Steroselective esterases and their use

Abstract

The application discloses an esterase with stereoselectivity and application thereof, and the esterase is: a) an esterase comprising a sequence as shown in SEQ ID NO: 2 or a sequence as shown in SEQ ID NO: 2; or b) a mutant based on SEQ ID NO: 2 comprising one or more than two mutations, and the esterase is used as a catalyst in the application of asymmetric resolution preparation of chiral oxacycloalkane carboxylate, has good substrate tolerance, high optical purity (ee s The reaction condition is mild, the environment is friendly, the operation is simple, the industrial amplification is easy, and the esterase has a good industrial application development prospect.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to a stereoselective esterase and its applications. Background Technology

[0002] (S)-Oxycyclic butane-2-carboxylic acid is an important chiral chemical raw material and pharmaceutical intermediate, widely used in chiral synthesis, particularly in the preparation of chiral drugs. It is also a crucial chiral building block for drugs, such as Danuglipron (PF-06882961), which is used to treat both type II diabetes and obesity, and its demand is growing rapidly. Because the two substituents attached to the chiral center of the oxycyclic butane-2-carboxylic ester are very similar, few enzymes have been reported to possess enantioselectivity to distinguish the two enantiomers of heterocyclic carboxylic esters. Furthermore, the small difference between the groups on both sides of the chiral center makes the chemical resolution of heterocyclic carboxylic esters via chiral auxiliaries challenging. Therefore, identifying novel esterases with high enantioselectivity is of great significance for the efficient synthesis of chiral oxycyclic carboxylic acids and their derivatives, and for elucidating the molecular mechanism of esterase enantioselectivity.

[0003] Biocatalysts for the biological preparation of (S)-oxetane-2-carboxylic acid and methyl (S)-oxetane-2-carboxylate can be either whole cells or enzymes. Because cells contain various enzymes, whole-cell catalysis often encounters side reactions, which reduce the yield of the target product. For a one-step reaction like the resolution of oxetane-2-carboxylate, enzymatic catalysis is more advantageous, as it can completely avoid side reactions and overcome the cell membrane barrier that hinders the transmembrane transport of substrate and product. Therefore, developing high-yield esterases through genetic engineering will lay a solid foundation for their application in the preparation of oxetane-2-carboxylate.

[0004] Esterases (EC 3.1.1.1) are a class of enzymes that catalyze the hydrolysis and synthesis of ester bonds (carboxyl ester bonds, amide bonds, thioester bonds, etc.). Most industrially used esterases are derived from microorganisms, primarily fungi, including 23 species from 12 genera such as *Aspergillus niger*, *Neurospora*, *Penicillium*, *Aspergillus flavus*, *Mucor*, *Pseudomonas*, *Monascus*, *Rhizopus*, *Geotrichum*, *Sclerotinia*, yeasts, and *Mucor*. Bacteria are also prevalent, including *Burkholderia*, *Staphylococcus*, *Pseudomonas*, and *Bacillus*. The esterase content in these wild-type fungi is low and varied, making large-scale industrial production using them as biocatalysts challenging. Therefore, constructing genetically engineered esterase-producing bacteria for mass production is of great significance. Thus, for methyl oxetane-2-carboxylate compounds, there is an urgent need to screen for highly efficient and selective biocatalysts to meet industrial requirements. Summary of the Invention

[0005] This application addresses the problem that there are few reported esterases available for the existing biocatalytic kinetic resolution of oxetane-2-carboxylate to prepare (S)-oxetane-2-carboxylate. It provides an esterase with excellent asymmetric catalytic activity and good stereoselectivity, as well as its applications. The esterase exhibits good stereoselectivity, high optical purity, and significant industrial application value.

[0006] The specific technical solution of this application is as follows:

[0007] 1. A stereoselective esterase, wherein the esterase is:

[0008] a) Contains a sequence as shown in SEQ ID NO:2 or a sequence as shown in SEQ ID NO:2; or

[0009] b) A mutant based on SEQ ID NO:2 containing one or more mutations.

[0010] 2. The esterase according to claim 1, wherein the amino acid sequence of the mutant contains an amino acid mutation corresponding to at least one of V144, S148 and E149 of SEQ ID NO:2, preferably containing an amino acid mutation corresponding to V144, S148 and E149 of SEQ ID NO:2.

