Method for large-scale purification of porphyrin compound from fermentation liquor and application of porphyrin compound

Abstract

The invention provides a method for large-scale purification of a porphyrin compound from fermentation liquor and application of the porphyrin compound. The method provided by the invention can efficiently, conveniently and flexibly purify and prepare the porphyrin compound in a large scale from the complex microbial fermentation liquor.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering. More specifically, the present invention relates to a method for large-scale purification of porphyrin compounds from fermentation broth and its applications. Background Art

[0002] Porphyrins are a class of aromatic macrocyclic molecules with a large π-conjugated structure and have a wide range of applications in the fields of biomedicine, functional materials, catalysis, and analytical chemistry. Due to their large π-conjugated structure, porphyrins have unique fluorescence absorption and photoexcited state energy, making porphyrins usable as photosensitizers in cancer cell localization and photodynamic therapy. In addition, porphyrins are easy to modify and self-assemble, enabling efficient photon capture, energy transfer, charge separation, and electron transfer. Therefore, porphyrins are also important monomer molecules for the synthesis of optoelectronic devices.

[0003] Traditional porphyrin production methods include chemical synthesis (such as phthalocyanine and chlorophyll) and isolation from animal blood or plant tissues. However, low yields, by-product interference, complex synthesis processes, and limited resources restrict the large-scale production of porphyrin compounds. Therefore, in recent years, people have begun to focus on using synthetic biology to produce porphyrins with microorganisms as cell factories. However, there is currently a lack of downstream methods for separating and purifying coproporphyrin III from complex fermentation broth, which limits the application of porphyrins.

[0004] Traditional porphyrins are extracted from blood and animal tissues and then refined by crystallization. However, the fermentation system is extremely different from tissues or blood, and a purification method for porphyrins suitable for the components of fermentation broth needs to be developed. So far, the separation and purification of porphyrins in fermentation broth remain at the analytical level.

[0005] Therefore, developing a method for large-scale purification of porphyrins from fermentation broth is crucial for the large-scale production of porphyrin compounds and the expansion of the application market of porphyrins. Summary of the Invention

[0006] The object of the present invention is to provide a method for large-scale purification of porphyrin compounds from fermentation broth and its applications.

[0007] In the first aspect of the present invention, a method for purifying porphyrin compounds from biological fermentation broth is provided, including:

[0008] (1) Obtaining the supernatant of the fermentation broth containing porphyrin compounds;

[0009] (2) Adjusting the pH value of the supernatant of the fermentation broth in (1) to pH 8 - 10 (preferably pH 8.5 - 9.5, more preferably pH 8.3 - 9; more preferably pH 8.4 - 8.7 or pH 8.3 - 8.6); separating and removing the precipitate to obtain the supernatant; adding isopropanol to the supernatant, separating and removing the precipitate to obtain the supernatant;

[0010] (3) Add acidic acetone to the supernatant in (2) to adjust the pH to 1 - 3 (preferably 1.5 - 2.8, more preferably 2 - 2.6; more preferably 2.3 - 2.7 or 2.4 - 2.6), separate and remove the precipitate, and obtain the supernatant.

[0011] (4) Add acetate to the supernatant in (3) to adjust the pH to 3 - 5 (preferably 3.5 - 5, more preferably 4 - 5, such as pH 4.2, pH 4.5, pH 4.7, etc.), and obtain the crystalline product.

[0012] (5) Dissolve the crystalline product in (4) in an alkaline solution to obtain a solution; add acetate to the solution to adjust the pH to 3 - 5 (preferably 3.5 - 5, more preferably 4 - 5, such as pH 4.2, pH 4.5, pH 4.7, etc.), and obtain the recrystallized product, which is the purified porphyrin compound.

[0013] In one or more embodiments, in (1), cells (bacteria) or other solids (such as cell debris, flocs, particulate matter, etc.) in the fermentation broth are removed by centrifugation.

[0014] In one or more embodiments, in (2), the pH value is adjusted to pH 8 - 10 with an alkaline solution; preferably, the alkaline solution is an NaOH solution (or other alkali metal hydroxides, such as a KOH solution).

[0015] In one or more embodiments, in (2), after adjusting the pH value, the separation and removal of the precipitate includes: allowing the supernatant to stand (standing at room temperature), and then centrifuging to remove the precipitate; preferably standing for 30 ± 20 min, more preferably standing for 30 ± 10 min.

[0016] In one or more embodiments, in (2), the final concentration of isopropanol is 15 - 70% by volume; preferably 18 - 50%; more preferably 20 - 35% (such as 20% (i.e., isopropanol to supernatant in a volume ratio of 1:4), 25% or 30%, etc.).

[0017] In one or more embodiments, in (2), after adding isopropanol, mix well (such as by stirring), allow to stand, and then centrifuging to remove the precipitate; preferably standing for 120 ± 60 min, more preferably standing for 1200 ± 30 min.

[0018] In one or more embodiments, in (3), the volume ratio of acidic acetone to the supernatant is 4:1 - 1:1; preferably 2:1 to 1:1 (most preferably 1:1).

[0019] In one or more embodiments, in (3), after adding acidic acetone, mix well and centrifuge to obtain the supernatant.

[0020] In one or more embodiments, in (4), the acetate is sodium acetate.

[0021] In one or more embodiments, in (4), the crystallization is carried out at 1-8 °C, in the dark and with static placement; preferably at 2-6 °C (such as 3, 4, 5 °C); preferably, static placement is for 16 ± 8 hours (more preferably for 16 ± 4 hours, 16 ± 3 hours or 16 ± 2 hours).

