Esterase mutant as well as preparation method and application thereof

Abstract

The invention discloses an esterase mutant as well as a preparation method and application thereof. In the invention, a plurality of esterase mutants are obtained by carrying out directional mutation modification on esterase from Acinetobacter sp.WCHAc010052, the esterase mutants have better stereoselectivity and can effectively improve the production efficiency of the (S)-3-cyclohexene-1-formic acid, the ee value of the obtained (S)-3-cyclohexene-1-formic acid is greater than 98%, the purity is greater than 99%, the theoretical yield can reach 50%, and the method is suitable for industrial production. And the method has important significance on the industrial large-scale production of the (S)-3-cyclohexene-1-formic acid.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to esterase mutants, their preparation methods, and applications. Background Technology

[0002] Edoxaban (trade name: Savaysa) is a small-molecule oral anticoagulant developed by Daiichi Sankyo Co., Ltd. of Japan, with a relative molecular mass of 738.27. In 2014, the Canadian guidelines for atrial fibrillation management recommended it for anticoagulation therapy in patients with non-valvular atrial fibrillation. Edoxaban's therapeutic efficacy is comparable to warfarin, a first-line anticoagulant used to prevent stroke and systemic thrombosis in patients with atrial fibrillation, with a lower incidence of overall bleeding events and cardiovascular death, showing excellent potential for application. Compared to similar factor Xa inhibitors (such as apixaban and rivaroxaban), edoxaban has a shorter half-life, thereby effectively reducing the risk of bleeding. Moreover, these similar coagulation factor Xa inhibitors have other usage drawbacks, such as requiring the kidneys to eliminate drug accumulation, which can easily induce other diseases; or significant antagonistic effects when used in combination (e.g., rivaroxaban and apixaban cannot be used in combination with P-pg and CYP3A4 inhibitors). Eduxaban, however, has not been found to have these usage drawbacks to date, thus giving it a broad market prospect.

[0003] Chiral 3-cyclohexene-1-carboxylic acid is an important chemical reagent and organic intermediate, widely used in pharmaceuticals, chemicals, and other fields. For example, (R)-3-cyclohexene-1-carboxylic acid can be used to synthesize various pharmaceutical intermediates, such as the intermediate for synthesizing the antitumor drug (+)-Phyllanthocin, and the C-terminal intermediate for FK 506 (Prograf: a macrolide antibiotic isolated from Streptomyces by Fujisawa Pharmaceutical Co., Ltd., Japan). 24 -C 34 Fragment C of Leustroducsin B, and the initial raw material for oseltamivir phosphate (Tamiflu: a drug for the prevention and treatment of influenza), etc. Generally, the early construction and introduction of chiral centers can reduce cumbersome and complex process steps in the synthesis. For example, (S)-3-cyclohexene-1-carboxylic acid, as a starting material for edoxaban, can generate key chiral intermediates of edoxaban through multiple chemical steps, and finally obtain edoxaban through the stacking of other intermediates. Therefore, how to efficiently prepare (S)-3-cyclohexene-1-carboxylic acid is key to the industrialization of edoxaban.

[0004] In existing technologies, diastereomeric resolution methods are used to obtain (S)-3-cyclohexene-1-carboxylic acid. For example, using chiral phenylethylamine as a chiral resolving agent yields a diastereomeric isomer of racemic (S)-3-cyclohexene-1-carboxylic acid formed by phenylethylamine. Based on the difference in solubility of these two isomers in acetone, (S)-3-cyclohexene-1-carboxylic acid is separated. This process requires slow cooling and recrystallization six times, but the final yield of (S)-3-cyclohexene-1-carboxylic acid is only 28.7%. Furthermore, this method requires the use of toxic acetone during resolution and is complex. Therefore, a greener, more environmentally friendly, and simpler preparation method is needed. In related technologies, enzymatic resolution is mainly used to produce (S)-3-cyclohexene-1-carboxylic acid. However, existing enzymatic resolution methods have significant drawbacks. For example, using animal-derived esterases (mainly porcine liver esterase) to produce (S)-3-cyclohexene-1-carboxylic acid is problematic because these enzymes are primarily extracted from animals. In vitro expression methods can only achieve efficient expression in Origami series of basal bacteria, which cannot yet be cultured at high densities, thus greatly limiting the large-scale application of enzymatic resolution. Secondly, the expression of porcine liver esterase requires molecular chaperones, further increasing the difficulty of in vitro expression. Existing microbial enzymatic processes can only hydrolyze (R)-3-cyclohexene-1-carboxylic acid from cyclohexene carboxylate, retaining (S)-3-cyclohexene-1-carboxylic acid, followed by further hydrolysis with NaOH to obtain (S)-3-cyclohexene-1-carboxylic acid. As described in Chinese patent CN112813131B, a hydrolysis conversion rate of over 62% is required to obtain a product that meets the chiral purity requirements. This demonstrates that although currently available enzymes generally possess high activity, their selectivity is too poor, thus affecting the yield of the final product and increasing production costs. Therefore, obtaining a highly active and stereoselective esterase / lipase for the industrial production of (S)-3-cyclohexene-1-carboxylic acid is of great significance. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the objective of this invention is to provide an esterase mutant, its preparation method, and its application. In this invention, various esterase mutants are obtained through targeted mutagenesis of an esterase derived from Acinetobacters p. WCHAc010052. These esterase mutants exhibit good stereoselectivity and can effectively improve the production efficiency of (S)-3-cyclohexene-1-carboxylic acid. The resulting (S)-3-cyclohexene-1-carboxylic acid has an ee value greater than 98%, a purity greater than 99%, and a theoretical yield of up to 50%. This is of great significance for the large-scale industrial production of (S)-3-cyclohexene-1-carboxylic acid.

