Method for producing long-chain dibasic acid by biological fermentation

Abstract

The application belongs to the field of biological fermentation and particularly relates to a method for producing long-chain dibasic acid through biological fermentation, which comprises: a first fermentation process: inoculating seed liquid of fermentation bacteria into a fermentation tank and starting fermentation, and adding a substrate after 6-10 hours of fermentation, wherein the pH is controlled to be 5.0-8.5 after 6 hours of fermentation, and the fermentation is performed for 100-160 hours; and a second fermentation process: further adjusting the pH of the fermentation product obtained in the first fermentation process to 7.0-9.0, adding fatty alcohol dehydrogenase and / or fatty alcohol oxidase, and performing fermentation for 2-8 hours; wherein the fermentation bacteria comprise at least one of Candida tropicalis, Candida viswanathii and Candida sake; and the substrate comprises at least one of C9-C22 normal alkanes. The method provided by the application has high yield and high conversion rate, and has few impurities such as fatty monoacid and fatty hydroxy acid, thereby reducing the work of removing impurities in the subsequent extraction.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation, specifically relating to a method for producing long-chain dicarboxylic acids by bio-fermentation. Background Technology

[0002] Dicarboxylic acids are acidic compounds containing two ionizable hydrogen atoms in a single molecule. They include inorganic acids such as sulfuric acid and carbonic acid, as well as a wide variety of organic dicarboxylic acids. In the field of organic chemistry, an important class of raw materials is called long-chain dicarboxylic acids (DCA), specifically referring to straight-chain compounds with carboxyl groups at both ends of the carbon chain and more than nine carbon atoms. Their structural formula is HOOC(CH2). n COOH, where n is an integer greater than 7. Long-chain dicarboxylic acids have a wide range of applications, and can be used as raw materials to synthesize specialty nylons, high-grade fragrances, high-grade hot melt adhesives, cold-resistant plasticizers, high-grade lubricants, high-grade rust inhibitors, high-grade paints and coatings, etc.

[0003] Long-chain dicarboxylic acids can typically be synthesized using chemical or biological methods. Chemical synthesis involves long routes, requires high temperatures and pressures, and places stringent demands on catalysts. Therefore, the variety of long-chain dicarboxylic acids that can be synthesized on an industrial scale is limited, with only a few varieties such as dodecanoic acid. Biological methods, on the other hand, use long-chain alkanes as substrates and convert them through microbial processes. Specifically, they utilize the unique double-terminal oxidation capabilities of certain microorganisms to ferment n-alkanes and produce long-chain dicarboxylic acids.

[0004] The current mainstream production route is the bio-fermentation method, which uses microorganisms such as Candida tropicalis, Candida sakei, and Candida viresis to oxidize the two methyl groups at both ends of straight-chain alkanes (n-decane, n-dodecane, n-tetrazane, etc.) to carboxyl groups at room temperature and pressure, generating dicarboxylic acids with the corresponding number of carbon atoms. This method is not only mild and environmentally friendly, but also has high yield and low cost.

[0005] Figure 1 The production mechanism of long-chain dicarboxylic acids is illustrated. By interpreting this mechanism, it is found that insufficient activity of cytochrome P450 monooxygenase / cytochrome P450 reductase (CYP / CPR) and fatty alcohol dehydrogenase / fatty alcohol oxidase (ADH / FAO) leads to the ineffective conversion of metabolic intermediates, fatty monocarboxylic acids and fatty hydroxy acids. These substances accumulate during fermentation, resulting in a high content of impurities and acids in the fermentation broth after fermentation (fatty monocarboxylic acids + fatty hydroxy acids / dicarboxylic acids account for approximately 3%~5%). Because their structure and chemical properties are very similar to long-chain dicarboxylic acids, they are difficult to separate effectively using conventional methods, increasing the difficulty of separation and purification, and causing the final product to be substandard. Even trace amounts of residual hydroxy acids can affect the later applications of long-chain dicarboxylic acids, such as the production yield and quality of nylon fibers and musk T.

[0006] To improve the above situation, relevant professionals have carried out technical research. For example, CN110684676B reduced the ratio of hydroxy acids / diacids in decadecanoic acid fermentation broth from 0.92% to 0.56% by enhancing the activity of alcohol dehydrogenase (ADH) in Candida tropicalis strain and knocking out the acyl-CoA oxidase POX4 gene. The ratio of hydroxy acids / diacids in decadecanoic acid fermentation broth was reduced from 1.41% to 0.72%, and the ratio of hydroxy acids / diacids in hexadecanoic acid fermentation broth was reduced from 3.08% to 1.96%. After purification through alkali dissolution, membrane filtration, decolorization, acid precipitation, filtration, acetic acid re-dissolution, re-decolorization, and cooling crystallization, the content of decadecanoic acid and dodecanoic acid was less than 200 ppm, and the content of hexadecanoic acid was less than 2000 ppm. CN110683947B discovered that controlling the pH value of the feed solution, the membrane temperature, and the concentration of long-chain dicarboxylic acids during membrane filtration can effectively retain hydroxy acid impurities. Combined with decolorization, acid precipitation, and acetic acid recrystallization steps, the hydroxy acid content in undecanoic acid, dodecanoic acid, tridecanoic acid, and tetradecanoic acid can be reduced to below 400 ppm.