[0022] In one or more embodiments, in (4), after forming crystals, the crystals are separated by centrifugation.

[0023] In one or more embodiments, in (5), the alkali solution is a NaOH solution (or other alkali metal hydroxides, such as a KOH solution); preferably, the alkali solution is a 0.1-0.6 M solution (more preferably 0.2-0.5 M or 0.3-0.4 M solution).

[0024] In one or more embodiments, in (5), after dissolving in the alkali solution, the obtained solution is centrifuged to remove the precipitate, and a supernatant solution is obtained.

[0025] In one or more embodiments, in (5), the recrystallization is carried out at 1-8 °C, in the dark and with static placement; preferably at 2-6 °C (such as 3, 4, 5 °C); preferably, static placement is for 30-80 minutes (more preferably for 35-60 minutes, 40-55 minutes or 45-50 minutes).

[0026] In one or more embodiments, in (5), after forming crystals, the crystals are separated by centrifugation.

[0027] In one or more embodiments, after obtaining crystals by crystallization or recrystallization and separating the crystals by centrifugation, it further includes washing the crystals, preferably by deionized water, such as washing 1-5 times.

[0028] In one or more embodiments, the centrifugation is carried out at 8000 ± 3000 rpm for 10 ± 5 minutes; preferably, the centrifugation is carried out at 8000 ± 2000 rpm for 10 ± 3 minutes; preferably, the centrifugation is carried out at 8000 ± 1000 rpm for 10 ± 2 minutes.

[0029] In one or more embodiments, after step (5), it further includes: freeze-drying the obtained recrystallization product.

[0030] In another aspect of the present invention, there is provided the use of the method described in any one of the foregoing in purifying porphyrin compounds.

[0031] In another aspect of the present invention, there is provided a kit for purifying porphyrin compounds from a biological fermentation broth, which includes: an alkali solution 1 for adjusting the pH value of the fermentation broth supernatant to pH 8 - 10; preferably, the alkali solution 1 is a NaOH solution; isopropanol; acidic acetone for adjusting the supernatant to pH 1 - 3; an acetate salt for adjusting the supernatant to pH 3 - 5; preferably, the acetate salt is sodium acetate; and an alkali solution 2 for dissolving the crystalline product; preferably, the alkali solution 2 is a NaOH solution; preferably, the concentration of the alkali solution 2 is 0.1 - 0.6 M.

[0032] In another aspect of the present invention, there is provided the use of the kit for purifying porphyrin compounds.

[0033] In one or more embodiments, the porphyrin compounds include (but are not limited to): coproporphyrin III, ferroprotoporphyrin III, Co-coproporphyrin III, Ni-coproporphyrin III, Cu-coproporphyrin III, Zn-coproporphyrin III, Mn-coproporphyrin III.

[0034] In one or more embodiments, the porphyrin compound is coproporphyrin III.

[0035] In one or more embodiments, the biological fermentation broth is a fermentation product of a porphyrin compound-producing bacterium.

[0036] In one or more embodiments, the porphyrin compound-producing bacteria include: photosynthetic bacteria, Paracoccus denitrificans, Bacillus subtilis, Agrobacterium tumefaciens, Mesorhizobium chinense, Methanobacterium barkeri, Escherichia coli or Corynebacterium glutamicum; preferably, the photosynthetic bacteria include: Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter azotoformans or Rhodobacter gelatinosus.

[0037] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Flow chart for the purification of porphyrin compounds.

[0039] Figure 2 Effect of removing impurities by the precipitation method;

[0040] (A) Effect of adjusting pH with NaOH on precipitation;

[0041] (B) Effects of methanol, ethanol, and isopropanol on precipitation;

[0042] (C) Effect of isopropanol dosage on precipitation.

[0043] Figure 3 Effect of acidic acetone on the extraction efficiency of coproporphyrin III;

[0044] (A) Extraction efficiency of coproporphyrin III after extraction with acetone at different pH values;

[0045] (B) Extraction efficiency of coproporphyrin III fermentation broth at different acidic acetone volume ratios (pH = 2.5);

[0046] The extraction efficiency is related to acidic acetone (pH = 0.5); the data shown are the mean ± SD (n > 3).

[0047] Figure 4 、Effect of crystallization conditions on the purity and recovery rate of coproporphyrin III. (A) Purity of coproporphyrin III crystallized at different pH values adjusted by sodium acetate. (B) Purity and recovery rate of coproporphyrin III under different recrystallization sedimentation conditions.

[0048] Figure 5 、Effect of each module in the purification process on the purity and recovery rate of coproporphyrin III. ① Centrifugation of fermentation broth, ② Precipitation method, ③ Extraction, ④ Crystallization, ⑤ Recrystallization, ⑥ Drying.

[0049] Figure 6 、Analysis results of the purity and recovery rate of different metal porphyrin compounds. Specific implementation mode

[0050] The present inventor has conducted in-depth research and revealed a method for efficiently and conveniently purifying porphyrin in batches from fermentation broth, which is used for the purification and preparation of porphyrin compounds in complex fermentation broth.