[0011] 3. A stereoselective esterase comprising the sequence shown in SEQ ID NO:4 or the sequence shown in SEQ ID NO:4.

[0012] 4. A nucleic acid molecule that encodes any one of the terms 1-3.

[0013] 5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or the sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0014] 6. An expression vector comprising the nucleic acid molecule described in item 4 or 5.

[0015] 7. The expression vector according to item 6, wherein the expression vector is a plasmid, granule, bacteriophage, or viral vector.

[0016] 8. A host cell comprising the expression vector described in item 6 or 7.

[0017] 9. The host cell according to claim 8, wherein the host cell is a bacterium, fungus, plant cell or animal cell.

[0018] 10. The use of any one of the esterases described in items 1-3, the nucleic acid molecule described in item 4 or 5, the expression vector described in item 6 or 7, or the host cell described in item 8 or 9 in the production of chiral oxacycloalkane carbamates.

[0019] 11. A method for producing chiral oxacycloalkane carbamates, comprising:

[0020] Chiral cycloalkane carbamates are obtained by resolving oxacycloalkane carbamates using any one of items 1-3.

[0021] 12. According to the method described in item 11, the structural formula of the oxacycloalkane formate compound is shown in formula (I):

[0022]

[0023] Where n is any integer from 1 to 3, and R is a group including methyl, ethyl, isopropyl or phenyl.

[0024] The effects of the invention

[0025] The esterase described in this application exhibits good substrate tolerance and high optical purity (ee) in the asymmetric resolution preparation of chiral oxacycloalkane carbamates. s With a purity of over 99%, the reaction conditions are mild, environmentally friendly, easy to operate, and readily scalable for industrial application, making it a promising candidate for industrial application development. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the crude enzyme solution analyzed by polyacrylamide gel electrophoresis. Lane 1 is the broken supernatant, and lane 2 is the precipitate.

[0027] Figure 2 A- Figure 2 B is the gas chromatogram of the racemic substrate and product from Example 8. Figure 2 Gas chromatogram of racemic oxetane carboxylate A. Figure 2 B is the gas chromatogram of (S)-oxetane carboxylate methyl ester prepared using a mutant. Detailed Implementation

[0028] The present application will now be described in detail. While specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0029] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0030] This application provides a stereoselective esterase, wherein the esterase is:

[0031] a) Contains a sequence as shown in SEQ ID NO:2 or a sequence as shown in SEQ ID NO:2; or

[0032] b) A mutant based on SEQ ID NO:2 containing one or more mutations.

[0033] Stereoselectivity refers to the chemical property in which one stereoisomer takes precedence over another in a chemical reaction. In this application, the stereoselective esterase refers to an esterase that preferentially acts on a specific stereoisomer, such as a chiral cycloalkane carbamate, thereby allowing the chiral cycloalkane carbamate to be obtained through resolution.

[0034] The amino acid sequence of SEQ ID NO:2 is as follows:

[0035] MTLDVKRWLALLKKVMQNDNKPFESLRVQETRDPAVQNFLKQLQGNMSEEEYTKAFEVPVTDGPMPIRDIFVRIYRPTNEEKLPVIIYFHGGGWVIGNIDTHDSLCRKLANQTNCVVISVDYRLAPEHKFPAAIEDCYDALKWVVENSEELGVDPNKI AVAGDSAGGNLAAVVTLMSRDKGGPKICFQILIYPVTDLEMETPSYEKYNEGYLLTKKAMEWFWDHYLPDPEDRQNPYVSPLLAEDFSNLPPAFIITAEYDPLRDEGEAYAEKLKEAGNPVTYKRYEGMIHGFINMSGVLDAAAEALEEIAEYLKKFFL

[0036] The mutant refers to an amino acid sequence relative to SEQ ID NO:2 that contains one or more changes at positions, namely substitution, insertion and / or deletion, while still retaining its activity.

[0037] The esterase described in this application may be an esterase ancestor enzyme constructed by ancestor sequence reconstruction, an esterase obtained by artificially synthesizing the full amino acid sequence, or an esterase obtained by cloning and expression through genetic engineering methods.