[0007] This shows that current long-chain dicarboxylic acid fermentation production technology still suffers from high levels of residual impurities such as hydroxy acids, which can only be effectively removed through complex gene editing and extraction methods. Summary of the Invention

[0008] The problem the invention aims to solve

[0009] To address the problems existing in current technologies, such as the high content of impurities like hydroxy acids in the fermentation broth during the production of long-chain dicarboxylic acids by microbial fermentation, which requires complex genetic modification of microbial strains and involves arduous post-processing, this invention has conducted extensive research and provides a new method for the production of long-chain dicarboxylic acids by bio-fermentation. This method improves the yield and conversion rate of long-chain dicarboxylic acids, reduces the content of impurities such as fatty monocarboxylic acids and fatty hydroxy acids in the fermentation products, and facilitates the separation and purification of long-chain dicarboxylic acids.

[0010] Solution for solving the problem

[0011] [1]. A method for producing long-chain dicarboxylic acids by bio-fermentation, wherein the method comprises:

[0012] First fermentation process: The seed liquid of fermentation bacteria is introduced into the fermenter and fermentation begins. Substrate is added after 6-10 hours of fermentation. The pH is controlled at 5.0-8.5 after 6 hours of fermentation, and the fermentation lasts for 100-160 hours.

[0013] Second fermentation process: The pH of the fermented product obtained in the first fermentation process is further adjusted to 7.0-9.0, and fatty alcohol dehydrogenase and / or fatty alcohol oxidase are added, and fermentation is carried out for 2-8 hours.

[0014] The fermentation bacteria include at least one of *Candida tropicalis*, *Candida viswanathii*, and *Candida sake*; the substrate includes at least one of n-alkane, straight-chain saturated fatty acid, straight-chain saturated fatty acid ester, and straight-chain saturated fatty acid salt.

[0015] The long-chain dicarboxylic acid includes aliphatic dicarboxylic acids with 10 or more carbon atoms.

[0016] [2]. According to the method of [1], wherein, during the first fermentation process, the OD of the seed liquid is... 600 The value is 10-15, and the amount of seed liquid added is 10% (v / v)-20% (v / v) of the initial fermentation broth.

[0017] [3]. According to the method of [1] or [2], wherein, during the first fermentation process, the concentration of the substrate in the fermentation broth is maintained at 1% (w / v) to 5% (w / v).

[0018] [4]. The method according to any one of [1]-[3], wherein the condition control in the first fermentation process includes one or more of the following:

[0019] The temperature should be controlled between 28-35℃.

[0020] Control the airflow to 0.5-1.4 vvm.

[0021] The control pressure is 0.05-0.1 MPa.

[0022] Dissolved oxygen should be controlled to be greater than 15%.

[0023] [5]. The method according to any one of [1]-[4], wherein, during the second fermentation process, the GenBank accession number of the coding sequence of the fatty alcohol dehydrogenase includes at least one of GU056282, GU056284, GU056283, GU056286 and GU056287.

[0024] [6]. The method according to any one of [1]-[5], wherein, during the second fermentation process, the GenBank accession number of the coding sequence of the fatty alcohol oxidase includes at least one of AY538780, GU056288, AY538781 and AY538782.

[0025] [7]. The method according to any one of [1]-[6], wherein, in the second fermentation process, the amount of fatty alcohol dehydrogenase and / or fatty alcohol oxidase added is 1-20 g / L.

[0026] [8]. The method according to any one of [1]-[7], wherein the fermenter contains a fermentation medium and a carbon source; the fermentation medium includes a nitrogen source, inorganic salts and growth factors.

[0027] [9]. The method according to any one of [1]-[8], wherein the preparation process of the seed liquid includes: activating and culturing the fermentation bacteria in a YPD shake flask, when OD 600 When the temperature reaches 8-15°C, inoculate with seed culture medium for cultivation, with the inoculation amount being 1% (v / v)-5% (v / v) of the seed culture medium.

[0028]

[10] . A method for preparing a polymer, wherein the method comprises:

[0029] Long-chain dicarboxylic acids are prepared according to any one of [1]-[9].

[0030] The polymerization step involves polymerizing the long-chain dicarboxylic acid with other monomers to prepare the polymer.

[0031] The other monomers include monomers containing active hydrogen, epoxy groups, and at least one group selected from isocyanate groups.

