A carboxylesterase mutant, its preparation method, and a method for preparing (S)-3-cyclohexene-1-carboxylic acid using the mutant.

Abstract

This invention discloses a carboxylesterase mutant, its preparation method, and a method for preparing (S)-3-cyclohexene-1-carboxylic acid using the same. The carboxylesterase mutant is based on the amino acid sequence SEQ ID NO.1 and undergoes any one or at least two of the following mutations: I232L, P234V, S235A, or F391L. This invention creatively discovers that by designing mutations at multiple different sites on the amino acid, the activity of the carboxylesterase can be improved, for example, significantly increasing its activity in catalyzing the production of (S)-3-cyclohexene-1-carboxylic acid from racemic 3-cyclohexene-1-carboxylic acid. This effectively reduces enzyme usage, lowers production costs, and increases the yield of the product (S)-3-cyclohexene-1-carboxylic acid. Simultaneously, it enhances the soluble expression capacity of the carboxylesterase, which is beneficial for large-scale production via microbial fermentation and is more suitable for commercial and industrial applications.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and relates to a carboxylesterase mutant, its preparation method, and a method for preparing (S)-3-cyclohexene-1-carboxylic acid using the mutant. Background Technology

[0002] Carboxylesterases are a superfamily of fatty acid esterases widely found in biological tissues and organs. They are used in the industrial synthesis of chiral chemicals such as pharmaceutical intermediates, exhibiting high stereoselectivity in the hydrolysis of compounds. When catalyzing chiral compounds, they selectively hydrolyze one of the corresponding isomers, yielding enantiomers of single purity, playing a crucial role in the industrial production of single-enantiomer functional compounds and pharmaceutical intermediates. Animal liver carboxylesterases, especially porcine liver esterases, have been extensively studied. These are serine hydrolases, and in production, a mixture of porcine liver esterases is typically obtained by extracting from porcine liver.

[0003] CN114410607A discloses a thermophilic carboxylesterase mutant and its applications. The mutant is used in a recombinant expression vector synthesized in the *E. coli* DH5α host and expressed via a *Pichia pastoris* system, induced by periodic addition of methanol. However, this method suffers from problems such as low intracellular expression levels of the mutant, the inability to commercially produce carboxylesterase in *E. coli*, and high methanol consumption costs.

[0004] Existing porcine liver carboxylesterases (PLEs) are mainly expressed through Pichia pastoris, with soluble expression of PLE5 achieved by co-transformation of molecular chaperone vectors in Escherichia coli. However, this method suffers from problems such as the inability of PLEs expressed in Pichia pastoris to be secreted into the culture medium and the resulting enzyme having low catalytic activity.

[0005] In summary, existing carboxylesterases suffer from unsatisfactory enzyme activity, hindering industrial-scale production. Therefore, developing a method for constructing carboxylesterase expression strains and mutants, effectively screening mutant enzymes, and obtaining soluble carboxylesterase mutants with high catalytic activity has become one of the most pressing problems to be solved in the field of genetic engineering. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a carboxylesterase mutant, its preparation method, and a method for preparing (S)-3-cyclohexene-1-carboxylic acid. This invention solves the problem of unsatisfactory activity of existing hepatic esterases, which makes industrial production difficult. It achieves soluble expression of carboxylesterase, significantly improves the enzyme activity of carboxylesterase, and effectively increases the yield of (S)-3-cyclohexene-1-carboxylic acid in the catalytic hydrolysis reaction using methyl 3-cyclohexene-1-carboxylate as a substrate.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a carboxylesterase mutant, wherein the carboxylesterase mutant is based on the amino acid sequence SEQ ID NO.1 and undergoes any one or a combination of at least two of the following mutations: I232L, P234V, S235A or F391L.

[0009] This invention creatively discovers that by designing mutations at multiple different sites of amino acids, the activity of carboxylesterase can be improved. For example, it significantly enhances the activity of carboxylesterase in producing (S)-3-cyclohexene-1-carboxylic acid from racemic 3-cyclohexene-1-carboxylic acid, effectively reducing enzyme usage, lowering production costs, and increasing the yield of (S)-3-cyclohexene-1-carboxylic acid. At the same time, it improves the soluble expression ability of carboxylesterase, which is beneficial for large-scale production through microbial fermentation and is more suitable for commercial and industrial applications.

