Method for catalyzing hydrolysis of oxetane by epoxy hydrolase
By screening highly efficient epoxide hydrolases using gene mining and heterologous expression technologies, the problem of generating chiral vicinal diols through the hydrolysis and ring-opening of oxetane was solved, enabling the efficient synthesis of chiral drug precursors with a catalytic efficiency of over 40%.
Patent Information
- Application Number
- CN202410968017.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-18
- Publication Date
- 2026-01-20
Smart Images
Figure HDA0004952287550000011 
Figure HDA0004952287550000012 
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biocatalysis, and particularly relates to a method for hydrolysis of oxetane catalyzed by a series of epoxide hydrolases. BACKGROUND
[0002] Oxetane, as a cyclic ether compound with large ring strain, has been found in many natural products, of which the most famous example is paclitaxel, which was first discovered in the bark of yew trees in 1971 and used for cancer treatment, which indicates that oxetane has obvious metabolic stability in vivo (Chemical Reviews, 2016, 116(19): 12150-12233; Angewandte Chemie International Edition, 2010, 49(48): 9052-9067.). In addition, in the field of pesticides, oxetane also has a wide range of uses, of which the most famous is the insecticide EDO, which has an activity 25 times that of the pesticide DDT and is biodegradable. Some are herbicides or biologically active molecules, such as oxasulfuron, norbornane derivatives, etc. However, the biosynthesis and transformation of oxetane is still a key problem.
[0003] Epoxide hydrolases (EHs, EC 3.3.2.) are widely derived from animals, plants and microorganisms, which can catalyze the kinetic resolution of racemic epoxides to generate chiral vicinal diols and chiral epoxides, and can also produce only enantioselective ring-opening vicinal diol products.
[0004] The catalytic efficiency is high, and the region and stereoselectivity are strong, which is conducive to the synthesis of high-purity chiral compounds. Therefore, microbial EHs have become a very important biological catalyst for the synthesis of chiral drugs, and also a powerful biosynthetic element. However, there are few reports on the reaction of epoxide hydrolase catalyzing the hydrolysis of oxetane, so it is of important application value to develop and study new synthesis methods and technologies, and then efficiently synthesize chiral drug molecule precursors. SUMMARY
[0005] In view of the deficiencies in the prior art, the present application synthesizes a series of epoxide hydrolases (EHs) through gene mining and data analysis, and expresses the engineering bacteria of the series of epoxide hydrolases and prepares whole cell lysate and crude enzyme, which are then used to screen corresponding 2-phenyloxetane to generate (S)-1-phenyl-1,3-propanediol.
[0006] The application provides a method for catalyzing oxetane hydrolysis by an epoxy hydrolase.
[0007] To this end, the application mines and characterizes 23 databases of epoxy hydrolases by gene mining, heterologous expression and the like, and further uses the epoxy hydrolases to catalyze 2-phenyloxetane to generate (S)-1-phenyl-1,3-propanediol, which is an intermediate of antidepressant (S)-duloxetine, male sexual dysfunction treatment (S)-dapoxetine and potential chiral drugs.
[0008] The recombinant expression vector is transformed into a suitable microbial host. The host microorganism is various host microorganisms in the art, as long as the recombinant expression vector can be stably self-replicated and the alcohol de-epoxy hydrolase gene can be effectively expressed. Preferably, the host microorganism is Escherichia coli, preferably Escherichia coli BL21 (DE3). The recombinant expression vector is transformed into E. coli BL21 (DE3) to obtain the genetically engineered strain of the application.
[0009] The application provides a method for catalyzing oxetane hydrolysis by an epoxy hydrolase, which uses an epoxy hydrolase derived from metagenomic DNA, Rhodococcus erythropolis, Rhizorhapis suberifaciens, Rhodococcus opacus or Actinomadura namibiensis as a catalytic enzyme to generate (S)-1-phenyl-1,3-propanediol from 2-phenyloxetane.