[0006] In a first aspect, the present invention provides an esterase mutant comprising:

[0007] (1) An esterase mutant obtained by substituting, deleting, or adding 1-10 amino acid residues in the sequence shown in SEQ ID NO: 2; or

[0008] (2) An esterase mutant that has at least 95% sequence identity and retains catalytic activity compared to the one shown in SEQ ID NO: 2.

[0009] In some embodiments of the present invention, the number of amino acid residues replaced, deleted, or added is 1-5.

[0010] In some embodiments of the present invention, the number of amino acid residues replaced, deleted, or added is 1, 2, 3, 4, or 5.

[0011] In some embodiments of the present invention, the esterase mutant is an esterase mutant obtained by replacing 1, 2, 3, 4 or 5 amino acid residues in the sequence shown in SEQ ID NO: 2.

[0012] In some embodiments of the present invention, the esterase mutant has at least 98% or 99% sequence identity.

[0013] In this invention, the term "retaining catalytic activity" refers to the preservation of catalytic activity against a substrate, in which the substrate is racemic 3-cyclohexene-1-carboxylate. In this invention, "retaining catalytic activity" includes having catalytic activity similar to or stronger than that of the esterase shown in SEQ ID NO: 2, including having catalytic efficiency or catalytic capacity of 50%-10000% compared to the esterase shown in SEQ ID NO: 2.

[0014] In some embodiments of the present invention, the positions where the amino acid residues are replaced, deleted, or added include: amino acid residues at positions 67, 78, 139, 230, and 249, based on the sequence shown in SEQ ID NO: 2.

[0015] In some embodiments of the present invention, the amino acid residue substitutions include at least one of I67A, F78L, H139M, V230M, and L249A.

[0016] In some embodiments of the present invention, the amino acid residues are replaced with a combination of I67A, F78L, H139M, V230M and L249A.

[0017] In some embodiments of the present invention, the esterase mutant includes: an esterase mutant having the sequence shown in SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27 or 29.

[0018] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-H139M, which is formed by mutating the H at position 139 of Est (i.e., the esterase shown in SEQ ID NO: 2) to M, and the amino acid sequence is shown in SEQ ID NO: 13.

[0019] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-L249A, which is formed by mutating L at position 249 of Est to A, and the amino acid sequence is shown in SEQ ID NO: 15.

[0020] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-V230M, which is formed by mutating V at position 230 of Est to M, and the amino acid sequence is shown in SEQ ID NO: 17.

[0021] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-I67A, which is formed by mutating I at position 67 of Est to A, and the amino acid sequence is shown in SEQ ID NO: 19.

[0022] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-F78L, which is formed by mutating F at position 78 of Est to L, and the amino acid sequence is shown in SEQ ID NO: 21.

[0023] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-H139M-L249A, which is formed by mutating the H at position 139 of Est to M and the L at position 249 to A, and the amino acid sequence is shown in SEQ ID NO: 23.

[0024] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-H139M-L249A-V230M, which is formed by mutating the H at position 139 of Est to M, the L at position 249 to A, and the V at position 230 to M, and the amino acid sequence is shown in SEQ ID NO: 25.

