A method for synthesizing a key chiral intermediate of speciosin p derivative

Abstract

The application discloses a synthesis method of a key chiral intermediate of Speciosin P derivative, and particularly relates to the technical field of bioengineering. Moesziomyces antarcticus The method adopts an esterase derived from Pseudomonas putida as a catalyst, immobilizes the esterase on a resin to obtain an immobilized enzyme, performs a transesterification reaction in a pure organic phase, performs chiral resolution on racemic 2-methylbut-3-yn-1,2-diol, synthesizes a chiral intermediate (2R)-2-methylbut-3-yn-1,2-diol, and the yield of the obtained product can reach 45%, and the ee value is greater than 99%. The application has good industrialization potential and commercial value in the application of efficiently catalyzing racemic 2-methylbut-3-yn-1,2-diol to prepare (2R)-2-methylbut-3-yn-1,2-diol.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for synthesizing a key chiral intermediate of a Speciosin P derivative. Background Technology

[0002] Speciosin P is a natural oxycyclohexane compound, first discovered in basidiomycetes. Hexagonia speciosa It was isolated from fermentation cultures. Its molecular structure is characterized by a hydroquinone core linked to an alkynyl side chain, and its chemical name is 2-(3,4-dihydroxybut-1-alkynyl)benzene-1,4-diol. In recent years, this compound has attracted attention due to its unique structural features and potential biological activities. The biological activity of Speciosin P is mainly reflected in the field of plant growth regulation. In wheat coleoptile bioassays, this compound exhibited significant allelopathic activity, with an IC50 value of [missing information]. 50 The value reached 14 µM, while the IC50 of its low-activity analogue, speciosin G, was... 50 With a molecular weight of only 2568 µM, studies have shown that the terminal hydroxyl group of the side chain is crucial for potent phytotoxic activity, making speciosin P an ideal reference standard for structure-activity relationship (SAR) studies and the discovery of plant growth regulators. Furthermore, speciosin P and its analogues have demonstrated potential pharmaceutical applications, including antibacterial and anticancer activities. Related studies have found that speciosins G and P exhibit cytotoxic activity against human cancer cell lines HeLa and HT29 and can inhibit mitochondrial respiration in Saccharomyces cerevisiae. These findings suggest that speciosin P has potential value in drug or pesticide development.

[0003] The first total synthetic route for Speciosin P was reported by Guerrero-Vásquez et al. in 2014. The core of this synthetic strategy is the Sonogashira coupling reaction, which constructs the core skeleton of the target molecule from readily available commercial starting materials. Furthermore, the related natural product siccayne can be synthesized in two steps from the same starting material. The synthesis involves the preparation of a halodiol intermediate and the final product obtained through steps such as epoxidation and regioselective transformation. However, chemical synthesis also has limitations: multi-step reactions involve the use of organic solvents and metal catalysts, resulting in poor environmental friendliness; the cost may be high for large-scale production; and, moreover, chemical synthesis yields a racemic mixture, while the natural product may possess specific optical activities.

[0004] Therefore, to further enrich the molecular diversity of Speciosin P compounds and optimize their pharmacological activity, researchers often modify the structure of Speciosin P to prepare a series of Speciosin P derivatives. Direct chemical modification using Speciosin P as a substrate often results in difficulty controlling site selectivity. Therefore, the synthesis of Speciosin P derivatives usually requires obtaining corresponding key intermediates. These intermediates are important precursors for constructing Speciosin P derivatives and have high research value in natural product derivatization studies. The chemical structures of Speciosin P derivatives are as follows: Figure 1 As shown.

[0005] Currently, the preparation of intermediates for this Speciosin P derivative mainly relies on chemical synthesis routes, with few reported biosynthetic methods. Existing chemical synthesis processes generally suffer from harsh reaction conditions, poor stereoselectivity, and numerous byproducts, resulting in difficulties in product separation and high preparation costs, making it difficult to meet the needs of research and large-scale preparation. Therefore, developing a synthetic method for intermediates of Speciosin P derivatives has significant research and application value. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a method for synthesizing a key chiral intermediate of a Speciosin P derivative. Specifically, it provides an esterase-catalyzed method for synthesizing the chiral intermediate (2R)-2-methylbut-3-yne-1,2-diol from racemic 2-methylbut-3-yne-1,2-diol, wherein the chiral alcohol is an intermediate of a Speciosin P derivative.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the invention provides the use of a lipase with the amino acid sequence shown in SEQ ID NO: 1 in the preparation of (2R)-2-methylbut-3-yne-1,2-diol.

[0008] In some embodiments of the present invention, the (2R)-2-methylbut-3-yne-1,2-diol is prepared by enantioselective transesterification of racemic 2-methylbut-3-yne-1,2-diol catalyzed by lipase.

[0009] In some embodiments of the present invention, the reaction system of the selective transesterification reaction further includes excipients.

[0010] In some embodiments of the present invention, the excipient is an ester compound.

[0011] In some embodiments of the present invention, the ester compound includes vinyl acetate.

[0012] In some embodiments of the present invention, the amount of the excipient added is 1-3 molar equivalents of racemic 2-methylbut-3-yne-1,2-diol.

[0013] In some embodiments of the present invention, the amount of the excipient added is twice the molar equivalent of racemic 2-methylbut-3-yne-1,2-diol.

[0014] In some embodiments of the present invention, the selective transesterification reaction is carried out in an organic solvent.

[0015] In some embodiments of the present invention, the organic solvent includes at least one selected from methyl ether, toluene, petroleum ether, DMSO, and cyclohexane.

[0016] In some embodiments of the present invention, the organic solvent includes at least one of methyl ether, toluene, and petroleum ether.

[0017] In some embodiments of the present invention, the organic solvent is specifically methyl ether.

[0018] In some embodiments of the present invention, the lipase is used in the form of a free enzyme or an immobilized enzyme.

[0019] In some embodiments of the present invention, the free enzyme is catalyzed in the form of fermentation supernatant, crude enzyme solution or purified enzyme.