[0038] In this application, the ancestral sequence reconstruction (ASR) described is discussed in Randall et al. (Nat. Commun. 7: 12847 doi: 10.1038 / ncomms 12847 (2016)). The authors define ASR as "the process of analyzing modern sequences in an evolutionary / phylogenetic context to infer ancestral sequences at specific nodes in a tree." Ancestor sequence reconstruction (ASR) is used in molecular evolutionary studies. Unlike traditional methods of studying protein evolution by horizontally comparing related protein homologs at the ends of different branches of a phylogenetic tree, ASR probes statistically inferred ancestral proteins within tree nodes in a vertical manner. A phylogenetic tree is a branching graph showing evolutionary relationships between multiple biological species or other entities based on similarities and differences in their physical or genetic characteristics. In a rooted phylogenetic tree, each node with offspring represents the inferred most recent common ancestor of those offspring. In ASR, multiple related homologs of the target protein are selected and aligned using multiple sequence alignment (MSA) to construct a phylogenetic tree with statistically inferred sequences at the nodes of the branches. These sequences are known as "ancestors." The process of synthesizing the corresponding DNA, converting it into cells, and producing proteins is called "reconstruction."

[0039] Ancestor sequences are typically calculated using maximum likelihood, although the Bayesian method can also be performed. Since ancestors are inferred from phylogeny, the topology and composition of the phylogeny play a major role in the output ASR sequence. An ASR does not claim to reconstruct the actual sequence of an ancient protein / DNA, but rather a sequence likely similar to the sequence at that node. Maximum likelihood (ML) works by generating sequences where residues at each position are predicted to be most likely to occupy that position using the inference method used. Typically, this is a score matrix calculated from existing sequences (similar to those used in BLAST or MSA). Alternative methods include maximum parsimony (MP), which constructs sequences based on sequence evolution models, where the concept of the minimum number of nucleotide sequence changes typically represents the most efficient and most probable evolutionary pathway. MP is often considered the least reliable reconstruction method because it can oversimplify evolution to a degree unsuitable for a billion-year scale. Other methods include the Bayesian method, which involves considering residue uncertainties. Such methods are sometimes used to supplement ML methods, but they typically produce more ambiguous sequences (i.e., sequences containing residue positions for which definitive substitutions cannot be predicted). In such cases, multiple ASR sequences covering most of the ambiguity are usually generated and compared with each other. In some implementations, ancestor sequence reconstruction is performed using the online software FireProt-ASR (FireProt-ASR(muni.cz)).

[0040] The esterase described in this application originates from the genus *Acinetobacter*. The esterase is obtained through ancestral sequence reconstruction, constructing an ancestral esterase, synthesizing the complete amino acid sequence of the esterase artificially, and cloning and expressing the resulting esterase using genetic engineering methods.

[0041] In this process, after constructing the esterase progenitor enzyme, the cloned enzyme was repeatedly compared and screened by measuring and comparing the activity of the hydrolase and its stereoselectivity to racemic oxobutane-2-carboxylate, and finally the esterase progenitor enzyme with the best catalytic performance was obtained, whose amino acid sequence is shown in SEQ ID NO:2.

[0042] After obtaining the optimal esterase progenitor enzyme, the full-length gene sequence was obtained through codon optimization based on the corresponding amino acid sequence. This gene sequence was then delivered to a gene synthesis company for artificial synthesis. After obtaining the gene, it was amplified using PCR, and the sequence was ligated into pET28a. The primers used are as follows:

[0043] Upstream primer:

[0044] 5'-gtgccgcgcggcagc catatgATGACCTTAGATGTGAAGCGTTGG-3'(SEQ ID NO:5)

[0045] Downstream primer:

[0046] 5'-acggagctcgaattc ggatcc TTACAGGAAAAATTTTTCAGATATTCG-3'(SEQ ID NO:6)

[0047] In this sequence, the underlined portion of the upstream primer nucleotide sequence represents the NdeI restriction site, and the underlined portion of the downstream primer represents the BamHI restriction site. Then, using the artificially synthesized gene as a template, polymerase chain reaction (PCR) was employed to amplify the gene, obtaining the complete full-length esterase gene DNA fragment. This full-length esterase gene (nucleotide sequence shown in SEQ ID NO. 1 of the sequence listing), named Est, is 951 nucleotides in length. Its coding sequence, from the first base to the 951st base, has a start codon of ATG and a stop codon of TAA. This sequence contains no introns, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO. 2 of the sequence listing.

