to a measuring method of 4.3 (p-nitrophenol butyrate is taken as a substrate). The effect of metal ions on esterase EstP00714 enzyme activity is shown in Table 3.2 mM Ba compared to control2+、Fe3+、Al3+、Cu2+、Mn2+、Mg2+、Ca2+Activating the activity of esterase EstP00714 catalyzing p-nitrophenolbutyrate, 2mM Mg2+The activity of the enzyme reaches 127.96 percent under the action of concentration. Low concentration of Li+、Ni2+、Na+、K+Has little influence on esterase EstP00714 enzyme activity, and Co has little influence on esterase EstP00714 enzyme activity2+And Zn2+has obvious inhibiting effect on the activity of esterase EstP 00714. The inhibition is enhanced with increasing metal ion concentration. 10mM Cu2+、Fe3+、Zn2+Has strong inhibition to esterase EstP00714 enzyme activity.
TABLE 3 Effect of Metal ions on esterase EstP00714 Activity
5.5 Effect of surfactants on esterase EstP00714 Activity
According to the types and corresponding final concentrations of the surfactants in the table 4, a surfactant-treated esterase EstP00714 is added into a Tris-HCl (pH 9.0) buffer solution of a reaction system, the enzyme activity in the reaction system without the surfactant is taken as 100 percent as a reference, incubation is carried out for 3h at 20 ℃, and the relative enzyme activity is measured according to a measuring method of 4.3 (p-nitrophenol butyrate is taken as a substrate). The effect of the surfactant on the enzymatic activity of esterase EstP00714 is shown in Table 4. TritonX-100 has little influence on enzyme activity under two mass concentrations of 0.1% and 0.5%, Tween-20 and S10 with 0.1% have little influence on enzyme activity, and the inhibition effect is enhanced along with the increase of the concentration. SDS, SDBS and CTAB have strong inhibition on the enzyme activity of esterase EstP00714 at the concentration of 0.1%.
TABLE 4 Effect of surfactants on esterase EstP00714 activity
5.6 Effect of organic solvents on esterase EstP00714 Activity
According to the types of organic solvents and the corresponding final concentrations shown in Table 5, adding an organic solvent to treat esterase EstP00714 enzyme solution into a Tris-HCl (pH 9.0) buffer solution of a reaction system, incubating for 3h under the treatment condition of 20 ℃, taking the esterase activity in the reaction system without the organic solvent as 100 percent as a reference, and then measuring the relative enzyme activity according to a measuring method of 4.3 (taking p-nitrophenol butyrate as a substrate), wherein the results are shown in Table 5. Under the concentration of 10%, n-hexane and isooctane have activating effect on esterase EstP00714, heptane and DMSO have no obvious effect, and other organic solvents have inhibiting effect on esterase EstP00714 to different degrees. Under high concentration (50%), esterase EstP00714 keeps higher enzyme activity in n-hexane, heptane and n-decanol, and residual enzyme activity is more than 50%. The above results indicate that the esterase EstP00714 is resistant to organic solvents.
TABLE 5 Effect of organic solvents on esterase EstP00714 activity
Example 6: splitting (+/-) -methyl mandelate by esterase EstP00714
6.1 influence of pH on splitting (+/-) -methyl mandelate by esterase EstP00714
Esterase EstP00714 pure enzyme with the final concentration of 60 mu g/mL and 30mM (+/-) -methyl mandelate substrate are added into reaction systems with different pH values, the reaction is carried out for 2h at 40 ℃, a gas chromatography chiral column is used for detection, and the enantiomeric excess value (e.e) of the substrate is calculated according to the peak area.s) And conversion (C), the results are shown in table 6. As can be seen from the table, at pH 8.5, e.e.s99 percent, but the conversion rate also reaches 99 percent, so the yield of the (S) -methyl mandelate is very low, and the optimized pH value is 7.0 by comprehensive consideration.
Equation 1:Equation 2:
In the formula: a. theSAnd ARRespectively represent the peak areas of (S) -methyl mandelate and (R) -methyl mandelate, A0And A represents the peak areas of methyl mandelate before and after the reaction, respectively.
TABLE 6 influence of pH on the resolution of (+ -) -methyl mandelate by esterase EstP00714
6.2 Effect of organic solvent on splitting (+/-) -methyl mandelate by esterase EstP00714
Adding esterase EstP00714 pure enzyme with the final concentration of 60 mu g/mL, 30mM (+/-) -methyl mandelate substrate and 10% (v/v) of organic solvent in the table 7 into a Tris-HCl (pH 7.0) buffer solution of a reaction system, reacting for 2 hours at 40 ℃ by taking the organic solvent as a control, detecting by using a gas chromatography chiral column, and calculating a substrate enantiomeric excess value (e.e) according to the peak area.s) And conversion (C), the results are shown in table 7.
TABLE 7 influence of organic solvents on the resolution of (+/-) -methyl mandelate by esterase EstP00714
It can be seen from Table 7 that the presence of most organic solvents reduced the stereoselectivity and the conversion of the esterase EstP 00714. The stereoselectivity of ethanol on esterase EstP00714 was relatively small.
