Biological degradation process of polyester, and use of product produced thereby

Abstract

The present application relates to the biological degradation process of a polyester, and a use of a product produced thereby, and, specifically, to a method in which an enzyme is reacted with a polyester that comprises a dicarboxylic acid and ethylene glycol (EG) as units in the presence of ammonia, thereby precipitating and recovering the dicarboxylic acid by means of sulfuric acid, and in which microorganisms are cultured in a medium comprising ethylene glycol and ammonium sulfate that have been produced by the enzyme and precipitation reaction, thereby producing a target product.

Description

Biological degradation process of polyester and uses of products produced thereby

[0001] The present application relates to a biological degradation process of polyester and the use of products produced thereby.

[0002]

[0003] More than 400 million tons of plastics are newly produced each year. As environmental concerns surrounding waste plastics grow, efforts are being made to reduce production through regulations on single-use products and the use of alternative plastics. However, production continues to increase annually. Polyethylene terephthalate (PET), which accounts for less than 10% of all plastics, produces approximately 360 million tons annually. Due to its primary use in disposable products, PET is considered the plastic with the shortest life cycle. Recycling of waste plastics includes mechanical recycling, pyrolysis, and chemical recycling, each of which is either commercialized or in the final stages of research toward commercialization. While each technology offers potential solutions to the waste plastic issue, none of the existing methods are perfect due to the impacts of downcycling on quality, carbon neutrality, resource depletion, and eutrophication of seawater and freshwater.

[0004] A series of research results have been published on the use of biological technology to decompose plastics in order to solve environmental problems caused by waste plastics, such as microplastics, greenhouse gas emissions, and resource depletion. However, an efficient decomposition process is still required, and to date, there has been no research on the application of byproducts generated during the decomposition process.

[0005] [Prior Art Literature]

[0006] (Patent Document 1) US 10851355 B2

[0007]

[0008] The present application relates to a biological degradation process for polyester and the use of products produced thereby.

[0009]

[0010] The purpose of the present application is to provide a method for producing a target product, which comprises the steps of precipitating a dicarboxylic acid converted from a polyester containing dicarboxylic acid and ethylene glycol (EG) as units by an enzymatic reaction in the presence of ammonia (NH3) with sulfuric acid and recovering the precipitated dicarboxylic acid, and culturing a microorganism that produces the target product in a medium containing ethylene glycol (EG) and ammonium sulfate (AMS) produced by the enzymatic and precipitation reactions.

[0011] Another object of the present application is to provide a method for preparing a medium, in which a dicarboxylic acid converted from a dicarboxylic acid and ethylene glycol as units by an enzymatic reaction in the presence of ammonia is precipitated by sulfuric acid and recovered, and ethylene glycol and ammonium sulfate produced by the enzymatic and precipitation reactions are added to a microbial culture medium.

[0012] Another object of the present application is to provide a method for decomposing polyester, comprising the step of reacting polyester with an enzyme in the presence of ammonia.

[0013]

[0014] According to the polyester decomposition process provided in the present application, the efficiency of polyester decomposition is increased, and by-products generated in the polyester decomposition process can be usefully used, thereby providing economic and environmental advantages.

[0015]

[0016] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0017] Furthermore, those skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments of the present application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0018] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the technical field to which this application pertains and the content of this application.

[0019]

[0020] One aspect of the present application is a method for decomposing polyester, comprising a step of reacting an enzyme with a polyester containing dicarboxylic acid and ethylene glycol (EG) as monomers in the presence of ammonia (NH3).

[0021] In one embodiment, ammonia present within the reactor where the enzymatic reaction occurs acts as a pH adjusting substance. That is, the pH lowering effect caused by dicarboxylic acid generated during the polyester decomposition process can be buffered by the presence of ammonia.

[0022] The term "polyester" refers to a polymer containing an ester functional group in the main chain of its structure. For example, polyethylene terephthalate (PET) is a semi-aromatic copolymer composed of two monomers, terephthalic acid (TPA) and ethylene glycol. The polyester may further contain monomers other than dicarboxylic acid and ethylene glycol.

