Microbacterium and application thereof in catalyzing 4-HBC to produce 1, 4-HBC
By screening out the highly efficient Microbacterium SYP-GBC-2025 strain, optimizing the fermentation medium and co-solvent, the problems of low 4-HBC yield and environmental pollution in the existing technology were solved, and efficient and low-cost 1,4-HBC production was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-04-03
AI Technical Summary
In existing technologies, the yield of 1,4-HBC produced by Mycobacterium strains catalyzing 4-HBC is low, and traditional chemical synthesis suffers from harsh reaction conditions and serious environmental pollution.
A strain of Microbacterium sp., SYP-GBC-2025, was screened out. This strain has high dehydrogenation activity and can catalyze the dehydrogenation of 4-HBC to synthesize 1,4-HBC under mild reaction conditions. The fermentation medium parameters were optimized to improve the conversion efficiency by using inexpensive culture medium raw materials and environmentally friendly cosolvent ethyl acetate.
It achieves efficient production of 1,4-HBC with a yield of up to 53.53%, under mild reaction conditions with no heavy metal pollution, which is in line with the trend of green chemical development and reduces production costs.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a strain of microbacterium and its application in the catalytic production of 1,4-HBC from 4-HBC. Background Technology
[0002] 1,4-Dien-21-hydroxy-20-methylpregn-3-one (1,4-HBC) is an important intermediate in the synthesis of steroid hormone drugs. Modification of the double bonds at the C1 and C2 positions in its molecular structure can significantly enhance its anti-inflammatory and antitumor biological activities, making it a key precursor for the preparation of glucocorticoids such as prednisone and dexamethasone. Traditional chemical synthesis of 1,4-HBC requires heavy metal catalysts (such as Pd / C), which presents problems such as harsh reaction conditions (high temperature and high pressure), severe environmental pollution, and numerous byproducts (such as the C6 and C7 double bond isomer).
[0003] Microbial transformation technology, with its high regioselectivity and stereoselectivity, has become the preferred method for steroid dehydrogenation reactions. In existing technologies, Mycobacterium strains, due to their content of 3-ketosteroid-Δ... 1 1,4-HBC dehydrogenases catalyze the dehydrogenation of steroids at the C1,2 positions, but their conversion to 4-HBC still suffers from low yields, with wild-type strains generally achieving yields below 10%. Therefore, screening for highly efficient dehydrogenating strains is a core requirement for the industrial production of 1,4-HBC. Summary of the Invention
[0004] This invention provides a strain of microbacterium and its application in the catalytic production of 1,4-HBC from 4-HBC.
[0005] Specifically, the present invention provides the following technical solutions.
[0006] In a first aspect, the present invention provides microbacteria ( Microbacterium sp. The strain SYP-GBC-2025 was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and is classified as Microbacterium. Microbacterium sp. The accession number is CGMCC No. 34395.
[0007] This invention obtains a strain of microbacterium (Bacillus) through screening. Microbacterium sp. This strain exhibits high dehydrogenation activity, enabling it to efficiently and specifically catalyze the dehydrogenation of 21-hydroxy-20-methylpregn-4-en-3-one (4-HBC) to synthesize 1,4-HBC, achieving a high 1,4-HBC yield. Furthermore, it can utilize inexpensive culture medium raw materials, which helps reduce production costs.
[0008] Secondly, the present invention provides a microbial preparation comprising the aforementioned microbacteria ( Microbacterium sp. SYP-GBC-2025.
[0009] Preferably, in the microbial preparation, microbacteria ( Microbacterium sp. SYP-GBC-2025 exists in live bacterial form.
[0010] The microbial preparations described above may be solid or liquid formulations. Except for those containing microbes (… Microbacterium sp. In addition to SYP-GBC-2025, the microbial preparation may also contain excipients permitted in the field of microbial preparations, including but not limited to carriers (such as wheat bran) and cryoprotectants (such as sugars, polyols).