[0048] The sequence of SEQ ID NO:1 is as follows:

[0049] ATGACCTTAGATGTGAAGCGTTGGTTGGCGTTACTGAAGAAAGTCATGCAAAATGATAATAAGCCCTTCGAGAGTCTGCGCGTCCAGGAAACGCGCGATCCTGCCGTCCAGAACTTTTTGAAACAGTTGCAAGGTAATATGTCTGAAGAGGAATATACTAAAGCCTTTGAAGTACCCGTGACTGACGGTCCTATGCCTATTCGTGACATCTTTGTCCGCATTTATCGCCCGACCAACGAGGAGAAATTACCCGTAATTATCTACTTCCATGGAGGAGGTTGGGTAATTGGCAACATTGATACGCACGATTCGTTATGCCGTAAACTTGCCAACCAGACTAATTGCGTTGTCATCTCAGTGGACTATCGTTTGGCCCCAGAACATAAATTTCCGGCGGCAATTGAAGATTGCTATGACGCCTTAAAATGGGTTGTAGAGAATTCAGAGGAGTTAGGAGTCGATCCTAACAAGATCGCTGTCGCGGGAGACTCAGCCGGCGGTAACTTAGCTGCGGTCGTAACCCTTATGTCGCGCGACAAGGGGGGCCCGAAGATCTGCTTTCAGATCTTAATTTACCCGGTGACGGACTTGGAGATGGAGACCCCTTCCTATGAGAAGTACAATGAAG

[0050] GGTACCTTTTGACCAAGAAAGCAATGGAGTGGTTCTGGGACCATTACT

[0051] TACCTGACCCAGAAGACCGTCAGAACCCATATGTGAGCCCGCTTCTTG

[0052] CAGAAGACTTCAGTAACCTTCCTCCTGCGTTTATTATTACTGCGGAGTA

[0053] CGATCCTTTGCGTGATGAAGGGGAAGCATATGCCGAAAAGTTGAAAGA

[0054] GGCGGGTAATCCAGTTACATATAAACGCTATGAGGGAATGATCCACGGA

[0055] TTCATTAATATGTCAGGGGTCTTGGATGCCGCTGAGGCCCTGGAAGAGA

[0056] TTGCCGAATATCTGAAAAAATTTTTCCTGTAA.

[0057] Due to codon degeneracy, nucleic acid molecules encoding the aforementioned esterases (amino acid sequences as shown in SEQ ID NO.2) are not limited to those with sequences as shown in SEQ ID NO.1. A polynucleotide homologue can also be provided by appropriately introducing substitutions, deletions, alterations, insertions, or additions of nucleotides.

[0058] In some embodiments, the amino acid sequence of the mutant contains an amino acid mutation at at least one of the sites V144, S148 and E149 of SEQ ID NO:2, preferably containing an amino acid mutation at the sites V144, S148 and E149 of SEQ ID NO:2.

[0059] The term "corresponds" has the meaning commonly understood by those skilled in the art. Specifically, "corresponds" means the position in one sequence that corresponds to a specified position in another sequence after two sequences have been aligned for homology or sequence identity.

[0060] In this application, the V at position 144 can be mutated to T, the S at position 148 to F, or the glutamic acid at position 149 can be mutated to A; preferably, the V at position 144 can be mutated to T, the S at position 148 can be mutated to F, and the glutamic acid at position 149 can be mutated to A, and the amino acid sequence is shown in SEQ ID NO:4.

[0061] The sequence of SEQ ID NO:4 is as follows:

[0062] MTLDVKRWLALLKKVMQNDNKPFESLRVQETRDPAVQNFLKQLQG

[0063] NMSEEEYTKAFEVPVTDGPMPIRDIFVRIYRPTNEEKLPVIIYFHGGGWVI

[0064] GNIDTHDSLCRKLANQTNCVVISVDYRLAPEHKFPAAIEDCYDALKWTV

[0065] ENFAELGVDPNKIAVAGDSAGGNLAAVVTLMSRDKGGPKICFQILIYPVT

[0066] DLEMETPSYEKYNEGYLLTKKAMEWFWDHYLPDPEDRQNPYVSPLLAE

[0067] DFSNLPPAFIITAEYDPLRDEGEAYAEKLKEAGNPVTYKRYEGMIHGFINM

[0068] SGVLDAAEALEEIAEYLKKFFL.

