CN103820416B - High-stereoselectivity esterolytic enzyme, encoding gene and application of encoding gene - Google Patents
High-stereoselectivity esterolytic enzyme, encoding gene and application of encoding gene Download PDFInfo
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- CN103820416B CN103820416B CN201410022841.2A CN201410022841A CN103820416B CN 103820416 B CN103820416 B CN 103820416B CN 201410022841 A CN201410022841 A CN 201410022841A CN 103820416 B CN103820416 B CN 103820416B
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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
- C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
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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 provides high-R-isomer-stereoselectivity esterolytic enzyme, an encoding gene of the esterolytic enzyme, a carrier containing the encoding gene, an engineering bacterium and application of the encoding gene. An amino acid sequence of the esterolytic enzyme is shown as SEQ ID No.2, and a nucleotide sequence of the encoding gene is shown as SEQ ID No.1. The esterolytic enzyme provided by the invention has higher catalysis activity and stereoselectivity, and can be used for preparing an optically pure chiral compound, particularly a levetiracetam intermediate, namely alpha-ethyl-2-oxygen-1-pyrrolidine methyl acetate.
Description
（One）Technical field
The present invention relates to a kind of highly-solid selectively ester hydrolase and its encoding gene, and the load containing the encoding gene
Body, engineering bacteria and its application.
（Two）Background technology
Ester hydrolase includes lipase and esterase, and it is the living things catalysis that a class has important use in chiral synthesis
Agent.These enzymes can recognize very wide substrate, and the biology of current ＞ 40% can not have ester hydrolase catalysis to complete to thing synthetic reaction,
The features such as these reactions are with mild condition, the regioselectivity of reflection and high stereo selectivity.Disappear outside Enzymatic kinetic resolution
The aspects such as rotation ester, amine and conversion prochirality alcohol wait until to be widely applied.In addition, they can be additionally used in selective esterification, transesterification
In polyreaction.
Due to the growth of chiral medicine and intermediate demand, increasingly obtained using bioanalysises or enzymatic clarification chipal compounds
Attention and industrial applications to people.The key issue of bioanalysises synthesizing chiral compound is to find with High level of stereoselectivity choosing
The biocatalyzer of selecting property catalysiss.Because ester hydrolase tool has been widely used, screening new ester hydrolase is just becoming enzyme
The study hotspot of engineering.Although ester hydrolase is distributed very extensively in microbial world, their stereo selectivity catalysiss differ
Sample.Often there are various ester hydrolases in vivo in same biology, due to having differences property of stereo selectivity between them, be utilized as
Biological cell or their thick enzyme preparation often reduce the optical purity of product when carrying out enzymatic clarification chipal compounds
（Geun-Joong Kim,Journal of Molecular Catalysis B:Enzymatic17,2002:29-38）.Solve
This problem can reduce the generation of side reaction by the control measures of engineering, it is also possible to obtain single by isolating and purifying
Enzyme preparation is difficult to be used carrying out catalytic reaction, but these processes often more complicated and high cost in reproduction.If logical
Cross engineered means, the gene of the enzyme required for Direct Cloning and expression, it becomes possible to easily reach the above object.
（Three）The content of the invention
It is an object of the present invention to provide it is a kind of screening obtain with the stereoselective ester hydrolase of higher R- isomers, contain
There are carrier, engineering bacteria and its application of the encoding gene.
The technical solution used in the present invention is：
One kind has the stereoselective ester hydrolase of R- isomers, and its aminoacid sequence is as shown in SEQ ID NO.2.
The present inventor has found that bacillus megaterium can produce a kind of with height R- isomeries by the substantial amounts of microorganism of screening
The stereoselective ester hydrolase of body, ester hydrolase of the present invention is from Bacillus cercuses（Bacillus cereus）WZZ001,
Deposit number CCTCC M2012403, disclose in CN102994429A.Inventor obtains bacillus cereuss by PCR amplifications
One kind of WZZ001 has the stereoselective esterase gene BCEST of R- isomers, determines its nucleotide sequence, such as sequence table
Shown in middle SEQ ID No.1, wherein coded sequence（CDS）Terminate to the 1458th from the 1st base of DNA, ATG is transcription initiation
Codon, TAA is tanscription termination password；And corresponding aminoacid sequence is obtained, as shown in SEQ ID No.2 in list.