[0010] In this invention, the amino acid sequence shown in SEQ ID NO.1 is as follows:

[0011] MTEDARPLLTTNYGQLLGKTVGAKETDRLIHVFMGVPFAKPPIGPLRFE

[0012] DPQPPEPWSSIREATENPPMCLQDKKGMEQLADFFKAKFDFPPVSEDCLYLN

[0013] VFTPADRGENPELPVMVFIHGGGLTMGGAGMFEGSALSAYENVVVVSIQYR

[0014] LGIMGFFSTGDKEARGNYGFLDQVAALRWVRDNIKDFGGNPQSVTIFGESAG

[0015] GLSVSAQVLSPLSKGLFQRAISESGVAILPSLMASKTEEILPILHVVANISSCSV

[0016] SSLADCLKKKTEDEIVEISAAMKFVAFPAVVDGVFLPKPAEEILASKESNPVPF

[0017] LIGVNNHEFGWILPLALNISGYREGMEKKDIQSILVALPFVHSFTSVVPFIMEE

[0018] YFGDTNDPKELRNNFLDLVGDIIFVIPALRTAKYHRDSGHPVYFYEFQHRPSM

[0019] YDDSKEDFVKADHGDELYFVVGGPFLKSGILFKSNGTEEEKILSKTIMKYWA

[0020] NFARNGDPNGLGLAEWPKYDEDEDYLEINLTQESSQRLKGGRFKFWTVTLP

[0021] DKIKEMMEEKEHVEL.

[0022] In a second aspect, the present invention provides a nucleic acid molecule containing the coding sequence of the carboxylesterase mutant described in the first aspect.

[0023] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0024] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecules described in the second aspect and / or the recombinant vector described in the third aspect.

[0025] Fifthly, the present invention provides a method for preparing the carboxylesterase mutant described in the first aspect, the method comprising the following steps:

[0026] (1) Insert the nucleic acid sequence SEQ ID NO.2 of the carboxylesterase into a plasmid to obtain a recombinant plasmid. Using the recombinant plasmid as a template, perform site-directed saturation mutagenesis on any one or at least two combinations of the 232nd, 234th, 235th and 391st sites of the carboxylesterase. The nucleic acid sequence of the primer for the 232nd site mutation includes the sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the nucleic acid sequence of the primer for the 234th site mutation includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the nucleic acid sequence of the primer for the 235th site mutation includes the sequences shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleic acid sequence of the primer for the 391st site mutation includes the sequences shown in SEQ ID NO.9 and SEQ ID NO.10.

[0027] (2) The mutant plasmid obtained in step (1) is transformed into the host bacteria, cultured and purified to obtain the carboxylesterase mutant.

[0028] In this invention, the nucleic acid sequence shown in SEQ ID NO.2 is as follows:

[0029] atgacggaggacgctagacctttgcttactacaaattatggtcaactgcttggtaaaactgttggagccaaggaaac

[0030] tgatcgactgatccacgtgtttatgggtgtcccattcgctaagccaccaattggtccacttcgtttcgaagacccacaaccac

[0031] cagaaccttggtcttcaattagagaggctacagagaacccaccaatgtgtttacaggacaaaaaaggtatggagcagttg

[0032] gctgatttttttaaagccaaattcgactttccccctgtttcagaggattgtctttatttgaatgtcttcactcctgcagacaggggt

[0033] gaaaaccctgaactgccagtcatggtgtttattcatggtggaggcttgacgatgggaggtgcaggtatgttcgagggatcc

[0034] gcattaagtgcttacgaaaacgtagtagttgtgtctatccaatacagattgggtattatgggtttttttagtactggagacaaag

[0035] aggcaaggggtaattatggcttcctggaccaagttgctgcattgcgatgggttagagataatataaaggacttcggtggtaa

[0036] tccacagtctgtaaccatttttggtgagtctgctggtggtttatctgtctccgcccaagtactatccccactgtctaaaggtttgt

[0037] ttcaaagggccatctctgaaagtggagtcgctatcttaccatcattgatggcatccaaaactgaggaaattttgccaatcctg