[0011] Thirdly, the present invention provides a method for preparing the above-described microbial preparation, the method comprising processing the microbacteria ( Microbacterium sp. The steps for culturing SYP-GBC-2025 are as follows: the culture temperature is preferably 25-30℃.
[0012] Fourthly, the present invention provides the aforementioned microbacteria ( Microbacterium sp. The use of SYP-GBC-2025 or the aforementioned microbial preparation in the preparation of 1,4-HBC.
[0013] Preferably, the 1,4-HBC is prepared using 4-HBC as a substrate.
[0014] Preferably, the application includes: using 4-HBC as a substrate, utilizing the microbacteria ( Microbacterium sp. SYP-GBC-2025 or the microbial preparation catalyzes the dehydrogenation of 4-HBC to synthesize 1,4-HBC.
[0015] Fifthly, the present invention provides the aforementioned microbacteria ( Microbacterium sp. The use of SYP-GBC-2025 or the aforementioned microbial preparation in the preparation of fermentation agents for the synthesis of 1,4-HBC or in the construction of production strains of 1,4-HBC.
[0016] Preferably, the fermentation agent contains the microbacteria ( Microbacterium sp. SYP-GBC-2025.
[0017] Preferably, the production strain for constructing 1,4-HBC is the microbacterium ( Microbacterium sp. SYP-GBC-2025 is the starting strain. A production strain of 1,4-HBC is constructed by gene editing (e.g., CRISPR-Cas9 gene editing) on the starting strain.
[0018] Sixthly, the present invention provides the microbacteria ( Microbacterium sp. The application of SYP-GBC-2025 or the aforementioned microbial preparation in catalyzing the C1,2-position dehydrogenation reaction of steroidal compounds.
[0019] Preferably, the steroid compound is 4-HBC.
[0020] In a seventh aspect, the present invention provides a method for preparing 1,4-HBC, the method comprising: using 4-HBC as a substrate, and utilizing the microbacterium ( Microbacterium sp. SYP-GBC-2025 or the aforementioned microbial preparation converts 4-HBC to 1,4-HBC.
[0021] Preferably, the concentration of 4-HBC in the conversion system is 1-50 g / L. More preferably, it is 1-30 g / L.
[0022] Preferably, the method includes: placing the microbacteria ( Microbacterium sp. SYP-GBC-2025 or the microbial preparation described herein may be inoculated into a fermentation medium containing 4-HBC for biotransformation.
[0023] Preferably, the fermentation medium used for the conversion includes a carbon source, a nitrogen source, and inorganic salts.
[0024] The carbon source is preferably maltose. The nitrogen source is preferably sodium nitrate. The inorganic salt preferably includes KH₂PO₄ and / or MgSO₄.
[0025] This invention targets microbacteria ( Microbacterium sp. SYP-GBC-2025 underwent fermentation medium optimization, revealing that using maltose as the carbon source and sodium nitrate as the nitrogen source was more effective in improving the conversion efficiency of 4-HBC to 1,4-HBC, thereby increasing the yield of 1,4-HBC. Systematic optimization of key parameters such as carbon and nitrogen sources, pH, and substrate concentration further improves yield and batch stability.
[0026] Because 4-HBC has poor water solubility (<0.1 g / L), a solvent is needed to aid dissolution. However, traditional solvents (such as DMSO) are highly toxic to the bacterial cells, thus limiting the transformation efficiency. This invention found that dissolving 4-HBC in ethyl acetate and then adding it to the transformation system does not produce toxic effects on the bacterial strain and has minimal impact on the transformation efficiency.
[0027] Preferably, the 4-HBC is dissolved in ethyl acetate.
[0028] Preferably, 4-HBC is prepared as a 1-3% solution using ethyl acetate and added to the conversion system.
[0029] Preferably, the fermentation medium comprises the following components: maltose 25-35 g / L, sodium nitrate 15-25 g / L, KH₂PO₄ 1-2 g / L, and MgSO₄·7H₂O 0.2-0.8 g / L. The pH of the fermentation medium is preferably 7.0-8.0.