[0025] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-H139M-L249A-V230M-I67A, which is formed by mutating the H at position 139 of Est to M, the L at position 249 to A, the V at position 230 to M, and the I at position 67 to A, with the amino acid sequence shown in SEQ ID NO: 27.

[0026] In some embodiments of the present invention, the esterase mutant includes: esterase mutant Est-H139M-L249A-V230M-I67A-F78L, which is obtained by mutating the H at position 139 of Est to M, the L at position 249 to A, the V at position 230 to M, the I at position 67 to A, and the F at position 78 to L, with the amino acid sequence shown in SEQ ID NO: 29.

[0027] In some embodiments of the present invention, the esterase mutant may be further modified, including but not limited to: glycosylation, phosphorylation, acetylation, methylation, ubiquitination or esterification; or the introduction of non-natural amino acids (such as α-aminoketo acid or β-aminoketo acid).

[0028] In a second aspect, the present invention provides a nucleic acid molecule encoding the esterase mutant described above.

[0029] In some embodiments of the present invention, the nucleic acid molecule has the sequence shown in SEQ ID NO:14, 16, 18, 20, 22, 24, 26, 28 or 30.

[0030] In some embodiments of the present invention, the nucleic acid molecule is further linked with a modifying sequence and / or a functional sequence.

[0031] In some embodiments of the present invention, the modified sequence and / or functional sequence includes at least one of the following: signal peptide, promoter, enhancer, terminator, tool enzyme recognition site, ribozyme, self-cleaving intron, miRNA binding site or ribosome binding site (RBS).

[0032] A third aspect of the present invention provides a biomaterial comprising any one of the following (1)-(3):

[0033] (1) An expression unit containing the nucleic acid molecules described above;

[0034] (2) Transformants containing the nucleic acid molecules described above;

[0035] (3) Transformers containing the expression in (1).

[0036] In some embodiments of the present invention, the expression unit includes a plasmid.

[0037] In this invention, the term "expression vector" refers to a vector or expression system used to integrate or insert a targeted exogenous gene.

[0038] In some embodiments of the present invention, the transformants include bacteria, fungi, viruses, plant cells, or animal cells.

[0039] In this invention, the term "transformant" refers to a recipient cell that acquires a new genetic marker after incorporation or introduction of a foreign gene.

[0040] In some embodiments of the present invention, the transformant does not involve plant or animal reproductive materials.

[0041] In some embodiments of the present invention, the transformants include common cell vectors such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pichia pastoris.

[0042] In some embodiments of the present invention, the construction of the expressants and transformants can be achieved based on any conventional techniques in the art.

[0043] A fourth aspect of the present invention provides a composition comprising at least one of the esterase mutant, nucleic acid molecule, and biological material described in the above aspects.

[0044] In some embodiments of the present invention, the composition further includes excipients.

[0045] In some embodiments of the present invention, the excipients are rationally selected based on factors such as the product form of the composition, its intended use, and the route of administration, and include, but are not limited to: diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, crospovidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0046] A fifth aspect of the invention provides the use of at least one of the esterase mutants, nucleic acid molecules, biomaterials, and compositions described above in biosynthesis.

[0047] In some embodiments of the present invention, the biosynthesis includes at least one of biocatalytic reaction, biofermentation and bioenzymatic hydrolysis.

[0048] In some embodiments of the present invention, the biosynthesis is a biocatalytic reaction.

[0049] In some embodiments of the present invention, at least one of the esterase mutant, nucleic acid molecule, biomaterial, and composition is used as a catalyst in the biocatalytic reaction.

[0050] A sixth aspect of the present invention provides the use of at least one of the esterase mutants, nucleic acid molecules, biomaterials and compositions described above in the preparation of edoxaban or an intermediate thereof.

[0051] In some embodiments of the present invention, the intermediate includes (S)-3-cyclohexene-1-carboxylic acid.

[0052] A seventh aspect of the present invention provides a method for preparing (S)-3-cyclohexene-1-carboxylic acid, comprising the following steps:

[0053] Catalysis is performed using at least one of the esterase mutants, nucleic acid molecules, biomaterials, and compositions described above, along with racemic 3-cyclohexene-1-carboxylate.

[0054] In some embodiments of the present invention, the catalytic conditions include: a catalytic temperature of 28-37°C and a catalytic time of 8-14 h.

[0055] In some embodiments of the present invention, the catalytic temperature is 30-35°C.

[0056] In some embodiments of the present invention, the catalytic temperature is 30°C.

