An ene reductase and its application in asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters

Abstract

The present invention relates to Swingsia samuiensi ene reductase and its application in asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters. The ene reductase SsER derived from Swingsia samuiensi, its gene, a recombinant expression vector containing the gene and a recombinant expression transformant, and a method for asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters using the recombinant ene reductase or the recombinant expression transformant as a catalyst are obtained by gene mining. Compared with the prior art, the present invention uses ene reductase to catalyze the preparation of saturated ketoesters, which has the significant advantages of simple process, high product optical purity and environmental friendliness, and has good industrial application prospects. The present invention provides a new ene reductase, which can stereoselectively catalyze the asymmetric reduction of α- / β-unsaturated ketoesters, mild reaction conditions, high conversion rate, high catalytic activity, good product optical purity, and has good industrial application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to an ene reductase derived from Swingsia samuiensi, a gene encoding the ene reductase, a recombinant expression vector and a recombinant expression transformant containing the gene, and a method for catalyzing the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters by using the recombinant ene reductase or the recombinant expression transformant as a catalyst. Background Art

[0002] γ-Ketoesters are widely used and are important intermediates for the synthesis of various heterocyclic compounds (such as pyrrole, furan, lactone, β-lactam antibiotics, etc.) and other natural products and drugs.

[0003] γ-Ketoesters are widely used in the synthesis of lactones. In 2014, Díaz-Rodríguez et al. used alcohol dehydrogenase to asymmetric reduce prochiral γ- / δ-ketoesters to generate corresponding chiral hydroxy esters, which spontaneously cyclized to form a series of optically pure lactones (ACS Catal, 2014, 4(2): 386-393). In 2014, Classen reported the use of enolate reductase to asymmetric reduce α- / β-unsaturated ketoesters to obtain saturated γ-ketoester intermediates, and used alcohol dehydrogenase combination cascade catalysis to synthesize β-methyl-substituted γ-valerolactone (ACS Catal. 2014, 4, 1321-1331), with an ee value of 98-99% and a maximum yield of 90%, but the substrate was limited to methyl-substituted short-chain lactones. In 2015, Brenna used the same synthetic route and a different oxidoreductase (OYE) to synthesize the same product (J.Mol.Catal.B 2015,114,77-85). In 2018, Kumru also used the same route to enrich the position and type of substituents on the synthesized γ-ketoesters, thereby enriching the types and positions of synthesized γ-lactones (ChemCatChem 2018,10,4917-4926).

[0004] In summary, γ-ketoesters are important intermediates for many lactone compounds and have very important application value. The biocatalytic synthesis of multi-substituted chiral γ-ketoesters and γ-lactones is the general trend. The above methods require the use of α- / β-unsaturated ketoesters, and the use of such compounds to biocatalytically synthesize α- / β-substituted γ-ketoesters, or further reduce carbonyl groups to synthesize multi-substituted chiral γ-lactones, face problems such as lack of enzyme resources, narrow catalytic substrate spectrum, poor product optical purity, and low yield, and have not yet been industrialized.

[0005] In response to the above-mentioned major needs and problems, this study explored the resources and carried out molecular modification of the ene(ketone) reductase required for the synthesis of polysubstituted chiral lactones, created enzyme catalysts with high stereoselectivity and high activity, and expanded the substrate spectrum. Summary of the invention

[0006] The purpose of the present invention is to provide an olefin reductase and its application in the asymmetric reduction of long-chain α- / β-unsaturated ketoesters in order to overcome the above-mentioned problems, that is, starting from α- / β-unsaturated γ-ketoesters, highly stereoselective α- / β-substituted γ-ketoesters are obtained by asymmetric reduction of olefin reductase.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] One of the technical solutions of the present invention:

[0009] The present invention provides an alkene reductase, wherein the alkene reductase is a protein of (a) or (b):

[0010] (a) a protein consisting of the amino acid sequence shown in SEQ ID No. 2;

[0011] (b) A protein derived from (a) which has a catalytic activity higher than that of (a) and has a catalytic activity of catalyzing the asymmetric reduction of α- / β-unsaturated ketoesters to prepare optically active α- / β-substituted γ-ketoesters in the amino acid sequence shown in SEQ ID No. 2 by substitution, deletion or addition of one or several amino acids.