Due to the particularity of aminoacid sequence, any piece containing the peptide protein of aminoacid sequence shown in SEQ ID NO.2
Section or its variant, such as its examples of conservative variations, bioactive fragment or derivant, as long as the fragment of the peptide protein or peptide protein variant
With aforementioned amino acid sequences homology more than 90%, the row of the scope of the present invention are belonged to.The specific change may include
The disappearance of aminoacid, insertion or replacement in aminoacid sequence；Wherein, for the conservative of variant sexually revises, the aminoacid replaced
With the structure or chemical property similar to original acid, such as isoleucine is replaced with leucine, variant also can have non-conservative
Sexually revise, such as replace glycine with tryptophan.
The invention further relates to the encoding gene of the ester hydrolase.Specifically, the gene nucleotide series such as SEQ ID
Shown in NO.1.
Due to the particularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ ID NO.1, as long as it is more with this
Nucleotide has more than 90% homology, belongs to the row of the scope of the present invention.The variant of the polynucleotide refers to a kind of tool
There is the polynucleotide sequence that one or more nucleotide change.The variant of this polynucleotide can make the displacement variant or non-of life
Raw variant, including substitution variants, Deletion variants and insert variation.As known in the art, allelic variant is
The alternative forms of one polynucleotide, it is probably the replacement of a polynucleotide, disappearance or inserts, but will not be from substantially changing
Become the function of the peptide protein of its coding.
The invention further relates to contain the recombinant vector of the encoding gene, and obtained using recombinant vector conversion
Recombination engineering bacteria.
The recombinant vector be with conventional method by the present invention ester hydrolase gene nucleotide sequence be connected to it is various
Built-up on carrier, the carrier can be commercially available plasmid, cosmid, phage or viral vector etc., such as pUC,
PBluescript (Stratagene), pLEX (Novagen, Inc., Madison, Wis.), PQE (Qiagen), pREP,
PSE420 and pLEX (Invitrogen）, but it is not limited to these carriers.The BCEST genes that preferably PCR is expanded
Product forms the expression vector of BCEST genes by TA clones and expression vector pEASY-E1 connections（Plasmid）pEASY-E1-
BCEST.Expression vector pEASY-E1-BCEST converts Trans1-T1 competent cells, expands plasmid.
The genetic engineering bacterium is that the expression vector comprising ester hydrolase gene nucleotide series of the present invention is transformed into into place
Main microorganism, such as competence escherichia coli --- colon bacillus（Escherichia coli）Obtain in BL21 (DE3)
Engineering strain, such as by above-mentioned plasmid pEASY-E1-BCEST conversion wherein obtain the large intestine containing pEASY-E1-BCEST
Escherichia E.coli BL21 (DE3)/pEASY-E1-BCEST.
The invention further relates to application of the gene in Prepare restructuring ester hydrolase.Specific application is included with above-mentioned
Invention expression vector transformed host cell, cultivates transformant, obtains the ester hydrolase of restructuring.Wherein, the host cell can be
Protokaryon, eukaryotic microorganisms or insecticide etc..Preferably escherichia coli, it is above-mentioned genetic engineering bacterium to obtain corresponding transformant
Strain：Colon bacillus E.coli BL21 (DE3)/pEASY-E1-BCEST.This bacterial strain can be under conventional IPTG inductions（
Add during OD600=0.5～0.6 or so, be 0.1～1mmol/L to its concentration）High efficient expression ester hydrolase albumen, as
The restructuring ester hydrolase of the present invention.
The invention further relates to described ester hydrolase prepares chiral compound in stereo selectivity catalysis resolution of racemic substrate
Application in thing.The racemic substrate is one of following：α-ethyl -2- oxygen -1- methyl pyrrolidineacetates, BOC- alanine first
Ester, BOC-2- aminobutyric acid methyl esters, BOC-2- aminopentanoic acid methyl esters.
The restructuring ester hydrolase of the present invention can be used for preparing optically pure chipal compounds（The acid of R configurations and S configuration esters),
Particularly levetiracetam intermediate (S)-α-ethyl -2- oxygen -1- methyl pyrrolidineacetates.