6.3 influence of reaction temperature on splitting (+/-) -methyl mandelate by esterase EstP00714
Adding esterase EstP00714 pure enzyme with the final concentration of 60 mu g/mL and 30mM (+/-) -methyl mandelate substrate into a Tris-HCl (pH 7.0) buffer solution of a reaction system, reacting for 2h at different temperatures, and measuring the stereoselectivity of splitting (+/-) -methyl mandelate by the esterase EstP00714 through chiral gas chromatography, wherein the results are shown in Table 8.
As can be seen from table 8, e.e.sAnd the conversion increases with increasing temperature, between 45 and 50 ℃, e.e.sEssentially unchanged, maintained at 95%, in order to obtain the maximum yield of (S) -methyl mandelate, chosen at the same e.e.sThe temperature at which the lower conversion is the smallest is the optimum temperature, i.e. 40 ℃.
TABLE 8 influence of temperature on the resolution of (+ -) -methyl mandelate by esterase EstP00714
6.4 Effect of surfactant on splitting (+/-) -methyl mandelate by esterase EstP00714
Under optimized conditions (40 ℃, pH7.0 Tris-HCl buffer solution), esterase EstP00714 pure enzyme with the final concentration of 60 mu g/mL, 30mM (+/-) -methyl mandelate substrate, 0.01% (w/v) of surfactant (Tween-20, Tween-80, TritonX-100 and sodium tripolyphosphate) in the table 9 are added into a reaction system, the surfactant is not added as a control, and after 2 hours of reaction, a sample is used for gas chromatography detection, and the result is shown in the table 9. As can be seen from Table 9, the presence of surfactant compared to the controlIn the case of (d), e.e.sThere was a different degree of reduction, with the conversion increasing in the presence of sodium tripolyphosphate, but with e.e.sIs obviously reduced. TritonX-100 has relatively small influence on splitting (+/-) -methyl mandelate by esterase EstP 00714.
TABLE 9 Effect of surfactants on the resolution of (+ -) -methyl mandelate by esterase EstP00714
6.5 influence of substrate concentration on splitting (+/-) -methyl mandelate by esterase EstP00714
Under optimized conditions (40 ℃, pH7.0 Tris-HCl buffer solution), esterase EstP00714 pure enzyme with the final concentration of 60 mu g/mL and 20-90mM (+/-) -methyl mandelate substrate are added into the reaction system, and after 2 hours of reaction, a sample is used for GC detection, and the result is shown in figure 7. As can be seen from fig. 7, as the substrate concentration increases, e.e.sAnd the conversion rate decreases rapidly. Indicating that at higher substrate concentrations, the conversion of the reaction decreased. E.e at a substrate concentration of 20 mM.sThe conversion rate was 99% or more, but the yield of methyl (S) -mandelate was very low, and 30mM was selected as the optimum substrate concentration.
6.6 Effect of reaction time on splitting (+/-) -methyl mandelate by esterase EstP00714
Under the optimized conditions (40 ℃, pH7.0 Tris-HCl buffer solution), esterase EstP00714 pure enzyme with the final concentration of 60 mu g/mL and 30mM (+/-) -methyl mandelate substrate are added into a reaction system, 500 mu L of esterase is taken out at different time intervals, ethyl acetate is used for extraction, anhydrous sodium sulfate is used for removing water, and the result is shown in figure 8 by gas chromatography chiral column detection. As can be seen from fig. 8, e.e. the reaction time is increased.sAnd C gradually increases. After 2h of reaction, e.e.sAnd the conversion rate is not obvious, so the optimum time of catalyzing (+/-) -methyl mandelate by esterase EstP00714 is 2h, and the optimum reaction condition is e.e.s95% with a conversion of 86%. The gas chromatograms before and after the reaction under the optimum conditions are shown in FIGS. 9 and 10.
Claims (9)
1. An esterase EstP00714, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
2. An esterase gene encoding the esterase EstP00714 of claim 1.
3. The esterase gene according to claim 2, characterized in that the sequence of the esterase gene is shown in SEQ ID No. 1.
4. A recombinant expression vector comprising the esterase gene of claim 2 or 3.
5. The recombinant expression vector of claim 4, wherein the expression vector is a pET-28a (+) vector.
6. a genetically engineered bacterium comprising the esterase gene of claim 2 or 3.
7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium is Escherichia coli BL21(DE 3).
8. The use of esterase EstP00714 in catalyzing ester hydrolysis as claimed in claim 1, wherein said use is the use of esterase EstP00714 in catalyzing and resolving (+/-) -methyl mandelate to obtain (S) -methyl mandelate.
9. The esterase of claim 1, EstP00714 resistant to Mg2+、Li+、Na+、K+The method comprises the following steps of catalyzing by using esterase EstP00714 in the catalytic resolution of (+/-) -methyl mandelate to obtain (S) -methyl mandelate, wherein the esterase EstP-20, TritonX-100, n-hexane, isooctane, heptane, DMSO or n-decanol are used for catalysis.
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