[0051] In this field, little is known about the production potential of producing porphyrin compounds by microbial fermentation, and there is no mature large-scale (scale larger than shake flask) fermentation process, let alone a large-scale purification process. In the previous research of the present inventor, the production process of porphyrin compounds was explored and optimized, providing a new way to establish a fermentation production process for the industrialization of the production of porphyrin compounds such as coproporphyrin III by microbial fermentation as soon as possible. Further, the present inventor optimized the purification process specifically for purifying porphyrin compounds from the fermentation products of porphyrin compound-producing bacteria.

[0052] Porphyrin compounds are a class of large heterocyclic compounds formed by the α-carbon atoms of four pyrrole-like subunits interconnected by methylene bridges (=CH-). Its parent compound is porphin, and porphin with substituents is called porphyrin.

[0053] As used in the present invention, the "porphyrin compound" may include but is not limited to: iron coproporphyrin III, coproporphyrin III, etc. Further, the "porphyrin compound" may also include protoporphyrin IX, vitamin B12, chlorophyll, etc. The "porphyrin compound" may also be a variant or derivative form based on the compounds disclosed in the present invention. For example, the parent nucleus structure of the compound remains unchanged, but groups (such as aliphatic hydrocarbon groups containing 1-4 carbon atoms (preferably 1-2 carbon atoms)) are substituted at individual (such as 1-3, 1-2) positions.

[0054] As used in the present invention, the "porphyrin compound"-producing bacteria may be, but are not limited to, the following bacteria: photosynthetic bacteria, Paracoccus denitrificans, Bacillus subtilis, Agrobacterium tumefaciens, Rhizobium mesoamericanum, Methanosarcina barkeri str. Fusaro. Preferably, the photosynthetic bacteria are selected from purple non-sulfur photosynthetic bacteria. The purple non-sulfur bacteria may be selected from the genera Rhodobacter and Rhodospirillum. More preferably, the photosynthetic bacteria include bacteria selected from the following group: Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter azotoformans, or Rubrivivax gelatinosus.

[0055] As used in the present invention, the terms "fermentation", "fermentation culture", or "culture" can be used interchangeably and refer to the process by which microbial cells grow under aerobic conditions using exogenously supplied nutrients (such as carbon sources, nitrogen sources, etc.).

[0056] The inventors of the present invention use microorganisms represented by Rhodobacter sphaeroides to ferment and produce porphyrin compounds. While obtaining the fermentation product, it further relates to the process of purifying porphyrin compounds from the fermentation broth, especially a purification process suitable for industrial production or large-scale production.

[0057] In the research and experiments of the inventors of the present invention, through in-depth analysis and research of the fermentation product, various factors affecting purification were analyzed, and the following main process was determined: ① centrifugation of the fermentation broth, ② precipitation method, ③ extraction, ④ crystallization, ⑤ recrystallization; optionally, ⑥ drying may further be carried out.

[0058] In a preferred embodiment of the present invention, taking coproporphyrin III as an example, the downstream recovery process undergoes 6 process steps. Several operations were explored for possible improvement methods, including three modules: (1) Precipitating impurities to remove proteins, organic salts, polysaccharides, etc. (2) Extracting porphyrin compounds from the protein-bound form with acidic acetone to obtain total porphyrin compounds, removing residual protein impurities, and more importantly, avoiding the co-crystallization of proteins and porphyrin compounds. (3) Further improving the purity of porphyrin compounds through recrystallization optimization. Finally, the effects of each operation unit on the purity and recovery rate of porphyrin compounds were explored. Through this downstream purification process, high-purity porphyrin compounds can be obtained, which can be used for subsequent metal modification and derivatization reactions, providing a rich substrate basis for the extended research on the application of porphyrin compounds.

[0059] Although specific steps are provided in the embodiments of the present invention, the purification process of the present invention can be scaled up for industrial production. Depending on the size of the system, based on the core concept provided by the present invention, those skilled in the art can make appropriate adjustments according to the general knowledge they have to facilitate the obtaining of high-purity and high-recovery products.

[0060] The present invention also provides the application of the method for obtaining high-purity porphyrin compounds and reducing product loss.

[0061] Compared with traditional natural separation means or chemical synthesis means, microbial fermentation has advantages such as fast speed and less influence by external factors; the yield of some compounds synthesized by microorganisms is much higher than that of plant extraction, and it has become an important means for obtaining natural products. Porphyrin compounds have a low natural abundance, and the chemical synthesis method is cumbersome and complex. In the present invention, the method of microbial fermentation is used to efficiently and directionally synthesize porphyrin compounds, and a purification process suitable for obtaining high-quality products is developed, which extremely effectively reduces the cost of separating and purifying such compounds.

[0062] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to the conditions described in, for example, Molecular Cloning: A Laboratory Manual, Third Edition, edited by J. Sambrook et al., published by Science Press, or according to the conditions recommended by the manufacturer.

[0063] Materials and Methods

[0064] 1. Purifying porphyrin compounds from fermentation broth

[0065] A series of purification process modules for porphyrin compounds were established as Figure 1As shown in the figure, it is divided into 6 operation units, including: fermentation, centrifugation to remove cells; sedimentation of precipitate; extraction; crystallization; recrystallization; drying. The above operation units are respectively labeled as ① fermentation, centrifugation to remove cells; ② sedimentation of precipitate; ③ extraction; ④ crystallization; ⑤ recrystallization; ⑥ drying. For key operation units including precipitation method, extraction, crystallization and recrystallization, strict optimization and evaluation have been carried out.