[0020] In some embodiments of the present invention, the lipase is used in the form of an immobilized enzyme for the selective transesterification reaction.

[0021] In some embodiments of the present invention, the immobilized enzyme includes a lipase and an immobilized carrier.

[0022] In some embodiments of the present invention, the immobilized carrier includes at least one of amino resin, epoxy resin and macroporous adsorption resin.

[0023] In some embodiments of the present invention, the amino resin is selected from at least one of LXTE-703, ESR-3, ES110, HA104F, EA104F, HA109M, LXTE-701, LXTE-704, and LXTE-700s.

[0024] In some embodiments of the present invention, the epoxy resin is selected from at least one of ES108, ES103B, GA611M and LXTE-1000HFA.

[0025] In some embodiments of the present invention, the macroporous adsorption resin includes ESQ-3.

[0026] In some embodiments of the present invention, the immobilization carrier is epoxy resin.

[0027] In some embodiments of the present invention, the immobilization carrier is specifically LXTE-1000HFA.

[0028] In some embodiments of the present invention, the method for preparing the immobilized enzyme includes: contacting the lipase with a pretreated immobilization carrier in a buffer solution to immobilize the lipase on the immobilization carrier.

[0029] In some embodiments of the present invention, the buffer solution is a phosphate buffer solution.

[0030] In some embodiments of the present invention, the buffer solution is a 0.1 M, pH 8.0 sodium phosphate buffer solution.

[0031] In some embodiments of the present invention, the immobilization temperature is 25-35°C and the immobilization time is 15-21 hours.

[0032] In some embodiments of the present invention, the immobilization temperature is 30°C and the immobilization time is 18 h.

[0033] In some embodiments of the present invention, the temperature of the selective transesterification reaction is 20-35°C.

[0034] In some embodiments of the present invention, the temperature of the selective transesterification reaction is 25°C.

[0035] In some embodiments of the present invention, the reaction time of the selective transesterification reaction is 16-20 h.

[0036] In some embodiments of the present invention, the reaction time for the selective transesterification reaction is 18 h.

[0037] In some embodiments of the present invention, when an immobilized enzyme is used as a catalyst, the mass ratio of the racemic 3-yne-1,2-diol to the immobilized enzyme is 1:1 to 5:1.

[0038] In some embodiments of the present invention, when an immobilized enzyme is used as a catalyst, the mass ratio of the racemic 3-yne-1,2-diol to the immobilized enzyme is 2:1.

[0039] In some embodiments of the present invention, 1.0 g of the immobilized enzyme is added to every 2.0 g of racemic 2-methylbut-3-yne-1,2-diol.

[0040] In some embodiments of the present invention, each 2.0 g of racemic 2-methylbut-3-yne-1,2-diol is dissolved in 15-25 mL of organic solvent.

[0041] In some embodiments of the present invention, each 2.0 g of racemic 2-methylbut-3-yne-1,2-diol is dissolved in 20 mL of organic solvent.

[0042] In some embodiments of the present invention, for every 2.0 g of racemic 2-methylbut-3-yne-1,2-diol, 20 mL of organic solvent, 1.0 g of immobilized enzyme, and 2 molar equivalents of excipients are added.

[0043] In some embodiments of the present invention, after the selective transesterification reaction is completed, the reaction product is separated to obtain (2R)-2-methylbut-3-yne-1,2-diol.

[0044] In some embodiments of the present invention, high performance liquid chromatography is used to detect the substrate and product in the reaction system, and the reaction conversion rate and the enantiomeric excess value (ee value) of the product are calculated based on the detection results of the substrate and product.

[0045] A second aspect of the present invention provides a method for preparing (2R)-2-methylbut-3-yne-1,2-diol, comprising the following steps: using a lipase with the amino acid sequence shown in SEQ ID NO: 1 to catalyze an enantioselective transesterification reaction of racemic 2-methylbut-3-yne-1,2-diol to obtain (2R)-2-methylbut-3-yne-1,2-diol.

[0046] In some embodiments of the present invention, the selective transesterification reaction is carried out in the presence of excipients and organic solvents.

[0047] In some embodiments of the present invention, the organic solvent includes at least one selected from methyl ether, toluene, petroleum ether, DMSO, and cyclohexane.

[0048] In some embodiments of the present invention, the organic solvent includes at least one of methyl ether, toluene, and petroleum ether.

[0049] In some embodiments of the present invention, the organic solvent is specifically methyl ether.

[0050] In some embodiments of the present invention, the excipient is an ester compound.

[0051] In some embodiments of the present invention, the ester compound includes vinyl acetate.

[0052] In some embodiments of the present invention, the amount of the excipient added is 1-3 molar equivalents of racemic 2-methylbut-3-yne-1,2-diol.

[0053] In some embodiments of the present invention, the amount of the excipient added is twice the molar equivalent of racemic 2-methylbut-3-yne-1,2-diol.

[0054] In some embodiments of the present invention, the lipase is used in the form of a free enzyme or an immobilized enzyme.

[0055] In some embodiments of the present invention, the lipase is used in the specific manner described above.

[0056] In some embodiments of the present invention, the temperature and reaction time of the selective transesterification reaction are as described above.

[0057] The beneficial effects of this invention are: This invention is the first to synthesize (2R)-2-methylbut-3-yne-1,2-diol via biocatalysis using a preferred enzyme. This biocatalytic method not only features mild reaction conditions and environmental friendliness, but also exhibits high regioselectivity and stereoselectivity. Furthermore, it avoids chiral resolution and heavy metal residues in the product, effectively compensating for the shortcomings of chemical methods. Simultaneously, the preferred lipase Lip-1 provided in this invention catalyzes the above reaction with a yield of 45% (theoretical yield 50%), producing an optical purity of 99.2% and a purity of 99.5%, demonstrating strong stereoselectivity, high resolution efficiency, and high product purity. Moreover, this synthetic method requires only one step to obtain the target product and boasts advantages such as simple reaction, few steps, mild reaction conditions, environmental friendliness, high catalytic activity, and low production cost, exhibiting significant industrial application potential and commercial value. Attached Figure Description

[0058] Figure 1 The structural formula of the Speciosin P derivative is given.

