CN103820416B - High-stereoselectivity esterolytic enzyme, encoding gene and application of encoding gene - Google Patents

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

A kind of highly stereoselective ester hydrolase, coding gene and application thereof

(1) Technical field

The invention relates to a highly stereoselective ester hydrolase and its encoding gene, as well as a carrier containing the encoding gene, engineering bacteria and application thereof.

(2) Background technology

Ester hydrolases include lipase and esterase, which are a class of biocatalysts with important uses in chiral synthesis. These enzymes can recognize a wide range of substrates. At present, more than 40% of biological object synthesis reactions can be catalyzed by ester hydrolases. These reactions have the characteristics of mild conditions, high regioselectivity and high stereoselectivity. It has been widely used in enzymatic kinetic resolution of racemic esters, amines and conversion of pre-chiral alcohols. In addition, they can be used in selective esterification, transesterification and polymerization reactions.

Due to the increasing demand for chiral drugs and intermediates, the use of biological or enzymatic synthesis of chiral compounds has increasingly attracted people's attention and industrial applications. The key problem of biological synthesis of chiral compounds is to find biocatalysts with highly stereoselective catalytic functions. Because ester hydrolases have a wide range of uses, screening new ester hydrolases is becoming a research hotspot in enzyme engineering. Although ester hydrolases are widely distributed in the microbial kingdom, their stereoselective catalytic functions are not the same. There are often multiple ester hydrolases in the same organism. Due to the differences in stereoselectivity between them, the optical purity of the product will often be reduced when using biological cells or their crude enzyme preparations for enzymatic synthesis of chiral compounds (Geun -Joong Kim, Journal of Molecular Catalysis B: Enzymatic 17, 2002:29-38). To solve this problem, engineering regulation means can be used to reduce the occurrence of side reactions, and a single enzyme preparation can be obtained through separation and purification to catalyze the reaction. However, these processes are often complicated and costly, and are not easy to be used in reproduction. If the genes of the required enzymes are directly cloned and expressed by means of genetic engineering, the above objectives can be easily achieved.

(3) Contents of the invention

The object of the present invention is to provide a screened ester hydrolase with higher R-isomer stereoselectivity, a vector containing the coding gene, engineering bacteria and application thereof.

The technical scheme adopted in the present invention is:

An ester hydrolase with R-isomer stereoselectivity, the amino acid sequence of which is shown in SEQ ID NO.2.

By screening a large number of microorganisms, the present inventors found that Bacillus megaterium can produce an ester hydrolase with high R-isomer stereoselectivity. The ester hydrolase of the present invention comes from Bacillus cereus (Bacillus cereus) WZZ001, and the deposit number is CCTCC M2012403 has been disclosed in CN102994429A. The inventor obtained a kind of esterase gene BCEST with R-isomer stereoselectivity of Bacillus sp. The sequence (CDS) starts from the first base of the DNA and ends at the 1458th base, ATG is the transcription initiation codon, and TAA is the transcription termination codon; and the corresponding amino acid sequence is obtained, as shown in SEQ ID No.2 in the list.

Due to the particularity of the amino acid sequence, any fragment or variant of the peptide protein containing the amino acid sequence shown in SEQ ID NO.2, such as its conservative variant, biologically active fragment or derivative, as long as the fragment of the peptide protein or the peptide The variants have more than 90% homology with the aforementioned amino acid sequence, and all belong to the protection scope of the present invention. Specifically, the changes may include deletion, insertion or substitution of amino acids in the amino acid sequence; wherein, for conservative changes in variants, the replaced amino acids have similar structures or chemical properties to the original amino acids, such as replacing isoamino acids with leucine Leucine, variants may also have non-conservative changes, such as replacing glycine with tryptophan.

The present invention also relates to the coding gene of the ester hydrolase. Specifically, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.1, as long as it has more than 90% homology with the polynucleotide, falls within the protection scope of the present invention. A polynucleotide variant refers to a polynucleotide sequence having one or more nucleotide changes. Variants of this polynucleotide may be biological variants or abiotic variants, including substitutional variants, deletion variants and insertional variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of a polynucleotide without substantially changing the function of the encoded peptide protein.

The invention also relates to a recombinant vector containing the coding gene, and a recombinant genetically engineered bacterium obtained by transforming the recombinant vector.

Described recombinant vector is constructed by linking the nucleotide sequence of the ester hydrolase gene of the present invention on various vectors by conventional methods, and this vector can be a 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 not limited to these vectors. Preferably, the BCEST gene product amplified by PCR is connected to the expression vector pEASY-E1 by TA cloning to form the expression vector (plasmid) pEASY-E1-BCEST of the BCEST gene. The expression vector pEASY-E1-BCEST was transformed into Trans1-T1 competent cells, and the plasmid was amplified.