[0057] In some embodiments of the present invention, the catalytic time is 8-12 hours.

[0058] In some embodiments of the present invention, the catalytic time is 8 hours.

[0059] In some embodiments of the present invention, the catalytic system further contains a solvent.

[0060] In some embodiments of the present invention, the solvent includes a buffer solution.

[0061] In some embodiments of the present invention, the buffer solution comprises a phosphate buffer solution.

[0062] In some embodiments of the present invention, the pH of the catalytic system is 5-10.

[0063] In some embodiments of the present invention, at least one of the esterase mutant, nucleic acid molecule, biomaterial, and composition is used as a catalyst.

[0064] In some embodiments of the present invention, the form of the catalyst is not limited, and may include wet bacterial cells, wet bacterial cells immobilized with cells, enzymes extracted from wet bacterial cells after ultrasonic disruption, or immobilized enzymes. When it is a wet bacterial cell, the water content is 70-90% by mass.

[0065] In some embodiments of the present invention, the culture medium for the bacteria can be any suitable culture medium in the art, including but not limited to LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0066] In some embodiments of the present invention, the weight ratio of substrate to catalyst is 50-160:30-100.

[0067] In some embodiments of the present invention, the bacterial activity in the catalyst is: 130-140 U / g for the parent (i.e., the esterase shown in SEQ ID NO: 2) and 100-110 U / g for the esterase mutant.

[0068] An eighth aspect of the present invention provides a method for preparing edoxaban, comprising the following steps:

[0069] (1) Using at least one of the esterase mutants, nucleic acid molecules, biomaterials and compositions described above, racemic 3-cyclohexene-1-carboxylate esters is catalyzed to obtain (S)-3-cyclohexene-1-carboxylic acid;

[0070] (2) Edoxaban was prepared using (S)-3-cyclohexene-1-carboxylic acid.

[0071] In some embodiments of the present invention, the preparation method in step (2) is not limited and can be prepared by any known preparation method in the art.

[0072] In some embodiments of the present invention, the catalytic conditions are limited as defined above.

[0073] In some embodiments of the present invention, the catalytic system is defined as described above.

[0074] In some embodiments of the present invention, the weight ratio of substrate to catalyst is limited as defined above.

[0075] In some embodiments of the invention, the bacterial activity in the catalyst is limited as defined above.

[0076] The beneficial effects of this invention are:

[0077] This invention utilizes directed evolution technology to successfully modify and obtain a variety of high-activity esterase mutants, which change the stereoselectivity of the parent esterase, reversing it from R-type selectivity to S-type selectivity. The product changes from (R)-3-cyclohexene-1-carboxylic acid to (S)-3-cyclohexene-1-carboxylic acid. Furthermore, through multiple rounds of iterative mutation, the selectivity of the mutants is improved, enabling the efficient generation of (S)-3-cyclohexene-1-carboxylic acid with a high ee value.

[0078] The emergence of the esterase mutant in this invention completely changes the existing production process of (S)-3-cyclohexene-1-carboxylic acid. The previous process of selectively producing R-type 3-cyclohexene-1-carboxylic acid to obtain S-type 3-cyclohexene-1-carboxylic acid ester and then further hydrolyzing it to obtain S-type 3-cyclohexene-1-carboxylic acid has been changed to a one-step direct production of S-type 3-cyclohexene-1-carboxylic acid. This greatly reduces production costs and complexity and has good application prospects in the production of (S)-3-cyclohexene-1-carboxylic acid. Attached Figure Description

[0079] Figure 1 This is a schematic diagram of the reaction process of catalyzing the hydrolysis of racemic 3-cyclohexene-1-carboxylate using the esterase mutant Est-H139M-L249A-V230M-I67A-F78L.

[0080] Figure 2 The results are based on the HPLC detection of the ee value of (S)-3-cyclohexene-1-carboxylic acid obtained by the method in the embodiments of the present invention.

[0081] Figure 3 The results are based on the HPLC analysis of the purity of (S)-3-cyclohexene-1-carboxylic acid obtained by the method described in the embodiments of this invention. Detailed Implementation

[0082] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0083] Example 1

[0084] In this embodiment, a total of 7 esterases were screened and recombinant plasmid vectors were constructed based on the commercially available pET28a plasmid. The recombinant plasmid vectors were transformed into Escherichia coli BL21 to obtain recombinant genetically engineered bacteria. The recombinant genetically engineered bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Then, 2% inoculum (v / v) was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 150 rpm until the bacterial concentration OD600 = 0.6 was reached. IPTG was added to a final concentration of 0.1 mM and induced at 20°C for 12 h. The bacterial cells were collected by centrifugation at 12000 rpm for 10 min at 4°C. The wet bacterial cells were washed with 0.85% physiological saline and used as a catalyst to catalyze racemic 3-cyclohexene-1-carboxylate to further screen for the optimal esterase.