[0059] Figure 2 The reaction formula is (2R)-2-methylbut-3-yne-1,2-diol.

[0060] Figure 3 The result is the HPLC detection result of the ee value of (2R)-2-methylbut-3-yne-1,2-diol. Detailed Implementation

[0061] 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.

[0062] In the preparation of the lipase of this invention, the host microorganism for protein expression is Escherichia coli. Escherichia coli BL21(DE3) or Pichia pastoris Pichia pastoris For GS115, the recombinant plasmids selected are pET28 (for E. coli) or pPIC9k (for Pichia pastoris). The molecular biology procedures involved are all well-known and routine experimental methods in the biological field, including gene acquisition (PCR), splicing of plasmids and target genes (i.e., vector construction), introduction of plasmids containing the target gene fragment into bacterial cells (i.e., transformation), and bacterial culture and enzyme production (i.e., fermentation).

[0063] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0064] LB solid medium formula: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 20g / L.

[0065] YPD medium composition: 20 g / L peptone, 10 g / L yeast extract, 22 g / L glucose monohydrate (for solid medium, add 20 g / L agar powder).

[0066] BMGY medium formulation: Dissolve 10g yeast extract and 20g peptone in 700mL water, sterilize at 121℃ for 20min, cool to room temperature, and then add the following mixture: 100mL 1M phosphate buffer (pH 6.0); 100mL 13.4% YNB (yeast nitrogen base, containing ammonium sulfate but no amino acids); 2mL 0.02% biotin; 100mL 10% glycerol.

[0067] BMMY medium formulation: Dissolve 10g yeast extract and 20g peptone in 700mL water, sterilize at 121℃ for 20min, cool to room temperature, and then add the following mixture: 100mL 1M phosphate buffer (pH 6.0); 100mL 13.4% YNB (yeast nitrogen base, containing ammonium sulfate but not amino acids); 2mL 0.02% biotin; 100mL 5% methanol.

[0068] The specific reaction formula for (2R)-2-methylbut-3-yne-1,2-diol is as follows: Figure 2 As shown.

[0069] Example 1 In this embodiment, a method for obtaining recombinant genetically engineered lipases from different sources is provided.

[0070] The specific experimental steps are as follows: This invention involves a total of 7 lipases, detailed information of which is shown in Table 1, and the above lipases are named Lip-1, Lip-2, Lip-3, Lip-4, Lip-5, Lip-6 and Lip-7 respectively.

[0071] Table 1. Lipases from different sources

[0072] The amino acid sequence of Lip-1 is as follows: APATETLDRRAALPNPYDDPFYTTPSNIGTFAKGQVIQSRKVPTDIGNANNAASFQLQYRTTNTQNEAVADVATVWIPAKPASPPKIFSYQVYEDATALDCAPSYSYLTGL DQPNKVTAVLDTPIIIGWALQQGYYVVSSDHEGFKAAFIAGYEEGMAILDGIRALKNYQNLPSDSKVALEGYSGGAHATVWATSLADSYAPELNIVGASHGGTPVSAKDTF TFLNGGPFAGFALAGVSGLSLLAHPDMESFIEARLNAKGQQTLKQIRRGRGFCLPQVVLTYPFLNVFSLVNDTNLLNEAPIAGILKQETVVQAEASYTVSVPKFPRFIWHAIP DEIVPYQPAATYVKEQCAKGANINFSPYPIAEHLTAEIFGLVPSLWFIKQAFDGTTPKVICGTPIPAAIAGITTPSADQVLGSDLANQLRSLNGKQSAFGKPFGPITPP (SEQ IDNO: 1).

[0073] The codon-optimized Lip-1 nucleotide sequence is as follows:

[0074] The amino acid and nucleotide sequences of Lip-2 are shown in SEQ ID NO: 3-4, the amino acid and nucleotide sequences of Lip-3 are shown in SEQ ID NO: 5-6, the amino acid and nucleotide sequences of Lip-4 are shown in SEQ ID NO: 7-8, the amino acid and nucleotide sequences of Lip-5 are shown in SEQ ID NO: 9-10, the amino acid and nucleotide sequences of Lip-6 are shown in SEQ ID NO: 11-12, and the amino acid and nucleotide sequences of Lip-7 are shown in SEQ ID NO: 13-14.

[0075] The amino acid sequence of Lip-2 is as follows: MIGDNKMLFQKILNRKHGVTARVLDHLPKRLQTSLADVLSYSYQYPQLDSYIKCILAIEHKQGKTKLIDTDLLHAREQFEKQMISIQFKPTKVK HVEDLRLPLQSGVIFARHYHPAPHKKLPMVIYYHGGGFIFGSVNSYDEVCWLLAINANVQVLSIEYPLAPEVGPEKIIQICEDALAWTYQNRKNL KILKNRIAVAGDSAGGNIATVIAQKSLKKNHVPCAQLLIYPKTDFKNKYPSYYAYKEGLFLTGQDVEMMTEQYVKKFQIELDHPLVSPMFGQLKN IAPAFIVTVTHDVLHDEGEIYAHRLQYSGVKIFYKNYIDQTHGFINLTPISNRSKKYSIEIFKEFRQFWDQQEGFFRGLFYIWKIFSLEIK (SEQ ID NO: 3).

[0076] The codon-optimized Lip-2 nucleotide sequence is as follows:

[0077] The amino acid sequence of Lip-3 is as follows: MVAFQRSIHDLVEKGQGSAGRALDRLPTFAQESLTKLLGYPYQFPQLDSFTKCLMAAQLKQGKVGFIGADVERSRKQFESQMQSIINKPTHIQFVEDIRLPLQSGSVYARHYHPAPNKKLPMIVFYHGGGFVVGSVDTHDEVCRLIAAHAKVQVLSIEYPLAPEVGPRDLIQSCEDALA WVYQNRRQFKILKNRIAVAGDSAGGNISAVVAQRTVNTVFAPQAQFLIYPAVDFKSRHPSFFAYKDGLVLTGSDIDYVTDYYATRFNMALDDPIISPTNGQLKKLAPAFVVTAGHDLLHDEGKIYAYKLRQNGVKVHYQDYEDQTHGFINLTPVSRKAKKYVIEMSKEFRKFWDKQ (SEQ ID NO: 5).