[0038] cacgttgtggcaaacatttcttcttgttccgtctcctccttggcagattgcttaaagaaaaaagactgaggacgaaatagttgaa

[0039] ataagtgctgcaatgaagttcgtcgcatttccagccgtcgtagacggaggttttttttgcctaagccagcagaagaaaatattggc

[0040] footaagaatccaacccagttcctttcttgatcggtgtcaacaaccacgagttcggatggatttttccctcttgcactaaatt

[0041] tcaggttaccgtgaaggtatggagaagaaagatattcaaagtattcttgtcgccctgcctttcgtacattcatttacttctgtgg

[0042] taccattcatcatggaagaatacttcggtgataccaacgatccaaaagagctaagaataaacttcttggatctagtgggtga

[0043] cattatttttgttattccagctttaagaactgctaagtatcatagggatagtggtcaccctgtttattttacgaatttcaacaccgt

[0044] ccatcaatgtacgacgattctaaaagaattttgttaaggccgatcacggtgatgaactatacttcgttgttggtggacctttct

[0045] tgaagtctggtattctgttcaaaagtaacggtaccgaaaaaattttatccaaaactattatgaagtactgggcaaatt

[0046] tcgctagaaacggtgaccccaatggattgggtttggctgagtggcctaaatacgatgaggatgaagaattacttagaaatca

[0047] atctgacccaagaaagttctcagagacttaaaggtggtcgttttaaattttggactgttacattacctgacaaaatcaaggag

[0048] atgatggaagagaaggagcacgttgaactt.

[0049] In this invention, the nucleic acid sequence shown in SEQ ID NO.3 is as follows:

[0050] gaaagtggagtcgctctcttaccatcattg.

[0051] In this invention, the nucleic acid sequence shown in SEQ ID NO.4 is as follows:

[0052] caatgatggtaagagagcgactccactttc.

[0053] In this invention, the nucleic acid sequence shown in SEQ ID NO.5 is as follows:

[0054] gtggagtcgctatcttagtatcattgatggcatcc.

[0055] In this invention, the nucleic acid sequence shown in SEQ ID NO.6 is as follows:

[0056] ggatgccatcaatgatactaagatagcgactccac.

[0057] In this invention, the nucleic acid sequence shown in SEQ ID NO.7 is as follows:

[0058] gtcgctatcttaccagcattgatggcatcc.

[0059] In this invention, the nucleic acid sequence shown in SEQ ID NO.8 is as follows:

[0060] ggatgccatcaatgctggtaagatagcgac.

[0061] In this invention, the nucleic acid sequence shown in SEQ ID NO.9 is as follows:

[0062] ggtgacattattctcgttattccagc.

[0063] In this invention, the nucleic acid sequence shown in SEQ ID NO.10 is as follows:

[0064] gctggaataacgagaataatgtcacc.

[0065] Preferably, the plasmid comprises pET28a(+).

[0066] Preferably, the host bacteria include Escherichia coli, Pichia pastoris, or Bacillus subtilis.

[0067] In this invention, the constructed carboxylesterase mutant can be efficiently expressed in a variety of host bacteria, which is beneficial for large-scale production through microbial fermentation and is more suitable for commercial and industrial applications.

[0068] In a sixth aspect, the present invention provides the application of the carboxylesterase mutant according to the first aspect in a catalytic hydrolysis reaction.

[0069] Preferably, the substrate for the hydrolysis reaction includes esters or amides.

[0070] Preferably, the substrate for the hydrolysis reaction includes methyl 3-cyclohexene-1-carboxylic acid.

[0071] In a seventh aspect, the present invention provides a method for preparing (S)-3-cyclohexene-1-carboxylic acid, the method comprising:

[0072] Water, phosphate buffer, methyl 3-cyclohexene-1-carboxylate, and the carboxylesterase mutant described in the first aspect are mixed and reacted (reaction equation as follows). Figure 1 As shown in the figure, after the reaction was completed, the product was purified to obtain the (S)-3-cyclohexene-1-carboxylic acid.