[0010] Preferably, the epoxy hydrolase derived from metagenomic DNA is AKK23712.1; the epoxy hydrolase derived from Rhodococcus erythropolis is Q9ZAG3.3; the epoxy hydrolase derived from Rhizorhapis suberifaciens is MBB4640159.1; the epoxy hydrolase derived from Rhodococcus opacus is BAH52894.1; and the epoxy hydrolase derived from Actinomadura namibiensis is MBA8954773.1.
[0011] More specifically, the catalytic enzyme participates in the reaction in the form of whole cells of a recombinant bacterium expressing the epoxy hydrolase, a prepared crude enzyme solution or a pure enzyme.
[0012] In a specific embodiment, the recombinant bacteria is Escherichia coli.
[0013] More specifically, the reaction system is as follows: the enzyme is used in an amount of 0.05-0.2 g / ml; the substrate concentration is 5-15 mM, specifically 8-12 mM, more specifically 10 mM; and the pH of the reaction system is 7-8, for example 7.2-7.6, specifically 7.4.
[0014] In a preferred embodiment, the whole cell is used as a catalyst in an amount of 0.05-0.2 g / ml, specifically 0.1 g / ml.
[0015] The reaction conditions are as follows: 25-35°C, preferably 30°C, 800-1200 rpm, preferably 1000 rpm.
[0016] The present application also provides the use of an epoxy hydrolase in catalyzing the hydrolysis of an oxetane, wherein the epoxy hydrolase is derived from Metagenomic DNA, Rhodococcus erythropolis, Rhizorhapis suberifaciens, Rhodococcus opacus or Actinomadura namibiensis, and is used as a catalytic enzyme to generate (S)-1-phenyl-1,3-propanediol from 2-phenyloxetane.
[0017] Specifically, the epoxy hydrolase is used to generate (S)-1-phenyl-1,3-propanediol or (R)-2-phenyloxetane from 2-phenyloxetane.
[0018] More preferably, the epoxy hydrolase derived from Metagenomic DNA is AKK23712.1; the epoxy hydrolase derived from Rhodococcus erythropolis is Q9ZAG3.3; the epoxy hydrolase derived from Rhizorhapis suberifaciens is MBB4640159.1; the epoxy hydrolase derived from Rhodococcus opacus is BAH52894.1; and the epoxy hydrolase derived from Actinomadura namibiensis is MBA8954773.1.
[0019] The present application is through the research screening, found that the source of metagenomic DNA (Metagenomic DNA) of epoxy hydrolase AKK23712.1, the source of Rhodococcus erythropolis (Rhodococcus erythropolis) of epoxy hydrolase Q9ZAG3.3, the source of Rhizorhapis suberifaciens (Rhizorhapis suberifaciens) of epoxy hydrolase MBB4640159.1, the source of Rhodococcus opacus (Rhodococcus opacus) of epoxy hydrolase BAH52894.1, the source of Actinomadura namibiensis (Actinomadura namibiensis) of epoxy hydrolase MBA8954773.1 c% reaches 40% or more. And especially the source of metagenomic DNA (Metagenomic DNA) of epoxy hydrolase (CH55-LEH) AKK23712.1, the source of Rhizorhapis suberifaciens (Rhizorhapis suberifaciens) of epoxy hydrolase (E1000) MBB4640159.1 can efficiently catalyze 2-phenyloxetane to generate product (S)-1-phenyl-1,3-propanediol. The product is an intermediate for synthesizing antidepressant (S)-duloxetine, treating male sexual dysfunction (S)-dapoxetine dapoxetine and potential chiral drugs. Therefore, the present application has higher practical value. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 The schematic diagram for epoxy hydrolase catalyzing 2-aryloxetane hydrolysis reaction.
[0021] Figure 2 Data mining new epoxy hydrolase phylogenetic analysis.
[0022] Figure 3 The HPLC detection spectrum of the reaction of crude enzyme powder of epoxy hydrolase catalyzing 2-phenyloxetane to generate (S)-1-phenyl-1,3-propanediol.