[0012] Furthermore, the protein (b) is a protein consisting of a new amino acid sequence formed by replacing one amino acid at position 314 of the amino acid sequence shown in SEQ ID No. 2.

[0013] Preferably, the protein (b) is:

[0014] (1) replacing the 314th alanine in the amino acid sequence shown in SEQ ID No. 2 with glycine;

[0015] (2) replacing the 314th alanine of the amino acid sequence shown in SEQ ID No. 2 with tyrosine;

[0016] (3) replacing the 314th alanine in the amino acid sequence shown in SEQ ID No. 2 with threonine;

[0017] The DNA sequence of the alkene reductase of the present invention is derived from the chromosome of Swingsia samuiensi AH83.

[0018] The present invention also provides a method for obtaining the ene reductase: that is, through gene mining and large-scale screening of laboratory preserved microbial strains, a gene on the chromosome of Swingsia samuiensi AH83 is found, and an ene reductase capable of catalyzing the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters is obtained by artificial synthesis and cloning and expression, and the ene reductase is named ene reductase SsER, which is NADPH-dependent. The amino acid sequence of the ene reductase is shown in SEQ ID No. 2.

[0019] The cloning and expression methods mentioned in the present invention are conventional biotechnology means in the art.

[0020] The ene reductase of the present invention has the following properties: it can catalyze the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters to prepare optically active α- / β-substituted γ-ketoesters.

[0021] The second technical solution of the present invention:

[0022] The present invention provides a nucleic acid encoding the olefin reductase SsER as described in technical solution 1, specifically, its nucleotide sequence is shown in SEQ ID No.1, with a total length of 1068 nucleotide bases. Its coding sequence (CDS) starts from the first base to the 1068th base, the start codon is ATG, the stop codon is TTT, and there is no intron. The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.2.

[0023] The present invention also provides the source of the DNA encoding the alkene reductase SsER: the DNA encoding the alkene reductase is obtained by an artificial full sequence synthesis method.

[0024] The third technical solution of the present invention:

[0025] The invention provides a recombinant expression vector comprising the alkene reductase SsER nucleic acid sequence.

[0026] The recombinant expression vector of the present invention can be constructed by connecting the olefin reductase SsER nucleic acid to various expression vectors by conventional methods in the art. The expression vectors include various conventional plasmid vectors in the art, preferably plasmid pET-28a.

[0027] Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the olefin reductase SsER gene sequence DNA fragment obtained by PCR amplification is double-digested with restriction endonucleases EcoR I and Hind III, and the empty plasmid pET-28a is double-digested with restriction endonucleases EcoR I and Hind III, the olefin reductase SsER gene DNA fragment and the pET-28a plasmid after the above digestion are recovered, and the T 4 The recombinant expression vector (pET28a-SsER) containing the olefin reductase SsER gene was constructed by DNA ligase ligation.

[0028] The fourth technical solution of the present invention:

[0029] The invention provides a recombinant expression transformant comprising the alkene reductase SsER recombinant expression vector.

[0030] The recombinant expression transformant of the present invention can be obtained by transforming the recombinant expression vector described in Technical Solution 3 into a host cell. The host cell is a conventional host cell in the art, as long as the recombinant expression vector can stably replicate itself and the gene of the olefin reductase SsER carried by it can be effectively expressed. The host cell is preferably Escherichia coli, more preferably Escherichia coli E. coli BL21 (DE3) or Escherichia coli E. coli DH5α. This scheme preferably transforms the recombinant expression vector into Escherichia coli E. coli BL21 (DE3) to obtain the preferred genetically engineered strain of the present invention (E. coli BL21 (DE3) / pET28a-SsER).

[0031] The fifth technical solution of the present invention is:

[0032] The present invention provides an alkene reductase catalyst, wherein the alkene reductase catalyst is in any of the following forms:

[0033] (1) culturing the recombinant expression transformant as described in technical solution 4, and isolating the transformant cells containing the olefin reductase SsER;

[0034] (2) culturing the recombinant expression transformant as described in technical solution 4, isolating transformant cells containing the olefin reductase SsER, and disrupting the transformant cells containing the olefin reductase SsER to obtain a cell disrupted liquid;

[0035] (3) freeze-drying the cell disrupted liquid of the olefin reductase SsER to obtain a freeze-dried enzyme powder;

[0036] (4) The olefin reductase SsER or a mutant of the olefin reductase SsER, wherein the mutant of the olefin reductase SsER refers to a protein having an amino acid sequence shown in SEQ ID No. 2 which has been substituted, deleted or added with several amino acids and can catalyze the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters to prepare optically active γ-ketoesters, while having improved catalytic activity.