[0078] The codon-optimized Lip-3 nucleotide sequence is as follows:

[0079] The amino acid sequence of Lip-4 is as follows: MMKIVPPKPFFFEAGERAVLLLHGFTGNSADVRMLGRFLESKGYTCHAPIYKGHGVPPEELVHTGPDDWWQDVMNGYEFLKNKGYEKIAVAGLSLGGVFSLKLGYTVPIEGIVTMCAPMYIKSEE TMYEGVLEYAREYKKREGKSEEQIEQEMEKFKQTPMKTLKALQELIADVRDHLDLIYAPTFVVQARHDEMINPDSANIIYNEIESPVKQIKWYEQSGHVITLDQEKDQLHEDIYAFLESLDW (SEQ ID NO: 7).

[0080] The codon-optimized Lip-4 nucleotide sequence is as follows: 5'-ATGATGAAAATTGTGCCGCCGAAACCGTTTTTTTTTGAAGCGGGCGAACGCGCGGTGCTGCTGCTGCATGGCTTTACCGGCAACAGCGCGGATGTGCGCATGCTGGGCCGCTTTCTGGAAAGCAAAGGCTATACCTGCCATGCGCCGATTTATAAAGGCCATGGCGTGCCGCCGGAAGAACTGGTGCATACCGGCCCGGATGATTGGTGGCAGGATGTGATGAACGGCTATGAATTTCTGAAAAACAAAGGCTATGAAAAAATTGCGGTGGCGGGCCTGAGCCTGGGCGGCGTGTTTAGCCTGAAACTGGGCTATACCGTGCCGATTGAAGGCATTGTGACCATGTGCGCGCCGATGTATATTAAAAGCGAAGAAACCATGTATGAAGGCGTGCTGGAATATGCGCGCGAATATAAAAAACGCGAAGGCAAAAGCGAAGAACAGATTGAACAGGAAATGGAAAAATTTAAACAGACCCCGATGAAAACCCTGAAAGCGCTGCAGGAACTGATTGCGGATGTGCGCGATCATCTGGATCTGATTTATGCGCCGACCTTTGTGGTGCAGGCGCGCCATGATGAAATGATTAACCCGGATAGCGCGAACATTATTTATAACGAAATTGAAAGCCCGGTGAAACAGATTAAATGGTATGAACAGAGCGGCCATGTGATTACCCTGGATCAGGAAAAAGATCAGCTGCATGAAGATATTTATGCGTTTCTGGAAAGCCTGGATTGG-3' (SEQ ID NO: 8).

[0081] Wherein, the amino acid sequence of Lip-5 is: MRSSLVLFFVSAWTALASPVRREVSQDLFDQFNLFAQYSAAAYCAKNNDAPAGANVTCRGSICPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGTLVPVTKNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL (SEQ ID NO: 9).

[0082] The codon-optimized Lip-5 nucleotide sequence is: 5'-ATGCGCAGCAGCCTGGTGCTGTTTTTTGTGAGCGCGTGGACCGCGCTGGCGAGCCCGGTGCGCCGCGAAGTGAGCCAGGATCTGTTTGATCAGTTTAACCTGTTTGCGCAGTATAGCGCGGCGGCGTATTGCGCGAAAAACAACGATGCGCCGGCGGGCGCGAACGTGACCTGCCGCGGCAGCATTTGCCCGGAAGTGGAAAAAGCGGATGCGACCTTTCTGTATAGCTTTGAAGATAGCGGCGTGGGCGATGTGACCGGCTTTCTGGCGCTGGATAACACCAACCGCCTGATTGTGCTGAGCTTTCGCGGCAGCCGCAGCCTGGAAAACTGGATTGGCAACATTAACCTGGATCTGAAAGGCATTGATGATATTTGCAGCGGCTGCAAAGGCCATGATGGCTTTACCAGCAGCTGGCGCAGCGTGGCGAACACCCTGACCCAGCAGGTGCAGAACGCGGTGCGCGAACATCCGGATTATCGCGTGGTGTTTACCGGCCATAGCCTGGGCGGCGCGCTGGCGACCGTGGCGGGCGCGAGCCTGCGCGGCAACGGCTATGATATTGATGTGTTTAGCTATGGCGCGCCGCGCGTGGGCAACCGCGCGTTTGCGGAATTTCTGACCGCGCAGACCGGCGGCACCCTGTATCGCATTACCCATACCAACGATATTGTGCCGCGCCTGCCGCCGCGCGAACTGGGCTATAGCCATAGCAGCCCGGAATATTGGATTACCAGCGGCACCCTGGTGCCGGTGACCAAAAACGATATTGTGAAAGTGGAAGGCATTGATAGCACCGATGGCAACAACCAGCCGAACACCCCGGATATTGCGGCGCATCTGTGGTATTTTGGCCTGATTGGCACCTGCCTG-3' (SEQ ID NO: 10).

[0083] Wherein, the amino acid sequence of Lip-6 is: MRSSLVLFFVSAWTALASPIRREVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFGLIGTCL (SEQ ID NO: 11).