[0073] Preferably, the reaction temperature is 25-30°C (e.g., 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C, etc.), and the time is 6-24 hours (e.g., 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 18 hours, 20 hours, 22 hours, or 24 hours, etc.).

[0074] Preferably, the pH of the reaction is 8.5 to 9.0 (e.g., 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, etc.).

[0075] Preferably, the mass concentration of the methyl 3-cyclohexene-1-carboxylate is 3% to 20%, including but not limited to 4%, 5%, 6%, 7%, 8%, 9%, 15%, 17%, 18% or 19%.

[0076] Preferably, the concentration of phosphate in the phosphate buffer solution is 5mM to 200mM, including but not limited to 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 50mM, 80mM, 90mM, 100mM, 150mM, 180mM, 190mM, 192mM, 195mM, 196mM, 198mM or 199mM.

[0077] Preferably, the phosphate comprises disodium hydrogen phosphate.

[0078] Preferably, the amount of the carboxylesterase mutant is 1% to 30% of the weight of methyl 3-cyclohexene-1-carboxylate, including but not limited to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 24%, 25%, 26%, 28%, or 29%.

[0079] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0080] It is understood that the purification methods commonly used in the field for (S)-3-cyclohexene-1-carboxylic acid are applicable to this invention. For example, an extraction method can be selected, specifically including: extracting with an organic solvent, merging the organic phases, retaining the aqueous phase, centrifuging the merged organic phases and allowing them to stand to separate into layers, removing the lower aqueous phase to obtain a clear organic phase, washing with a detergent, separating into layers, discarding the aqueous phase, drying with a desiccant, and vacuum concentrating until no fractions are distilled off.

[0081] Preferably, the organic solvent includes ethyl acetate, the detergent includes a 3% sodium carbonate solution, and the desiccant includes anhydrous sodium sulfate.

[0082] Preferably, the dried organic layer is vacuum concentrated at a temperature of 50°C.

[0083] Compared with the prior art, the present invention has the following beneficial effects:

[0084] This invention creatively discovers that by designing mutations at multiple different sites of amino acids, the activity of carboxylesterase can be improved. For example, it significantly enhances the activity of carboxylesterase in producing (S)-3-cyclohexene-1-carboxylic acid from racemic 3-cyclohexene-1-carboxylic acid, effectively reducing enzyme usage, lowering production costs, and increasing the yield of (S)-3-cyclohexene-1-carboxylic acid. At the same time, it improves the soluble expression ability of carboxylesterase, which is beneficial for large-scale production through microbial fermentation and is more suitable for commercial and industrial applications. Attached Figure Description

[0085] Figure 1 The chemical equation diagram for the hydrolysis of racemic 3-cyclohexene-1-carboxylic acid methyl ester by carboxylesterase. Detailed Implementation

[0086] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0087] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0088] Example 1

[0089] Construction and expression of XlCARe-pET28a(+) genetically engineered strain.

[0090] (1) Constructing the XlCARe-pET28a(+) genetically engineered strain

[0091] (a) The liver carboxylesterase XlCARe fragment synthesized by digestion of the whole gene with restriction endonucleases NdeⅠ and XhoⅠ (New England Biolabs) (the sequence is shown in SEQ ID NO.2, synthesized by Changzhou Gene Biotechnology Co., Ltd.);

[0092] (b) Recombined into vector pET28a(+), and transformed into E.coli Top10 competent cells (purchased from Beijing TransGen Biotech Co., Ltd.);

[0093] (c) Place E. coli Top10 in LB liquid medium (5g yeast extract, 10g peptone, 10g sodium chloride, adjust pH to 7.0; add water to make up to 1L), culture overnight at 37℃ with shaking at 200rpm, and extract recombinant plasmid XlCARe-pET28a(+);

[0094] (d) The positive recombinant plasmid XlCARe-pET28a(+) was transformed into the expression host strain BL21(DE3) (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain the original strain XlCARe-pET28a(+) / BL21(DE3).