[0032] The effects of the invention

[0033] This invention provides a method for producing long-chain dicarboxylic acids through bio-fermentation. By controlling and adjusting the pH value of the fermentation broth and adding fatty alcohol dehydrogenase and / or fatty alcohol oxidase exogenously in the later stage of fermentation, the solubility of heteroacids such as fatty monocarboxylic acids and fatty hydroxy acids is increased. This allows heteroacids to more easily enter cells and come into contact with intracellular enzymes or with exogenously added alcohol dehydrogenase and / or alcohol oxidase, thereby converting them into the target product, long-chain dicarboxylic acids. This effectively reduces impurities such as fatty monocarboxylic acids and fatty hydroxy acids in the fermentation product, not only increasing the yield of long-chain dicarboxylic acids but also facilitating the subsequent separation and purification of long-chain dicarboxylic acids, reducing the workload of impurity removal in the later extraction process, and also benefiting the production and preparation of downstream products. Attached Figure Description

[0034] Figure 1 Mechanism of bio-fermentation production of long-chain dicarboxylic acids. Detailed Implementation

[0035] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0036] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0037] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0038] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0039] In this invention, the numerical range represented by "value A ~ value B", "value A - value B", and "value A above / below" refers to the range that includes the endpoint values ​​A and B.

[0040] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0041] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0042] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] This invention provides a method for producing long-chain dicarboxylic acids through bio-fermentation. By controlling and adjusting the pH value of the fermentation broth and adding fatty alcohol dehydrogenase and / or fatty alcohol oxidase exogenously in the later stage of fermentation, the solubility of heteroacids such as fatty monocarboxylic acids and fatty hydroxy acids is increased, making it easier for heteroacids to enter the cell and come into contact with intracellular enzymes or with exogenously added alcohol dehydrogenase and / or alcohol oxidase, thereby converting them into the target product, long-chain dicarboxylic acids. This method increases the yield of long-chain dicarboxylic acids and reduces the workload of removing impurities such as fatty monocarboxylic acids and fatty hydroxy acids during subsequent separation and purification of long-chain dicarboxylic acids.

[0044] Fermentation broth

[0045] The fermentation broth of this invention contains fermenting bacteria, substrate, and fermentation culture medium, and the fermentation broth can be stored in a fermenter. During the fermentation process, the target product, a long-chain dicarboxylic acid, will be generated in the fermentation broth, as well as possible impurities such as fatty monocarboxylic acids and fatty hydroxy acids.

[0046] In some embodiments, the fermentation strain used in this invention is Candida tropicalalis, Candida viswanathii, or Candida sake; preferably Candida viswanathii.

[0047] The present invention does not specifically limit the specific strain of the fermenting bacteria mentioned above. In some specific embodiments, the fermenting strain selected in the present invention is Candida virescens ATCC 20962.

[0048] In some embodiments, the fermentation bacteria are added to the fermenter in the form of a seed culture, the preparation process of which includes: activating and culturing the fermentation bacteria in a YPD shake flask, and when the OD... 600 At 8-15 hours (generally after 16-24 hours of culture), inoculate with seed culture medium at a rate of 1% (v / v)-5% (v / v) of the seed culture medium (e.g., 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0% (v / v)).

[0049] The present invention does not make any special requirements for the seed culture medium, and commonly used seed culture media in the art can be used.

[0050] In some embodiments, the seed culture medium comprises a carbon source, a nitrogen source, and an inorganic salt; preferably, the carbon source comprises at least one of sucrose and glucose, the nitrogen source comprises at least one of yeast extract, corn steep liquor powder, peptone, and urea, and the inorganic salt comprises potassium dihydrogen phosphate; more preferably, the seed culture medium comprises sucrose, glucose, yeast extract, corn steep liquor powder, peptone, potassium dihydrogen phosphate, and urea; even more preferably, the seed culture medium comprises 10 g / L sucrose, 20 g / L glucose, 5 g / L yeast extract, 5 g / L corn steep liquor powder, 5 g / L peptone, 5 g / L potassium dihydrogen phosphate, and 3 g / L urea.

[0051] In some embodiments, the substrate includes at least one of n-alkane, straight-chain saturated fatty acid, straight-chain saturated fatty acid ester, and straight-chain saturated fatty acid salt.

[0052] In some embodiments, the substrate comprises at least one of C10-C22 n-alkanes; preferably, the substrate comprises any one of C10-C22 n-alkanes, such as n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, n-eicosane, or n-docosahexadecane; more preferably, the substrate comprises any one of C10-C18 n-alkanes.

[0053] The present invention does not impose any particular limitation on the source and preparation method of the above-mentioned substrates. For example, they can be obtained from petroleum or synthesized by chemical means.

[0054] Corresponding to the above-mentioned substrates, the long-chain dicarboxylic acids that can be produced by the present invention include at least one of the long-chain dicarboxylic acids of C10-C22 (for example, at least one of decadecanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, icosanoic acid, or docosanoic acid); preferably, the long-chain dicarboxylic acids that can be produced include at least one of the long-chain dicarboxylic acids of C10-C18.