[0030] Preferably, the conversion temperature is 25-30℃ (more preferably 28-30℃). The pH of the bioconversion is 7.0-8.0. The bioconversion rotation speed is preferably 180-230 rpm. The bioconversion time is 100-180 h (more preferably 120-168 h).
[0031] Preferably, the conversion rate is 5%-15%. More preferably, it is 8%-12%.
[0032] The seed culture medium used for preparing the seed solution for transplantation comprises the following components: 2-8 g / L peptone, 1-5 g / L beef extract, and 2-8 g / L NaCl. The preferred pH is 6.8-7.5.
[0033] Preferably, the temperature for seed culture is 25-30℃.
[0034] The above method further includes, after the conversion is completed, the steps of extracting the obtained conversion solution with dichloromethane and semi-preparative HPLC purification of 1,4-HBC.
[0035] In some embodiments of the present invention, the method for preparing 1,4-HBC includes the following steps: (1) Activation of bacterial strains: Microbacteria ( Microbacterium sp. SYP-GBC-2025 was inoculated into NA medium and cultured at 28-30℃ for 5-7 days; (2) Seed culture: The activated strain was inoculated into seed culture medium and cultured at 28-30℃ and 180-230 rpm for 40-50 h until the dry weight of the cells was 7-9 g / L. (3) Fermentation and conversion: Inoculate the seed liquid into the fermentation medium at a conversion rate of 8%-12%, add 4-HBC (final concentration 1-5 g / L) with 1%-3% ethyl acetate as a solvent, and convert at 28-30℃ and 200-230 rpm for 120-168 h; (4) Product purification: The conversion solution was extracted with dichloromethane and purified by semi-preparative HPLC. The elution peak with a retention time of 12.6 min was collected to obtain pure 1,4-HBC.
[0036] Preferably, in step (4), the conversion liquid is separated by centrifugation, the precipitate is extracted with dichloromethane, the organic phase is concentrated by rotary evaporation, and then purified by semi-preparative HPLC.
[0037] Preferably, the semi-preparative HPLC purification conditions are as follows: chromatographic column: SinoChrom ODS-BP 5 μm×250mm×10 mm; mobile phase: acetonitrile-water = 60:40; flow rate: 3.5 mL / min; detection wavelength: 254 nm.
[0038] Preferably, after purification, lyophilization yields a white powder of 1,4-HBC with a purity ≥95%.
[0039] The beneficial effects of this invention include at least the following: This invention provides a strain of microbacterium ( Microbacterium sp. This strain can catalyze the dehydrogenation of 4-HBC (21-hydroxy-20-methylpregn-4-en-3-one) to produce 1,4-HBC (1,4-diene-21-hydroxy-20-methylpregn-3-one), and has the following advantages: (1) High strain specificity: It specifically catalyzes the dehydrogenation of 4-HBC at the C1,2 position, and the resulting product 1,4-HBC has high purity, which solves the problem of double bond position isomerism in chemical synthesis; (2) High conversion efficiency: It can efficiently catalyze the dehydrogenation of 4-HBC to synthesize 1,4-HBC with a yield of up to 53.53%; (3) Low production cost: It can use inexpensive raw materials such as maltose and sodium nitrate, and can use ethyl acetate, a co-solvent that can be recycled (recovery rate >80%). (4) Environmentally friendly: The reaction conditions are mild (normal temperature and pressure), with no heavy metal pollution, which is in line with the trend of green chemical development.
[0040] Based on this strain, this invention provides a method for producing 1,4-HBC, which has the advantages of high efficiency, specificity and low cost, and can be used for industrial production of 1,4-HBC, providing key technical support for the green synthesis of steroidal drug intermediates. Attached Figure Description
[0041] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0042] Figure 1 The microbacteria in Example 1 of this invention ( Microbacterium sp. The colony characteristics of SYP-GBC-2025 are shown in the image. The left image shows the result on day 5 of culture, and the right image shows the result on day 7 of culture.