[0023] In one embodiment, the polyester of the present application may include terephthalic acid as a monomer. In any one of the above-described embodiments, the polyester may be selected from polyethylene terephthalate, polyethylene terephthalate glycol (PETG), polyethylene co-isosorbide terephthalate (PEIT), polytrimethylene terephthalate (PTT), polybutylene adipate terephthalate (PBAT), polycyclohexylenedimethylene terephthalate (PCT), and polybutylene terephthalate (PBT). Specifically, the dicarboxylic acid may be, but is not limited to, terephthalic acid.

[0024] In any one of the embodiments described above, the enzymatic reaction is performed under conditions of about pH 6 to 10. For example, it may be performed under conditions of about pH 7 to 9.

[0025] In any one of the above-described embodiments, the enzymatic reaction is performed under a temperature condition of about 20°C to 80°C. For example, it may be performed under a temperature condition of about 30°C to 70°C, or about 40°C to 60°C.

[0026] In any one of the above-described embodiments, the ammonia is in a gaseous state. For the purposes of the present application, since the ammonia is in a gaseous state, the water level of the reactor does not change, so that the initial input amount of polyethylene terephthalate substrate during polyethylene terephthalate decomposition can be increased, thereby increasing the production of terephthalic acid, a useful product. Meanwhile, the gaseous ammonia can dissolve in water to form an aqueous solution.

[0027] In any one of the above-described embodiments, the ammonia gas injection rate is 0.05 vvm (vessel volume per minute) to 5 vvm.

[0028] In any one of the embodiments described above, the enzyme is an enzyme having polyethylene terephthalate decomposing activity. With respect to polyethylene terephthalate decomposing enzymes, the disclosures in US 2020-0048621 A1 and Three-directional engineering of IsPETase with enhanced protein yield, activity, and durability, Journal of Hazardous Materials, Volume 459, 2023, 132297, ISSN 0304-3894 are incorporated herein by reference in their entirety. As an example, the enzyme may be, but is not limited to, Z1-PETase.

[0029] Meanwhile, in relation to the amino acid sequence in the present application, even if it is described as a polypeptide "comprising" the amino acid sequence described in a specific sequence number, a polypeptide "consisting of" the amino acid sequence described in a specific sequence number, or a polypeptide or protein "having" the amino acid sequence described in a specific sequence number, it is obvious that a protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added can also be used in the present application, as long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, it may be a case in which the amino acid sequence has an addition of a sequence that does not alter the function of the protein at the N-terminus and / or C-terminus, a mutation that may occur naturally, a silent mutation thereof, or a conservative substitution, but is not limited thereto.

[0030] For example, the polyethylene terephthalate degrading enzyme may comprise an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% homology or identity thereto with the Z1-PETase. For example, in the sequence of the polyethylene terephthalate degrading enzyme of the present application, the amino acid corresponding to amino acid 121 from the N-terminus of the polyethylene terephthalate degrading enzyme (IsPETase) sequence derived from Ideonella sakaiensis is glutamic acid, the amino acid corresponding to amino acid 186 is histidine, the amino acid corresponding to amino acid 242 is threonine, the amino acid corresponding to amino acid 246 is aspartic acid, the amino acid corresponding to amino acid 233 is cysteine, the amino acid corresponding to amino acid 282 is cysteine, the amino acid corresponding to amino acid 181 is valine, the amino acid corresponding to amino acid 180 is valine, the amino acid corresponding to amino acid 37 is aspartic acid, the amino acid corresponding to amino acid 132 is glutamic acid, the amino acid corresponding to amino acid 224 is glutamic acid, and the amino acid corresponding to amino acid 171 is It is cysteine, and the amino acid corresponding to amino acid number 193 may be cysteine.

[0031] In any one of the embodiments described above, the enzyme may be used at 0.01% to 1% (w / w) per polyester medium.

[0032]

[0033] Another aspect of the present application is a method for producing a target product, comprising the steps of (a) recovering a dicarboxylic acid converted from a polyester containing a dicarboxylic acid and ethylene glycol as units by an enzymatic reaction in the presence of ammonia by precipitating the dicarboxylic acid with sulfuric acid, and (b) culturing a microorganism that produces the target product in a medium containing ethylene glycol and ammonium sulfate produced by the enzymatic and precipitation reactions.