[0095] (2) Expression of XlCARe-pET28a(+) genetically engineered strain

[0096] (a) Select single colonies and inoculate them into 50 mL of TB medium containing 50 μg / mL kanamycin sulfate. Incubate at 37°C with shaking at 200 rpm until OD. 600 =0.6;

[0097] (b) Add 0.2 mM IPTG as an inducer, incubate at 25°C with shaking, then centrifuge to collect the bacterial cells and resuspend them in 50 mM Tris-HCl buffer (20 mM, pH 8.0);

[0098] (c) After ultrasonic disruption, the supernatant was obtained by centrifugation at 12,000 rpm for 15 min for activity determination and as a control for mutant screening.

[0099] Example 2

[0100] In this embodiment, the XlCARe mutant strain was obtained.

[0101] (1) Using the Fast Mutagenesis System kit from Total Gold, four pairs of primers were designed (as shown in Table 1). PCR amplification was performed on the XlCARe-pET28a(+) wild-type plasmid constructed in Example 1 using these four pairs of primers. The isoleucine at position 232 of XlCARe was mutated to leucine (I232L), the proline at position 234 to valine (P234V), the serine at position 235 to alanine (S235A), and the phenylalanine at position 391 to leucine (F391L). Different combinations of mutations were also performed, such as I232L+P234V, which indicates simultaneous mutations of I232L and P234V. The resulting plasmids expressing the mutants were named XlCARe-I232L, XlCARe-P234V, and XlCARe-PET28a(+), respectively. ARe-S235A, XlCARe-F391L, XlCARe-(I232L+P234V), XlCARe-(I232L+S235A), XlCARe-(I232L+F391L), XlCARe-(P234V+S235A), XlCARe-(P234V+F391L), XlC ARe-(S235A+F391L), XLCARE-(I232L+P234V+S235A), XlCARe-(I232L+P234V+F 391L), XlCARe-(P234V+S235A+F391L), XlCARe-(I232L+P234V+S235A+F391L). PCR reaction conditions: 95℃ for 3 min, 25 cycles (95℃ for 20 s, 55℃ for 20 s, 72℃ for 2 min, 72℃ for 10 min), PCR amplification system (50 μL): template 1 μL, forward and reverse primers 1 μL each, 2×TransStar 25 μL of Fly PCRSuperMix and 22 μL of sterile double-distilled water;

[0102] (2) Add 1 μL of DMT enzyme to 50 μL of PCR product, mix well, incubate at 37°C for 1 hour, and purify and recover the PCR product using an Aspirin gel extraction kit. Transformants were sequenced by Changzhou Jiyu Company. Plasmids with correct sequences were transformed into BL21(DE3) to obtain the engineered strains XlCARe mutants XlCARe-I232L-pET28a(+) / BL21(DE3) and XlCARe-P234V-pET28a(+) / BL21(DE3). ), XlCARe-S235A-pET28a(+) / BL21(DE3), XlCARe-F391L-pET28a(+) / BL21(DE3), P234V-pET28a(+) / BL21(DE3), XlCARe-I232L+S235A-pET28a(+) / BL21(DE3), XlCARe-I232L+F391 L-pET28a(+) / BL21(DE3), XlCARe-P234V+S235A-pET28a(+) / BL21(DE3), XlCARe-P234V+F391L-p ET28a(+) / BL21(DE3),XlCARe-S235A+F391L-pET28a(+) / BL21(DE3),XlCARe-I232L+P234V+S235 A-pET28a(+) / BL21(DE3), XlCARe-I232L+P234V+F391L-pET28a(+) / BL21(DE3), XlCARe-P234V+S 235A+F391L-pET28a(+) / BL21(DE3), XlCARe-I232L+P234V+S235A+F391L-pET28a(+) / BL21(DE3).

[0103] Table 1

[0104] Primer name Serial Number Primer sequence (5'-3') I232L-F SEQ ID NO.3 GAAAGTGGAGTCGCTCTCTTACCATCATTG I232L-R SEQ ID NO.4 CAATGATGGTAAGAGAGCGACTCCACTTTC P234V-F SEQ ID NO.5 GTGGAGTCGCTATCTTAGTATCATTGATGGCATCC P234V-R SEQ ID NO.6 GGATGCCATCAATGATACTAAGATAGCGACTCCAC S235A-F SEQ ID NO.7 GTCGCTATCTTACCAGCATTGATGGCATCC S235A-R SEQ ID NO.8 GGATGCCATCAATGCTGGTAAGATAGCGAC F391L-F SEQ ID NO.9 GGTGACATTATTCTCGTTATTCCAGC F391L-R SEQ ID NO.10 GCTGGAATAACGAGAATAATGTCACC

[0105] Example 3

[0106] This embodiment purifies and screens the activity of the carboxylesterase mutant obtained in Example 2.