[0055] Considering the toxic effects of the aforementioned substrates on the fermenting bacteria, this invention employs a fed-batch fermentation process to improve production rate while ensuring the activity of the fermenting bacteria. In some embodiments, the initial addition amount of the substrate is 0.5% (w / v)-5% (w / v) of the initial fermentation broth (e.g., 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), or 5.0% (w / v), etc.), and then every 4-8 hours (e.g., every 5-6 hours). The substrate is added at 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, or 8h, etc., and its concentration is controlled at 1% (w / v)-5% (w / v) of the fermentation broth (e.g., 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), or 5.0% (w / v), etc.).

[0056] In some implementations, the total amount of substrate added throughout the production process is 24% (w / v) to 40% (w / v) of the initial fermentation broth.

[0057] The present invention does not make any special requirements for the fermentation medium, and common fermentation media in the art can be used.

[0058] In some embodiments, the fermentation medium includes a nitrogen source, inorganic salts, and growth factors.

[0059] In some specific embodiments, the nitrogen source includes at least one of yeast extract powder, corn steep liquor powder, urea, ammonium salt, and nitrate; preferably, the nitrogen source includes at least one of yeast extract powder, corn steep liquor powder, urea, ammonium sulfate, and potassium nitrate; more preferably, the nitrogen source content in the fermentation medium is 15-35 g / L; even more preferably, the nitrogen source content in the fermentation medium is 18-30 g / L.

[0060] In some specific embodiments, the inorganic salt includes at least one selected from sulfate, hydrochloride, molybdate, and phosphate; preferably, the inorganic salt includes at least one selected from potassium dihydrogen phosphate, ferrous sulfate, zinc sulfate, magnesium sulfate, sodium molybdate, and manganese chloride; more preferably, the content of inorganic salt in the fermentation medium is 4.5-10.5 g / L; even more preferably, the content of inorganic salt in the fermentation medium is 5-10 g / L.

[0061] In some specific embodiments, the growth factor includes at least one of vitamin B1, vitamin B2, vitamin B5, vitamin B7 and citric acid; preferably, the content of the growth factor in the fermentation medium is 0.01-1 mg / L; more preferably, the content of the growth factor in the fermentation medium is 0.05-0.5 mg / L.

[0062] In some more specific embodiments, the fermentation medium comprises yeast extract, corn steep liquor powder, urea, potassium nitrate, potassium dihydrogen phosphate, ammonium sulfate, vitamin B1, vitamin B2, vitamin B5, vitamin B7, citric acid, ferrous sulfate, zinc sulfate, magnesium sulfate, sodium molybdate, and manganese chloride; preferably, the fermentation medium comprises 5 g / L yeast extract, 12 g / L corn steep liquor powder, 3 g / L urea, 8 g / L potassium nitrate, 5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, 0.05 mg / L vitamin B1, 0.02 mg / L vitamin B2, 0.02 mg / L vitamin B5, 0.05 mg / L vitamin B7, 0.1 mg / L citric acid, 0.1 g / L FeSO4, 2.27 mg / L ZnSO4, 0.1 g / L MgSO4, 0.1 mg / L Na2MoO4, and 5.3 mg / L MnCl2.

[0063] In some embodiments, the fermentation medium further contains an antifoaming agent; preferably, it may contain 0.2-1 g / L of antifoaming agent; more preferably, it may contain 0.5 g / L of antifoaming agent. The specific type of antifoaming agent is not particularly limited; commonly used antifoaming agents in the art, such as polyether antifoaming agent XPJ999, can be selected.

[0064] In addition, to maintain the activity of the fermenting bacteria and to produce long-chain dicarboxylic acids, the fermentation broth also contains a carbon source. The concentration of the carbon source in the fermentation broth is maintained at 0.1% (w / v) to 5.0% (w / v), for example, it can be 0.1% (w / v), 0.5% (w / v), 1.0% (w / v), 2.0% (w / v), 3.0% (w / v), 4.0% (w / v), or 5.0% (w / v).

[0065] <Fermentation Process>

[0066] This invention produces long-chain dicarboxylic acids through a first fermentation process and a second fermentation process as shown below:

[0067] First fermentation process: The seed liquid of fermentation bacteria is introduced into the fermenter and fermentation begins. Substrate is added after 6-10 hours of fermentation. The pH is controlled at 5.0-8.5 after 6 hours of fermentation, and the fermentation lasts for 100-160 hours.

[0068] Second fermentation process: The pH of the fermented product obtained from the first fermentation process is further adjusted to 7.0-9.0, and fatty alcohol dehydrogenase and / or fatty alcohol oxidase are added, and fermentation is carried out for 2-8 hours.

[0069] As described in the pre-fermentation broth section, the fermentation bacteria, seed liquid, and substrate involved in the above fermentation process are inoculated into a fermenter containing fermentation culture medium, and the substrate and carbon source are added to form the initial fermentation broth.

[0070] In some implementations, when the OD of the seed solution 600 The seed liquid is introduced into the fermenter at 10-15 hours. The amount of seed liquid introduced can be 10% (v / v)-20% (v / v) of the initial fermentation liquid, for example, it can be 10% (v / v), 11% (v / v), 12% (v / v), 13% (v / v), 14% (v / v), 15% (v / v), 16% (v / v), 17% (v / v), 18% (v / v), 19% (v / v) or 20% (v / v), etc.