[0069] This application does not impose any restrictions on the mutation method. Mutation can be carried out according to conventional methods in the art, such as directed mutagenesis, random mutagenesis, or construction of synthetic oligonucleotides, and then the mutated DNA sequence can be expressed in the host cell to obtain mutants with amino acid sequence substitution, insertion and / or deletion.

[0070] The high esterase activity described in this application enhances the enzyme's potential for industrial application.

[0071] The mutant described in this application has more than 90% homology with SEQ ID NO:2.

[0072] This application provides a nucleic acid molecule encoding the esterase described above. In some embodiments, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or the sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0073] The sequence of SEQ ID NO:3 is as follows:

[0074] ATGACCTTAGATGTGAAGCGTTGGTTGGCGTTACTGAAGAAAGTCATGCAAAATGATAATAAGCCCTTCGAGAGTCTGCGCGTCCAGGAAACGCGCGATCCTGCCGTCCAGAACTTTTTGAAACAGTTGCAAGGTAATATGTCTGAAGAGGAATATACTAAAGCCTTTGAAGTACCCGTGACTGACGGTCCTATGCCTATTCGTGACATCTTTGTCCGCATTTATCGCCCGACCAACGAGGAGAAATTACCCGTAATTATCTACTTCCATGGAGGAGGTTGGGTAATTGGCAACATTGATACGCACGATTCGTTATGCCGTAAACTTGCCAACCAGACTAATTGCGTTGTCATCTCAGTGGACTATCGTTTGGCCCCAGAACATAAATTTCCGGCGGCAATTGAAGATTGCTATGACGCCTTAAAATGGACCGTAGAGAATTTTGCAGAGTTAGGAGTCGATCCTAACAAGATCGCTGTCGCGGGAGACTCAGCCGGCGGTAACTTAGCTGCGGTCGTAACCCTTATGTCGCGCGACAAGGGGGGCCCGAAGATCTGCTTTCAGATCTTAATTTACCCGGTGACGGACTTGGAGATGGAGACCCCTTCCTATGAGAAGTACAATGAAGGGTACCTTTTGACCAAGAAAGCAATGGAGTGGTTCTGGGACCATTACTTACCTGACCCAGAAGACCGTCAGAACCCATATGTGAGCCCGCTTCTTGCAGAAGACTTCAGTAACCTTCCTCCTGCGTTTATTATTACTGCGGAGTACGATCCTTTGCGTGATGAAGGGGAAGCATATGCCGAAAAGTTGAAAGAGGCGGGTAATCCAGTTACATATAAACGCTATGAGGGAATGATCCACGGATTCATTAATATGTCAGGGGTCTTGGATGCCGCTGAGGCCCTGGAAGAGATTGCCGAATATCTGAAAAAATTTTTCCTGTAA

[0075] This application provides an expression vector comprising the nucleic acid molecules described above.

[0076] In this application, the expression vector is constructed by cloning the above-mentioned esterase gene into the expression vector using conventional methods in the art. The expression vector includes various conventional vectors in the art, such as commercially available plasmids, granules, bacteriophages or viral vectors, etc., preferably pET-28a plasmid.

[0077] In this application, the term "clay particle" refers to a sticky particle.

[0078] For example, expression vectors can be prepared using the following methods:

[0079] The esterase gene product obtained by PCR amplification is digested with restriction endonucleases NdeI and BamHI. At the same time, the expression vector, such as pET-28a, is also digested with restriction endonucleases NdeI and BamHI to form complementary sticky ends. The digested esterase gene product and the digested expression vector, such as pET-28a plasmid, are recovered and ligated using T4 DNA ligase to construct an expression vector, such as pET28a-est, containing the esterase gene.

[0080] This application provides a host cell that includes the expression vector described above.

[0081] In this application, the host cell is a conventional host cell in the art, as long as the expression vector can stably replicate itself and the esterase gene it carries can be effectively expressed. The host cell can be, for example, bacteria, fungi, plant cells, animal cells, etc.

[0082] The bacteria are preferably Escherichia coli, more preferably Escherichia coli BL21(DE3) or Escherichia coli DH5α.

[0083] In this application, the expression vector, such as pET28a-est, can be transformed into a host cell, such as Escherichia coli BL21(DE3), to obtain the host cell, namely Escherichia coli BL21(DE3) / pET28a-est.