[0107] (1) Purification of the carboxylesterase mutant obtained in Example 2

[0108] (a) 1g of wet bacterial cells: 10mL (reaction buffer) resuspended, sonicated to break the bacteria, 1s ON, 2s OFF, total time 10min, the lysate was centrifuged at 10000rpm for 10min, the supernatant was collected and filtered for subsequent purification.

[0109] (b) The purification method was nickel column affinity chromatography: the purification column was a 5 mL pre-packed column. The entire purification system was equilibrated with equilibration buffer (20 mM Tris-HCl buffer, 500 mM NaCl, 5 mM imidazole, pH 8.0). The sample was loaded at a flow rate of 1 mL / min. The elution buffer (20 mM Tris-HCl buffer, 500 mM NaCl, 500 mM imidazole, pH 8.0) was used for gradient elution. The target protein was collected, concentrated and desalted to a final volume of 1 mL using an ultrafiltration tube (10 kDa cutoff). The protein solution was aliquoted and stored at -80°C for later use. This method was used to purify the carboxylesterase mutant constructed in Example 2.

[0110] (2) Screening of mutant enzyme activity

[0111] At 30°C, 10 mg of purified carboxylesterase mutant and methyl 3-cyclohexene-1-carboxylate were added to 10 mL of phosphate buffer (20 mM, pH 8.5) to a final concentration of 100 mM. The pH of the system was maintained at 8.5 with 20% sodium hydroxide. The reaction was carried out with wild-type carboxylesterase (refer to Example 1) as a control. The samples were analyzed after 8 h. Substrate detection method: methyl 3-cyclohexene-1-carboxylate was analyzed by GC using a Phenomen FBX-DexDET gas chromatograph with a flame ionization detector (GC). Purity was calculated from the peak area ratio of the GC chromatogram. The chiral purity of cyclohexenecarboxylic acid was analyzed by high-performance liquid chromatography (HPLC) using a variable UV detector at a detection wavelength of 205 nm. The column was a Daicel chiralpakAY-H, and the mobile phase was n-hexane:ethanol = 100:1 (containing 0.1% TFA). Chiral purity was calculated from the peak area ratio of HPLC chromatography. Conversion and selectivity were calculated, and the results are shown in Table 2.

[0112] Table 2

[0113]

[0114]

[0115] The results showed that, compared with wild-type carboxylesterases, carboxylesterases with single point mutations (I232L, P234V, S235A, F391L) exhibited significantly improved selectivity, as did those with combined site mutations (I232L+P234V, I232L+S235A, I232L+F391L, P234V+S235A, P234V+F391L, S235A+F391L, I232L+P234V). The selectivity and conversion rate of carboxylesterases (+S235A, I232L+P234V+F391L, P234V+S235A+F391L, I232L+P234V+S235A+F391L) are superior to or significantly superior to wild-type, with a conversion rate of up to 58% and a selectivity of up to 98.0%. This indicates that the present invention can significantly improve the activity of carboxylesterases by designing mutations at multiple different sites of amino acids.

[0116] Example 4

[0117] In this embodiment, the XlCARe(I232L+P234V+S235A+F391L) carboxylesterase mutant was prepared.

[0118] (1) Shake-flask fermentation

[0119] (a) Preparation of shake flask seed culture medium: 5g yeast extract, 10g peptone, 10g NaCl were dissolved in 800mL distilled water, the pH was adjusted to 7, and the volume was brought up to 1000mL with distilled water. The solution was kept at 121℃ for 15min. After the solution was cooled to 60℃, kanamycin (abbreviated as kanamycin) was added to a final concentration of 50μg / mL.