The genetically engineered bacterium is a gene obtained by transforming an expression vector comprising the nucleotide sequence of the ester hydrolase gene of the present invention into a host microorganism, such as a competent Escherichia coli——Escherichia coli (Escherichia coli) BL21 (DE3) Engineering strains, such as transforming the above-mentioned plasmid pEASY-E1-BCEST to obtain Escherichia coli E. coli BL21(DE3)/pEASY-E1-BCEST containing pEASY-E1-BCEST.

The invention also relates to the application of the gene in the preparation of recombinant ester hydrolase. Specific applications include transforming host cells with the above-mentioned expression vector of the present invention, culturing transformants, and obtaining recombinant ester hydrolase. Wherein, the host cell may be prokaryotic, eukaryotic microorganisms or insects. Escherichia coli is preferred, and the corresponding transformant obtained is the above-mentioned genetic engineering strain: Escherichia coli E.coli BL21(DE3)/pEASY-E1-BCEST. This strain can efficiently express the ester hydrolase protein under the induction of conventional IPTG (add when OD600=0.5-0.6, and the concentration can be 0.1-1 mmol/L), which is the recombinant ester hydrolase of the present invention.

The invention also relates to the application of the ester hydrolase in stereoselective catalytic resolution of racemic substrates to prepare chiral compounds. The racemic substrate is one of the following: α-ethyl-2-oxo-1-pyrrolidine acetate methyl ester, BOC-alanine methyl ester, BOC-2-aminobutyric acid methyl ester, BOC-2 - Methyl valerate.

The recombinant ester hydrolase of the present invention can be used to prepare optically pure chiral compounds (R configuration acid and S configuration ester), especially levetiracetam intermediate (S)-α-ethyl-2-oxygen - Methyl 1-pyrrolidine acetate.

The beneficial effects of the present invention are mainly reflected in: the ester hydrolase of the present invention has higher catalytic activity and stereoselectivity, and can be used to prepare optically pure chiral compounds, especially levetiracetam intermediate (S)-α - Methyl ethyl-2-oxo-1-pyrrolidine acetate.

(4) Description of drawings

Fig. 1 is the PCR amplification electrophoresis pattern of the ester hydrolase gene BCEST of the present invention, wherein, 1, the PCR amplification product of BCEST; 2, DNA Marker (λ-Hind III digest, TakaRa company).

Figure 2 is the electrophoretic pattern of the plasmid pEASY-E1-BCEST PCR verification of the present invention, wherein, 1, the PCR product of the recombinant plasmid pEASY-E1-BCEST; 2, DNA Marker (λ-Hind III digest, TakaRa company).

Fig. 3 is the PAGE electrophoresis figure of the expression product of the genetic engineering strain E.coli BL21 (DE3)/pEASY-E1-BCEST of the present invention, wherein, 1, before the induction of the genetic engineering strain E.coli BL21 (DE3)/pEASY-E1-BCEST Product; 2. Product of genetically engineered strain E.coli BL21(DE3)/pEASY-E1-BCEST induced by IPTG; 3. Low molecular weight standard protein (TakaRa Company).

Fig. 4 is the gas phase detection spectrum of the substrate racemic α-ethyl-2-oxo-1-pyrrolidine acetate methyl ester.

Fig. 5 is a gas phase detection spectrum of the hydrolysis of methyl α-ethyl-2-oxo-1-pyrrolidine acetate by the recombinant esterase of the present invention.

Figure 6 is a schematic diagram of the construction of the expression plasmid pEASY-E1-BCEST.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Materials and methods in the embodiment are:

For the molecular cloning technique used, see "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

TransStartTM Taq DNA Polymerase was purchased from TransGen Biotech. DNA markers, genome extraction kits, plasmid extraction kits, DNA recovery and purification kits, and agarose electrophoresis reagents were all purchased from TaKaRa, Dalian Bao Biological Company, and the usage methods refer to the product instructions.

Trans1-T1 Competent Cells (Beijing Quanshijin Biotechnology Co., Ltd.)

Escherichia coli BL21 (DE3) (TakaRa, Dalian Treasure Biological Company)

Ni-NTA Sefinose Kit (Bio Basic INC)

Example 1: Cloning of the BCEST gene from Bacillus cereus

Degenerate primers 1 and 2 were designed according to the genome DNA sequence of Bacillus cereus ATCC10876 (GenBank: ACLT01000051.1).