[0034] In the method for producing the above target product, the enzymatic reaction of step (a), the polyester and the dicarboxylic acid, and the method for decomposing the polyester described above can be applied.

[0035] The method of the present invention can reduce waste from the polyester decomposition process. For example, the decomposition of polyethylene terephthalate produces terephthalic acid, a useful product, while the byproducts, ethylene glycol and ammonium sulfate, can be directly used for microbial culture without additional purification, thereby reducing waste from the polyethylene terephthalate decomposition process.

[0036]

[0037] In any one of the embodiments described above, the ethylene glycol and ammonium sulfate may be present in the solution remaining after the dicarboxylic acid is recovered in step (a).

[0038] In any one of the embodiments described above, the dicarboxylic acid may be terephthalic acid.

[0039] In one embodiment, the method for producing the target product may comprise two steps: (i) an enzymatic reaction and (ii) a terephthalic acid precipitation reaction. The method for decomposing polyester described above may be applied to the enzymatic reaction (i).

[0040] The type of acid used in the above (ii) terephthalic acid precipitation reaction is sulfuric acid (H2SO4), and ammonium sulfate is produced when sulfuric acid reacts with ammonia. In the terephthalic acid precipitation reaction, the pH of the medium can be adjusted to about 1 to 2 by sulfuric acid, but is not limited thereto.

[0041]

[0042] In any one of the above-described embodiments, ethylene glycol (EG) and ammonium sulfate (AMS), which are by-products produced during the decomposition of terephthalic acid, can be transferred to the medium without an additional purification process.

[0043] In any one of the embodiments described above, the method further comprises a step of filtering the solution containing terephthalic acid (TPA). The filter cutoff can be applied by a person skilled in the art. After the filtering step, terephthalic acid is recovered from the solution, and the remaining retentate can be subjected to an enzymatic decomposition reaction for the remaining polyethylene terephthalate that has not been decomposed, or recycled to the reactor so that the remaining terephthalic acid can be precipitated.

[0044] In any one of the embodiments described above, the solution containing a salt of terephthalic acid may be subjected to a concentration step in which water contained in the solution is removed and terephthalic acid precipitation can be induced.

[0045]

[0046] In one embodiment, the microorganism producing the target product of the present application may be a microorganism that naturally possesses the ability to produce the target product, or may be a microorganism that has been genetically modified to have the ability to produce the target product enhanced or enhanced. Such genetic modifications may be appropriately selected depending on the type of target product. For example, reference may be made to the disclosures in US 11499173 B2, US 10947495 B2, and others.

[0047] In any one of the embodiments described above, the target product is an L-amino acid. Examples thereof include, but are not limited to, threonine, isoleucine, lysine, and cysteine.

[0048] In any one of the embodiments described above, the target product is a precursor of an L-amino acid, such as, but not limited to, O-phosphoserine.

[0049] In any of the aforementioned embodiments, the microorganism may be a microorganism capable of utilizing ammonium sulfate as a nitrogen source. For example, the microorganism may be a microorganism that naturally possesses the ability to utilize ammonium sulfate as a nitrogen source, or a microorganism that has been genetically modified to have the ability to utilize ammonium sulfate as a nitrogen source enhanced.

[0050] In any one of the embodiments described above, the microorganism may be a microorganism capable of utilizing ethylene glycol as a carbon source. For example, the microorganism may be a microorganism that naturally has the ability to utilize ethylene glycol as a carbon source, or may be a microorganism that has been genetically modified to have or have enhanced the ability to utilize ethylene glycol as a carbon source. For example, the microorganism may include a modification in which one or more genes from among foreign fucO, aldA, glcD, glcE, glcF, gcl, and glxR genes are introduced, or one or more genes from among endogenous fucO, aldA, glcD, glcE, glcF, gcl, and glxR are enhanced. For example, foreign fucO, aldA, glcD, glcE, and glcF may be introduced, or expression of endogenous fucO, aldA, glcD, glcE, and glcF may be enhanced. As another example, foreign fucO, aldA, glcD, glcE, glcF, gcl and glxR may be introduced, or endogenous fucO, aldA, glcD, glcE, glcF, gcl and glxR may be enhanced.