[0066] The fermentation uses Rhodobacter sphaeroides (Rhodobacter sphaeroides 2.4.1RSI-ΔfnrL with the global regulatory factor fnrL knocked out, and this strain has applied for a patent with the application number 201910999107.4). During fermentation, inoculate at a ratio of 15% in a 5L tank. Specifically, the strain is first diluted and spread on an agar plate (0.8% yeast extract, 0.3% glucose, 0.2% NaCl, 0.13% KH2PO4, 0.0125% MgSO4, 1.5% agar, adding 15mg / L biotin, 1mg / L nicotinic acid, and 1mg / L thiamine hydrochloride), and inverted in an incubator at 32°C for 6-7 days to grow monoclonal colonies. Pick 8-10 monoclonal colonies from the agar plate and inoculate them into 100mL of seed culture medium (0.2% yeast extract, 1% glucose, 0.08% corn steep powder, 0.08% sodium glutamate, 0.35% (NH4)2SO4, 0.2% NaCl, 0.08% K2HPO4, 0.08% KH2PO4, 0.013% MgSO4, 0.02% FeSO4 and adding 0.2% CaCO3, 1mg / L thiamine hydrochloride, 1mg / L nicotinic acid and 15μg / L biotin), and place it on a shaker at 32°C and 220rpm until its optical density value (OD700) is 5-8. Finally, inoculate the seed liquid at 15% into 2.5L of fermentation broth medium (4% glucose, 0.4% corn steep powder, 0.3% sodium glutamate, 0.3% (NH4)2SO4, 0.28% NaCl, 0.3% KH2PO4, 0.63% MgSO4, and adding 1mg / L thiamine hydrochloride, 1mg / L nicotinic acid and 15μg / L biotin). Centrifuge at 8000rpm for 10 minutes to obtain the supernatant of the fermentation broth. In order to pretreat the fermentation broth, first use 1mol / L NaOH to adjust the supernatant to different pH values, which are 6.5 (initial pH), 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 respectively, then let it stand at room temperature for 30min, and then centrifuge at 8000rpm for 10 minutes. Retain the supernatant.

[0067] When performing alcohol analysis, add equal volumes of methanol, ethanol or isopropanol to the supernatant respectively, and stir for 10 minutes. After the mixture stands at room temperature for 2 hours, centrifuge at 8000rpm for 10 minutes to obtain the supernatant for subsequent operations.

[0068] When exploring the effect of the addition ratio of isopropanol on impurity removal, isopropanol was added to the supernatant at different volume ratios, including volume ratios of isopropanol to the fermentation broth of 2:1, 1:1, 1:2, 1:4, 1:8, 1:12. After centrifugation at 8000 rpm for 10 minutes, the supernatant was obtained for subsequent operations.

[0069] Equal volumes of acidic acetone with different pH values were added to the supernatant. Meanwhile, acidic acetone with different volume ratios (pH = 2.5) was evaluated, including 4:1, 2:1, 1:1, 1:1.5, 1:2, 1:4 (volume ratio of acidic acetone to the fermentation broth, v / v). After adding the acidic acetone, after thorough shaking and mixing (shaking for 30 s), centrifugation was carried out at 8000 rpm for 10 minutes, and the coproporphyrin III content in the supernatant was measured, and the extraction efficiency was calculated.

[0070] In the crystallization unit, different amounts of sodium acetate were added to the extracted supernatant, and its pH was adjusted to 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6 respectively, and it was placed at 4 °C and allowed to stand in the dark for 16 hours to slowly form crystals. After centrifugation at 8000 rpm for 10 minutes, the coproporphyrin III crystals were separated by centrifugation, rinsed three times with deionized water, and then freeze-dried.

[0071] In recrystallization, first, the dried coproporphyrin III crystals were completely dissolved in 0.3 M NaOH, and then centrifuged at 8000 rpm for 10 minutes to remove the precipitate. The supernatant was adjusted to pH 4.5 with acetic acid, and then allowed to stand at 4 °C for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes respectively. After crystallization, centrifugation was carried out at 8000 rpm for 10 minutes, and it was rinsed three times with deionized water to obtain crystals.

[0072] In addition, the recrystallized coproporphyrin III can be subjected to a second recrystallization. The steps are the same as the first one.

[0073] In the evaluation, analysis, and optimization process by the inventor, the above steps were fully or partially selected.

[0074] All the above coproporphyrin III crystals can be freeze-dried, and then the purity and recovery rate can be determined according to their weights and the HPLC analysis results.

[0075] 2. Evaluate each operation unit in the CPIII purification process

[0076] Evaluate the influence of each module on the purity and recovery rate of coproporphyrin III crystals:

[0077] ①④⑥ represent obtaining the fermentation broth and centrifuging to obtain the supernatant of the fermentation broth; directly adjusting the pH of the fermentation broth supernatant to 4.5 with acetic acid and allowing it to stand overnight; freeze-drying the obtained crystals.

[0078] ①② (only NaOH) ④⑥ indicate obtaining the fermentation broth and centrifuging to obtain the supernatant of the fermentation broth; adjusting the pH of the supernatant of the fermentation broth to 8.5 with 1M NaOH, removing the precipitate after standing; adding sodium acetate to the supernatant to adjust the pH = 4.5 to cause crystallization; then performing freeze-drying.

[0079] ①② (only isopropanol) ④⑥ indicate obtaining the fermentation broth and centrifuging to obtain the supernatant of the fermentation broth; adding 20% isopropanol to the supernatant of the fermentation broth, removing the precipitate after standing; adding sodium acetate to the supernatant to adjust the pH = 4.5 to cause crystallization; then performing freeze-drying.