Primer 1 sequence: TCTAAAATTGAAACACCTGTTATG

Primer 2 sequence: TTATTTAATTTTCTTAAATGTAAGC

The genomic DNA of Bacillus cereus CCTCC M2012403 was used as a template for PCR amplification (using Takala kit). The PCR reaction system and reaction conditions are shown in Tables 1 and 2. Steps 2 to 4 are repeated 29 times.

Table 1: PCR amplification reaction system

Table 2: PCR amplification reaction conditions

The PCR amplification product was detected by 0.8% agarose gel electrophoresis, and the product was a single band with a size of about 1500bp (as shown in Figure 1). Purify and recover the PCR product. For specific steps, refer to the instructions of the Beijing Biotech Genomic DNA Rapid Purification and Recovery Kit, and then use a micro-nucleic acid and protein measuring instrument to detect the concentration and purity.

Embodiment 2: Construction of expression vector and transformant

A-T ligation of the target fragment with the pEASY-E1 expression vector, the ligation system is as follows in Table 3:

Table 3: Connection system of pEASY-E1Expression Vector

Mix gently and react at room temperature for 5 min. The ligation product was transformed into 50 μL of primary melted Trans1-T1 competent cells. The positive recombinants with the correct expression direction were identified by PCR (as shown in Figure 2), and transferred to the LB medium amplification vector. The recombinant plasmid pEASY-E1-BCEST was extracted from Trans1-T1 cells with TaKaRa plasmid extraction kit, transformed into E.coli BL21(DE3) competent cells, and positive clones were obtained by screening with Amp-resistant medium and identified by PCR cloning. Containing the correct recombinant plasmid, thereby obtaining a transformant—a genetic engineering strain E.coliBL21(DE3)/pEASY-E1-BCEST expressing the esterase of the present invention.

Embodiment 3: Expression of recombinant ester hydrolase

The genetically engineered strain E.coli BL21(DE3)/pEASY-E1-BCEST was inoculated into 50 mL of LB medium containing 80 μg/mL ampicillin, and cultured overnight at 37° C. with shaking. Take 1 mL of the culture solution and connect it to 50 mL of fresh LB medium containing 80 μg/mL ampicillin, culture to about OD600=0.6, add IPTG to its concentration of 0.2 mmol/L for induction, and continue to culture for 8 h. Bacteria were collected by centrifugation.

Embodiment 4: the application of recombinant ester hydrolase

The recombinant Escherichia coli in Example 3 was taken, and the wall was broken by ultrasound, and the bacterial cells were removed by centrifugation, and the supernatant was passed through a HisTrapTMFF affinity chromatography column to obtain a partially purified recombinant ester hydrolase. The obtained recombinant ester hydrolase hydrolyzes racemic α-ethyl-2-oxo-1-pyrrolidine methyl acetate and other substrates, the reaction solvent is 50mM pH8.0 Tris hydrochloric acid buffer solution, and the substrate concentration is 2 % (volume ratio), the enzyme dosage is 30mg/L buffer solution, and the reaction temperature is 30°C. After reacting for 60min, the reaction product was analyzed by gas chromatography (the 6890N gas chromatograph and BGB-175 chiral gas chromatograph produced by Agilent) and the optical purity of the product (as shown in Figure 4 and Figure 5, where (R) The gas chromatographic retention times of -α-ethyl-2-oxo-1-pyrrolidine acetate and (S)-α-ethyl-2-oxo-1-pyrrolidine acetate were 28.9min and 29.5min, respectively ), the specific operation method is as follows: the carrier gas is N2. Injection port temperature: 220°C. The air flow rate is 300mL/min, and the makeup gas flow rate is 30mL/min. The split ratio is 30:1, and the injection volume is 1 μL. Oven heating program: the initial temperature is 120°C and kept for 3 minutes, then the temperature is raised to 175°C at 2°C/min and kept for 1 minute. FID detects its temperature: 250°C.

Table 4: Reaction results of chiral compounds catalyzed by recombinant ester hydrolase

It can be seen from the results that after the hydrolysis of different substrates by recombinant ester hydrolase, the corresponding products of S configuration with optical purity of 99.9% were obtained respectively. Compared with the crude ester hydrolase isolated from wild-type Bacillus cereus CCTCC M2012403, which catalyzes the reaction of the same substrate, the optical purity and yield of (S)-α-ethyl-2-oxo-1-pyrrolidine acetate methyl ester rate is improved. It shows that the use of recombinant ester hydrolase can eliminate the side reactions of other isoenzymes produced by wild bacteria, and improve the optical purity and yield of chiral compounds.

The above descriptions are only preferred embodiments of the present invention, and do not limit the technical content of the present invention in any form. All simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention fall within the 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.