[0051] The above modifications may confer or enhance the ability of microorganisms to utilize ethylene glycol as a carbon source. For example, the fucO, aldA, glcD, glcE, glcF, gcl, and glxR genes may be derived from E. coli, but are not limited thereto, and any genes with the same function may be included.

[0052] In any one of the embodiments described above, the microorganism may be a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia.

[0053] For example, the above Corynebacterium genus microorganisms include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, and Corynebacterium. It may be, but is not limited to, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens.

[0054] For example, the above Escherichia genus microorganism may be Escherichia coli.

[0055] The term "cultivation" in this application refers to growing microorganisms under appropriately controlled environmental conditions. The culturing process of this application can be performed according to appropriate culture conditions known in the art. These culturing processes can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0056] In this application, the term "medium" means a material mixed with nutrients as the main component necessary for culturing the microorganism of this application, and supplies nutrients and growth factors, including water essential for survival and growth.

[0057] The medium used for culturing the microorganism of the present application may contain ethylene glycol and ammonium sulfate produced by the above-described enzyme and precipitation reaction as a carbon source and a nitrogen source.

[0058] For example, the culture medium used for culturing the microorganism of the present application may be prepared by adding ethylene glycol and ammonium sulfate produced by the enzyme and precipitation reaction described above to a commercially available medium or a commonly used medium.

[0059] For example, the medium used for culturing microorganisms of the present invention may further include other carbon sources and nitrogen sources in addition to ethylene glycol and ammonium sulfate. For example, the carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto. The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0060] In addition, the culture medium used for culturing the microorganism of the present application may be a conventional medium containing other appropriate nutrients, inorganic compounds, amino acids, and / or vitamins.

[0061] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0062] In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but is not limited thereto.

[0063] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0064] The target product produced by the culture of the present application may be secreted into the medium or remain within the cells.

[0065] The method for producing the target product of the present application may additionally include a step of recovering the target product from the culture medium or microorganism. The recovery step may be included after the culturing step.

[0066] The above recovery may be performed by collecting the target product using a suitable method known in the art according to the culture method of the microorganism of the present application, such as a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the target product may be recovered from the medium or microorganism using a suitable method known in the art.

[0067]

[0068] Another aspect of the present application provides a method for preparing a medium, wherein a dicarboxylic acid converted from polyethylene terephthalate by an enzymatic reaction in the presence of ammonia is precipitated with sulfuric acid and recovered, and ethylene glycol and ammonium sulfate produced by the enzyme and precipitation reaction are added to a microbial culture medium. The enzymatic reaction, precipitation, recovery, microorganisms, and culture medium are as described above.

[0069]

[0070] Hereinafter, this application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0071]

[0072] Example 1. Comparison of alkaline substances used for pH control in biological PET degradation reactions.

[0073] For the experiment, PET-decomposing enzyme, 10 mM phosphate buffer, and PET medium were added to stirred reactors with a total volume of 3000 ml, and the enzyme reaction was performed. Each reactor was equipped with a heating / cooling device to maintain temperature during the reaction and a pH probe (Mettler Toledo 405-DPAS-SC-K8S) to maintain pH. During the reaction, stirring was performed at 500 rpm to ensure good mixing of the PET medium, buffer, and PET-decomposing enzyme, and 5 N NaOH and ammonia gas (0.5 vvm) were used as a base substance to maintain pH. The temperature and pH were maintained at 50 degrees and 8, respectively, during the reaction.

[0074]

[0075] For the above experiment, 75 g to 600 g of B-PET (2.5% to 20% of the total weight of the initial reaction medium) was prepared and reacted with the enzyme for 24 hours. B-PET (PET sample derived from PET bottles) was obtained through the following processing. Transparent PET bottles were crushed using a crusher, and then the crushed PET was melted in a high-temperature oven at 270°C. The molten PET was immediately immersed in 4°C water to harden. The obtained hardened PET was subjected to a cryogenic crushing process and then passed through a steel mesh to obtain PET powder of 300 μm or less.