[0043] Figure 2 The microbacteria in Example 1 of this invention (C Microscopic images of SYP-GBC-2025. The left image shows the preparation of the microbacterium SYP-GBC-2025 specimen using acid-fast staining, where the bacteria are stained red under the microscope. The right image shows a partial field of view.
[0044] Microbacterium sp. The image shows the HPLC chromatogram of 1,4-HBC in Example 4 of this invention. After purification of the conversion solution, the retention time of the target product was 12.6 min.
[0045] Figure 3 This is the ESI-MS spectrum of the conversion product in Example 4 of the present invention.
[0046] Figure 4 For example, 1,4-HBC in Embodiment 4 of the present invention 1 H-NMR spectrum (CDCl3, 400 MHz, δH 7.05, 6.22 are double bond proton signals).
[0047] Figure 5 For example, 1,4-HBC in Embodiment 4 of the present invention 13 C-NMR spectrum (100 MHz, CDCl3). Detailed Implementation
[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0049] In the following examples, the formula for calculating the 1,4-HBC yield is as follows: .
[0050] Example 1: Acquisition and Preservation of the Strains A bacterial strain was isolated from a soil sample and identified by 16S rRNA sequencing. Its 16S rRNA sequence was similar to that of *Microbacterium* (*Microbacterium*). Figure 6 The homology with the bacteria reached 99.8%, and it was identified as a microbacterium ( ). Microbacterium sp. The strain was named SYP-GBC-2025.
[0051] Microbes ( Microbacterium sp.SYP-GBC-2025 was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Microbacterium. Microbacterium sp. The accession number is CGMCC No. 34395.
[0052] Microbes SYP-GBC-2025 were cultured on NA medium for 7 days. The center of the colonies turned orange and deepened in color compared to before, while the edges remained irregular and pale yellow. Microbacterium sp. As shown.
[0053] A specimen of Microbe bacillus SYP-GBC-2025 was prepared using acid-fast staining. Under a microscope, the bacterial cells were observed to be stained red. Figure 1 Microbes appear red, while other bacteria appear blue. Figure 2 Almost all the bacteria in the sample are red, therefore it is a microbacterium.
[0054] Furthermore, it has been verified that Microbacterium SYP-GBC-2025 can specifically catalyze the dehydrogenation of 4-HBC at the C1,2 position to generate 1,4-HBC, with a byproduct content of <3%.
[0055] Example 2: Optimization of conditions for the production of 1,4-HBC using Microbes SYP-GBC-2025 This embodiment optimized the fermentation medium for Microbacterium SYP-GBC-2025. The optimal conditions were determined through single-factor experiments and orthogonal experiments, as detailed below.
[0056] 1. Carbon source screening: Adding 30 g / L of different carbon sources to the basal culture medium showed that maltose (30 g / L) increased the yield by 32.5% compared with glucose and sucrose. Therefore, maltose was determined to be the optimal carbon source.
[0057] 2. Nitrogen source screening: Sodium nitrate (20 g / L) reduced by-products by 18.3% compared with ammonium sulfate and yeast extract, therefore sodium nitrate was determined to be the optimal nitrogen source.
[0058] 3. Orthogonal Experiment (Table 1): Through L9(3 4 The optimal combination was determined by orthogonal experiment: maltose 30 g / L, sodium nitrate 20 g / L, pH 7.5, and inoculum amount 10%; where A, B, and C represent maltose concentration, sodium nitrate concentration, and initial pH of the culture medium, respectively.
[0059] Table 1 L9(3) 4 Orthogonal experimental design and results
[0060] Based on the data presented in Table 1, the influence of the three factors is A > B > C, meaning that maltose concentration has the greatest effect, followed by sodium nitrate concentration, while the initial pH of the culture medium has the least impact. Range analysis shows that the optimal combination of these three factors is A2B2C1, which means setting the maltose concentration to 30 g / L, the sodium nitrate concentration to 20 g / L, and the initial pH of the fermentation medium to 7.5.