The beneficial effects are mainly as follows：Ester hydrolase of the present invention has higher catalysis activity and three-dimensional selection
Property, can be used for preparing optically pure chipal compounds, particularly levetiracetam intermediate (S)-α-ethyl -2- oxygen -1- pyrroles
Cough up alkane methyl acetate.
（Four）Description of the drawings
Fig. 1 is the PCR amplification electrophoresis patterns of ester hydrolase gene BCEST of the present invention, wherein, 1, the PCR of BCEST amplification produces
Thing；2、DNA Marker（The digest of λ-Hind III, TakaRa companies）.
Fig. 2 is plasmid pEASY-E1-BCEST PCR of the present invention checking electrophoresis patterns, wherein, 1, recombiant plasmid pEASY-
E1-BCEST PCR primers；2nd, DNA Marker (digest of λ-Hind III, TakaRa companies）.
Fig. 3 is the PAGE of engineering strain E.coli BL21 (DE3)/pEASY-E1-BCEST expression products of the present invention
Electrophoretogram, wherein, product before 1, engineering strain E.coli BL21 (DE3)/pEASY-E1-BCEST inductions；2nd, gene work
Journey bacterial strain E.coli BL21 (DE3)/pEASY-E1-BCEST products Jing after IPTG inductions；3rd, low-molecular-weight standard protein
（TakaRa companies）.
Fig. 4 is substrate racemic ' alpha '-ethyl -2- oxygen -1- methyl pyrrolidineacetate vapor detection collection of illustrative plates.
Fig. 5 is the vapor detection collection of illustrative plates of Recombinant esterase hydrolyzing alpha of the present invention-ethyl -2- oxygen -1- methyl pyrrolidineacetates.
Fig. 6 is that expression plasmid pEASY-E1-BCEST builds schematic diagram.
（Five）Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Materials and methods in embodiment are：
The molecule clone technology for being adopted is referring to volumes such as J. Pehanorm Brookers《Molecular Cloning:A Laboratory guide》.
TransStartTM Taq DNA Polymerase are purchased from TransGen Biotech companies.DNA marker, base
Because a group extracts kit, plasmid extraction kit, DNA recovery purifying test kits, sepharose electrophoresis reagent are purchased from TaKaRa, greatly
Lian Bao biotech firms, using method refers to catalogue.
Trans1-T1 competent cells (Beijing Quanshijin Biotechnology Co., Ltd）
Colon bacillus BL21 (DE3) (TakaRa, Dalian treasured biotech firm）
Ni-NTA Sefinose Kit（Bio Basic INC companies）
Embodiment 1：BCEST genes are cloned from Bacillus cercuses
According to Bacillus cereus ATCC10876 genomic dna sequences（GenBank:ACLT01000051.1）If
Meter degenerate primer 1 and primer 2.
The sequence of primer 1：TCTAAAATTGAAACACCTGTTATG
Primer 2 sequence：TTATTTAATTTTCTTAAATGTAAGC
Enter performing PCR as template with Bacillus cercuses Bacillus cereus CCTCC M2012403 genomic DNAs to expand
Increase（Using Takala test kits）, PCR reaction systems and reaction condition are as shown in Tables 1 and 2.Step 2 to 4 repeats 29 times.
Table 1：Pcr amplification reaction system
Table 2：Pcr amplification reaction condition
Pcr amplification product is detected with 0.8% agarose gel electrophoresiies, product is single band, and size is about 1500bp（Such as
Shown in Fig. 1).Purification recovery is carried out to PCR primer, concrete steps are reclaimed with reference to the vast Tyke genomic DNA fast purifying in Beijing
Kit specification, then with trace dna protein measurement instrument detectable concentration and purity.
Embodiment 2：The structure of expression vector and transformant
Purpose fragment and pEASY-E1 expression vectors are carried out into A-T to be connected, linked system such as table 3 below：
Table 3：PEASY-E1Expression Vector linked systems
It is gently mixed, under room temperature 5min is reacted.Connection product is converted and melts Trans1-T1 competent cells at the beginning of 50 μ L.PCR
The positive recombinant in the correct expression direction of method identification（As shown in Figure 2）, it is forwarded to LB culture medium amplification vectors.Use TaKaRa plasmids
Extracts kit extracts recombiant plasmid pEASY-E1-BCEST, Transformed E .coli BL21 (DE3) senses from Trans1-T1 thalline
By state cell, the screening of Jing Amp resistance cultures base obtains positive colony and Jing PCR clone identifications, containing correct recombiant plasmid, from
And obtain engineering strain E.coliBL21 (DE3)/pEASY-E1-BCEST that transformant one expresses ester hydrolase of the present invention.