[0076] The enzyme used is a variant of PET hydrolase (US 2020-0048621 A1) known to decompose PET, which is an IsPETase variant Z1-PETase (IsPETase (S121E / D186H / N246D / S242T / N233C / S282C / P181V / A180V / N37D / R132E / R224E / A171C / S193C) described in Three-directional engineering of IsPETase with enhanced protein yield, activity, and durability, Journal of Hazardous Materials, Volume 459, 2023, 132297, ISSN 0304-3894, and this enzyme was used at 1:1000 (0.1% w / w) per PET medium used.

[0077]

[0078] The above conditions are summarized and shown in Table 1.

[0079] Experimental group A-1A-2B-1B-2B-PET Input amount 75g600g Enzyme input amount 75mg600mg Basic substance for pH maintenance 5N NaOH Ammonia gas (0.5vvm) 5N NaOH Ammonia gas (0.5vvm)

[0080] Biological PET degradation reactions were performed under the above four conditions, and the concentrations of PET degradation products, MHET (Mono(2-hydroxyethyl) terephthalate), TPA, and EG, were measured. The volume of the final reaction solution was also measured. The results for each experiment are summarized and presented in Table 2.

[0081]

[0082] Experimental group A-1A-2B-1B-2MHET production concentration (g / L)9.041069.278.5TPA production concentration (g / L)5.56.241.143.0EG production concentration (g / L)1.922.0115.416.8Volume of reaction solution after reaction (ml)3130300039103000

[0083]

[0084] The above results confirmed the potential use of ammonia gas as a pH-adjusting alkaline agent for biological PET degradation reactions. Furthermore, when a gaseous alkaline agent was used rather than a liquid one to obtain the final degradation product, TPA, the degradation rate was higher than when using NaOH solution. Furthermore, the addition of a gaseous alkaline agent did not significantly affect the volume increase of the reaction solution after the reaction, thereby increasing productivity per batch.

[0085]

[0086] Example 2. Recovery of TPA and residual substances after biological PET degradation reaction using ammonia gas.

[0087] In the reaction of Example 1, the PET decomposition reaction using NaOH and ammonia gas was terminated, and the remaining PET was removed by filtration. (Small basket centrifuge)

[0088] To recover the TPA salt from the remaining liquid after filtration, sulfuric acid (99%, Daejung Chemical) was added to adjust the pH of the solution to approximately 1 to 2, thereby precipitating the TPA salt. During this process, the PET decomposition reaction was completed, and the generated MHET was naturally converted to TPA.

[0089] Only the TPA salt was recovered, and the components of the remaining solution were analyzed. Cations and anions were analyzed using ion chromatography, and EG was analyzed using HPLC. The analysis results showed that sodium sulfate (SS) and EG were detected in the residual solution of the reaction solution using NaOH, and AMS and EG were detected in the residual solution of the reaction solution using ammonia gas. The results described above are summarized and presented in Table 3.

[0090]

[0091] Experimental group A-1A-2B-1B-2Ammonium sulfate (AMS) concentration (g / L)04.16038.13Ethylene glycol (EG) concentration (g / L)2.022.2316.117.4

[0092] From the above results, it was possible to identify substances present in the solution remaining after TPA recovery according to the alkaline substances added to correct the pH of TPA formed during the biological PET degradation process. The solution formed in the B-2 experimental group, which had the highest AMS and EG concentrations, was designated PET-BY and further experiments were conducted.

[0093]

[0094] Example 3. Production of L-amino acids using E. coli strains utilizing PET degradation byproducts.

[0095] Example 3-1. Production of L-threonine using an E. coli strain utilizing PET degradation byproducts.