[0085] The information on the seven esterases used is shown in the table below.

[0086] Table 1. Esterase Information and Sources

[0087]

[0088]

[0089] The esterase sequences described above were all synthesized by Beijing Qingke Biotechnology Co., Ltd. based on database records.

[0090] The specific steps for using recombinant genetically engineered bacteria as a catalyst to catalyze racemic 3-cyclohexene-1-carboxylate are as follows: 1.0 g of racemic 3-cyclohexene-1-carboxylate (final concentration of 50 g / L) and 0.6 g of the recombinant genetically engineered bacteria prepared in the above steps are added to 20 mL of phosphate buffer (100 mM, pH 7.0) and incubated at 30 °C for 12 h.

[0091] The enantiomeric excess (ee value) of (S)-3-cyclohexene-1-carboxylic acid was determined using liquid chromatography.

[0092] The results are shown in the table below.

[0093] Table 2. Catalytic effects of recombinant genetically engineered bacteria producing different esterases on racemic 3-cyclohexene-1-carboxylate.

[0094]

[0095] It was found that esterases 1, 2, and 3 exhibited R-type selectivity for the substrate racemic 3-cyclohexene-1-carboxylate and could catalyze the hydrolysis of the R-type substrate, while esterases 4, 5, 6, and 7 showed no activity for the substrate. Moreover, esterase 1 showed the highest conversion rate (enzyme activity of 138.7 U / g, and the activities of the esterases used subsequently were all between 130-140 U / g) and a lower E value, making it more suitable for stereoselective inversion modification. Therefore, esterase 1 (hereinafter referred to as Est) derived from Acinetobacters p. WCHAc010052 was selected as the parent enzyme for modification.

[0096]

[0097] The amino acid sequence is: MVAFNTKIQKMMEKGQGAAARTLDRLPGIAQETLSKALGYPYHYPDLDPFIKCMMAAQIKQGKIGFIGDDPAHSRKVFDQQMQSIRAQATPVKRIEDLRLPLHSGTIFARHYHPAPHKKLPMIVFYHGGGFVVGGMDSHDEACRLIAVHAGAQVLSIDYPLAPEASPKQLIQTCEDA LAWVYQNRRQFKILKNRIAVAGDSAGGNISAVVAQRSANKVYAPEAQFLIYPVVDFKSRHPSFYAYKDGLVLTGADVDYVTDYYATQHDIQLDDPMISPTYGNLKRQPPAFVVTAGHDLLHDEGEIYAHKLRHQGNKVEYQEYSDQTHGFLNLTPVSRRAKKITIEISKNFRKFWDRQRAA(SEQ ID NO:2).

[0098] Example 2

[0099] In this embodiment, Est (esterase-1) is modified by targeted point mutations, wherein the point mutation sites include His at site 139, Leu at site 249, Val at site 230, Ala at site 67, and Phe at site 78.

[0100] The specific method is as follows:

[0101] Using the recombinant plasmid vector containing Est (named pET28a-Est) constructed in the above embodiment as a template, the template was amplified by overlapping extension PCR to obtain a single mutant.

[0102] The overlap extension PCR reaction system is shown in the table below.

[0103] Table 3 Overlap Extension PCR Reaction System

[0104] Components content template 0.1ng-1ng 2×Phanta Max buffer 25μL dNTPs (10 mM / species) 1μL Mutant primers 1 μL each upstream and downstream Phanta Max Super-Fidelity DNA Polymerase 1U ddH2O Add to 50μL

[0105] The overlap extension PCR reaction program is as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 30s, 65℃ annealing for 30s, 72℃ extension for 6min, 30 cycles; 72℃ complete extension for 7min to obtain the amplification product (i.e., the plasmid sequence containing the mutant sequence).

[0106] The mutation primers used are shown in the table below.