[0084] This application provides a method for preparing esterase, which includes inoculating the host cells described above into a culture medium for fermentation to obtain a fermentation broth, centrifuging the fermentation broth to collect the cell bodies, and breaking the cell bodies to obtain the esterase.

[0085] The culture medium can be any culture medium in the art that can grow the transformant and produce esterase. For example, the culture medium can be LB medium, preferably, the components of which include: 5-15 g / L peptone, 1-10 g / L yeast extract, 5-15 g / L NaCl, and pH 6.0-8.0.

[0086] In this application, there are no special restrictions on culture methods and conditions. Appropriate selections can be made according to general knowledge in the art, based on factors such as host cell type and culture method, as long as the transformant can grow and produce esterase. Other specific operations for culturing transformants can be performed according to conventional procedures in the art.

[0087] For example, the strain culture method includes: inoculating the host cells (e.g., E. coli BL21(DE3)) into LB medium containing kanamycin and culturing them until the optical density OD of the culture medium reaches a certain level. 600 When the esterase concentration reaches 0.6-0.8 (preferably 0.6), it can be efficiently expressed under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1-1.0 mmol / L (preferably 0.2 mmol / L).

[0088] This application provides the use of the esterase, nucleic acid molecule, expression vector, or host cell described above in the production of chiral oxacycloalkane carbamates.

[0089] The esterase described in this application, due to its stereoselectivity, can be resolved to yield chiral oxacycloalkane carbamates.

[0090] This application provides a method for producing chiral oxacycloalkane carbamates, comprising:

[0091] The esterase described above is used to resolve oxacycloalkane carbamate compounds to obtain chiral cycloalkane carbamates. In some embodiments, the structural formula of the oxacycloalkane carbamate compounds is shown in formula (I):

[0092]

[0093] Where n is any integer from 1 to 3, and R is a group including methyl, ethyl, isopropyl or phenyl.

[0094] For example, the oxacycloalkane formate compounds mentioned can be methyl oxacyclobutane-2-carboxylate, methyl oxacyclopentane-2-carboxylate, methyl oxacyclohexane-2-carboxylate, etc.

[0095] In some embodiments, the temperature of the separation reaction is 20-60°C, preferably 30-50°C.

[0096] For example, the temperature for the splitting reaction can be 20℃, 25℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 55℃, 60℃, etc.

[0097] In some embodiments, the pH of the resolution reaction is 5.0-10.0, preferably 6.0-8.0.

[0098] For example, the pH of the resolution reaction can be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, etc.

[0099] In some embodiments, the esterase described above is used to resolve oxacycloalkane carbamate compounds to obtain chiral cycloalkane carbamates in a buffer system. Preferably, the buffer is sodium phosphate buffer, Tris-HCl buffer, or glycine-NaOH buffer.

[0100] In some embodiments, the esterase described above is dissolved in a buffer solution, and an oxetine carbamate compound is added to a final concentration of 100-1000 mM. The reaction is carried out at 20-60°C with mechanical stirring, and the pH is controlled by adding 1.0 M NaOH until the substrate ee is close to 99%. After the reaction, the remaining methyl oxetane-2-carboxylate is extracted three times with dichloromethane. The extracts are combined, dried overnight with anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain (S)-oxetane-2-carboxylate.

[0101] Using the esterase separation described above, optical purity can be achieved to 99%, and the reaction conditions are mild and the operation is relatively simple, showing great promise for industrial applications.

[0102] Example

[0103] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0104] Example 1 Cloning of the esterase gene

[0105] The esterase ancestor enzyme, constructed using conventional ancestor sequence reconstruction methods in this field, has the amino acid sequence shown in SEQ ID NO:2. Its encoding gene was obtained through codon optimization, and the full-length sequence was further synthesized artificially by a gene synthesis company. The PCR primers are designed as follows:

[0106] Upstream primer:

[0107] 5'-gtgccgcgcggcagc catatg ATGACCTTAGATGTGAAGCGTTGG-3'

[0108] Downstream primer:

[0109] 5'-acggagctcgaattc ggatcc TTACAGGAAAAATTTTTCAGATATTCG-3'

[0110] In this design, the underlined portion of the upstream primer represents the NdeI restriction site, and the underlined portion of the downstream primer represents the BamHI restriction site.