[0023] Figure 4 The GC-MS detection spectrum of the reaction of crude enzyme powder of epoxy hydrolase catalyzing 2-phenyloxetane to generate (S)-1-phenyl-1,3-propanediol. Wherein, T A3 = 6.6 min, T OH = 8.5 min, FW = 152. DETAILED DESCRIPTION
[0024] The present application will be further described in detail below in combination with specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.
[0025] The experimental methods in the following examples are all routine methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.
[0026] In the quantitative tests in the following examples, three repeated experiments are set up, and the results are averaged.
[0027] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.
[0028] In the following examples, Re-LEH is a hydrolase derived from Rhodococcus erythropolis, and the Accession Number of the amino acid sequence is Q9ZAG3.3.
[0029] CH55-LEH is a hydrolase derived from Metagenomic DNA, and the Accession Number of the amino acid sequence is AKK23712.1.
[0030] E996 is a hydrolase derived from Mycolicibacterium fortuitum, and the Accession Number of the amino acid sequence is SUA03756.1.
[0031] E997 is a hydrolase derived from Rhodococcus opacus, and the Accession Number of the amino acid sequence is BAH52894.1.
[0032] E998 is a hydrolase derived from Actinomadura namibiensis, and the Accession Number of the amino acid sequence is MBA8954773.1.
[0033] E999 is a hydrolase derived from Rhodococcus aetherivorans, and the Accession Number of the amino acid sequence is UYF92534.1.
[0034] E1000 is a hydrolase derived from Rhizorhapis suberifaciens, and the Accession Number of the amino acid sequence is MBB4640159.1.
[0035] E1026 is a hydrolase derived from Rhodococcus imtechensis RKJ300, with Accession Number EID76986.1 for the amino acid sequence;
[0036] E1027 is a hydrolase derived from Williamsia limnetica, with Accession Number PYE12450.1 for the amino acid sequence;
[0037] E1028 is a hydrolase derived from Pseudonocardia sp. Ae707_Ps1, with Accession Number OLM08926.1 for the amino acid sequence;
[0038] E1029 is a hydrolase derived from Actinomycetia bacterium, with Accession Number MSY70684.1 for the amino acid sequence;
[0039] E1030 is a hydrolase derived from Sphingobium czechense LL01, with Accession Number KMS57740.1 for the amino acid sequence
[0040] E1031 is a hydrolase derived from Rhodococcus kyotonensis, with Accession Number OAK56221.1 for the amino acid sequence
[0041] 2x High-fidelity Master Mix is a product of Takara Bio Inc., with product number TP001.
[0042] Example 1, Gene mining of epoxy hydrolases
[0043] With the hydrolytic enzyme (Re-LEH) derived from Rhodococcus erythropolis as a template, a series of amino acid sequences of epoxy hydrolase are obtained through NCBI data mining, and the sequences are analyzed to obtain the corresponding evolutionary tree, and the sequences in the evolutionary tree are synthesized by a company, and the sequences in the examples are as follows: Re-LEH, CH55-LEH, pET-22b-E996, pET-22b-E997, pET-22b-E998, pET-22b-E999, pET-22b-E1000, pET-22b-E1026, pET-22b-E1027, pET-22b-E1028, pET-22b-E1029, pET-22b-E1030, pET-22b-E1031, a total of 13.
[0044] Example 2, preparation of the engineering bacteria of the epoxy hydrolase
[0045] The coding gene of the epoxy hydrolase is synthesized by Jin Kai Rui Biological Technology Co., Ltd. (codon optimization is performed according to the need with Escherichia coli as the host), and the synthesized gene is connected to various expression vectors to be constructed. The expression vector is various vectors in the prior art. The vector in the present application is specifically pET-22b, the DNA small fragment between the enzyme digestion recognition sites NcoI and XhoI of pET-22b is replaced by the coding gene of the related enzyme after the whole gene synthesis, to obtain a recombinant expression vector, including: pET-22b-Re-LEH, pET-22b-CH55-LEH, pET-22b-E996, pET-22b-E997, pET-22b-E998, pET-22b-E999, pET-22b-E1000, pET-22b-E1026, pET-22b-E1027, pET-22b-E1028, pET-22b-E1029, pET-22b-E1030, pET-22b-E1031, a total of 13.