[0071] In some embodiments, the present invention controls the pH of the fermentation broth during the first fermentation process to be 5.0-8.5, preferably controlling the pH to always be a value within the range of 5.0-8.5 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, etc.), more preferably controlling the pH to always be a value within the range of 5.5-8.0, and even more preferably controlling the pH to always be a value within the range of 5.5-7.3.

[0072] By properly controlling the pH value of the fermentation broth during the first fermentation process, the yield of long-chain dicarboxylic acids can be effectively increased.

[0073] In some embodiments, the present invention controls the fermentation temperature during the first fermentation process to be 28-35℃ (e.g., 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, or 35℃, etc.), and / or controls the airflow to be 0.5-1.4 vvm (e.g., 0.5 vvm, 0.6 vvm, 0.7 vvm, 0.8 vvm, 0.9 vvm, 1.0 vvm, 1.1 vvm, 1.2 vvm). (e.g., m, 1.3vvm or 1.4vvm), and / or, control the pressure to be 0.05-0.1MPa (e.g., 0.05MPa, 0.06MPa, 0.07MPa, 0.08MPa, 0.09MPa or 0.1MPa), and / or, control the dissolved oxygen to be greater than 15% (e.g., 16%, 19%, 22%, 25%, 28%, 30% or 33%, preferably greater than or equal to 30%).

[0074] The first fermentation process lasts 100-160 hours, preferably 100-140 hours, to produce and accumulate a large amount of long-chain dicarboxylic acids.

[0075] As fermentation progresses, the activity of the fermenting bacteria and their internal enzymes may become insufficient, leading to the production of impurities such as fatty monoacids and / or fatty hydroxy acids. To address this, in the later stages of fermentation, after the first fermentation process, the pH of the fermentation broth is adjusted to increase the solubility of fatty monoacids and fatty hydroxy acids, allowing them to pass through the cell membrane more easily. Simultaneously, additional fatty alcohol dehydrogenases and / or fatty alcohol oxidases are added to compensate for the lack of intracellular enzymes. This process increases the contact between impurities such as fatty monoacids and / or fatty hydroxy acids and intracellular enzymes or exogenously added alcohol dehydrogenases and / or alcohol oxidases, thereby enabling their conversion into the target product, long-chain dicarboxylic acids.

[0076] In some specific embodiments, after the first fermentation process of the present invention is completed, the pH of the fermented product obtained from the first fermentation process is further adjusted to 7.0-9.0, preferably to a value among 7.0-9.0 (for example, it can be adjusted to 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0, etc.), more preferably to a value among 7.0-8.5, even more preferably to a value among 7.0-8.0, and even more preferably to a value among 7.3-7.6.

[0077] In some specific embodiments, the GenBank accession number of the coding sequence of the fatty alcohol dehydrogenase selected in this invention includes at least one of GU056282, GU056284, GU056283, GU056286, and GU056287; preferably any one of GU056282, GU056284, GU056283, GU056286, and GU056287; more preferably any one of GU056282, GU056286, and GU056287.

[0078] In some specific embodiments, the GenBank accession number of the coding sequence of the fatty alcohol oxidase selected in this invention includes at least one of AY538780, GU056288, AY538781, and AY538782; preferably any one of AY538780, GU056288, AY538781, and AY538782; more preferably any one of AY538780 and GU056288.

[0079] The present invention does not particularly limit the acquisition method of the above-mentioned fatty alcohol dehydrogenase and fatty alcohol oxidase, for example, they can be produced by fermentation of Escherichia coli. In some embodiments, the fatty alcohol dehydrogenase and fatty alcohol oxidase are produced by the following steps: (1) construction of recombinant Escherichia coli carrying the encoding genes of the fatty alcohol dehydrogenase and / or the fatty alcohol oxidase; (2) fermentation culture of recombinant Escherichia coli; (3) induction and expression of fatty alcohol dehydrogenase and / or the fatty alcohol oxidase; optionally (4) isolation and purification of fatty alcohol dehydrogenase and / or the fatty alcohol oxidase.

[0080] In some embodiments, the amount of fatty alcohol dehydrogenase and / or fatty alcohol oxidase added is 1-20 g / L, for example, it can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L, etc., preferably 5-15 g / L.

[0081] The second fermentation process lasts 2-8 hours. This short fermentation time can promote the conversion of fatty monocarboxylic acids and fatty hydroxy acids into long-chain dicarboxylic acids, while minimizing the degradation of long-chain dicarboxylic acids by entering cells through the β-oxidation pathway due to the simultaneous increase in the solubility of these acids.

[0082] Through the first and second fermentation processes, this invention can achieve the effects of large alkane addition, high yield of long-chain dicarboxylic acids, high conversion rate, and low impurities (fatty monoacids and fatty hydroxy acids) in the fermentation broth, thus meeting the requirements of production and subsequent extraction.