[0107] Table 4. Primers for Est point mutation

[0108]

[0109] After confirming the PCR amplification product was correct by 0.9% agarose gel electrophoresis, the mutant sequence was obtained by digestion with Dpn I restriction enzyme. The digestion reaction system was as follows: 1 μL of Dpn I restriction enzyme was added to 20 μL of PCR amplification product, digested at 37°C for 3 h to fully remove plasmid DNA, followed by inactivation at 65°C for 10 min. The obtained mutant sequence was transformed into *E. coli* BL21(DE3) competent cells by heat shock. The transformed cells were plated on LB agar plates containing kanamycin and cultured overnight, yielding a mutant library of approximately 300 clones per plate. Four to five clones were selected from these clones and cultured on LB medium at 37°C for 8 h, followed by sequencing verification. After verification, the corresponding recombinant engineered bacteria were named esterase recombinant engineered bacteria E. coli BL21(DE3) / Est-H139M, E. coli BL21(DE3) / Est-L249A, E. coli BL21(DE3) / Est-V230M, E. coli BL21(DE3) / Est-I60A and E. coli BL21(DE3) / Est-F78L, respectively.

[0110] The amino acid sequence of Est-H139M is shown in SEQ ID NO:13, and the nucleotide sequence is shown in SEQ ID NO:14.

[0111] The amino acid sequence of Est-L249A is shown in SEQ ID NO:15, and the nucleotide sequence is shown in SEQ ID NO:16.

[0112] The amino acid sequence of Est-V230M is shown in SEQ ID NO:17, and the nucleotide sequence is shown in SEQ ID NO:18.

[0113] The amino acid sequence of Est-I60A is shown in SEQ ID NO:19, and the nucleotide sequence is shown in SEQ ID NO:20.

[0114] The amino acid sequence of Est-F78L is shown in SEQ ID NO:21, and the nucleotide sequence is shown in SEQ ID NO:22.

[0115] Further, the corresponding mutant plasmids containing a single point mutation were extracted, and point mutations were introduced according to the method described in the above embodiments to obtain mutants with multiple mutation sites, namely Est-H139M-L249A, Est-H139M-L249A-V230M, Est-H139M-L249A-V230M-I67A, and Est-H139M-L249A-V230M-I67A-F78L, and their recombinant Escherichia coli. The mutation primers and PCR reaction system used are as described in the steps above.

[0116] The amino acid sequence of Est-H139M-L249A is shown in SEQ ID NO:23, and the nucleotide sequence is shown in SEQ ID NO:24.

[0117] The amino acid sequence of Est-H139M-L249A-V230M is shown in SEQ ID NO:25, and the nucleotide sequence is shown in SEQ ID NO:26.

[0118] The amino acid sequence of Est-H139M-L249A-V230M-I67A is shown in SEQ ID NO:27, and the nucleotide sequence is shown in SEQ ID NO:28.

[0119] The amino acid sequence of Est-H139M-L249A-V230M-I67A-F78L is shown in SEQ ID NO:29, and the nucleotide sequence is shown in SEQ ID NO:30.

[0120] Following the methods described in the above embodiments, the catalytic effect of recombinant engineered bacteria based on these mutants on the hydrolysis reaction of racemic 3-cyclohexene-1-carboxylate was tested.

[0121] The results are shown in the table below.

[0122] Table 5. Catalytic effects of recombinant genetically engineered bacteria producing different esterase mutants on racemic 3-cyclohexene-1-carboxylate.

[0123]

[0124] As shown in the results above, after reacting at 30℃ for 12 h, the final conversion rate of racemic 3-cyclohexene-1-carboxylate catalyzed by parental Est was 73.9%, with (R)-3-cyclohexene-1-carboxylate almost completely converted. The ee value of (S)-3-cyclohexene-1-carboxylate was 95.1%, and the ratio of (R)-3-cyclohexene-1-carboxylic acid to (S)-3-cyclohexene-1-carboxylic acid in the product was close to 1.8:1, with an ee value of only 29.1%. Therefore, the calculated E value of parental Est was only 4.5. However, an E value greater than 100 or even 200 is generally required for significant industrial application value. This indicates that the yield of (S)-3-cyclohexene-1-carboxylate ester prepared from unmodified parental Est and then hydrolyzed to (S)-3-cyclohexene-1-carboxylic acid is extremely low, the cost is too high, and there are significant drawbacks. Recombinant engineered bacteria based on single-point mutants such as Est-H139M, Est-L249A, Est-V230M, Est-I67A, and Est-F78L all showed an E value of around 1 when the bacterial concentration was the same. However, after performing superimposed mutations at the five sites, it was found that the stereoselectivity was reversed, changing from R-type selectivity to S-type selectivity, and the product generated became (S)-3-cyclohexene-1-carboxylic acid. The E values ​​of the multi-site mutants Est-H139M-L249A, Est-H139M-L249A-V230M, Est-H139M-L249A-V230M-I67A, and Est-H139M-L249A-V230M-I67A-F78L are 19.5, 52.1, 105.5, and greater than 200, respectively. The ee values ​​of the generated (S)-3-cyclohexene-1-carboxylic acid are 77.9%, 87.3%, 92.5%, and 98.1%, respectively, which meet the requirements for industrial application. In summary, the 5-site mutant Est-H139M-L249A-V230M-I67A-F78L produces (S)-3-cyclohexene-1-carboxylic acid with the highest ee value (but the enzyme activity is slightly reduced, around 100-110 U / g), and has relatively higher potential for industrial applications.