[0046] The above-mentioned 13 recombinant expression vectors are transformed into Escherichia coli competent cells, and the Escherichia coli is cultured and preserved to obtain the genetically engineered strain of the present application.
[0047] Example 3, expression of the epoxy hydrolase and preparation of the whole cell lysate and crude enzyme
[0048] 1. The recombinant expression vector constructed in Example 1 or the recombinant expression plasmid constructed in Example 2 is transformed into Escherichia coli BL21 (DE3) competent cells to obtain a recombinant cell.
[0049] 2. The transformants were inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37°C, 220 rpm for 12 h. Then, the culture was inoculated into TB liquid medium containing 50 μg / mL kanamycin at a volume percentage of 1%, and incubated at 37°C until the OD 600nm When the OD was 0.6-0.8, IPTG was added to a final concentration of 0.1 mmol / L, and the expression was induced at 20°C, 220 rpm for 12 h. Then, the precipitated bacteria (i.e., whole cells) were collected by centrifugation at 4°C, 4000 rpm for 10 min, and resuspended in phosphate buffer (50 mmol / L, pH 7.4) to obtain a bacterial suspension. Then, the bacterial cells were broken by ultrasonic treatment under ice bath conditions to obtain a broken sample (i.e., crude enzyme solution).
[0050] 3. The crude enzyme solution was centrifuged at 4°C, 8000 rpm for 30 min, and the supernatant was collected. The obtained supernatant was frozen at -80°C and then freeze-dried by a vacuum freeze dryer to obtain crude enzyme powder.
[0051] Example 4. Catalysis of 2-phenyloxetane to (S)-1-phenyl-1,3-propanediol by the epoxide hydrolase
[0052] The crude enzyme powder of the epoxide hydrolase prepared in Example 3 was used to catalyze 2-phenyloxetane to (S)-1-phenyl-1,3-propanediol.
[0053] The reaction system was: crude enzyme powder of the epoxide hydrolase (0.1 g / mL), substrate 10 mM, PBK buffer (50 mM, pH 7.4), and the total volume was 5 mL. After the reaction system was reacted at 30°C, 1000 rpm for 24 h, the product was detected by gas chromatography and liquid chromatography.
[0054] Detection conditions: GC method: (column BGB-176, 25 m x 0.25 mm ID, temperature program: 90°C, hold for 0.1 min; 16°C / min, 135°C, 20°C / min, 220°C, hold for 2 min, carrier: N2); HPLC method: (chiral liquid chromatography column OD-H; Hexane: IPA = 90:10; flow rate V = 1 mL / min; wavelength: λ = 220 nm.
[0055] The conversion rate was calculated and the results are shown in Table 1. The results show that the c% of the epoxy hydrolase AKK23712.1 derived from metagenomic DNA, the epoxy hydrolase Q9ZAG3.3 derived from Rhodococcus erythropolis, the epoxy hydrolase MBB4640159.1 derived from Rhizorhapis suberifaciens, the epoxy hydrolase BAH52894.1 derived from Rhodococcus opacus, the epoxy hydrolase MBA8954773.1 derived from Actinomadura namibiensis reached 40% or more, and in particular, the epoxy hydrolase (CH55-LEH) AKK23712.1 derived from metagenomic DNA and the epoxy hydrolase (E1000) MBB4640159.1 derived from Rhizorhapis suberifaciens can efficiently catalyze 2-phenyloxirane to generate (S)-1-phenyl-1,3-propanediol.