[0080] ①②④⑥ indicate obtaining the fermentation broth and centrifuging to obtain the supernatant of the fermentation broth; adjusting the pH of the supernatant of the fermentation broth to 8.5 with 1M NaOH, removing the precipitate after standing, adding 20% isopropanol to the supernatant, and centrifuging to discard the obtained precipitate; adding sodium acetate to the supernatant to adjust the pH = 4.5 to cause crystallization; then performing freeze-drying.

[0081] ①③④⑥ indicate obtaining the fermentation broth and centrifuging to obtain the supernatant of the fermentation broth; without precipitation treatment, directly adding an equal volume of acidic acetone with a pH of 2.5, mixing well, centrifuging at 8000 rpm for 10 minutes to remove the precipitate; adding sodium acetate to the supernatant to adjust the pH = 4.5 to cause crystallization; then performing freeze-drying.

[0082] ①②③④⑥ indicate obtaining the fermentation broth and centrifuging to obtain the supernatant of the fermentation broth; adjusting the pH of the supernatant of the fermentation broth to 8.5 with 1M NaOH, removing the precipitate after standing, adding 20% (v / v) isopropanol to the supernatant, and centrifuging to discard the obtained precipitate; adding an equal volume of acidic acetone with a pH of 2.5 to the supernatant, mixing well, centrifuging at 8000 rpm for 10 minutes to remove the precipitate; adding sodium acetate to the supernatant to adjust the pH = 4.5 to cause crystallization; then performing freeze-drying.

[0083] ①②③④⑤once⑥ indicate obtaining the fermentation broth and centrifuging to obtain the supernatant of the fermentation broth; adjusting the pH of the supernatant of the fermentation broth to 8.5 with 1M NaOH, removing the precipitate after standing, adding 20% isopropanol to the supernatant, and centrifuging to discard the obtained precipitate; adding an equal volume of acidic acetone with a pH of 2.5 to the supernatant, mixing well, and centrifuging to remove the precipitate; adding sodium acetate to the supernatant to adjust the pH = 4.5 to cause crystallization; dissolving the crystallized coproporphyrin III crystals completely with NaOH, adjusting the pH value to 4.5 with acetic acid to cause recrystallization; then performing freeze-drying.

[0084] ①②③④⑤twice⑥ indicates that on the basis of ①②③④⑤once⑥, after redissolving the recrystallized coproporphyrin III, performing the second recrystallization.

[0085] All the crystals obtained after the above purification were freeze-dried, and the crystal purity and the recovery rate of this process were measured.

[0086] 3. Analytical methods

[0087] For the determination of the precipitate mass of each unit during the purification process, the mass of the centrifuge cup was weighed in advance. After each unit operation, the supernatant obtained by centrifugation was poured into another centrifuge cup as completely as possible. The total mass of the precipitate and the centrifuge cup was weighed, and the wet weight of the precipitate was calculated. The precipitate was placed in an oven at 65 °C, and the mass of the centrifuge tube and the precipitate was weighed every other day until the mass remained unchanged. After weighing, the dry weight of the precipitate was calculated.

[0088] Measurement of crystal purity and recovery rate: Weigh 3 portions of a certain amount of freeze-dried coproporphyrin III crystal powder, prepare solutions with concentrations of 0.125 mg / mL and 0.0625 mg / mL respectively, measure using HPLC and calculate the true concentration of coproporphyrin III with the coproporphyrin III standard curve. The ratio of the true concentration to the concentration of the prepared coproporphyrin III is the purity of coproporphyrin III recovered in the current batch. Measure the coproporphyrin III concentration of the untreated fermentation broth and calculate the total amount of coproporphyrin III in the treated sample. After the coproporphyrin III in the fermentation broth crystallizes and is freeze-dried, measure the mass of the coproporphyrin III crystal powder and calculate the true mass according to the purity. The ratio of the true mass of coproporphyrin III to the total amount of coproporphyrin III in the original fermentation broth is the recovery rate of coproporphyrin III obtained in the current batch.

[0089] Example 1. Preliminary purification of fermentation broth

[0090] The fermentation broth obtained through biological fermentation has a complex composition, and its impurities include both soluble substances and insoluble substances. Especially for soluble substances such as proteins, amino acids, organic acids, inorganic salts, and sugars, it is very difficult to remove them efficiently, which has a great impact on the separation of coproporphyrin III secreted outside the cells. In addition, the fermentation broth contains various metal ions such as Mg 2+ , Co 2+ , Fe 2+ etc. These metal ions are prone to form complexes with organic macromolecules such as proteins, amino acids, and nucleic acids in alkaline solutions. When hoping to obtain a high-purity and high-quality final product, how to effectively remove impurities efficiently is very crucial.

[0091] The inventors conducted a purification condition analysis from multiple perspectives and with multiple focuses, including the application of centrifugation, precipitation method, extraction method, crystallization / recrystallization method, etc., and at the same time analyzed the corresponding preferred reagents, preferred addition times, etc.