[0096] E. coli strains can naturally grow using EG as a carbon source, but two genes were introduced to utilize EG effectively. The two genes fucO (NCBI Gene ID: 947273) and aldA (NCBI Gene ID: 945672) were cloned into the vector pEG03 (Addgene, #154138) into the E. coli strain KFCC10718 (Korean Patent Publication No. 1992-0008365), which produces L-threonine, to create the KFCC10718 / pEG03 strain. Specifically, the vector was transformed into the KFCC10718 strain through electroporation and spread on LB (Luria-Bertani) plate medium (tryptone 10 g, yeast extract 5 g, NaCl 10 g, and agar 1.5% / L) containing the selection marker chloramphenicol, and cultured overnight at 37°C, and then strains showing resistance to chloramphenicol were selected. PET-BY produced in Example 2 was used as a carbon source and a nitrogen source, respectively, in the culture of the manufactured E. coli strain with enhanced EG utilization ability to produce L-threonine, and the possibility of increasing the growth of E. coli and L-amino acid productivity was confirmed. The strain was cultured overnight in LB solid medium in an incubator at 33°C, and one platinum ear was inoculated into a baffle flask containing 25 mL of a titer medium containing glucose with the following composition. This was cultured in an incubator at 33°C and 200 rpm for 50 hours, and the strain growth (OD562), L-threonine production concentration, and EG consumption were analyzed, which are shown in Table 4. The experimental group used a medium with 100 ml of PET-BY prepared in Example 2 added. The EG concentration of the medium used in the experimental group was approximately 1.88 g / L.

[0097]

[0098] <Production medium (pH 7.0)>

[0099] Glucose 70 g, (NH4)2SO4 25 g, KH2PO4 1 g, MgSO4·H2O 0.5 g, FeSO4·H2O 5 mg, MnSO4·H2O 5 mg, yeast extract 2 g, CaCO3 30 g (based on 1 liter of distilled water).

[0100] Culture conditions OD562L-threonine concentration (g / L)EG concentration (g / L)Production medium 22.127.60PET-BY containing production medium 23.529.10.73

[0101] As shown in the above results, we confirmed that strain growth and increased L-threonine concentration were observed in the medium containing PET-BY. This suggests that EG and AMS contained in PET-BY were used as carbon and nitrogen sources, respectively, for microbial growth and amino acid production.

[0102]

[0103] Example 3-2. Production of OPS (O-phosphoserine) using E. coli strains utilizing PET degradation byproducts.

[0104] Additionally, in order to verify the possibility of production for other amino acids, the pEG03 vector was introduced into the E. coli strain KCCM11815P (US 10947495 B2) producing OPS, a precursor of L-cysteine, using the same method as Example 3-1, and the strain was cultured using the same method as Example 3-1. The results were analyzed and shown in Table 5.

[0105] Culture conditions OD600 OPS concentration (g / L) EG concentration (g / L) Production medium 20.11.680 PET-BY containing production medium 22.61.810.83

[0106]

[0107] From the above results, it was confirmed that PET-BY can be used as a carbon source and nitrogen source to produce L-threonine and OPS, and further L-amino acids, using E. coli.

[0108]

[0109] Example 4. Production of L-amino acids based on a Coryne strain endowed with EG utilization ability using PET degradation by-products.

[0110]

[0111] Corynebacterium glutamicum ATCC13032 is a strain widely used for L-amino acid production, but it does not naturally have a metabolic pathway that can utilize EG. Therefore, a strain with a metabolic pathway that can utilize EG was created in a Corynebacterium strain used for L-amino acid production, and it was cultured in an amino acid production medium supplemented with PET-BY, verifying that the production concentration of amino acids increased.

[0112]

[0113] Example 4-1. Production of L-lysine using a Corynebacterium strain with EG utilization ability utilizing PET degradation byproducts.