[0120] (b) Fermentation steps: The original bacterial strain (XLCARE-I232L+P234V+S235A+F391L-pET28a(+) / BL21(DE3)) was streaked on an LB agar plate and incubated overnight at 37°C with the plate inverted position. Single colonies were picked from the plate and inoculated into 3 mL of seed culture medium (10 mL test tubes), and incubated at 37°C with shaking at 200 rpm for 16 hours. OD 600 The culture medium was inoculated at a rate of 1% into 300 mL of seed culture medium (1 L Erlenmeyer flask) and incubated at 37°C with shaking at 200 rpm for 6 hours. OD 600 It grows to 1.

[0121] (2) Fermentation culture

[0122] (a) Preparation of fermentation medium: 6 g / L Na2HPO4, 3 g / L KH2PO4, 0.246 g / L MgSO4·7H2O, 2.24 g / L (NH4)2SO4, 0.5 g / L NaCl, and 20 g / L glucose were stirred and dissolved. The mixture was kept at 121℃ for 30 min. After cooling, kanamycin filtered through a sterile membrane was added.

[0123] (b) Preparation of fed-batch culture medium: Dissolve 600 g / L glucose in water, keep at 115 °C for 30 min, and cool before use;

[0124] (c) Fermentation control in the fermenter: DO is controlled at 25%, the aeration ratio is 1:1 (VVM), the fermentation temperature is controlled at 37℃, pH 7.0, dissolved oxygen spikes after 8 hours of cultivation, then feeding begins, OD... 600 =30 induction, final IPTG concentration of 1mM, induction temperature controlled at 25℃, cultured for 21 hours and then removed from the container.

[0125] (3) Fermentation broth treatment and enzyme preparation

[0126] The concentrated bacterial cells were homogenized using a homogenizer, with the bacterial concentration controlled at 100 g / L and the homogenization pressure controlled at 40 MPa. After centrifugation, the supernatant was obtained from the cell lysate. The supernatant was then concentrated by ultrafiltration to obtain the carboxylesterase mutant enzyme solution.

[0127] Example 5

[0128] In this embodiment, a carboxylesterase mutant is used to catalyze the synthesis of (S)-3-cyclohexene-1-carboxylic acid.

[0129] Add 750g of water, 4g of disodium hydrogen phosphate, 90g of methyl 3-cyclohexene-1-carboxylate, and 13.5g of the carboxylesterase mutant enzyme solution from Example 4 to a 1L reaction flask. Maintain the system temperature at 30℃ and the pH at 8.5 with 20% sodium hydroxide. After reacting for 8 hours, take a sample for chiral GC until the GC results show selectivity ee. s When the concentration is greater than 98%, the reaction is terminated immediately. Extraction is performed twice with 280g of ethyl acetate each time. The organic phases are combined, and the collected organic layer is dried with anhydrous sodium sulfate. The dried organic layer is concentrated under vacuum at 50°C until no fractions are distilled off, yielding the resolved product (S)-3-cyclohexene-1-carboxylate.

[0130] The obtained methyl (S)-3-cyclohexene-1-carboxylate was added to 150 mL of 30% alkali solution, stirred, and reacted at 50 °C. After 3 hours, the reaction was marked by TLC with no substrate residue, which was the endpoint. After cooling to room temperature, 30% hydrochloric acid was added to adjust the pH of the reaction solution to 2. The mixture was extracted twice with 150 g of ethyl acetate each time. The organic phases were combined, and the collected organic layer was dried with anhydrous sodium sulfate. The dried organic layer was concentrated under vacuum at 50 °C until no fraction was distilled off, yielding 30.78 g of the resolved product (S)-3-cyclohexene-1-carboxylic acid. The overall yield of the two steps was 38%.

[0131] The experimental results of Examples 3 and 5 show that the resolution yield of the product (S)-3-cyclohexene-1-carboxylic acid is significantly improved, indicating that the enzyme activity of the carboxylesterase mutant constructed in this invention is significantly improved, effectively increasing the catalytic synthesis rate of (S)-3-cyclohexene-1-carboxylic acid by carboxylesterase.