[0092] 1. Precipitation method

[0093] The inventors considered precipitation as a step in purification. Multiple different precipitation methods were adopted, and the amount (wet weight) of the obtained precipitate was analyzed:

[0094] (A) Obtain the fermentation broth and centrifuge to obtain the supernatant of the fermentation broth; adjust 8 identical fermentation supernatants to different pH values (pH to 6.5 - 10, as shown on the Figure 2 A abscissa) with 1M NaOH; let stand, and then centrifuge; obtain the precipitate and weigh the mass of the precipitate;

[0095] (B) Organic solvent precipitation method: Obtain the supernatant of the fermentation broth, add 1M NaOH to adjust the pH in the fermentation broth supernatant to 8.5, let stand and then remove the precipitate; then divide the fermentation broth supernatant into 3 portions, and independently add equal volumes of methanol 50% (v / v), ethanol 50% (v / v), or isopropanol 50% (v / v) to each portion; let stand, and then centrifuge; obtain the precipitate and weigh the mass of the precipitate;

[0096] (C) Obtain the supernatant of the fermentation broth, first add 1M NaOH to adjust the pH in the fermentation broth supernatant to 8.5, let stand and then remove the precipitate; treat with isopropanol, and vary the different dosages of isopropanol (the volume ratio of isopropanol to the fermentation broth is 2:1, 1:1, 1:2, 1:4, 1:8, 1:12); then let stand, and then centrifuge; obtain the precipitate and weigh the mass of the precipitate.

[0097] After precipitation by the above methods respectively, the inventors first studied the optimal pH conditions for precipitating impurities and measured the weight of the precipitate. As Figure 2 shown in A, when the pH was adjusted above 8, the amount of precipitate increased rapidly; when the pH was adjusted to 8.5 and above, the wet weight of the precipitate was the largest. The small amount of residual protein might be because under such conditions, some proteins could not easily bind to metal cations.

[0098] The inventors also compared the method of adjusting the above pH to alkaline with other methods (acid precipitation method and salting - out method). For acid precipitation, proteins were made to form precipitates with some anions (trichloroacetic acid / salicylic acid), and the anions were added externally to the fermentation broth. For the salting - out precipitation method, external cations (NH4 + ) were added to change the surface charge of the proteins. However, the anion method and the salting - out method had unsatisfactory effects, and it was difficult for them to precipitate a variety of impurity proteins with different isoelectric points. In addition, both of these methods required the addition of external ions, which caused interference and was not conducive to the subsequent purification process. Therefore, using NaOH to adjust the pH of the fermentation broth to 8.5 could effectively and conveniently remove impurities such as proteins and other organic macromolecules, and relatively more impurities could be removed. Therefore, precipitating impurity proteins in an alkaline environment is superior to the other two commonly used precipitation methods (acid precipitation and salting - out precipitation strategies).

[0099] After analyzing the supernatant after precipitation in an alkaline environment, the inventors found that there were still various types of impurities, including soluble impurities such as sugars, organic acids and inorganic salts, so it was necessary to further remove the impurities. After analysis and comparison, the inventors selected organic solvents (alcohols) to further precipitate the impurities in the supernatant. The effects of alcohols with different numbers of carbon atoms on impurity removal were analyzed, and tests were conducted on commonly used reagents such as methanol, ethanol and isopropanol. The effect of different alcohols on impurity removal is measured by the weight of the resulting precipitate. Figure 2 As shown in B, isopropanol can precipitate impurities most effectively, with the precipitation amount being 6 times and 2 times more than that of methanol and ethanol, respectively.

[0100] The results of evaluating the optimal volume ratio of fermentation broth to isopropanol are as follows Figure 2 As shown in C, when the volume ratio of isopropanol to fermentation broth is 2:1 to 1:4, a large amount of impurities can be collected; when the volume ratio of isopropanol to fermentation broth is 1:4, a large amount of impurity precipitates are obtained while using relatively minimal organic reagents and having the lowest cost.

[0101] Example 2: Extraction of porphyrin compounds with acidic acetone

[0102] The secreted coproporphyrin III in the fermentation broth exists in the form of free coproporphyrin III and non-covalently bound protein-bound coproporphyrin III. In order to crystallize and purify coproporphyrin III in the subsequent crystallization and purification process, it is crucial to obtain as much free form of coproporphyrin III as possible. Thus, after analysis and research, the inventor added an extraction unit at the appropriate time of the whole recovery process of coproporphyrin III. The inventor analyzed and showed that the free form of coproporphyrin III can be efficiently separated and obtained by extracting total coproporphyrin III with acidic acetone.

[0103] After fermentation, the fermentation liquid was obtained and the supernatant of the fermentation liquid was obtained by centrifugation; after precipitation treatment with NaOH and isopropanol, the obtained supernatant was directly added with an equal volume of acidic acetone to make the solution pH = 2.5, after thorough shaking and mixing, centrifuged at 8000rpm for 10 minutes, and the coproporphyrin III content in the supernatant was determined to calculate the extraction efficiency. Figure 3 As shown in Figure A, acidic acetone (pH 0.1-3, preferably pH 0.1-2.5) has a high extraction efficiency for coproporphyrin III. When the acidity decreases, the extraction efficiency decreases.

[0104] At the same time, the inventor's analysis showed that ( Figure 3 B), when the ratio of acidic acetone to the treated supernatant is 4:1 to 1:1, a high extraction efficiency is achieved. Among them, when the ratio of acidic acetone to the treated supernatant is 1:1, a very high extraction efficiency is obtained, the organic reagent used is relatively minimal, and the cost is the lowest, so the extraction efficiency is optimal under this ratio.

