Pseudomonas mendocina and application thereof in catalyzing (+ / -)-alpha-pinene to prepare optically pure trans-(+)-pinyl hydrate
By using marine Pseudomonas mendocina S761 to catalyze (±)-α-pinene, the low yield and high cost problems of optically pure trans-(+)-hydrated pinanol in the existing technology are solved, and efficient and low-cost optical purity preparation is achieved, which is suitable for the fields of medicine and fine chemicals.
Patent Information
- Application Number
- CN202510797171.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-09-09
AI Technical Summary
The existing methods for preparing optically pure trans-(+)-pinenol hydrate suffer from low yield, high cost, and insufficient optical purity, and lack stereoselective catalytic ability for (+)-α-pinene and (-)-α-pinene.
Marine-derived Pseudomonas mendocina S761 was used to catalyze the production of (±)-α-pinene using whole cells or crude enzyme solution. The specific steps included culturing the cells in marine LB medium to the logarithmic phase, resuspending the cells in PBS buffer, and reacting them at 30°C for 96 hours. Finally, the product was extracted using acetonitrile-sodium chloride aqueous two-phase extraction, and the reaction conditions such as substrate ratio and pH value were optimized to improve selectivity.
The efficient preparation of trans-(+)-pinenol hydrate with high optical purity was achieved, with a yield of 282.68 mg/L and a conversion rate of 72.25%. Only a single enantiomer was generated. It has the advantages of high product purity, low cost and mild reaction conditions, and is suitable for the fields of medicine and fine chemicals.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biocatalysis, in particular to Pseudomonas mendocina and application thereof in catalyzing (±)-α-pinene to prepare optically pure trans-(+)-pinenol hydrate. Background Art
[0002] Trans-sobrerol, also known as trans-sobrerol, belongs to the terpene diol class of compounds. It is a mucolytic drug with expectorant and antiasthmatic effects. It is mainly used to treat diseases such as bronchitis and asthma. Recent studies have found that this compound has the pharmacological effect of inducing tumor cell apoptosis and is expected to become a new anti-cancer drug. In addition, this compound can be used as an important chiral source reagent in organic synthesis.
[0003] Optically pure trans-sobrerol is an important pharmaceutical and fine chemical intermediate, but existing preparation methods, such as plant extraction, chemical synthesis, or lipase resolution, suffer from low yields, high costs, or insufficient optical purity. Existing reports on microbial catalysis of (±)-α-pinene mostly produce mixtures (such as verbenol and terpineol), and lack stereoselective catalytic ability for (+)-α-pinene and (-)-α-pinene.
[0004] Based on this, the present application proposes a Pseudomonas mendocina and its application in catalyzing the preparation of optically pure trans-(+)-pinene hydrate from (±)-α-pinene to solve the above problems. Summary of the Invention
[0005] (1) Technical problems solved
[0006] In response to the deficiencies in the prior art, the present invention provides a Pseudomonas mendocinae bacterium and its use in catalyzing the preparation of optically pure trans-(+)-sobrerol from (±)-α-pinene. The Pseudomonas mendocinae bacterium is screened and obtained from a marine environment. It can selectively catalyze (+)-α-pinene to produce highly optically pure trans-(+)-sobrerol, with a product ee value of >99%. It also provides a "one-pot" biocatalytic system that directly uses inexpensive (±)-α-pinene as a substrate, avoiding the complex process of traditional multi-step synthesis.
[0007] (2) Technical solution
[0008] To achieve the above object, the present invention provides the following technical solution: a Pseudomonas mendocina strain, the strain is named Pseudomonas mendocina S761, and the deposit number of the strain is CGMCC NO: 24715.
[0009] The strain can be used to catalyze (±)-α-pinene to prepare optically pure trans-(+)-pinenol hydrate.