[0037] The present invention also provides a method for semi-rational construction of an olefin reductase SsER mutant. The amino acid sequence of SsER is input into SWISS-MODEL, homology modeling is performed, and then the modeled protein model is used for molecular docking with 4-oxopentenoic acid ethyl ester (E-2a), and a suitable docking posture model is selected according to the catalytic mechanism and binding energy of the short-chain dehydrogenase. The activity of the enzyme is further improved by performing site-directed saturation mutation and combined mutation on the amino acids near the substrate pocket. In the three-dimensional structure of the SsER amino acid sequence shown in SEQ ID No.2, the amino acid residues around the substrate 4-oxopentenoic acid ethyl ester (E-2a) binding site include: asparagine at position 293, glutamine at position 294, aspartic acid at position 295, tyrosine at position 296, serine at position 297, alanine at position 314, phenylalanine at position 315, glycine at position 316, arginine at position 317, and lysine at position 318. Site-directed saturation mutation technology is used to perform site-directed saturation mutation on the amino acid residues at these sites. The primers used are shown in Table 1:

[0038] Table 1 Primers for amino acid mutation sites

[0039]

[0040]

[0041] Only the mutant formed by replacing alanine at position 314 with an amino acid is an effective mutant, and preferably the SsER-A314Y mutant in which alanine at position 314 is replaced with tyrosine is a mutant that significantly improves the activity of ethyl 4-oxopentenoate (E / Z-2a) and ethyl 4-oxooctenoate (E-2b).

[0042] The present invention also provides a method for preparing the olefin reductase catalyst, which is preferably: culturing the recombinant expression transformant as described in technical solution four, and isolating and obtaining the recombinantly expressed olefin reductase SsER. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art that can grow the transformant and produce the recombinant olefin reductase of the present invention. The culture medium is preferably LB culture medium, and its formula is: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0. There are no special restrictions on the culture method and culture conditions, and they can be appropriately selected according to the common knowledge in the art according to factors such as the host cell type and the culture method, as long as the transformant can grow and produce the olefin reductase SsER. The specific operation of culturing the recombinant expression transformant can be carried out according to the conventional operation in the art. Preferably, the recombinant Escherichia coli E.coli BL21(DE3) / pET28a-SsER described in the present invention is inoculated into LB culture medium containing kanamycin and cultured at 37°C. When the optical density OD 600 Reach 0.5~1.0 (preferably OD 600 When the concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) is 0.1-1.0mmol / L (preferably 0.5mmol / L) at a final concentration is added to induce enzyme production, and the culture is continued at 16°C for 24 hours to efficiently express the olefin reductase SsER of the present invention. After the culture is completed, the precipitated bacterial cells are collected by centrifugation, which are the resting cells of the recombinant expression transformant; the harvested cells are suspended in PBS buffer (100mM, pH 7.0), ultrasonically disrupted, the disrupted liquid is centrifuged, and the supernatant is collected to obtain the crude enzyme solution of the recombinant olefin reductase SsER; the cell precipitate harvested by centrifugation is freeze-dried to obtain freeze-dried cells, which is conducive to long-term storage and convenient for future use.

[0043] Activity determination of olefin reductase SsER: preheat 1 ml reaction system (100 mmol / L sodium phosphate buffer, pH 7.0) containing 2 mmol / L α- / β-unsaturated ketoester and 0.1 mmol / L NADPH to 30°C, then add appropriate amount of olefin reductase SsER pure enzyme, mix well, and keep the reaction at 30°C. Detect the absorbance change of NADPH at 340 nm on a spectrophotometer, and record the absorbance change value within a certain period of time.

[0044] The enzyme activity was calculated according to the following formula:

[0045] Enzyme activity (U) = EW × V × 10 3 / (6220×l)

[0046] Where, EW is the change in absorbance at 340 nm within 1 min; V is the volume of the reaction solution, in mL; 6220 is the molar extinction coefficient of NADPH, in L / (mol·cm); l is the optical path length, in cm. One enzyme activity unit (U) corresponds to the amount of enzyme required to oxidize 1 μmol NADPH per minute under the above conditions.