[0105] Example 3, Crystallization of Porphyrin Compound

[0106] Crystallization is a method that is easy to scale up and can further purify compounds. Therefore, it is a preferred option in the industrial downstream recovery process. However, considering the individual characteristics of different compounds, when dealing with a specific compound, it is still necessary to explore suitable crystallization means; moreover, quite a number of compounds are not suitable for purification by crystallization due to the lack of well-explored crystallization conditions, such as being unable to crystallize or having co-crystallization of multiple impurities with the target compound.

[0107] In order to explore suitable crystallization conditions and achieve better reproducibility between batches, the inventors analyzed coproporphyrin III and conducted repeated experiments, and chose to crystallize coproporphyrin III under acidic conditions. Before crystallization, the treatment solvent was removed and the solution was concentrated to facilitate the coproporphyrin III reaching the crystallization saturation more easily. At the same time, different pH values were adjusted to analyze the suitable acidic environment.

[0108] The purification was carried out by the method as described in “①②③④⑥” above. The results are as Figure 4 shown in A. It is preferred to crystallize coproporphyrin III under acidic conditions and using sodium acetate as the crystallization reagent. A relatively high purity of coproporphyrin III can be obtained when the pH is 3.5 - 5; among them, when the pH is 4.5, the highest purity of coproporphyrin III is obtained, exceeding 86%. Nevertheless, there are still impurities that have not been removed and co-crystallize with coproporphyrin III, affecting the purity of coproporphyrin III.

[0109] To further improve the purity of coproporphyrin III, the inventors conducted further optimization research. The analysis showed that coproporphyrin III can be completely dissolved in an alkaline solution, and the inventors further adopted the strategy of dissolving in an alkaline environment and acid crystallization. The purification was carried out by the method as described in “①②③④⑤once⑥” above, but the pH value of the recrystallization system was adjusted.

[0110] The crystallized coproporphyrin III was completely dissolved in NaOH, and the pH was adjusted to 4.5 with sodium acetate for recrystallization. First, the inventors studied the standing time of the recrystallization. It should be noted that compared with single crystallization (86%), the purity of coproporphyrin III was significantly improved ( Figure 4 B). Moreover, unexpectedly, compared with the single crystallization step (16 hours), the standing time was greatly shortened to within 1 hour.

[0111] On the other hand, the inventors found that another advantage of dissolving in an alkaline environment is that the remaining impurities after the previous steps can also be further removed through the redissolution step with NaOH, and relatively pure coproporphyrin III can be recrystallized faster.

[0112] As Figure 4B. When the standing time is extended to 30 to 40 minutes or even longer, the recovery rate of coproporphyrin III reaches more than 88% and stabilizes at this level. A time of 30 minutes to 60 minutes is suitable. Considering the recovery rate, purity, and efficiency comprehensively, a standing time of about 40 minutes (such as 40 - 50 minutes, 45 - 50 minutes, 50 - 55 minutes, 50 - 60 minutes) for recrystallization is most suitable.

[0113] such as Figure 4 B. Through the processing steps of precipitation, extraction, crystallization, and recrystallization, the inventor can purify coproporphyrin III with a purity higher than 94% (even higher than 95% or 96%) from the fermentation broth.

[0114] Example 4. Influence of each module on the purity and recovery rate of the crystallized product

[0115] Since the optimal conditions for coproporphyrin III crystallization have been obtained, in order to explore the influence of different operating units involved in this purification process on the purity of coproporphyrin III crystals and the recovery process, the inventor evaluated the 4 key modules of precipitation method, extraction, crystallization, and recrystallization involved in the whole process. The purification was carried out respectively using the schemes of "①④⑥", "①②④ (only NaOH)⑥", "①②④ (only isopropanol)⑥", "①②④ (NaOH + isopropanol)⑥", "①③④⑥", "①②③④⑥", "①②③④⑤once⑥", "①②③④⑤twice⑥", and the purity and recovery rate of the products were analyzed.

[0116] The results are as Figure 5 shown as follows:

[0117] Using the "①④⑥" scheme, after direct crystallization of the fermentation broth supernatant, other impurities precipitate and crystallize together with coproporphyrin III, and the purity of the obtained coproporphyrin III is only about 35%, and the recovery rate is only about 72%.

[0118] Using the schemes of ①② (only NaOH)④⑥", "①② (only isopropanol)④⑥" or "①③④⑥" respectively, that is, adding the precipitation method or extraction unit respectively, the crystal purities are 40%, 60% and 70% respectively; and the recovery rates show a certain increase, which are 65%, 85% and 95% respectively.

[0119] The schemes of "①②④ (NaOH + isopropanol)⑥" and "①③④⑥" are at a medium level in both purity and recovery rate, and there is still a need for optimization.

[0120] Using the "①②③④⑥" scheme, when the precipitation method and extraction unit are introduced simultaneously, the purity of the obtained crystals is further increased to 86%. Therefore, this module can remove the precipitation to a great extent, help most of the coproporphyrin III to crystallize out, and further increase the recovery rate to 90%.

[0121] In order to further obtain coproporphyrin III crystals with higher purity, the obtained crystals were dissolved and recrystallized. Using the "①②③④⑤once⑥" scheme, the crystal purity was further increased to 94%, and the recovery rate was maintained at a relatively high level (about 88%).

[0122] However, using the "①②③④⑤twice⑥" scheme, that is, after recrystallization twice, the crystal purity did not increase significantly, and the recovery rate decreased to 72% instead.