[0083] <Polymer Preparation Methods>

[0084] The present invention also provides a method for preparing a polymer, the method comprising:

[0085] The method described above for producing long-chain dicarboxylic acids by bio-fermentation is used to prepare long-chain dicarboxylic acids.

[0086] The polymerization step involves polymerizing the long-chain dicarboxylic acid with other monomers to prepare the polymer, wherein the other monomers include monomers containing at least one of the following groups: active hydrogen, epoxy group, and isocyanate.

[0087] For example, the other monomers include, but are not limited to, diols (e.g., ethylene glycol, 1,4-butanediol, 1,6-hexanediol, etc.), diamines (e.g., hexamethylenediamine, decanediamine, etc.), diepoxides (e.g., ethylene glycol diglycidyl ether, etc.), and diisocyanates (e.g., HDI, MDI, IPDI, etc.).

[0088] Example

[0089] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0090] In all the following embodiments of the present invention, the following experimental materials are used:

[0091] 1. Microbial strains

[0092] The fermentation strain selected for this patent is Candida virescens ATCC 20962 (purchased from Shanghai Microbiology Center (SHMCC)).

[0093] 2. Culture medium

[0094] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose. For solid YPD medium, an additional 20 g / L agar powder is added.

[0095] Seed culture medium: 10 g / L sucrose, 20 g / L glucose, 5 g / L yeast extract, 5 g / L corn steep liquor powder, 5 g / L peptone, 5 g / L potassium dihydrogen phosphate, 3 g / L urea.

[0096] Fermentation medium: 5 g / L yeast extract powder, 12 g / L corn steep liquor powder, 3 g / L urea, 8 g / L potassium nitrate, 5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, 0.1 g / L FeSO4, 0.5 g / L polyether defoamer XPJ999, 0.1 g / L growth factors (vitamin B1 0.05 mg / L, vitamin B2 0.02 mg / L, vitamin B5 0.02 mg / L, vitamin B7 0.05 mg / L, citric acid 0.1 mg / L), ZnSO4 2.27 mg / L, MgSO4 0.1 g / L, Na2MoO4 0.1 mg / L, MnCl2 5.3 mg / L;

[0097] Long-chain alkanes (total addition amount is 30% (w / v) of the initial fermentation volume) and glucose are sterilized separately.

[0098] In all the following embodiments of the present invention, the following detection method is used:

[0099] 1. OD detection method during fermentation process

[0100] n-Butanol:ethanol:water:chloroform = 10:10:10:1 (volume ratio v / v), dilute the fermentation broth 10-30 times, and at OD 600 Under the conditions.

[0101] 2. Gas-phase quantitative methods for long-chain dicarboxylic acids and hydroxy acids

[0102] Weigh 1g of fermentation broth, add 0.4mL of 6M NaOH solution, sonicate for 30min, and dry in a 105℃ oven. Add 5mL of 10% (v / v) sulfuric acid-methanol solution, incubate in a 70℃ water bath for 1h, cool, add 5mL of water and 5mL of internal standard solution (1mg / mL methyl undecanoate in chloroform), shake well, centrifuge at 4000rpm for 5min, take 1mL of the chloroform layer (lower layer solution) into a centrifuge tube, add 0.5g of anhydrous sodium sulfate to remove water, and filter through a membrane. The gas chromatography conditions were as follows: Column: 100% dimethyl polysiloxane column, ZB-130m×0.32mm×1μm or other equivalent column; Injection volume: 1μL; Split ratio: 5:1; Injector temperature: 270℃; Flow rate: 1mL / min; Column temperature: Programmed temperature: initial temperature 35℃, hold for 6 min, increase to 90℃ at 10℃ / min, hold for 2 min, increase to 180℃ at 20℃ / min, hold for 2 min, increase to 260℃ at 20℃ / min, hold for 8 min; Detector: FID; Detector temperature: 300℃; Carrier gas: N2: 25mL / min; H2: 30mL / min; Air: 300mL / min.

[0103] 3. Substrate detection methods

[0104] Accurately weigh 1.5g of fermentation broth into a 2ml centrifuge tube, incubate in a 95℃ metal bath for 10min, cool to room temperature, centrifuge at 12000r / min for 5min, transfer the upper oil layer to a 15mL centrifuge tube, add 5mL of internal standard solution, vortex to mix, and then filter the upper supernatant through an organic filter membrane for injection (or accurately weigh 0.5g of fermentation broth into a 15ml centrifuge tube, incubate in a boiling water bath for 10min, cool to room temperature, add 3mL of internal standard solution, vortex to mix, centrifuge, and then filter the upper supernatant through an organic filter membrane for injection). The chromatographic column used was a 100% dimethyl polysiloxane column, ZB-1 30m×0.32mm×1μm or other equivalent column; injection volume: 1μL; split ratio: 50:1; injection port temperature: 250℃; flow rate: 1mL / min; column temperature: programmed temperature rise: initial temperature 50℃, hold for 5min, increase to 230℃ at 20℃ / min, hold for 3min; detector: FID; detector temperature: 280℃; carrier gas: N2: 25mL / min; H2: 30mL / min; air: 300mL / min.