[0084] The codon-optimized Lip-6 nucleotide sequence is: 5'-ATGCGCAGCAGCCTGGTGCTGTTTTTTGTGAGCGCGTGGACCGCGCTGGCGAGCCCGATTCGCCGCGAAGTGAGCCAGGATCTGTTTAACCAGTTTAACCTGTTTGCGCAGTATAGCGCGGCGGCGTATTGCGGCAAAAACAACGATGCGCCGGCGGGCACCAACATTACCTGCACCGGCAACGCGTGCCCGGAAGTGGAAAAAGCGGATGCGACCTTTCTGTATAGCTTTGAAGATAGCGGCGTGGGCGATGTGACCGGCTTTCTGGCGCTGGATAACACCAACAAACTGATTGTGCTGAGCTTTCGCGGCAGCCGCAGCATTGAAAACTGGATTGGCAACCTGAACTTTGATCTGAAAGAAATTAACGATATTTGCAGCGGCTGCCGCGGCCATGATGGCTTTACCAGCAGCTGGCGCAGCGTGGCGGATACCCTGCGCCAGAAAGTGGAAGATGCGGTGCGCGAACATCCGGATTATCGCGTGGTGTTTACCGGCCATAGCCTGGGCGGCGCGCTGGCGACCGTGGCGGGCGCGGATCTGCGCGGCAACGGCTATGATATTGATGTGTTTAGCTATGGCGCGCCGCGCGTGGGCAACCGCGCGTTTGCGGAATTTCTGACCGTGCAGACCGGCGGCACCCTGTATCGCATTACCCATACCAACGATATTGTGCCGCGCCTGCCGCCGCGCGAATTTGGCTATAGCCATAGCAGCCCGGAATATTGGATTAAAAGCGGCACCCTGGTGCCGGTGACCCGCAACGATATTGTGAAAATTGAAGGCATTGATGCGACCGGCGGCAACAACCAGCCGAACATTCCGGATATTCCGGCGCATCTGTGGTATTTTGGCCTGATTGGCACCTGCCTG-3' (SEQ ID NO: 12).

[0085] Wherein, the amino acid sequence of Lip-7 is: MRPVRRAVPQDLLDQFELFSQYSAAAYCAANNHAPVGSDVTCSENVCPEVDAADATFLYSFEDSGLGDVTGLLALDNTNKLIVLSFRGSRSVENWIANLAADLTEISDICSGCEGHVGFVTSWRSVADTIREQVQNAVNEHPDYRVVFTGHSLGGALATIAAAALRGNGYNIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRDWGYSHSSPEYWVTSGNDVPVTANDITVVEGIDSTDGNNQGNIPDIPSHLWYFGPISECD (SEQ ID NO: 13).

[0086] The codon-optimized Lip-7 nucleotide sequence is: 5’-ATGCGCCCGGTGCGCCGCGCGGTGCCGCAGGATCTGCTGGATCAGTTTGAACTGTTTAGCCAGTATAGCGCGGCGGCGTATTGCGCGGCGAACAACCATGCGCCGGTGGGCAGCGATGTGACCTGCAGCGAAAACGTGTGCCCGGAAGTGGATGCGGCGGATGCGACCTTTCTGTATAGCTTTGAAGATAGCGGCCTGGGCGATGTGACCGGCCTGCTGGCGCTGGATAACACCAACAAACTGATTGTGCTGAGCTTTCGCGGCAGCCGCAGCGTGGAAAACTGGATTGCGAACCTGGCGGCGGATCTGACCGAAATTAGCGATATTTGCAGCGGCTGCGAAGGCCATGTGGGCTTTGTGACCAGCTGGCGCAGCGTGGCGGATACCATTCGCGAACAGGTGCAGAACGCGGTGAACGAACATCCGGATTATCGCGTGGTGTTTACCGGCCATAGCCTGGGCGGCGCGCTGGCGACCATTGCGGCGGCGGCGCTGCGCGGCAACGGCTATAACATTGATGTGTTTAGCTATGGCGCGCCGCGCGTGGGCAACCGCGCGTTTGCGGAATTTCTGACCGCGCAGACCGGCGGCACCCTGTATCGCATTACCCATACCAACGATATTGTGCCGCGCCTGCCGCCGCGCGATTGGGGCTATAGCCATAGCAGCCCGGAATATTGGGTGACCAGCGGCAACGATGTGCCGGTGACCGCGAACGATATTACCGTGGTGGAAGGCATTGATAGCACCGATGGCAACAACCAGGGCAACATTCCGGATATTCCGAGCCATCTGTGGTATTTTGGCCCGATTAGCGAATGCGAT-3’(SEQ ID NO:14)。

[0087] The synthesis of the complete genes of Lip-1, Lip-2, Lip-3, Lip-4, Lip-5, Lip-6, and Lip-7 was completed by Beijing Qingke Biotechnology Co., Ltd., and the codons of the genes were optimized by adding corresponding restriction enzyme sites to both ends of the genes. The plasmid of Lip-1 is pPIC9k with EcoRI and NotI restriction sites, and the plasmid of Lip-2, Lip-3, Lip-4, Lip-5, Lip-6, and Lip-7 is pET28a with EcoRI and NotI restriction sites.

[0088] Meanwhile, since Lip-1 has poor solubility in E. coli, the Lip-1 target gene fragment was inserted into the vector pPIC9k to complete the construction of the expression vector, resulting in the recombinant plasmid pPIC9k-Lip-1 for lipase; the target gene fragments of Lip-2, Lip-3, Lip-4, Lip-5, Lip-6, and Lip-7 were inserted into the vector pET28 to complete the construction of the expression vector, resulting in the recombinant plasmids pET28a-Lip-2, pET28a-Lip-3, pET28a-Lip-4, pET28a-Lip-5, pET28a-Lip-6, and pET28a-Lip-7 for lipase.

[0089] The obtained recombinant plasmid pPIC9k-Lip-1 was transformed into... Pichia pastoris In GS115, the obtained recombinant plasmids pET28a-Lip-2, pET28a-Lip-3, pET28a-Lip-4, pET28a-Lip-5, pET28a-Lip-6, and pET28a-Lip-7 were transformed into Escherichia coli BL21(DE3), and the specific transformation steps were conventional methods in the art.

[0090] This yielded recombinant genetically engineered bacteria. Pichia pastoris GS115 / pPIC9k-Lip-1, BL21(DE3) / pET28a-Lip-2, BL21(DE3) / pET28a-Lip-3, BL21(DE3) / pET 28a-Lip-4, BL21(DE3) / pET28a-Lip-5, BL21(DE3) / pET28a-Lip-6, BL21(DE3) / pET28a-Lip-7.