[0047] Technical solution six of the present invention:

[0048] The invention provides an application of the ene reductase catalyst in asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters.

[0049] The chemical structure of the medium- and long-chain α- / β-unsaturated ketoester is shown below:

[0050]

[0051] Furthermore, the invention provides an application of the ene reductase catalyst in catalyzing the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters.

[0052] The asymmetric reduction catalytic route of the medium- and long-chain α- / β-unsaturated ketoester by olefin reductase SsER is as follows:

[0053]

[0054] The asymmetric reduction of the α- / β-unsaturated ketoester can be carried out according to the following preferred method: in a phosphate buffer at pH 7.0, in the presence of glucose dehydrogenase, glucose and NADP + In the presence of, under the action of the olefin reductase catalyst, the asymmetric reduction reaction of the α- / β-unsaturated ketoester is catalyzed.

[0055] In the application, the concentration of the substrate in the reaction solution can be 0.1 to 100 mmol / L. According to the reaction system used, the amount of the ene reductase (preferably the SsER-A314Y mutant) is 1 to 500 U / L. When the enzymatic asymmetric reduction of the medium- and long-chain α- / β-unsaturated ketoester is carried out, the coenzyme NADPH is oxidized to generate NADP + In order to recycle the coenzyme NADPH, glucose and glucose dehydrogenase from Bacillus megaterium were added to the reaction system (J Ind Microb Biotechnol, 2011, 38: 633–641). Depending on the reaction system, the activity unit loading of glucose dehydrogenase can be equal to that of the ene reductase. The molar ratio of glucose to substrate is 1.0 to 1.5, and the additional NADP +The dosage is 0 - 1.0 mmol / L. The buffer solution is sodium phosphate buffer, preferably with a pH range of 6.0 - 8.0, more preferably pH 7.0. The concentration of the phosphate buffer solution is 0.05 - 0.2 mol / L. The temperature of the enzymatic asymmetric reduction reaction is 25 - 40°C, preferably 30°C. During the reaction process, samples are taken intermittently to measure the reaction conversion rate. The reaction time is based on the time when the substrate is completely converted or the reaction conversion rate stops increasing, generally 1 - 24 h. In this reaction, the reaction is carried out in a 2 mL centrifuge tube. 8 mM of different prochiral substrates, 12 mM of glucose, 0.2 mM of NADP + , 1 U of the pure enzyme of the SsER-A314Y mutant, and 4 U of glucose dehydrogenase are added to 1 mL of sodium phosphate buffer (100 mM, pH 7.0). The reaction is carried out on an oscillator at 1000 rpm and 30°C. After reacting for 12 h, the reaction is terminated with 2 M sulfuric acid solution, extracted with an equal volume of ethyl acetate, and then dried over anhydrous sodium sulfate overnight. The substrate conversion rate and the ee value of the reduction product are measured, as shown in Table 2.

[0056] Table 2 Results of asymmetric reduction of different substrates catalyzed by SsER-A314Y

[0057]

[0058] The reaction conversion rate and the enantiomeric excess value (ee) of the product can be analyzed by gas chromatography. Preferably, a -5Sil MS capillary chromatographic column (30 m × 0.25 mm × 0.25 μm) is used for the analysis of the conversion rate. The carrier gas is nitrogen, and the detector is a flame ionization detector (FID). The inlet temperature is 280°C, and the detector temperature is 280°C. A GC / CP-Chirasil-Dex CB capillary chromatographic column (25 m × 0.25 mm × 0.25 μm) is used for the analysis of the ee value of the reduction product. The carrier gas is nitrogen, and the detector is a flame ionization detector (FID). The inlet temperature is 280°C, the detector temperature is 280°C, and the column temperature is programmed as shown in Table 3.

[0059] Table 3 Analysis conditions for the ee value of the product of asymmetric reduction of different substrates by SsER-A314Y

[0060]

[0061]

[0062] After the enzymatic asymmetric reduction reaction is completed, an equal amount of a conventional water-insoluble organic solvent in the art, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, methyl tert-butyl ether, etc., is used for extraction, and the extraction is repeated twice, and the extracts are combined and dried by adding anhydrous sodium sulfate. The solvent is removed by rotary evaporation to obtain the corresponding optically active medium- and long-chain multi-substituted alkyl lactone crude product.

[0063] The various reactions or detection conditions described in the present invention can be combined or modified according to common knowledge in the art and can be verified through experiments.