[0114]

[0115] To impart EG utilization ability to Corynebacterium glutamicum CJ3P strain (US 11499173 B2) producing L-lysine, a strain was constructed in which the fucO (NCBI Gene ID: 947273), aldA (NCBI Gene ID: 945672), glcD (NCBI Gene ID: 947353), glcE (NCBI Gene ID: 2847718), glcF (NCBI Gene ID: 2847717), gcl (NCBI Gene ID: 945394), and glxR (NCBI Gene ID: 945146) genes of Escherichia coli were introduced. To amplify the above genes, the E. coli-Corynebacterium shuttle vector pCES208 (J. Microbiol. Biotechnol., 18:639-647, 2008) was used as a parent vector. The pCES208-PgapA-glcDEF-fucO-aldA-gcl-glxR vector was constructed by operably linking the gapA promoter (SEQ ID NO: 13) and the above seven genes. Specifically, the gapA promoter was amplified using the chromosome of Corynebacterium ATCC13032 as a template and sequence numbers 1 and 2, the glcD, glcE, and glcF genes were amplified using the chromosome of Escherichia coli MG1655 as a template and sequence numbers 3 and 4, the fucO gene was amplified using sequence numbers 5 and 6, the aldA gene was amplified using sequence numbers 7 and 8, the gcl gene was amplified using sequence numbers 9 and 10, and the glxR gene was amplified using sequence numbers 11 and 12 by PCR. Sequence numbers 1 to 12 used for gene amplification are shown in Table 6.

[0116]

[0117] SEQ ID NO: 1AGGGAACAAAAGCTGGGTACAAGCCTAAAAACGACCGAGCSEQ ID NO: 2TCTTCGTACAAGATGCTCATGTTGTGTCTCCTCTAAAGATSEQ ID NO: 3ATCTTTAGAGGAGACACAACATGAGCATCTTGTACGAAGASEQ ID NO: 4atcattctgttagccatcatAATGGAAACAGACCAGTTATTTSEQ ID NO: 5ATAACTGGTCTGTTTCCATTatgatggctaacagaatgattctSEQ ID NO: 6tgttgaacgggtactgacatTTTGCATATCGACGGCAATTSEQ ID NO: 7AATTGCCGTCGATATGCAAAatgtcagtacccgttcaacaSEQ ID NO: 8acggctctcatttttgccatttaagactgtaaataaaccaSEQ ID NO: 9tggtttatttacagtcttaaatggcaaaaatgagagccgtSEQ ID NO: 10ccaataaatcccagtttcatttattcatagtgcatgaagcSEQ ID NO: 11gcttcatgcactatgaataaatgaaactgggatttattggSEQ ID NO: 12AACTAGTGGATCCCCCGGGCtcaggccagtttatggttag

[0118]

[0119] Meanwhile, the pCES208 vector, which was treated with restriction enzymes PstI and KpnI and heat-treated at 65°C for 20 minutes, was ligated with the inserted DNA fragment amplified through the PCR using an Infusion Cloning Kit and then transformed into Escherichia coli DH5α. The strain was spread on LB solid medium containing kanamycin (25 mg / l). Colonies transformed with the vector into which the desired gene was inserted were selected through PCR, and the plasmid was obtained using a conventionally known plasmid extraction method. The constructed vector was transformed into the lysine-producing Corynebacterium glutamicum CJ3P strain by the electric pulse method (Appl. Microbiol. Biotecenol. (1999) 52:541-545), and colonies were obtained by spreading on a complex plate medium containing kanamycin (25 mg / l). The L-lysine-producing Corynebacterium glutamicum strain with EG utilization ability was cultured using the following method, and the strain growth (OD562) and L-lysine production concentration were analyzed, which are shown in Table 7. Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and cultured at 30˚C for 20 hours with shaking at 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured at 32˚C for 72 hours with shaking at 200 rpm. The compositions of the seed medium and the production medium are as follows. The experimental group used the medium to which 100 ml of PET-BY prepared in Example 2 was added. The EG concentration of the medium used in the experimental group was approximately 1.5 g / L.

[0120]

[0121] <Seed medium (pH 7.0)>

[0122] Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 100 ㎍, thiamine HCl 1000 ㎍, calcium-pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)

[0123]

[0124] <Production medium (pH 7.0)>

[0125] Glucose 100 g, (NH4)2SO4 40 g, Soy protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH2PO4 1 g, MgSO4·7H2O 0.5 g, Biotin 100 μg, Thiamine hydrochloride 1000 μg, Calcium-pantothenic acid 2000 μg, Nicotinamide 3000 μg, CaCO3 30 g (per 1 liter of distilled water).