[0091] Example 2 This embodiment provides a method for obtaining wet cells of recombinant genetically engineered bacteria containing lipases from different sources, as well as the preparation of immobilized enzymes.

[0092] The specific experimental steps are as follows: The recombinant lipase-producing bacteria (Lip-2-Lip-7) obtained in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Then, 2% (v / v) inoculation was carried out into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 150 rpm until the bacterial concentration OD600 = 0.6. IPTG was added to a final concentration of 0.1 mM and cultured at 28°C for 12 h. The bacteria were collected by centrifugation at 4°C and 12000 rpm for 10 min. The wet bacteria were washed with 0.85% physiological saline and stored at -20°C for later use.

[0093] Lip-1 was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of BMGY medium. After culturing at 28°C and 250 rpm for 18 h, the supernatant was removed by centrifugation at 3000 × g for 10 min. The cells were collected and resuspended in 5 mL of BMMY medium. The resuspended cells were then added to 100-200 mL of BMMY liquid medium for shake-flask culture, with the volume limited to less than 10%. The culture temperature was 28°C, the shaker speed was 250 rpm, and 0.5% anhydrous methanol was added for induction. Anhydrous methanol was added every 24 h (still at 0.5% v / v). The culture time was 2-3 days, depending on enzyme production. After successful culture, the cells were centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was collected for later use (the enzyme expressed by Pichia pastoris is in the supernatant).

[0094] Example 3 In this embodiment, a method for screening lipases from various sources is provided.

[0095] Using various esterases / lipases prepared in Example 2 as catalysts, the catalytic reaction of racemic 2-methylbut-3-yne-1,2-diol was carried out.

[0096] The CAS number of the substrate 2-methylbut-3-yne-1,2-diol of this invention is 21433-93-2.

[0097] The specific experimental steps are as follows: 2.0 g of racemic 2-methylbut-3-yne-1,2-diol was dissolved in 20 mL of methyl ether. Parallel reaction groups with different enzyme sources were set up: the Lip-1 group was added with 4 mL of Lip-1 fermentation supernatant prepared in Example 2; the Lip-2-Lip-7 groups were added with 1.0 g of wet Escherichia coli expressing Lip-2, Lip-3, Lip-4, Lip-5, Lip-6 or Lip-7 prepared in Example 2, and vinyl acetate in a molar equivalent of 2 times that of racemic 2-methylbut-3-yne-1,2-diol was added as an excipient for the transesterification reaction (i.e., 4.65 g of vinyl acetate was added for every 2 g of racemic 2-methylbut-3-yne-1,2-diol; vinyl acetate was purchased from Aladdin, CAS No.: 108-05-4). The reaction was carried out at 30 °C for 18 h. After the reaction was completed, the reaction solution was analyzed by high performance liquid chromatography, and the conversion rate and the enantiomeric excess value (ee value) of the product were calculated based on the peak areas of the substrate and product to evaluate the catalytic activity and selectivity of lipases from different sources.

[0098] The conversion rate calculation formula is as follows: First, calculate the substrate enantiomeric excess value (ee). s ) and the excess value of product enantiomeric expression (ee) p ): ee s = ×100%; Among them, A 1S For the peak area of ​​the S-configuration substrate, A 1R This represents the peak area of ​​the R-configuration substrate.

[0099] ee p = ×100%; Among them, A 2R The peak area of ​​the R-configuration product; A 2S This represents the peak area of ​​the S-configuration product.

[0100] Then based on ee s andee p Calculate the conversion rate (c): c = .

[0101] The results are shown in Table 2.

[0102] Table 2. Activity and selectivity of different esterases / lipases catalyzing racemic 2-methylbut-3-yne-1,2-diol

[0103] In Table 2, the " / " indicates no effect.

[0104] It was observed that Lip-1, 2, and 3 exhibited S-type selectivity for the substrate racemic 2-methylbut-3-yne-1,2-diol, catalyzing the S-type alcohol transesterification while retaining (2R)-2-methylbut-3-yne-1,2-diol. Lip-1 showed the highest activity and selectivity, while Lip-4 and 7 exhibited no substrate activity; therefore, the selectivity stemmed from... Moesziomyces antarcticus Lip-1 was used as the parent for subsequent studies.

[0105] Example 4 Since the subsequent enzyme-catalyzed reaction is carried out in an organic phase system, in order to improve the stability of Lip-1 in the reaction system and facilitate enzyme recovery and reuse, this embodiment immobilizes Lip-1 and screens different types of resin carriers.

[0106] The resins used in this embodiment include: amino resins LXTE-703, ESR-3, ES110, HA104F, EA104F, HA109M, LXTE-701, LXTE-704, and LXTE-700s; epoxy resins ES108, ES103B, GA611M, and LXTE-1000HFA; and macroporous adsorption resin ESQ-3. All resins were purchased from Xi'an Lanxiao Technology Co., Ltd.

[0107] The specific experimental steps are as follows: First, the different types of resins mentioned above are pretreated respectively.

[0108] For the amino resin, weigh appropriate amounts of resin and soak them in deionized water for 2–4 hours to allow the resin to fully swell. Pour off the supernatant and remove any floating fine particles. Then, place the swollen amino resin in a 2% (w / v) glutaraldehyde solution, adding 25 mL of the solution per 1 g of resin. Stir for 12 hours to activate the amino groups on the resin surface and introduce aldehyde functional groups. After treatment, pre-activated amino resin is obtained and ready for use.

[0109] For epoxy resin, weigh an appropriate amount of resin and soak it in deionized water for 2-4 hours to allow the resin to fully swell. Pour off the supernatant and remove any floating fine particles. Then, place the swollen epoxy resin in 0.1 M phosphate buffer (25 mL buffer per 1 g resin) and stir for 2 hours. After treatment, rinse repeatedly with distilled water 3-4 times to obtain pretreated epoxy resin for later use.