[0111] Using the artificially synthesized esterase ancestor DNA as a template, PCR amplification was performed. The PCR system consisted of: 10 μL of 2×Taq PCRMasterMix, 1 μL each of upstream and downstream primers (0.3 μmol / L), 1 μL (0.1 μg) of DNA template, and 7 μL of ddH2O. The PCR amplification program was as follows: (1) 95℃ for 3 min pre-denaturation; (2) 94℃ for 30 s denaturation; (3) 55℃ for 30 s annealing; (4) 72℃ for 2 min extension; steps (2) to (4) were repeated for 30 cycles; (5) 72℃ for 10 min extension, followed by cooling to 4℃. The PCR product was purified by agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit. A complete full-length esterase gene sequence was obtained, which was sequenced and was 951 bp in length, named Est. The nucleotide sequence of the gene is shown in SEQ ID NO.1 in the sequence listing.

[0112] Example 2 Preparation of esterase expression vector and expression transformant

[0113] The esterase gene DNA fragment obtained in Example 1 and the pET-28a empty plasmid were incubated at 37°C with restriction endonucleases NdeI and BamH. I The DNA was digested with two enzymes for 2 hours, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The target fragment was ligated overnight at 4°C using T4 DNA ligase to obtain the expression plasmid pET28a-est.

[0114] The expression plasmid was transformed into *Escherichia coli* DH5α competent cells. Positive clones were screened on kanamycin-containing resistant plates, and single clones were selected for colony PCR verification. The cells were cultured, and after plasmid amplification, the plasmid was extracted and re-transformed into *Escherichia coli* BL21(DE3) competent cells. The transformation solution was plated on LB agar plates containing kanamycin and incubated overnight at 37°C inverted mode to obtain the positive transformant *Escherichia coli* BL21(DE3) / pET28a-est. Colony PCR and gene sequencing verified the positive clones.

[0115] Example 3: Expression of esterase

[0116] The *E. coli* obtained in Example 2 were inoculated into LB medium containing kanamycin (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and cultured overnight at 37°C with shaking. Then, 1% (v / v) inoculation was added to a 500 mL Erlenmeyer flask containing 100 mL of LB medium and cultured at 37°C and 180 rpm with shaking. When the OD of the culture medium... 600 When the esterase concentration reached 0.6, IPTG was added to a final concentration of 0.2 mmol / L as an inducer. After induction at 25°C for 12 hours, the culture medium was centrifuged, cells were collected, and washed twice with physiological saline to obtain resting cells. These were then freeze-dried for 24 hours to obtain frozen stem cells, which were then stored at 4°C. Alternatively, the obtained resting cells could be suspended in a pH 7.0 buffer solution, sonicated in an ice bath, and the supernatant collected by centrifugation to obtain the crude esterase solution. The crude enzyme solution was analyzed by polyacrylamide gel electrophoresis, and the results are shown below. Figure 1 As shown, lane 1 contains the broken supernatant, and lane 2 contains the precipitate, indicating that the esterase exists in a soluble form.

[0117] Example 4: Determination of esterase activity

[0118] The hydrolytic activity of the esterase obtained in Example 3 was determined using a microplate reader by detecting changes in absorbance at 405 nm. The activity assay method was as follows: 1 mmol / L p-nitrophenol acetate was added to 200 μL of the reaction system (100 mmol / L sodium phosphate buffer, pH 8.0), incubated at 30°C for 2 min, and then an appropriate amount of the crude enzyme solution prepared in Example 3 was added. The mixture was quickly mixed, and the change in absorbance at 405 nm was detected. Enzyme activity (U) was defined as the amount of enzyme required to catalyze 1 μmol of p-nitrophenol acetate per minute under the above conditions. The specific activity of the esterase for p-nitrophenol acetate was determined to be 18 U / mg.

[0119] Example 5: Asymmetric resolution reaction of different esters catalyzed by esterases

[0120] 20 U of the crude enzyme solution prepared in Example 3 was added to 10 mL of sodium phosphate buffer (100 mmol / L, pH 8.0) and the reaction was carried out at 30 °C and 120 rpm with shaking. Samples were taken at regular intervals to monitor the reaction. After the reaction was completed, the enzyme was extracted twice with an equal volume of ethyl acetate. The extracts were combined, dried overnight with anhydrous sodium sulfate, and the substrate conversion rate and EE value were analyzed. The results are shown in Table 1.