[0064] Unless otherwise specified, the raw materials or reagents used in the present invention are commercially available.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] (1) The olefin reductase SsER and its preferred SsER-A314Y mutant provided by the present invention are novel olefin reductases that can efficiently catalyze the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters (especially (E)-2a and (E)-2b), and the mutant activity is as high as 8.5 U / mg.

[0067] (2) The saturated ketoester compound of the present invention can be used as an intermediate for perfume products and has a wide range of application value in the perfume market.

[0068] (3) The reaction conditions of the present invention are mild, the conversion rate is high, the catalytic activity is high, the optical purity of the product is good, and the ee value can be higher than 99%, which has a good industrial application prospect. DETAILED DESCRIPTION

[0069] The present invention is described in detail below through specific examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The various reactions or detection conditions described in the summary of the invention can be combined or changed according to common knowledge in the art, and can be verified by experiments.

[0070] The sources of materials in the following examples are:

[0071] The parent recombinant plasmid pET28a-SsER contains the nucleic acid sequence shown in SEQ ID No. 1 in the sequence table, and is a sequence constructed by Shanghai Qingke Biotechnology Co., Ltd.

[0072] The expression plasmid pET28a was purchased from Novagen.

[0073] E. coli DH5α and E. coli BL21 (DE3) competent cells, 2× Taq PCR MasterMix, and agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0074] Restriction endonucleases EcoR I and Hind III are both commercially available products from New England Biolabs (NEB).

[0075] Example 1 Gene cloning of olefin reductase SsER

[0076] According to the open reading frame of olefin reductase SsER, upstream and downstream primers were designed, and PCR amplification was performed using the DNA of the recombinant plasmid pET28a-SsER as a template.

[0077] The designed upstream and downstream primers are as follows:

[0078] Upstream primer SEQ ID No.23:

[0079] 5'-CCG G.A.A.T.C. ATGGCAGACTTATTT-3′;

[0080] Downstream primer SEQ ID No.24:

[0081] 5'-CCC AAAGCTT AAACGGATAATCAAT-3';

[0082] The underlined portion of the upstream primer is the restriction endonuclease EcoR I cleavage site, and the underlined portion of the downstream primer is the restriction endonuclease Hind III cleavage site.

[0083] The PCR system was as follows: 25 μL of 2× Taq PCR MasterMix, 2.5 μL of upstream primer and downstream primer (10 ng / μL), 1 μL of recombinant plasmid pET28a-SsER (100 ng / μL), and 19 μL of ddHO. 2 O. The PCR amplification procedure is: 95℃ pre-denaturation for 3min followed by 20-32 cycles of: 95℃ denaturation for 30s, 50℃ annealing for 30s, 72℃ extension for 1min; after the cycle is completed, the final extension is 72℃ for 5min. The PCR amplification product is purified by gel electrophoresis, and the target fragment is recovered using a DNA recovery kit. After DNA sequencing, the open reading frame encoded by the sequence is 1068bp in length, and its base sequence is shown in SEQ ID No.1.

[0084] Example 2: Preparation of recombinant expression plasmid and recombinant expression transformant of olefin reductase SsER

[0085] The DNA fragment of olefin reductase SsER obtained by PCR amplification in Example 2 and the pET-28a empty vector plasmid were double-digested with restriction endonucleases EcoR I and Hind I for 10-20 minutes, and then purified by agarose gel electrophoresis and recovered by a DNA purification kit. The recovered DNA fragment of olefin reductase SsER and the empty vector plasmid were separated and plated on T 4 Under the action of DNA ligase, the cells were connected overnight at 16°C for 12 hours to obtain the recombinant plasmid pET28a-SsER.

[0086] The obtained recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells, spread on LB medium plates containing 50 μg / mL kanamycin, and cultured at 37°C for more than 8 hours. The grown colonies were verified by colony PCR, and positive clones that successfully amplified the target band of about 1068 bp were picked. After sequencing verification, positive clones were picked to obtain the recombinant expression transformant E.coli BL21 (DE3) / pET28a-SsER.