[0110] For the macroporous adsorption resin ESQ-3, weigh an appropriate amount of resin and add 2-3 times the volume of 95% ethanol. Soak for 4-12 hours, stirring continuously during the soaking process to remove residual unpolymerized monomers, pore-forming agents, and other organic impurities from the resin. After soaking, pour off the ethanol and rinse the resin repeatedly with deionized water until the washing solution is clear and has no obvious ethanol odor. The pretreated macroporous adsorption resin is then ready for use.

[0111] The Pichia pastoris fermentation broth expressing Lip-1 according to the method in Example 2 was centrifuged to remove the cell cells, and the supernatant was collected. The obtained supernatant was used directly as the crude Lip-1 enzyme solution for subsequent immobilization. During immobilization, 1.0 g of the pretreated epoxy resin, amino resin, or macroporous adsorption resin was weighed, washed with 0.1 M, pH 8.0 sodium phosphate buffer, and filtered. Subsequently, 10 mL of Lip-1 crude enzyme solution supernatant and 10 mL of 0.1 M, pH 8.0 sodium phosphate buffer were added to each resin sample, and the mixture was immobilized by shaking at 30°C for 18 h.

[0112] After immobilization, the resin was collected by filtration and washed with 0.1 M, pH 8.0 sodium phosphate buffer to remove unbound free enzymes. The resulting resin loaded with Lip-1 after washing and filtration is the immobilized Lip-1.

[0113] Example 5 In this embodiment, the Lip-1 immobilized enzyme obtained by immobilization with different resin carriers in Example 4 was used as a catalyst to investigate the effect of different immobilization resins on the enzyme activity and selectivity of Lip-1 immobilized enzyme.

[0114] The specific experimental steps are as follows: 2.0 g of racemic 2-methylbut-3-yne-1,2-diol was dissolved in 20 mL of methyl ether to prepare 14 portions, which were then poured into 50 mL reaction flasks. Next, 1.0 g of each Lip-1 immobilized enzyme prepared using different types of resins was weighed and added to each of the 14 reaction flasks containing the substrate. For each reaction, vinyl acetate was added in a molar equivalent of 2 times that of racemic 2-methylbut-3-yne-1,2-diol as an excipient for the transesterification reaction. After reacting at 30 °C for 18 h, the conversion rate and the ee value of the product were measured. The formulas for calculating the conversion rate and ee value are shown in Example 3.

[0115] The results are shown in Table 3 below.

[0116] Table 3. Effects of different immobilization resins on the activity and selectivity of Lip-1 immobilized enzymes.

[0117] The results showed that the immobilized enzyme prepared with LXTE-1000HFA resin as the carrier had the best effect, with a conversion rate of up to 50.8% and an ee value of up to 99.15%. Therefore, the immobilized enzyme prepared with LXTE-1000HFA resin was selected as the catalyst.

[0118] Example 6 In this embodiment, the Lip-1 immobilized enzyme obtained by immobilization on the LXTE-1000HFA resin carrier in Example 4 was used as a catalyst to investigate the effect of different types of organic solvents in the reaction system on the enzyme activity and selectivity of the Lip-1 immobilized enzyme.

[0119] The specific experimental steps are as follows: 2.0 g of racemic 2-methylbut-3-yne-1,2-diol was dissolved in 20 mL of different organic solvents (including methyl ether, toluene, acetonitrile, tetrahydrofuran, DMSO, petroleum ether, isopropanol, cyclohexane, and isooctane). 1.0 g of the Lip-1 immobilized enzyme prepared in Example 4 was added as a catalyst, and vinyl acetate in a molar equivalent to 2 moles of racemic 2-methylbut-3-yne-1,2-diol was added as an excipient for the transesterification reaction. The reaction was carried out at 30 °C for 18 h. After the reaction, the reaction solution was analyzed by high-performance liquid chromatography (HPLC). The conversion rate and enantiomeric excess (ee) of the product were calculated based on the peak areas of the substrate and product to evaluate the catalytic activity and selectivity of the Lip-1 immobilized enzyme under different organic solvent conditions. The formulas for calculating the conversion rate and ee value are shown in Example 3.

[0120] The results are shown in Table 4 below.

[0121] Table 4. Effects of different organic solvents on the activity and selectivity of Lip-1 immobilized enzymes.

[0122] The results showed that the product obtained by using methyl ether as an organic solvent had the highest ee value, therefore methyl ether was chosen as the organic solvent for the substrate.

[0123] Example 7 In this embodiment, the Lip-1 immobilized enzyme obtained by immobilization on the LXTE-1000HFA resin carrier in Example 4 was used as a catalyst, and methyl ether in Example 6 was used as an organic solvent for the substrate. The effects of different temperatures in the reaction system on the enzyme activity and selectivity of the Lip-1 immobilized enzyme were investigated.

[0124] The specific experimental steps are as follows: 2.0 g of racemic 2-methylbut-3-yne-1,2-diol was dissolved in 20 mL of organic solvent (methyl ether). 1.0 g of the Lip-1 immobilized enzyme prepared in Example 4 was added as a catalyst, and vinyl acetate in a molar equivalent of 2 times that of racemic 2-methylbut-3-yne-1,2-diol was added as an excipient for the transesterification reaction. The reaction was carried out at 25-50 °C for 18 h. After the reaction, the reaction solution was analyzed by high-performance liquid chromatography (HPLC). The conversion rate and enantiomeric excess (ee) of the product were calculated based on the peak areas of the substrate and product to evaluate the catalytic activity and selectivity of the Lip-1 immobilized enzyme under different temperature conditions. The formulas for calculating the conversion rate and ee value are shown in Example 3.

[0125] The results are shown in Table 5 below.

[0126] Table 5. Effects of different temperatures on the activity and selectivity of Lip-1 immobilized enzymes.

[0127] The results showed that the conversion rate and the ee value of the product were not significantly different at 25-35℃, but the ee value of the product decreased significantly with increasing temperature. Therefore, 25℃ was selected as the optimal temperature (the stability of enzymes is generally better at low temperatures).