[0123] Therefore, considering the purity and recovery rate of the obtained coproporphyrin III comprehensively, the downstream separation and purification process of coproporphyrin III is as follows: the first fermentation broth is centrifuged to remove bacteria and insoluble precipitates, a precipitant is added to the supernatant to remove impurities such as proteins and sugars, and acidic acetone is added to extract coproporphyrin III. Subsequently, coproporphyrin III is crystallized. After washing with water to remove impurities, the crystals are redissolved and recrystallized once to further improve the crystallization purity. The crystal purity of coproporphyrin III obtained by this process can reach 94%, and the recovery rate can reach 88%.

[0124] Example 5. Analysis of Metal Porphyrin Compounds

[0125] Verify the purification efficiency of this method for other porphyrin compounds. Using the "①②③④⑤once⑥" scheme, the analysis object is the porphyrin compounds prepared by the method for which a patent has been applied (Patent Application No.: 202310754364.8, and all the corresponding contents are incorporated into the present invention), that is, the enzymatic catalysis converts coproporphyrin III in the fermentation broth into different metal porphyrin compounds Fe-coproporphyrin III, Co-coproporphyrin III, Ni-coproporphyrin III, Cu-coproporphyrin III, Zn-coproporphyrin III, Mn-coproporphyrin III.

[0126] The measurement results of purity and recovery rate are as Figure 6 shown in A - B. The purity and recovery rate of these porphyrin compounds are also very high. This is because of the similar macrocyclic conjugated structure of porphyrin compounds, which can be crystallized under the same conditions. In addition, when porphyrin compounds are produced by microbial fermentation, a large amount of impurities in the fermentation broth can be removed by this method, which helps to improve the purity. Therefore, this method is applicable to the large-scale purification of porphyrin compounds from fermentation broth.

[0127] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the invention patent shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited in this application for reference, just as if each document is cited separately for reference.

Claims

1. A method for purifying porphyrin compounds from biological fermentation broth, comprising: (1) Obtaining the supernatant of the fermentation broth containing porphyrin compounds; (2) Adjusting the pH value of the supernatant of the fermentation broth in (1) to pH 8 - 10; Separating and removing the precipitate to obtain the supernatant; adding isopropanol to the supernatant, separating and removing the precipitate to obtain the supernatant; (3) Adding acidic acetone to the supernatant in (2) to make the pH 1 - 3, separating and removing the precipitate to obtain the supernatant; (4) Adding acetate to the supernatant in (3) to adjust the pH to 3 - 5 to obtain a crystalline product; (5) Dissolving the crystalline product in (4) in an alkali solution to obtain a solution; Adding acetate to the solution to adjust the pH to 3 - 5 to obtain a recrystallized product, which is the purified porphyrin compound.

2. The method according to claim 1, characterized in that In (1), cells or other solids in the fermentation broth are removed by centrifugation.

3. The method according to claim 1, characterized in that, In (2), the pH value is adjusted to pH 8 - 10 with an alkali solution; preferably, the alkali solution is an NaOH solution; Or In (2), after adjusting the pH value, the separation and removal of the precipitate include: allowing the supernatant to stand, and then centrifuging to remove the precipitate; Preferably, standing for 30 ± 20 min, more preferably standing for 30 ± 10 min.

4. The method according to claim 1, wherein In (2), the final concentration of the isopropanol is 15 - 70% by volume ratio; preferably 18 - 50%; more preferably 20 - 35%; or In (2), after adding isopropanol, mixing well, standing, and then centrifuging to remove the precipitate; preferably standing for 120 ± 60 min, more preferably standing for 1200 ± 30 min.

5. The method according to claim 1, wherein In (3), the volume ratio of acidic acetone to the supernatant is 4:1 - 1:1; preferably 2:1 to 1:1; or In (3), after adding acidic acetone, mixing well and centrifuging to obtain the supernatant.

6. The method according to claim 1, characterized in that, In (4), the acetate is sodium acetate; or In (4), the crystallization is carried out at 1 - 8°C, standing in the dark; preferably carried out at 2 - 6°C (such as 3, 4, 5°C); preferably, standing for 16 ± 8 hours; or In (4), after forming crystals, centrifuging to separate the crystals.

7. The method according to claim 1, characterized in that In (5), the alkali solution is an NaOH solution; preferably, the alkali solution is a 0.1 - 0.6 M solution; or In (5), after dissolving in the alkali solution, centrifuging the obtained solution to remove the precipitate to obtain a supernatant solution; or In (5), the recrystallization is carried out at 1 - 8°C, standing in the dark; preferably carried out at 2 - 6°C; preferably, standing for 30 - 80 minutes; or In (5), after forming crystals, centrifuging to separate the crystals.

8. The method according to claim 1, wherein After step (5), it further includes: freeze-drying the obtained recrystallized product.

9. Use of the method according to any one of claims 1 - 8 in purifying porphyrin compounds.

10. A kit for purifying porphyrin compounds from biological fermentation broth, which includes: Alkali solution 1, used to adjust the pH value of the supernatant of the fermentation broth to pH 8 - 10; Preferably, the alkali solution 1 is an NaOH solution; Isopropanol; Acidic acetone, used to adjust the supernatant to pH 1 - 3; Acetate, used to adjust the supernatant to pH 3 - 5; preferably, the acetate is sodium acetate; And Alkali solution 2, used to dissolve the crystalline product; Preferably, the alkaline solution 2 is a NaOH solution; preferably, the concentration of the alkaline solution 2 is 0.1 - 0.6 M.

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