[0121] The specific analytical conditions for substrate conversion rate and substrate ees value are as follows:

[0122] Analysis was performed using a gas chromatograph with a chiral capillary column, CP-Chirasil-DEX (25m × 0.25mm × 0.25μm, Sigma), using nitrogen as the carrier gas. The injection port temperature was 280℃, the detector temperature was 280℃, the initial column temperature was 50℃, and the temperature was maintained at 160℃ for 3 min at a rate of 2℃ / min.

[0123] Table 1. Esterase activity and optical purity of products for different cycloalkane carbamates

[0124]

[0125] Example 6: Preparation of esterase mutants

[0126] Structural models of esterases were constructed using conventional methods in the art. After inserting substrate pairs into the active site, the interaction between the esterase and the substrate was analyzed. Mutations were introduced to enhance the interaction between the enzyme and the substrate using conventional methods in the art.

[0127] The full-length gene sequence of the esterase obtained in Example 1 (nucleotide sequence as shown in SEQ ID NO.1) was mutated by 3 bases. The mutation positions were as follows: V at position 144 was mutated to T, S at position 148 was mutated to F, and E at position 149 was mutated to A. The sequence of the mutant gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4. Crude enzyme solution of the mutant was prepared according to the method described in Examples 2-3.

[0128] Example 7: Asymmetric resolution reactions of different esters catalyzed by esterase mutants.

[0129] 20 U of the mutant crude enzyme solution prepared in Example 6 was added to 10 mL of sodium phosphate buffer (100 mmol / L, pH 8.0) and the reaction was carried out at 30 °C with shaking at 120 rpm. Samples were taken at regular intervals to monitor the reaction. After the reaction was completed, the enzyme was extracted twice with an equal volume of ethyl acetate. The extracts were combined, dried overnight with anhydrous sodium sulfate, and the substrate conversion rate and EES value were analyzed according to the method in Example 5. The results are shown in Table 2.

[0130] Table 2. Activity of mutants against different cycloalkane carbamates and optical purity of products.

[0131]

[0132] Example 8: Esterase mutant catalyzes the reaction of methyl oxacyclohexane-2-carboxylate.

[0133] The crude enzyme solution of the mutant enzyme prepared in Example 6 was added to 100 mL of sodium phosphate buffer (100 mmol / L, pH 8.0) to final concentrations of 0.2, 0.5, 1, or 2 mol / L, respectively. Conversion was continued until the substrate ee > 99.0%. After the reaction, the remaining methyl (S)-oxetane-2-carboxylate was extracted three times with dichloromethane. The extracts were combined and dried overnight with anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain methyl (S)-oxetane-2-carboxylate. The total yield of methyl (S)-oxetane-2-carboxylate after separation was 32.3%, with an optical purity of 99% ee.

[0134] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. An esterase with stereoselectivity, the amino acid sequence of the esterase being as set forth in SEQ ID NO: 2 or as set forth in SEQ ID NO:

4.

2. A nucleic acid molecule encoding the esterase of claim 1.

3. The nucleic acid molecule of claim 2, wherein, the nucleotide sequence of the nucleic acid molecule being as set forth in SEQ ID NO: 1 or SEQ ID NO:

3.

4. An expression vector comprising the nucleic acid molecule of claim 2 or 3.

5. The expression vector of claim 4, wherein the expression vector is a plasmid, a cosmid, a phage or a viral vector.

6. A host cell comprising the expression vector of claim 4 or 5.

7. The host cell of claim 6, wherein, the host cell is a bacterial, fungal, plant or animal cell.

8. Use of the esterase of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4 or 5, or the host cell of claim 6 or 7 in the production of a chiral oxacycloalkane carboxylate.

9. A method for producing a chiral oxacycloalkane carboxylate, comprising: resolving an oxacycloalkane carboxylate compound to obtain a chiral cycloalkane carboxylate using the esterase of claim 1.

10. The method of claim 9, wherein the oxacycloalkane carboxylate compound has a structure as set forth in formula (I): （I） wherein n is any integer from 1 to 3, and R is a group comprising a methyl group, an ethyl group, an isopropyl group or a phenyl group.

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