[0087] Example 3: Inducible expression of olefin reductase SsER

[0088] The recombinant expression transformant E. coli BL21 (DE3) / pET28a-SsER obtained in Example 2 was inoculated into LB medium containing 50 μg / mL kanamycin and cultured in a shaking incubator at 37 ° C for 12 h. Then, the inoculum was inoculated into a 500 ml conical flask containing 100 ml LB medium (containing 50 μg / mL kanamycin) at a rate of 1% (v / v), and the inoculum was placed in a shaking incubator at 37 ° C and 180 rpm. When the OD of the culture solution reached 600 When it reached 0.6, IPTG was added to a final concentration of 0.2 mmol / L for induction. After induction at 16°C for 24 h, the culture solution was centrifuged at 7500 rpm, the cell precipitate was collected, and washed with physiological saline to obtain resting cells.

[0089] 0.5 g of resting cells obtained by the above method are suspended in 100 mL of sodium phosphate buffer (100 mM, pH 6.0), ultrasonically disrupted in an ice-water bath, and the supernatant is collected by centrifugation to obtain a crude enzyme solution of the recombinant ene reductase SsER. The obtained crude enzyme solution is analyzed by polyacrylamide gel electrophoresis, and the recombinant ene reductase SsER exists in a soluble form. In addition, the obtained crude enzyme solution of the recombinant ene reductase SsER can be freeze-dried to obtain a crude enzyme powder of the recombinant ene reductase SsER.

[0090] Example 4 Site-directed saturation mutagenesis of olefin reductase SsER

[0091] Using pET28a-SsER as template, PrimeStar HS DNA Polymerase was used for PCR amplification. The PCR system was: pET28a-SsER plasmid (100nM, 1uL), upstream and downstream primers (10nM, 1uL), 5× PrimeStar Buffer (Mg 2+ plus, 4uL), dNTP Mixture (2.5mM, 1.6uL), PrimeStar HS DNA Polymerase (0.2uL), add sterile distilled water to make up to 20uL. PCR reaction program: (1) 95℃ pre-denaturation for 3min; (2) 98℃ denaturation for 10s; (3) set gradient temperature annealing for 10s; (4) 72℃ extension for 6min40s. Steps (2) to (4) were performed for a total of 30 cycles; and finally extended at 72℃ for 10min. Add 0.5μL of Dpn I enzyme to 8.5μL of PCR product and 1uL of rSmart cut buffer and incubate at 37℃ for 3h to digest the template. The digestion product was transformed into E. coli BL21 (DE3) competent cells and evenly spread on LB agar plates containing 50μg / ml kanamycin and placed in a 37℃ incubator for about 12h. The obtained monoclonal colonies were picked up and cultured in 96-well deep-well plates. The cultured cells were broken by lysozyme, and the expressed proteins were screened for high-throughput activity in 96-well plates using NADPH as a coenzyme. The mutants with higher activity were purified and characterized, and the corresponding genes were sequenced.

[0092] Through high-throughput screening with an ELISA instrument, it was found that replacing the 314th alanine with tyrosine (A314Y), glycine (A314G) and threonine (A314T) improved the activity of 4-oxopentenoic acid ethyl ester-(E) and 4-oxoctenoic acid ethyl ester-(E). It was purified and characterized, and the enzyme activity and kinetic parameters of the mutants were determined (as shown in Tables 4-7). Among them, SsER-A314Y was the best mutant, with an enzyme activity of 8.5 U / mg and a catalytic efficiency (K cat / K m ) was 1.3 times higher than that of SsER, and the catalytic efficiency (K cat / K m) was 4 times higher than that of SsER, and had better catalytic performance than other mutants, making it the best mutant. Method for determining the enzyme activity of SsER mutants: Preheat 1 mL of reaction system (100 mmol / L sodium phosphate buffer, pH 7.0) containing 2 mmol / L 4-oxopentenoic acid ethyl ester and 0.1 mmol / L NADPH to 30°C, then add an appropriate amount of SsER mutant enzyme solution, keep the reaction at 30°C, detect the absorbance change at 340 nm on a spectrophotometer, record the absorbance change within 1 min, and calculate the enzyme activity. High-throughput activity screening assay for SsER mutants: Sodium phosphate buffer (100 mmol / L, pH 7.0) containing 2 mmol / L 4-oxopentenoic acid ethyl ester and 0.1 mmol / L NADPH was dispensed into a 96-well plate and preheated to 30°C. Then, appropriate amounts of SsER mutant enzyme solution were added, and the reaction was oscillated at 30°C. The absorbance change of NADPH at 340 nm was detected on a microplate reader, the absorbance change within 10 min was recorded, and the corresponding enzyme activity was calculated.