[0061] In addition, the present invention screened the cosolvents for 4-HBC and finally determined that ethyl acetate was used to dissolve 4-HBC to prepare a 2% solution, which was then added to the transformation system until the final concentration of 4-HBC was 2.0 g / L, at which point the cell survival rate was >92%.
[0062] The fermentation medium for Microbeobacterium SYP-GBC-2025 was prepared according to the optimal combination mentioned above (maltose 30 g / L, sodium nitrate 20 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L, initial pH 7.5). The transformation conditions were: 28℃, 220 rpm shaking, initial pH 7.5, 10% inoculum, and transformation time 144 h. Three parallel verification experiments were conducted, yielding transformation yields of 46.39%, 48.27%, and 52.76%, respectively. The results showed that the transformation yield of 1,4-HBC reached 49.14%, an increase of 39.56% compared to the initial transformation yield of 9.71%. The initial conversion yield was the yield obtained by fermenting *Microbes SYP-GBC-2025* in an unoptimized fermentation medium, which had the following composition: sucrose: 30.00 g / L, yeast extract: 10.00 g / L, corn steep liquor powder: 10.00 g / L, potassium dihydrogen phosphate (K2HPO4): 2.00 g / L, potassium monohydrogen phosphate (KH2PO4): 1.60 g / L, magnesium sulfate (MgSO4·7H2O): 0.50 g / L, ferrous sulfate (FeSO4·7H2O): 0.05 g / L, pH 6.5.
[0063] Example 3: Production of 1,4-HBC using Microbes SYP-GBC-2025 This embodiment provides a method for producing 1,4-HBC using Microbes SYP-GBC-2025, the method comprising the following steps: (1) Activation of bacterial strain: Microbes SYP-GBC-2025 were streaked on NA medium (5 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 20 g / L agar, pH 7.0) and incubated at 28℃ for 6 days until the colony diameter was 3-4 cm. (2) Seed culture: single colonies were picked and inoculated into 50 mL of seed culture medium (NA liquid medium) and cultured at 28℃ and 200 rpm for 48 h until the dry weight of the cells reached 8.2 g / L; (3) Fermentation conversion: The seed culture was inoculated into the fermentation medium at a conversion rate of 10% (v / v), and 4-HBC was added with 2% ethyl acetate as a dissolving agent (the final concentration of 4-HBC in the conversion system was 2.0 g / L). The conversion was carried out at 28℃ and 220 rpm for 144 h with shaking. The fermentation medium formula was as follows: maltose 30 g / L, sodium nitrate 20 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L, and initial pH 7.5. (4) Product purification: The conversion solution was centrifuged at 5000 rpm for 10 min, and the precipitate was extracted three times with dichloromethane (equal volume each time). The organic phases were combined and concentrated by rotary evaporation. Semi-preparative HPLC purification was performed (chromatographic column: SinoChrom ODS-BP 5 μm×250 mm×10 mm; mobile phase acetonitrile-water=60:40; flow rate 3.5 mL / min; detection wavelength 254 nm). The elution peak with a retention time of 12.6 min was collected and lyophilized to obtain a white powder of 1,4-HBC with a purity ≥95%.
[0064] The yield of 1,4-HBC was found to be 53.53%.
[0065] Example 4: Identification of the structure of products from biotransformation The structure of the biotransformation product prepared using the method of Example 3 was identified as follows.
[0066] 1. HPLC analysis: The retention time of 1,4-HBC in the conversion solution was 12.6 min ( Figure 2 The results were consistent with those of the 1,4-HBC standard. The HPLC detection conditions are shown in Table 2.
[0067] Table 2
[0068] 2. MS identification ( Figure 3 ): ESI-MS m / z [M+Na) + =351.24, molecular weight 328.24, conforms to 1,4-HBC (C 22 H32 The theoretical value of O2.