[0056] Table 1. Results of epoxy hydrolase catalyzed hydrolysis of 2-phenyloxirane
[0057] Code Source c％ e.r GenBank Re-LEH Rhodococcus erythropolis 40 45：55 Q9ZAG3.3 CH55-LEH metagenomic DNA 50 86：14 AKK23712.1 E996 Mycolicibacterium fortuitum 31 50：50 SUA03756.1 E997 Rhodococcus opacus 52 50：50 BAH52894.1 E998 Actinomadura namibiensis 40 50：50 MBA8954773.1 E999 Rhodococcus aetherivorans _ _ MBA8954773.1 E1000 Rhizorhapis suberifaciens 40 85：15 MBB4640159.1 E1026 Rhodococcus imtechensis RKJ300 34 50：50 EID76986.1 E1027 Williamsia limnetica 38 50：50 PYE12450.1 E1028 Pseudonocardia sp. Ae707_Ps1 37 33：67 OLM08926.1 E1029 Actinomycetia bacterium - _ MSY70684.1 E1030 Sphingobium czechense LL01 16 50：50 KMS57740.1 E1031 Rhodococcus kyotonensis 28 50：50 OAK56221.1
[0058] Note: Conversion rate = A1 / A2 x 100%; A1: peak area value of sample 2-phenyloxirane obtained by gas chromatography analysis; A2: peak area value of (S)-1-phenyl-1,3-propanediol of standard obtained by liquid chromatography analysis.
Claims
1. A method for the hydrolysis of oxacyclobutane catalyzed by an epoxide hydrolase, characterized in that, Using metagenomics ( Metagenomic DNA Epoxyhydrolase from Rhodococcus erythropolis, derived from Rhizorhapis suberifaciens. Rhodococcus opacus or Actinomadura namibiensis Using epoxide hydrolase as a catalytic enzyme, 2-phenyloxetane is used as a substrate to generate ( S )-1-Pheny-1,3-propanediol.
2. The method as described in claim 1, characterized in that, The source is metagenomics ( Metagenomic DNA The epoxide hydrolase is AKK23712.1; the epoxide hydrolase derived from *Rhodococcus erythropolis* is Q9ZAG3.3; the epoxide hydrolase derived from *Rhizorhapis suberifaciens* is MBB4640159.1; the source... Rhodococcus opacus The epoxide hydrolase is BAH52894.1; the source is... Actinomadura namibiensis The epoxide hydrolase is MBA8954773.
1.
3. The method as described in claim 1, characterized in that, The catalytic enzyme participates in the reaction in the form of whole cells of recombinant bacteria expressing the epoxidase, or in the form of a prepared crude enzyme solution, or in the form of a pure enzyme.
4. The method as described in claim 3, characterized in that, The recombinant bacteria is *Escherichia coli* (Escherichia coli). Escherichia coli ).
5. The method as described in claim 3, characterized in that, The reaction system is as follows: the amount of enzyme used is 0.05-0.2 g / ml; the substrate concentration is 5-15 mM, specifically 8-12 mM, more specifically 10 mM; the pH of the reaction system is 7-8, for example 7.2-7.6, specifically 7.
4.
6. The method as described in claim 5, characterized in that, The amount of whole cells used as catalyst is 0.05-0.2 g / ml, specifically 0.1 g / ml.
7. The method as described in claim 1, characterized in that, The reaction conditions are: 25-35℃, preferably 30℃, 800-1200 rpm, preferably 1000 rpm.
8. The application of epoxide hydrolase in the catalytic hydrolysis of oxetane, characterized in that, The epoxide hydrolase is derived from metagenomics ( Metagenomic DNA ), Rhodococcus rubrum ( Rhodococcus erythropolis ), Rhodococcus opacus or Actinomadura namibiensis or rhizobium ( Rhizorhapis suberifaciens Epoxyhydrolase is used as the catalytic enzyme.
9. The application as described in claim 8, characterized in that, It is generated using 2-phenyloxetane as a substrate ( S )-1-phenyl-1,3-propanediol or ( R )-2-phenyloxetane.
10. The method as described in claim 8, characterized in that, The source is metagenomics ( Metagenomic DNA The epoxide hydrolase is AKK23712.1; the one derived from Rhodococcus rubrum ( Rhodococcus erythropolis The epoxide hydrolase is Q9ZAG3.3; the one derived from rhizobium ( Rhizorhapis suberifaciens The epoxide hydrolase in the sample is MBB4640159.1; the source is... Rhodococcus opacus The epoxide hydrolase is BAH52894.1; the source is... Actinomadura namibiensis The epoxide hydrolase is MBA8954773.1.