[0105] In all the following embodiments of the present invention, the following fermentation process and enzyme preparation process are used:

[0106] 1. Fermentation process control

[0107] The bacterial strain was revitalized by streak plating on YPD plates from the preservation tube. After incubation for approximately 16-24 hours, single colonies were picked and grown in primary seed (YPD) shake flasks for 16 hours. OD 600 When the measured value is 8, 2% of the inoculum is transferred to a primary seed tank (30℃, 0.06MPa, airflow 0.8vvm, DO > 15%) for cultivation. The inoculum is then cultured until the OD of the seed tank reaches 8%. 600 When the culture reaches approximately 15%, transfer it to a fermenter. Add glucose to the fermentation medium to a concentration of 2% (w / v), maintaining a glucose concentration no lower than 0.5% (w / v) throughout the process. Inoculate at a 15% (v / v) inoculation rate into the fermentation medium. Incubate at 30°C with dissolved oxygen at 30% and stirring at 300 rpm. Allow the initial cell growth to occur naturally at pH. After 8 hours of fermentation, add n-dodecane, maintaining an alkane concentration of 3% (w / v) to 5% (w / v) throughout the process. After 6 hours of fermentation, maintain the pH at 7.1. After 140 hours of fermentation, adjust the pH to 7.5, add 1-20 g / L of enzyme preparation, and continue fermentation for another 6 hours before transferring to the fermenter.

[0108] 2. Process control of shake-flask fermentation enzyme preparations

[0109] Take 30 μl of the constructed enzyme culture and inoculate it into LB medium containing Kan resistance. Incubate at 37°C for 15 h. Take 2 ml of the bacterial culture and transfer it to 100 ml of LB medium. Incubate to increase OD. 600When the bacterial growth reaches 0.4-0.6, IPTG is added for induction, the temperature is adjusted to 25℃, and after 20 hours, the fermentation broth is centrifuged to obtain cell precipitate, which is then stored in a -20℃ refrigerator for later use.

[0110] Example 1: Effects of different pH adjustments on the potency and conversion rate of long-chain dicarboxylic acids

[0111] Keeping other conditions constant during fermentation, the pH was maintained at 5.5, 5.9, 6.1, 6.5, 6.9, 7.1, 7.5, 7.7, 7.9, and 8.5 after 6 hours of fermentation. No further pH adjustments or enzyme additions were performed, and fermentation continued. The effect of 140 hours of fermentation on the potency and conversion rate of dodecanoic acid was detected, as shown in Table 1 below. When the pH was kept constant at 7.1 during fermentation, the potency reached its highest level, but the content of miscellaneous acids was also significantly higher.

[0112] Conversion rate calculation = (potency × fermentation broth mass) / ((total substrate addition - substrate residue) × fermentation broth mass)

[0113] Table 1. Effects of different pH adjustments on fermentation

[0114]

[0115] Example 2: Solubility effect of impurities under different pH conditions

[0116] Keeping other conditions constant in the fermentation process, 10 ml of fermentation broth fermented for 140 h was taken and the pH was adjusted to 5.5, 5.7, 5.9, 6.1, 6.5, 6.9, 7.1, 7.5, 7.7, 7.9, 8.1, 8.5, 8.9, and 9.0 respectively within 2 min using 10%-30% sodium hydroxide. After centrifugation, the supernatant was collected, and the content of hydroxy acids and fatty monoacids in the supernatant was detected by gas chromatography. The final concentration of each substance in the supernatant was calculated based on the dilution factor caused by the amount of sodium hydroxide added. As shown in Table 2 below, when the pH was adjusted to 7.5, the content of dodecanoic acid and dodecanoic hydroxy fatty acids in the supernatant gradually increased.

[0117] Table 2. Changes in the solubility of heteroacids under different pH conditions

[0118]

[0119] Example 3: Construction of expression vectors for alcohol dehydrogenase and alcohol oxidase and their effect on removing impurities

[0120] The following CDS sequences were selected from the database: GU056282, GU056284, GU056283, GU056286, GU056287; and GU056287 CDS sequences for fatty alcohol dehydrogenase (ADH): AY538780, GU056288, AY538781, AY538782. Using Escherichia coli as the host strain, the above enzyme genes were integrated into the pET28a vector using a one-step cloning technique. The enzyme preparations were obtained by shaking flask / fermentation tank culture and were encoded as A1, A2, A3, A4, A5, F1, F2, F3, and F4, respectively. Keeping other conditions constant during the fermentation process, in the later stage of fermentation (after 140 hours), the fermentation broth was dispensed into 1L parallel reactors, the pH was adjusted to 7.5, and 10g / L of the enzyme preparation prepared above was added. The temperature was 29℃, the pressure was 0.05MPa, the pH was 7.5, the air volume was 1vvm, the DO% was >30%, the initial rotation speed was 300rpm, and the reaction was catalyzed for 4-8 hours. As shown in Table 3 below, the enzyme coded as F2 (GU056288) had a better effect on reducing hydroxy acids.