[0125] Example 3

[0126] In this embodiment, the 5-site mutant Est-H139M-L249A-V230M-I67A-F78L obtained in the above embodiment was used as the experimental object to test the effects of cell concentration and substrate concentration on the catalytic effect in the catalytic reaction.

[0127] The steps for catalyzing racemic 3-cyclohexene-1-carboxylate with recombinant genetically engineered bacteria are the same as in Example 1 above, except that the final concentration of recombinant genetically engineered bacteria (20-100 g / L, i.e., the amount added is 0.4-2.0 g) and the final concentration of racemic 3-cyclohexene-1-carboxylate (50-160 g / L, i.e., the amount added is 1.0-3.2 g) are adjusted respectively.

[0128] The results are shown in the table below.

[0129] Table 6. Effects of different bacterial cell concentrations and substrate concentrations on catalytic efficacy.

[0130] Substrate concentration (g / L) Bacterial cell concentration (g / L) Conversion rate (%) (S)-3-cyclohexene-1-carboxylic acid ee value (%) 50 20 42.7 98.7 50 30 48.5 98.1 60 30 48.9 97.9 60 40 47.3 98.2 80 50 48.5 98.3 100 60 48.9 97.8 120 70 48.6 98.0 140 80 48.4 98.2 160 80 48.2 98.2 160 100 48.8 98.0

[0131] It was observed that at a substrate concentration of 50 g / L, the 20 g / L Est-H139M-L249A-V230M-I67A-F78L recombinant genetically engineered bacteria could not completely transform the R-type substrate. However, when the bacterial concentration was increased to 30 g / L, after reacting at 30°C for 12 h, the R-type substrate was completely transformed, and the ee value of (S)-3-cyclohexene-1-carboxylic acid was greater than 99%. Furthermore, as the substrate concentration increases, the required amount of bacterial cells also increases significantly. Substrate concentrations of 60 g / L, 80 g / L, 100 g / L, 120 g / L, 140 g / L, and 160 g / L require bacterial cells of 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, and 100 g / L, respectively. Considering factors such as cost and operational procedures, a substrate concentration of 140 g / L and a bacterial cell concentration of 80 g / L were chosen as the conditions for subsequent large-scale reactions.

[0132] Example 4

[0133] In this embodiment, the 5-site mutant Est-H139M-L249A-V230M-I67A-F78L obtained in the above embodiment was used as the experimental object to test the effect of reaction time on catalytic effect in the catalytic reaction.

[0134] The steps for catalyzing racemic 3-cyclohexene-1-carboxylate with recombinant genetically engineered bacteria are the same as in Example 1 above, except that the final concentration of recombinant genetically engineered bacteria (80 g / L, i.e., the amount added is 1.6 g) and the final concentration of racemic 3-cyclohexene-1-carboxylate (140 g / L, i.e., the amount added is 2.8 g) were adjusted, and the reaction times were 1, 2, 4, 6, 8, 10, and 12 h, respectively.

[0135] The results are as follows Figure 1 As shown.

[0136] It was observed that at a concentration of 80 g / L of recombinant genetically engineered bacteria containing the esterase mutant Est-H139M-L249A-V230M-I67A-F78L, a transformation rate of approximately 30% could be achieved in about 2 hours. The reaction rate then decreased, and the transformation rate only approached 49% after 8 hours. Therefore, with a substrate concentration of 140 g / L and a bacterial cell concentration of 80 g / L, a reaction time of 8 hours is the optimal reaction time for subsequent large-scale reactions.

[0137] Example 5

[0138] In this embodiment, the 5-site mutant Est-H139M-L249A-V230M-I67A-F78L obtained in the above embodiment was used as the experimental object to test its catalytic effect in a real large-scale reaction environment.