[0093] Table 4 Specific activity of different mutants against ethyl 3-methyl-4-oxopentenoate-(E)

[0094]

[0095] Table 5 Specific activity of different mutants against ethyl 3-methyl-4-oxooctenoate-(E)

[0096]

[0097]

[0098] Table 6 Kinetic parameters of different mutants for ethyl 3-methyl-4-oxopentenoate-(E)

[0099]

[0100] Table 7 Kinetic parameters of different mutants for ethyl 3-methyl-4-oxooctenoate-(E)

[0101]

[0102] Example 5 Effect of pH on the catalytic activity of olefin reductase SsER

[0103] The effect of pH on the activity of recombinant carbonyl reductase SsER was determined by standard methods in the range of pH 4.5 to 8.5. The buffers were citric acid-sodium citrate buffer (4.5 to 6.0), sodium phosphate buffer (6.0 to 7.5), and Tris-HCl buffer (7.5 to 8.5).

[0104] In 1 mL of the above buffer system, ethyl 4-oxooctenoate-(E) and NADPH were added to final concentrations of 2 mmol / L and 0.1 mmol / L, respectively, preheated to 30°C, and then an appropriate amount of ene reductase SsER was added, mixed evenly, and kept at 30°C for reaction. The absorbance change of NADPH at 340 nm was detected on a spectrophotometer, and the activity difference of ene reductase SsER in buffer solutions of different pH was determined. The results are shown in Table 8. The preferred pH range of the enzymatic reaction is 6.0 to 7.0, and more preferably pH 6.5.

[0105] Table 8 Effect of pH on the activity of SsER catalyzing the asymmetric reduction of ethyl 4-oxooctenoate-(E)

[0106]

[0107] Example 6 Effect of temperature on the catalytic activity of olefin reductase SsER

[0108] In 1 mL of sodium phosphate buffer (100 mM, pH 6.5), ethyl 4-oxooctenoate (E) and NADPH were added to final concentrations of 2 mmol / L and 0.1 mmol / L, respectively, and preheated for 5 min at 25-50°C, then an appropriate amount of ene reductase was added, mixed evenly, and kept in the same temperature as the preheating temperature for reaction, and the absorbance change of NADPH at 340 nm was detected on a spectrophotometer to determine the activity difference of ene reductase SsER under different temperature conditions, and the results are shown in Table 9. The preferred temperature range for the enzymatic reaction is 30-40°C.

[0109] Table 9 Effect of temperature on SsER asymmetric catalytic reduction

[0110]

[0111] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

[0112] East China University of Science and Technology

[0113] An ene reductase and its application in asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters twenty four

[0115] SIPOSequenceListing 1.0 1 1068

[0118] DNA

[0119] Swings in Samui 1

[0121] 2 356

[0124] PRT

[0125] Swingsia samuiensi 2

[0127] MADLFSSIKIGDIITKNRIFMAPLTRARVERDAVPVPMMAEYYAQRAQAGLIISEATGISREGLGWPYAPGIWTDEQVEAWKSVTQAVHDKGGKIVCQLCHMGRAVHSSVTGLQPVSASETTAPDEVHTYDGKKPYEKARALTKSDITRILNDYEQAARNAMRAGFDGVQIHAANGYLIDQFLRDGTNHRSDEYGGSLENRVRLLVEVTQRVIATVGAEKTGVRLSPNGDSQGVIDSAPEKIFVLAAQELERLGVAWLELRENSTTGTFLAPTDQPKLSPEIRKVFHRSLVLNQDYSFEEAQAAIRDGHADAIAFGRKFISNPDLPDRFAKNIPLQESNVSTWYSRGEEGYIDYPF 3 33