[0121] Table 3. Concentration variations of hydroxy fatty acids in dodecanoic acid fermentation broth under different enzyme preparations

[0122]

[0123] Example 4: The effect of pH adjustment and enzyme preparation addition on dodecanoic acid in the later stage of fermentation

[0124] With other conditions in the fermentation process kept constant, the control group (CK) did not adjust the pH to 7.5 in the later stages of fermentation and did not add enzyme preparation F2 to produce dodecanoic acid; the experimental group adjusted the pH to 7.5 in the later stages of fermentation and added enzyme preparation F2 (10 g / L). Both the control and experimental groups fermented for 146 hours before being discharged. The formula for calculating the percentage of hydroxy acids is: hydroxy acid concentration / potency. As shown in Table 4 below, the experimental group had a significant advantage in reducing hydroxy acids.

[0125] Table 4. Effects of pH adjustment and enzyme addition on dodecanoic acid production

[0126]

[0127] Example 5: Application of pH adjustment and enzyme preparation in the production of different long-chain dicarboxylic acids during the later stage of fermentation

[0128] n-Decadecane, n-tetrazane, and n-tetradecane were added to the fermenter of Candida virescens ATCC 20962, respectively. The experimental procedure was the same as in Example 4. After the fermenter was placed in the tank, samples were taken to detect the contents of the corresponding dicarboxylic acid, monocarboxylic acid, and hydroxy acid. The conversion rate and the proportion of miscellaneous acids were calculated. Fermentation was carried out for 140 hours without adjusting the pH to 7.5 and without adding 10 g / L of enzyme preparation as a control experiment. Fermentation was carried out for 150-160 hours.

[0129] Table 5. Effects of pH adjustment and enzyme addition on the production of different long-chain dicarboxylic acids.

[0130]

[0131] The results are shown in Table 5. Adjusting the pH and adding enzyme preparations can effectively reduce the content of hydroxy acids and monoacids in the fermentation broth.

Claims

1. A method for producing long-chain dicarboxylic acids by bio-fermentation, characterized in that, The method includes: First fermentation process: The seed liquid of fermentation bacteria is introduced into the fermenter and fermentation begins. Substrate is added after 6-10 hours of fermentation. The pH is controlled at 5.0-8.5 after 6 hours of fermentation, and the fermentation lasts for 100-160 hours. Second fermentation process: The pH of the fermented product obtained in the first fermentation process is further adjusted to 7.0-9.0, and fatty alcohol dehydrogenase and / or fatty alcohol oxidase are added, and fermentation is carried out for 2-8 hours. The fermentation bacteria include at least one of *Candida tropicalis*, *Candida viswanathii*, and *Candida sake*; the substrate is any one of C10-C18 n-alkanes. The long-chain dicarboxylic acid is any one of the C10-C18 long-chain dicarboxylic acids. The GenBank accession number for the coding sequence of the fatty alcohol dehydrogenase is any one of GU056282, GU056286, and GU056287, and the GenBank accession number for the coding sequence of the fatty alcohol oxidase is any one of AY538780 and GU056288.

2. The method according to claim 1, characterized in that, During the first fermentation process, the OD of the seed liquid 600 The value is 10-15, and the amount of seed liquid added is 10% (v / v)-20% (v / v) of the initial fermentation broth.

3. The method according to claim 1, characterized in that, During the first fermentation process, the concentration of the substrate in the fermentation broth is maintained at 1% (w / v) to 5% (w / v).

4. The method according to claim 1, characterized in that, The conditions controlled during the first fermentation process include one or more of the following: The temperature should be controlled between 28-35℃. Control the airflow to 0.5-1.4 vvm. The control pressure is 0.05-0.1 MPa. Dissolved oxygen should be controlled to be greater than 15%.

5. The method according to any one of claims 1-4, characterized in that, In the second fermentation process, the amount of fatty alcohol dehydrogenase and / or fatty alcohol oxidase added is 1-20 g / L.

6. The method according to any one of claims 1-4, characterized in that, The fermenter contains a fermentation medium and a carbon source; the fermentation medium includes a nitrogen source, inorganic salts, and growth factors.

7. The method according to any one of claims 1-4, characterized in that, The preparation process of the seed culture includes: activating and culturing the fermentation bacteria in a YPD shake flask, and when the OD... 600 When the temperature reaches 8-15°C, inoculate with seed culture medium for cultivation, with the inoculation amount being 1% (v / v)-5% (v / v) of the seed culture medium.

8. A method for preparing a polymer, characterized in that, The method includes: The method according to any one of claims 1-7 is used to prepare long-chain dicarboxylic acids. The polymerization step involves polymerizing the long-chain dicarboxylic acid with other monomers to prepare the polymer. The other monomers include monomers containing active hydrogen, epoxy groups, and at least one group selected from isocyanate groups.

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