[0069] 3. NMR identification: 1 1H NMR (400 MHz, CDCl3) spectrum Figure 4 The values given are: δH 0.72 (3H, s, H-18), 1.22 (3H, s, H-19), 1.03 (3H, d, J = 6.8 Hz, H-22), 3.62 (1H, dd, J = 10.5, 3.3Hz, H-21), 3.35 (1H, dd, J = 10.5, 6.8 Hz, H-21), which conform to the 4-HBC characteristic signal. 1 H NMR (CDCl3, 400 MHz) showed characteristic signals δH 7.05 (d, J=10.1 Hz, H-1) and 6.22 (dd, J=10.1, 1.9 Hz, H-2), proving the formation of a double bond at C1,2. 13 C NMR (100 MHz, CDCl3) spectrum ( Figure 5 Figure 6 The CCP provided 22 carbon signals. Comparing these with the HSQC spectrum, it was deduced that the substance contains 3 methyl groups (δC 12.2, 16.8, 18.8), 7 methylene groups (δC 67.9, 39.4, 33.7, 32.8, 27.7, 24.6, 22.9), 5 tertiary carbons (δC 87.0, 77.3, 46.6, 38.5, 38.6), 2 quaternary carbons (δC 43.7, 38.8), 1 carbonyl carbon (δC 186.5), and 4 double-bonded carbons (δC 169.5, 156.1, 127.5, 123.9).
[0070] The microbacterium SYP-GBC-2025 provided by this invention can achieve efficient and specific biotransformation of 1,4-HBC. After condition optimization, the yield of 1,4-HBC reaches 53.53%, providing a feasible solution for the industrial production of steroidal drug intermediates. This strain and method have broad application prospects and can be extended to the dehydrogenation reaction of other steroidal compounds.
[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. Microbes ( Microbacterium sp. SYP-GBC-2025, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34395.
2. A microbial preparation, characterized in that, The microbial preparation comprises the microbacteria described in claim 1 ( Microbacterium sp. SYP-GBC-2025.
3. The microbacteria described in claim 1 ( Microbacterium sp. The application of the microbial preparation described in SYP-GBC-2025 or claim 2 in the preparation of 1,4-HBC; Preferably, the 1,4-HBC is prepared using 4-HBC as a substrate.
4. The microbacteria described in claim 1 ( Microbacterium sp. The use of the microbial preparation described in SYP-GBC-2025 or claim 2 in the preparation of fermentation agents for the synthesis of 1,4-HBC or in the construction of production strains of 1,4-HBC.
5. The microbacteria described in claim 1 ( Microbacterium sp. The application of the microbial preparation described in SYP-GBC-2025 or claim 2 in catalyzing the dehydrogenation reaction at the C1,2 position of steroidal compounds.
6. A method for preparing 1,4-HBC, characterized in that, The method includes: using 4-HBC as a substrate, and utilizing the microbacterium described in claim 1 (…). Microbacterium sp. The microbial preparation described in SYP-GBC-2025 or claim 2 converts 4-HBC into 1,4-HBC.
7. The method according to claim 6, characterized in that, In the conversion system, the concentration of 4-HBC is 1-50 g / L; Preferably, the 4-HBC is dissolved in ethyl acetate.
8. The method according to claim 6 or 7, characterized in that, The fermentation medium used for the conversion includes a carbon source, a nitrogen source, and inorganic salts; Preferably, the carbon source is maltose, and / or the nitrogen source is sodium nitrate.
9. The method according to claim 8, characterized in that, The fermentation medium comprises the following components: maltose 25-35 g / L, sodium nitrate 15-25 g / L, KH2PO4 1-2 g / L, and MgSO4·7H2O 0.2-0.8 g / L.
10. The method according to any one of claims 6 to 9, characterized in that, The conversion temperature is 25-30℃; and / or, the pH is 7.0-8.0; and / or, the conversion amount is 5%-15%.