[0128] Example 8 In this embodiment, the Lip-1 immobilized enzyme obtained by immobilization on the LXTE-1000HFA resin carrier in Example 4 was used as a catalyst, and methyl ether in Example 6 was used as an organic solvent for the substrate. The reaction system temperature was fixed at 25°C, and the effects of different reaction times on the enzyme activity and selectivity of the Lip-1 immobilized enzyme were investigated.

[0129] 2.0 g of racemic 2-methylbut-3-yne-1,2-diol was dissolved in 20 mL of organic solvent (methyl ether). 1.0 g of the Lip-1 immobilized enzyme prepared in Example 4 was added as a catalyst, and vinyl acetate in a molar equivalent to 2 times that of racemic 2-methylbut-3-yne-1,2-diol was added as an excipient for the transesterification reaction. The reaction was carried out at 25 °C for 1 h, 2 h, 4 h, 6 h, 10 h, 14 h, 18 h, 24 h, and 30 h. After the reaction, the reaction solution was analyzed by high-performance liquid chromatography (HPLC). The conversion rate and enantiomeric excess (ee) of the product were calculated based on the peak areas of the substrate and product to evaluate the catalytic activity and selectivity of the Lip-1 immobilized enzyme under different reaction durations. The formulas for calculating the conversion rate and ee value are shown in Example 3.

[0130] The results are shown in Table 6.

[0131] Table 6. Effects of different reaction times on the activity and selectivity of Lip-1 immobilized enzymes.

[0132] It can be observed that the ee value of the product increases continuously with time, exceeding 99% after 18 hours. However, the conversion rate still increases at 24 hours and 30 hours, indicating that some (2R)-2-methylbut-3-yne-1,2-diol is converted, resulting in a lower yield. Therefore, 18 hours of reaction time is chosen as the optimal reaction time (not exceeding 20 hours).

[0133] Example 9 In this embodiment, a method for preparing (2R)-2-methylbut-3-yne-1,2-diol by catalyzing racemic 2-methylbut-3-yne-1,2-diol using an immobilized enzyme containing lipase Lip-1 is provided.

[0134] The specific experimental steps are as follows: Using the same methods as in Examples 2 and 4 above, the recombinant engineered lipase Lip-1 bacteria were cultured, induced, and immobilized for enzyme preparation, with the culture system scaled up to 3L proportionally. The resin carrier used for immobilization was LXTE-1000HFA.

[0135] Subsequently, (2R)-2-methylbut-3-yne-1,2-diol was prepared: 300 g of racemic 2-methylbut-3-yne-1,2-diol was dissolved in 3 L of methyl tertiary ether, and 150 g of the Lip-1 immobilized enzyme prepared in Example 4 was added. The mixture was incubated at 25 °C for 10 min, and vinyl acetate (twice the molar equivalent of the substrate racemic 2-methylbut-3-yne-1,2-diol) was added. The mixture was reacted at 300 rpm and 25 °C for 18 h. 500 μL of the mixture was taken as a sample, and the product was extracted with an organic reagent (ethyl acetate). The mixture was centrifuged at 12000 rpm to separate the layers, and the organic phase was analyzed by HPLC and the conversion rate was calculated.

[0136] The results are as follows Figure 3 As shown.

[0137] It can be observed that the S-type substrate 2-methylbut-3-yne-1,2-diol reacted almost completely after 18 h of reaction, with a conversion rate of 51.6%. Subsequently, the immobilized enzyme was removed by filtration, the organic phase was washed multiple times with water, and the diol was transferred to the aqueous phase. The aqueous phase was then extracted twice with ethyl acetate (1 / 2 volume of total volume). After centrifugation and separation, the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate and rotary evaporated to finally obtain 135.6 g of crude (2R)-2-methylbut-3-yne-1,2-diol, with a yield of 45.2% (theoretical yield 50%, 150 g), an ee value of 99.21%, and a purity of 99.5%.

[0138] 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. Application of lipase with the amino acid sequence shown in SEQ ID NO: 1 in the preparation of (2R)-2-methylbut-3-yne-1,2-diol.

2. The application according to claim 1, characterized in that, The (2R)-2-methylbut-3-yne-1,2-diol was prepared by enantioselective transesterification of racemic 2-methylbut-3-yne-1,2-diol catalyzed by lipase.

3. The application according to claim 2, characterized in that, The reaction system for the selective transesterification reaction also includes excipients; Preferably, the excipient is an ester compound; Preferably, the ester compound includes vinyl acetate.

4. The application according to claim 3, characterized in that, The amount of the excipient added is 1-3 molar equivalents of racemic 2-methylbut-3-yne-1,2-diol.

5. The application according to claim 4, characterized in that, The selective transesterification reaction is carried out in an organic solvent; Preferably, the organic solvent includes at least one selected from methyl ether, toluene, petroleum ether, DMSO, and cyclohexane.

6. The application according to claims 1-5, characterized in that, The lipase is used in the form of a free enzyme or an immobilized enzyme. Preferably, the immobilized enzyme includes a lipase and an immobilization carrier; Preferably, the immobilization carrier includes at least one of amino resin, epoxy resin, and macroporous adsorption resin.

7. The application according to any one of claims 6, characterized in that, The temperature for the selective transesterification reaction is 20-35℃; Preferably, the reaction time for the selective transesterification reaction is 16-20 h.

8. The application according to claim 7, characterized in that, When an immobilized enzyme is used as a catalyst, the mass ratio of the racemic 3-yne-1,2-diol to the immobilized enzyme is 1:1 to 5:

1.

9. A method for preparing (2R)-2-methylbut-3-yne-1,2-diol, characterized in that, Includes the following steps: (2R)-2-methylbut-3-yne-1,2-diol was obtained by enantioselective transesterification of racemic 2-methylbut-3-yne-1,2-diol catalyzed by lipase as shown in SEQ ID NO:

1.

10. The method according to claim 9, characterized in that, The selective transesterification reaction is carried out in the presence of excipients and organic solvents.

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