Embodiment 3：The expression of restructuring ester hydrolase
Engineering strain E.coli BL21 (DE3)/pEASY-E1-BCEST is connected to into 50mL, blue or green containing 80 μ g/mL ammonia benzyls
In the LB culture medium of mycin, 37 DEG C of concussion and cultivates are overnight.Take 1mL culture fluid be connected to 50mL, containing the new of 80 μ g/mL ampicillin
Fresh LB culture medium, cultivate to OD600=0.6 or so, add IPTG to be 0.2mmol/L inductions to its concentration, continue to cultivate 8h.From
Heart collects thalline.
Embodiment 4：The application of restructuring ester hydrolase
Recombination bacillus coli in Example 3, Jing ultrasonications, centrifugation removes thalline, supernatant Jing HisTrapTMFF
Partially purified restructuring ester hydrolase is obtained after affinity column.α-the second of the restructuring ester hydrolase hydrolysis of racemic for obtaining
The substrates such as base -2- oxygen -1- methyl pyrrolidineacetates, reaction dissolvent is 50mM pH8.0Tris hydrochloride buffers, concentration of substrate point
Wei 2%（Volume ratio）, enzyme dosage be 30mg/L buffer, 30 DEG C of reaction temperature.After reaction 60min, product Jing gas phase color
Spectrum（The 6890N gas chromatograpies and BGB-175 Chiral gas chromatographies that Agilent companies produce is lived）Analysis conversion ratio and product
Optical purity（As shown in Figure 4 and Figure 5, wherein (R)-α-ethyl -2- oxygen -1- methyl pyrrolidineacetates and (S)-α-ethyl -2-
The gas chromatography retention time of oxygen -1- methyl pyrrolidineacetates is respectively 28.9min and 29.5min）, carrying out practically method is such as
Under：Carrier gas is N2.Injector temperature：220℃.Air mass flow 300mL/min, make-up gas flow 30mL/min.Split ratio 30:1,
The μ L of sample size 1.Column temperature rise program：120 DEG C of holding 3min of initial temperature, 2 DEG C/min are warming up to 175 DEG C, keep 1min.FID
Detect its temperature：250℃.
Table 4：Restructuring ester hydrolase catalysis resolving chiral compound reaction result
As can be known from the results, different substrates respectively obtains optical purity 99.9% after ester hydrolase catalyzing hydrolysis of recombinating
S configurations corresponding product.The thick ester hydrolysis isolated with the Bacillus cereus CCTCC M2012403 of wild type
The reaction result of enzyme catalysiss same substrate compares, (S) optical purity and product of-α-ethyl -2- oxygen -1- methyl pyrrolidineacetates
Rate is improved.Illustrate to carry out the pair for reacting other isozymes that can eliminate wild mushroom generation instead using restructuring ester hydrolase
Should, improve the optical purity and yield of chipal compounds.
The above, is only presently preferred embodiments of the present invention, not makees any form to the technology contents of the present invention
On restriction.It is every according to the present invention technical spirit above example is made any simple modification, equivalent variations with repair
Decorations, each fall within protection scope of the present invention.
Claims (4)
1. application of a kind of gene with the stereoselective ester hydrolase of R- isomers in Prepare restructuring ester hydrolase, its
The aminoacid sequence of coding is as shown in SEQ ID NO.2.
2. application as claimed in claim 1, it is characterised in that the gene nucleotide series are as shown in SEQ ID NO.1.
3. application as claimed in claim 1, it is characterised in that described ester hydrolase splits outer disappearing in stereo selectivity catalysis
Rotation substrate prepares the application in chipal compounds.
4. application as claimed in claim 3, it is characterised in that the racemic substrate is one of following：α-ethyl -2- oxygen -1-
Methyl pyrrolidineacetate, BOC- methyl lactamines, BOC-2- aminobutyric acid methyl esters, BOC-2- aminopentanoic acid methyl esters.
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