[0130] DNA

[0131] Artificial Sequence 3

[0133] cgtagcctgg ttctgnnkca ggattatagc ttt 33 4 33

[0136] DNA

[0137] Artificial Sequence 4

[0139] aaagctataa tcctgmnnca gaaccaggct acg 33 5 33

[0142] DNA

[0143] Artificial Sequence 5

[0145] agcctggttc tgaatnnkga ttatagcttt gaa 33 6 33

[0148] DNA

[0149] Artificial Sequence 6

[0151] ttcaaagcta taatcmnnat tcagaaccag gct 33 7 33

[0154] DNA

[0155] Artificial Sequence 7

[0157] ctggttctga atcagnnkta tagctttgaa gaa 33 8 33

[0160] DNA

[0161] Artificial Sequence 8

[0163] ttcttcaaag ctatamnnct gattcagaac cag 33 9 33

[0166] DNA

[0167] Artificial Sequence 9

[0169] gttctgaatc aggatnnkag ctttgaagaa gca 33 10 33

[0172] DNA

[0173] Artificial Sequence 10

[0175] tgcttcttca aagctmnnat cctgattcag aac 33 11 33

[0178] DNA

[0179] Artificial Sequence 11

[0181] ctgaatcagg attatnnktt tgaagaagca cag 33 12 33

[0184] DNA

[0185] Artificial Sequence 12

[0187] ctgtgcttct tcaaamnnat aatcctgatt cag 33 13 33

[0190] DNA

[0191] Artificial Sequence 13

[0193] catgcagatg caattnnktt tggtcgtaaa ttt 33 14 33

[0196] DNA

[0197] Artificial Sequence 14

[0199] aaatttacga ccaaamnnaa ttgcatctgc atg 33

[0200] 15. 33

[0202] DNA

[0203] Artificial Sequence 15

[0205] gcagatgcaa ttgcannkgg tcgtaaattt att 33 16 33

[0208] DNA

[0209] Artificial Sequence 16

[0211] aataaattta cgaccmnntg caattgcatc tgc 33 17 33

[0214] DNA

[0215] Artificial Sequence 17

[0217] gatgcaattg catttnnkcg taaatttatt agc 33 18 33

[0220] DNA

[0221] Artificial Sequence 18

[0223] gctaataaat ttacgmnnaa atgcaattgc atc 33 19 33

[0226] DNA

[0227] Artificial Sequence 19

[0229] gcaattgcat ttggtnnkaa atttattagc aat 33 20 33

[0232] DNA

[0233] Artificial Sequence 20

[0235] attgctaata aatttmnnac caaatgcaat tgc 33 twenty one 33

[0238] DNA

[0239] Artificial Sequence twenty one

[0241] attgcatttg gtcgtnnktt tattagcaat ccg 33 twenty two 33

[0244] DNA

[0245] Artificial Sequence twenty two

[0247] cggattgcta ataaamnnac gaccaaatgc aat 33 twenty three twenty four

[0250] DNA

[0251] Artificial Sequence twenty three

[0253] ccggaattca tggcagactt attt 24 twenty four twenty four

[0256] DNA

[0257] Artificial Sequence twenty four

[0259] cccaagctta aacggataat caat

Claims

1. An ene reductase, It is characterized in that The alkene reductase has one of the following sequences: (1) replacing the 314th alanine in the amino acid sequence shown in SEQ ID No. 2 with glycine; (2) replacing the 314th alanine of the amino acid sequence shown in SEQ ID No. 2 with tyrosine; (3) The alanine at position 314 of the amino acid sequence shown in SEQ ID No. 2 is replaced by threonine.

2. An isolated nucleic acid, It is characterized in that The nucleic acid encodes the alkene reductase of claim 1.

3. A recombinant expression vector, It is characterized in that Comprising the nucleic acid as described in claim 2.

4. A recombinant expression transformant, It is characterized in that Comprising the recombinant expression vector as described in claim 3.

5. An olefin reductase catalyst, It is characterized in that Choose from any of the following: (1) culturing the recombinant expression transformant according to claim 4, and isolating transformant cells containing the olefin reductase according to claim 1; (2) culturing the recombinant expression transformant according to claim 4, isolating transformant cells containing the olefin reductase according to claim 1, and disrupting the transformant cells containing the olefin reductase to obtain a cell disrupted liquid; (3) Cultivating the recombinant expression transformant according to claim 4, isolating transformant cells containing the ene reductase according to claim 1, disrupting the transformant cells containing the ene reductase, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the ene reductase to obtain a lyophilized enzyme powder.

6. Use of the olefin reductase catalyst as claimed in claim 5, It is characterized in that The ene reductase catalyst is used in catalyzing the asymmetric reduction of α- / β-unsaturated ketoesters to prepare optically active α- / β-substituted γ-ketoesters.

7. Use of the olefin reductase catalyst as claimed in claim 6, It is characterized in that The chemical structure of the α- / β-unsaturated ketoester is any of the following: , , , or .

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