[0010] Specifically, the whole cells or crude enzyme solution of the above strain is used to catalyze (±)-α-pinene, and the specific steps are as follows:
[0011] S1 and strain S761 were cultured in marine LB medium to the logarithmic phase;
[0012] S2, collect resting cells and resuspend them in PBS buffer;
[0013] S3, adding (±)-α-pinene, reacting at 30°C for 96 hours;
[0014] S4, acetonitrile-sodium chloride aqueous two-phase extraction product.
[0015] Preferably, the substrate ratio: (±)-α-pinene: cells (OD600) is ≤0.5%, the pH value of the PBS buffer is 6.0-8.0, and the reaction temperature is 20-35°C.
[0016] Preferably, the substrate ratio is (±)-α-pinene: cells (OD600) = 1:4, the pH value of the PBS buffer is 7.4, and the reaction temperature is 30°C.
[0017] (3) Beneficial effects
[0018] Compared with the prior art, the present invention provides a xxxxx, which has the following beneficial effects:
[0019] The marine-sourced Pseudomonas mendocinae S761 provided by the present invention can efficiently and selectively catalyze (±)-α-pinene to produce optically pure trans-(+)-pinenol hydrate, with a trans-(+)-sobrerol yield of 282.68 mg / L (conversion rate of 72.25%), and only produces trans-(+)-sobrerol (without the (-)-enantiomer). It has strong tolerance to the toxicity of α-pinene (concentration can reach 0.5%), and has the advantages of high product purity, mild reaction conditions, and low cost. In addition, the obtained trans-(+)-sobrerol can be purified and used for carvone synthesis with a yield of >90%, and is suitable for the fields of medicine and fine chemicals. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The colony morphology and micrograph of the strain S761 of the present invention are shown;
[0021] Figure 2 is the phylogenetic tree of strain S761 in the present invention;
[0022] Figure 3This is the GC-MS spectrum of (+)-α-pinene catalyzed by the strain S761 of the present invention;
[0023] Figure 4 Chiral GC analysis was performed to prove the optical purity of the product. DETAILED DESCRIPTION
[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0025] The present invention provides a Pseudomonas mendocina strain named Pseudomonas mendocina S761. The strain was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on April 19, 2022, and the strain deposit number is CGMCC NO: 24715.
[0026] The strain S761 was obtained by enrichment culture from the sediment of Sanniang Bay using (±)-α-pinene as the sole carbon source. Its morphological and physiological and biochemical characteristics are as follows: Figure 1 As shown, the strain was plated on LB agar plates, revealing pale yellow colonies with smooth edges. Gram staining confirmed its Gram-negative nature. Microscopic observation revealed rod-shaped, nonflagellated colonies. Physiological and biochemical tests revealed positive catalase and oxidase tests.
[0027] The growth experiment of strain S761 is shown in Table 1 below:
[0028] Table 1 Growth experiment of strain S761
[0029]
[0030]
[0031] The chemical sensitivity test of strain S761 is shown in Table 2 below:
[0032] Table 2 Chemical sensitivity test of strain S761
[0033] Phylogenetic tree of strain Pseudomonas mendocina S761 Figure 2 As shown, the genome of strain Pseudomonas mendocina S761 was sequenced:
[0034] The results of 16S rRNA gene sequencing are as follows:
[0035]
[0036] The results of gyrB gene sequencing are as follows:
[0037]
[0038] Genome sequencing revealed that it contained a unique oxygenase gene (NCBI accession number CP158721), which was associated with stereoselective epoxidation. The strain was identified as Pseudomonas mendocina by sequencing of the 16S rRNA and gyrB genes.
[0039] The above strain can be used to catalyze (±)-α-pinene to produce optically pure trans-(+)-pinenol hydrate. Specifically, whole cells or crude enzyme solution of the above strain can be used to catalyze (±)-α-pinene. The specific steps are as follows:
[0040] S1 and strain S761 were cultured in marine LB medium to the logarithmic phase;
[0041] S2, collect resting cells and resuspend them in PBS buffer;
[0042] S3, adding (±)-α-pinene, reacting at 30°C for 96 hours;
[0043] S4, acetonitrile-sodium chloride aqueous two-phase extraction product.