[0126]

[0127] Culture conditions OD562L-Lysine concentration (g / L)EG concentration (g / L)Production medium 80.510.10PET-BY containing production medium 84.911.50.58

[0128]

[0129] Example 4-2 Production of L-threonine and L-isoleucine based on a Corynebacterium strain endowed with EG utilization ability using PET degradation by-products

[0130]

[0131] In order to impart EG utilization ability to the L-threonine-producing Corynebacterium glutamicum CJ3P::hom(G378E) strain and the L-isoleucine-producing Corynebacterium glutamicum CJ3P::hom(G378E)-ilvA(V323A) strain (US 11499173 B2), strains into which the fucO, aldA, glcD, glcE, glcF, gcl, and glxR genes of E. coli were introduced were constructed using the same method as in Example 4-1. The strains were cultured using the same method as in Example 4-1, and the growth (OD562) of the strains and the production concentrations of L-threonine and L-isoleucine were measured, and the results are shown in Tables 8 and 9.

[0132] Culture conditions OD562L-threonine concentration (g / L)EG concentration (g / L)Production medium75.63.60PET-BY containing production medium81.14.20.54

[0133] Culture conditions OD562L-Isoleucine concentration (g / L)EG concentration (g / L)Production medium73.50.70PET-BY containing production medium79.81.10.72

[0134]

[0135] The above results confirmed that PET-BY can be used as a carbon and nitrogen source for the production of L-amino acids using a Corynebacterium strain endowed with EG utilization ability. The results of Examples 3 and 4 above demonstrate that byproducts generated through the biological decomposition of waste PET can be utilized for the production of amino acids using microorganisms.

[0136]

[0137] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. (a) A step in which a dicarboxylic acid converted from a polyester containing dicarboxylic acid and ethylene glycol (EG) as units by an enzymatic reaction in the presence of ammonia (NH3) is precipitated and recovered with sulfuric acid, and (b) a step of culturing a microorganism producing a target product in a medium containing ethylene glycol and ammonium sulfate (AMS) produced by the enzyme and precipitation reaction, Method of producing the target product.

2. A production method according to claim 1, wherein the enzymatic reaction is performed under conditions of pH 6 to 10.

3. A production method according to claim 1, wherein the ammonia is a gas.

4. A production method according to claim 1, wherein the polyester contains terephthalic acid (TPA) as a unit.

5. A production method according to claim 1, wherein the polyester is selected from among polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyethylene co-isosorbide terephthalate (PEIT), polytrimethylene terephthalate (PTT), polybutylene adipate terephthalate (PBAT), polycyclohexylenedimethylene terephthalate (PCT), and polybutylene terephthalate (PBT).

6. A production method in the first paragraph, wherein the ethylene glycol and ammonium sulfate are present in the solution remaining after the dicarboxylic acid is recovered in step (a).

7. A production method in claim 6, wherein the dicarboxylic acid is terephthalic acid.

8. A production method according to claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia.

9. A production method according to claim 1, wherein the target product is an L-amino acid.

10. A production method in the third paragraph, wherein the ammonia gas concentration is 0.05 vvm to 5 vvm.

11. A production method in the first paragraph, wherein the amount of enzyme used in the enzyme reaction is 0.01% to 1% (w / w) per polyester medium.

12. A production method in which ethylene glycol (EG) and ammonium sulfate (AMS), which are by-products of the polyester decomposition step, are transferred to the medium without an additional purification process.

13. In the presence of ammonia, dicarboxylic acid is converted from polyester containing dicarboxylic acid and ethylene glycol as monomers by an enzymatic reaction and is recovered by precipitation with sulfuric acid. A method for preparing a culture medium for microorganisms, comprising adding ethylene glycol and ammonium sulfate produced by the above enzyme and precipitation reactions to a culture medium for microorganisms.

14. A method for decomposing polyester, comprising a step of reacting polyester with a polyester-decomposing enzyme in the presence of ammonia.

15. A method for decomposing polyester, characterized in that in paragraph 14, the enzymatic reaction occurs in a reaction tank, and the water level of the reaction tank does not change because the ammonia is a gas.