[0139] The specific experimental steps are as follows:

[0140] 7000 g of racemic 3-cyclohexene-1-carboxylate and 4000 g of recombinant genetically engineered bacteria containing the esterase mutant Est-H139M-L249A-V230M-I67A-F78L were added to 50 L of phosphate buffer (100 mM, pH 7.0) and incubated at 30 °C for 8 h. A total of 3 batches were performed.

[0141] After incubation, the bacterial cell layer was removed by centrifugation at 12000 rpm for 10 min. Unreacted (R)-3-cyclohexene-1-carboxylic acid was extracted from the aqueous phase with petroleum ether. The mixture was centrifuged again at 12000 rpm for 10 min to separate the layers. The remaining aqueous phase was extracted with ethyl acetate to obtain (S)-3-cyclohexene-1-carboxylic acid. Anhydrous sodium sulfate was added to remove excess water. After three extractions, the organic phase was rotary evaporated to obtain (S)-3-cyclohexene-1-carboxylic acid (product ee value > 98%). Testing showed that 2696.4 g, 2715.3 g, and 2718.5 g of (S)-3-cyclohexene-1-carboxylic acid were obtained from three large-scale reactions, respectively. Figure 2 and Figure 3 As shown, their ee values ​​are all greater than 98%, their purities are all greater than 99%, and their yields are 42.8%, 43.1%, and 43.2%, respectively (the highest theoretical yield can reach 50%).

[0142] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An esterase mutant, characterized in that, The esterase mutants include: (1) An esterase mutant obtained by substituting, deleting, or adding 1-10 amino acid residues in the sequence shown in SEQ ID NO: 2; or (2) An esterase mutant that has at least 95% sequence identity with SEQ ID NO: 2 and retains catalytic activity; Preferably, the number of amino acid residues replaced, deleted, or added is 1-5; Preferably, the esterase mutant has at least 98% sequence identity.

2. The esterase mutant according to claim 1, characterized in that, The positions where the amino acid residues are replaced, deleted, or added include: amino acid residues at positions 67, 78, 139, 230, and 249, based on the sequence shown in SEQ ID NO: 2; Preferably, the amino acid residue substitutions include: I67A, F78L, H139M, V230M, and L249A.

3. The esterase mutant according to claim 1 or 2, characterized in that, The esterase mutants include: Esterase mutants having the sequence shown in SEQ ID NO:13, 15, 17, 19, 21, 23, 25, 27 or 29.

4. A nucleic acid molecule encoding the esterase mutant according to any one of claims 1-3; Preferably, the nucleic acid molecule has the sequence shown in SEQ ID NO:14, 16, 18, 20, 22, 24, 26, 28 or 30.

5. A biomaterial, characterized in that, The biomaterial includes any one of the following (1)-(3): (1) Contains an expression unit of the nucleic acid molecule of claim 4; (2) A transformant containing the nucleic acid molecule of claim 4; (3) Transformants containing the expression in (1); Preferably, the expression unit includes a plasmid; Preferably, the transformant includes bacteria, fungi, viruses, plant cells, or animal cells.

6. A composition, characterized in that, The composition contains at least one of the esterase mutant according to any one of claims 1-3, the nucleic acid molecule according to claim 4, and the biomaterial according to claim 5; Preferably, the composition further includes excipients.

7. The use of at least one of the esterase mutants of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, and the composition of claim 6 in biosynthesis; Preferably, the biosynthesis includes at least one of biocatalytic reaction, biofermentation and bioenzymatic hydrolysis.

8. The use of at least one of the esterase mutant of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, and the composition of claim 6 in the preparation of edoxaban or an intermediate thereof; Preferably, the intermediate comprises (S)-3-cyclohexene-1-carboxylic acid.

9. A method for preparing (R)-3-cyclohexene-1-carboxylic acid, comprising the following steps: Catalysis was performed using at least one of the esterase mutants of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, and the composition of claim 6, on racemic 3-cyclohexene-1-carboxylate. Preferably, the catalytic conditions include: The catalytic temperature is 28-37℃, and the catalytic time is 8-14h.

10. A method for preparing edoxaban, comprising the following steps: (1) Using at least one of the esterase mutants of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, and the composition of claim 6 to catalyze the production of (S)-3-cyclohexene-1-carboxylic acid by means of racemic 3-cyclohexene-1-carboxylic acid; (2) Edoxaban was prepared using (S)-3-cyclohexene-1-carboxylic acid.

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