[0044] The substrate ratio (±)-α-pinene:cell (OD600) is ≤0.5%, the pH value of the PBS buffer is 6.0-8.0, and the reaction temperature is 20-35° C. Preferably, the substrate ratio (±)-α-pinene:cell (OD600) is 1:4, the pH value of the PBS buffer is 7.4, and the reaction temperature is 30° C.
[0045] Further investigation of the transformation of (-)-α-pinene and (+)-α-pinene by strain S761 was carried out. The structures of the products were determined by GC-MS and compared with the standards. The results are as follows: Figure 3 As shown in Figure 2, S761 can utilize (-)-α pinene for growth. However, when (-)-α pinene is used as a substrate, only a small amount of thujone is converted into the product. The main process is degradation, with a degradation rate of 30%. Figure 3 a. S761 can also utilize (+)-α-pinene for growth. The products transformed by resting cells using (+)-α-pinene as substrate include small amounts of thujone, pine carvone, and borneol. The main product is hydrated pinanol. Figure 3 b. These substances can all be obtained by the ring-opening isomerization of epoxide pinane, indicating that strain S761 first stereoselectively epoxidizes (±)-α-pinene to produce (+)-α-epoxide pinane, and then further produces other substances.
[0046] To further explore the cis-trans configuration and optical structure of the product hydrated pinol sobrerol, the chiral chromatographic column BetaDex120 gas chromatography was used to compare the standard tran-(±)-sobrerol and the product. The results are as follows Figure 4 As shown, the retention time 13.945 is tran-(-)-sobrerol, and the retention time 14.107 is tran-(+)-sobrerol, see Figure 4 a. By Figure 4 b It can be seen that the product of (±)-α-pinene catalyzed by Pseudomonas mendocina S761 is a single peak with a retention time of 14.107. After comparison with the standard, the substance was determined to be tran-(+)-sobrerol.
[0047] The marine-sourced Pseudomonas mendocinae S761 provided by the present invention can efficiently and selectively catalyze (±)-α-pinene to produce optically pure trans-(+)-pinenol hydrate, with a trans-(+)-sobrerol yield of 282.68 mg / L (conversion rate of 72.25%), and only produces trans-(+)-sobrerol (without the (-)-enantiomer). It has strong tolerance to the toxicity of α-pinene (concentration can reach 0.5%), and has the advantages of high product purity, mild reaction conditions, and low cost. In addition, the obtained trans-(+)-sobrerol can be purified and used for carvone synthesis with a yield of >90%, and is suitable for the fields of medicine and fine chemicals.
[0048] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A Pseudomonas mendocinae, characterized in that The strain was named as Pseudomonas mendocina S761, and the deposit number of the strain was CGMCC NO: 24715.
2. Use of the strain according to claim 1 in catalyzing the preparation of optically pure trans-(+)-pinenol hydrate from (±)-α-pinene.
3. A method for preparing trans-(+)-pinenol hydrate using the strain according to claim 1, characterized in that: The whole cells or crude enzyme solution of the strain according to claim 1 are used to catalyze (±)-α-pinene.
4. The method for preparing trans-(+)-pinenol hydrate according to claim 3, wherein The following steps are involved: S1 and strain S761 were cultured in marine LB medium to the logarithmic phase; S2, collect resting cells and resuspend them in PBS buffer; S3, adding (±)-α-pinene, reacting at 30°C for 96 hours; S4, acetonitrile-sodium chloride aqueous two-phase extraction product.
5. The method for preparing trans-(+)-pinenol hydrate according to claim 4, wherein The substrate ratio is (±)-α-pinene:cell (OD600) ≤ 0.5%, the pH value of the PBS buffer is 6.0-8.0, and the reaction temperature is 20-35°C.
6. The method for preparing trans-(+)-pinenol hydrate according to claim 5, characterized in that: The substrate ratio was (±)-α-pinene:cells (OD600) = 1:4, the pH value of the PBS buffer was 7.4, and the reaction temperature was 30°C.