[0132] In summary, this invention creatively discovers that by designing mutations at multiple different sites of amino acids, the activity of carboxylesterase can be improved. For example, it significantly enhances the activity of carboxylesterase in catalyzing the production of (S)-3-cyclohexene-1-carboxylic acid from racemic 3-cyclohexene-1-carboxylic acid, effectively reducing enzyme usage and production costs, while simultaneously increasing the yield of the product (S)-3-cyclohexene-1-carboxylic acid. Furthermore, it improves the soluble expression capacity of carboxylesterase, facilitating large-scale production through microbial fermentation and making it more suitable for commercial and industrial applications.

[0133] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A carboxylesterase mutant, characterized in that, The carboxylesterase mutant undergoes any one of the following mutations based on the amino acid sequence SEQ ID NO.1: (1) I232L; (2) P234V; (3) S235A; (4) F391L; (5) Combination of I232L and P234V; (6) Combination of I232L and S235A; (7) Combination of I232L and F391L; (8) P234V and S235A combination; (9) P234V and F391L combination; (10) Combination of S235A and F391L; (11) Combination of I232L, P234V and S235A; (12) Combination of I232L, P234V and F391L; (13) P234V, S235A and F391L combination; (14) Combination of I232L, P234V, S235A and F391L.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains the coding sequence of the carboxylesterase mutant according to claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecules of claim 2 and / or the recombinant vector of claim 3.

5. A method for preparing the carboxylesterase mutant of claim 1, characterized in that, The method includes the following steps: (1) Insert the nucleic acid sequence SEQ ID NO.2 of the carboxylesterase into the plasmid to obtain a recombinant plasmid. Using the recombinant plasmid as a template, perform site-directed saturation mutagenesis on any one or at least two combinations of the 232nd, 234th, 235th and 391st sites of the carboxylesterase. The nucleic acid sequence of the primer for the 232nd site mutation includes the sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the nucleic acid sequence of the primer for the 234th site mutation includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the nucleic acid sequence of the primer for the 235th site mutation includes the sequences shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleic acid sequence of the primer for the 391st site mutation includes the sequences shown in SEQ ID NO.9 and SEQ ID NO.

10. (2) The mutant plasmid obtained in step (1) is transformed into the host bacteria, cultured and purified to obtain the carboxylesterase mutant.

6. The method for preparing a carboxylesterase mutant according to claim 5, characterized in that, The plasmid is pET28a(+).

7. The method for preparing a carboxylesterase mutant according to claim 5, characterized in that, The host bacteria are Escherichia coli, Pichia pastoris, or Bacillus subtilis.

8. The application of the carboxylesterase mutant according to claim 1 in catalytic hydrolysis reactions.

9. The application according to claim 8, characterized in that, The substrate for the hydrolysis reaction is an ester or an amide.

10. The application according to claim 9, characterized in that, The substrate for the hydrolysis reaction is methyl 3-cyclohexene-1-carboxylic acid.

11. A method for preparing (S)-3-cyclohexene-1-carboxylic acid, characterized in that, The preparation method includes: Water, phosphate buffer, methyl 3-cyclohexene-1-carboxylate, and the carboxylesterase mutant of claim 1 were mixed and reacted. After the reaction was completed, the product was purified to obtain (S)-3-cyclohexene-1-carboxylic acid.

12. The method for preparing (S)-3-cyclohexene-1-carboxylic acid according to claim 11, characterized in that, The reaction is carried out at a temperature of 25-30°C for 6-24 hours.

13. The method for preparing (S)-3-cyclohexene-1-carboxylic acid according to claim 11, characterized in that, The pH of the reaction is 8.5 to 9.

0.

14. The method for preparing (S)-3-cyclohexene-1-carboxylic acid according to claim 11, characterized in that, The mass concentration of the methyl 3-cyclohexene-1-carboxylic acid is 3% to 20%.

15. The method for preparing (S)-3-cyclohexene-1-carboxylic acid according to claim 11, characterized in that, The phosphate concentration in the phosphate buffer solution is 5 mM to 200 mM.

16. The method for preparing (S)-3-cyclohexene-1-carboxylic acid according to claim 11, characterized in that, The phosphate is disodium hydrogen phosphate.

17. The method for preparing (S)-3-cyclohexene-1-carboxylic acid according to claim 11, characterized in that, The amount of the carboxylesterase mutant used is 1% to 30% of the weight of methyl 3-cyclohexene-1-carboxylate.

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