A high-efficiency enzymatic hydrolysis process of polycaprolactam
By combining sulfuric acid pretreatment with the synergistic effect of a multi-enzyme system, the problem of the difficult degradation of PA6 waste was solved, achieving efficient and low-cost recovery of aminocaproic acid monomers and addressing the environmental risks and high energy consumption issues of traditional methods.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YUANTIAN BIOTECHNOLOGY (TIANJIN) CO LTD
- Filing Date
- 2026-03-12
- Publication Date
- 2026-05-29
AI Technical Summary
Polycaprolactam (PA6) waste is difficult to degrade, and traditional treatment methods pose environmental risks and high energy consumption. Furthermore, bio-enzymatic hydrolysis is inefficient and cannot meet the recycling needs of high-performance materials.
The crystal structure of PA6 was destroyed by sulfuric acid pretreatment, and enzymatic hydrolysis was carried out by a multi-enzyme system. The high efficiency degradation of PA6 was achieved by the synergistic effect of sulfuric acid and hydrolases NylB-DNY and NylCk-TS. The high purity of the product was achieved by adjusting the calcium hydroxide and oxalic acid solutions.
It significantly improved the degradation efficiency of PA6, reaching over 93%, simplified the operation process, reduced costs, and achieved the recovery of high-purity aminocaproic acid monomer.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of polymer material recycling technology, specifically relating to a highly efficient enzymatic hydrolysis process for polycaprolactam. Background Technology
[0002] Polycaprolactam (PA6) has been widely used in various industrial fields such as textiles, automobile manufacturing, and electronics due to its excellent mechanical properties, wear resistance, and chemical stability. However, it is precisely this inherent chemical stability that makes waste PA6 difficult to degrade in the natural environment, thus causing a serious problem of "white pollution".
[0003] Traditional treatment methods for this problem, such as landfilling or incineration, pose environmental risks, including occupying land resources and releasing toxic and harmful gases, making them unsuitable for green development. Current PA6 recycling pathways mainly fall into three categories: mechanical recycling, chemical recycling, and biological recycling. While mechanical recycling offers the advantage of ease of operation, material properties gradually deteriorate with each recycling cycle, leading to a "degradation cycle" and failing to meet the requirements for high-performance material preparation. Chemical recycling can transform PA6 waste into high-value-added chemicals such as monomers and oligomers, providing a technological possibility for its closed-loop recycling. Current chemical recycling methods are mainly based on reaction mechanisms such as hydrolysis, pyrolysis, alcoholysis, aminolysis, and glycolysis. However, these methods generally rely on harsh reaction conditions (such as strong acid, strong alkali media, or high-temperature and high-pressure environments), resulting in high energy consumption and recycling costs, as well as secondary environmental pollution, limiting their large-scale application.
[0004] Enzymatic hydrolysis, as a green degradation technology with good environmental compatibility, mild reaction conditions, and low energy consumption, has shown broad application prospects in the field of polymer recycling. Previous studies have confirmed that specific enzymes such as proteases, keratinases, and amidases have hydrolytic activity against PA6. However, due to the high crystallinity and strong hydrophobicity of PA6, its macroscopic solid form greatly limits the accessibility of enzyme molecules to their substrate, resulting in low efficiency and lengthy reaction cycles for direct enzymatic hydrolysis. Summary of the Invention
[0005] In view of this, the present invention provides a high-efficiency enzymatic hydrolysis process for polycaprolactam. This process combines sulfuric acid with a multi-enzyme system to treat PA6 with high substrate concentration. The sulfuric acid pretreatment significantly changes the physical form and chemical accessibility of PA6, creating conditions for subsequent enzymatic hydrolysis reactions, and achieving efficient, deep, and green degradation of PA6.
[0006] To address the above technical problems, this invention provides a highly efficient enzymatic hydrolysis process for polycaprolactam, comprising acidic pretreatment, enzymatic depolymerization, and product post-treatment, the specific steps of which are as follows: Acidic pretreatment: The polycaprolactam waste to be treated is added to a sulfuric acid aqueous solution and stirred at 90~130℃ for 2~5h. After the reaction is completed, it is cooled to room temperature and the pH is adjusted to 7~7.5 with calcium hydroxide solution to obtain a slurry containing aminocaproic acid and aminocaproic acid polymer. Enzymatic depolymerization: Hydrolytic enzymes NylB-DNY and NylC were added to the slurry. k -TS was stirred at 40~55℃ for 40~60h to carry out catalytic depolymerization, and an enzymatic hydrolysate containing aminocaproic acid was obtained. Product post-processing: The pH of the enzymatic hydrolysate was adjusted to 9-10 with calcium hydroxide solution, and calcium sulfate was removed by filtration. The pH of the filtrate was adjusted to 7-8 with oxalic acid solution, and calcium oxalate was removed by filtration again. The filtrate was concentrated and recrystallized to obtain the high-quality aminocaproic acid monomer.
[0007] The high-efficiency enzymatic hydrolysis process for polycaprolactam provided by this invention first pretreats PA6 with sulfuric acid solution under heating conditions. The sulfuric acid molecules disrupt the crystal structure of PA6, reducing its crystallinity and causing PA6 to depolymerize into soluble aminocaproic acid monomers and soluble or insoluble aminocaproic acid polymers. This significantly improves the substrate's accessibility to the enzyme, providing a foundation for the hydrolase to efficiently contact and hydrolyze the substrate. Then, a specific hydrolase is added to further catalyze the depolymerization of the soluble or insoluble aminocaproic acid polymers into aminocaproic acid monomers, achieving high-efficiency enzymatic hydrolysis of PA6. Specifically, the hydrolase NylB-DNY degrades linear dimers and linear oligomers into aminocaproic acid monomers using an exonuclease mode, while the hydrolase NylC... k -TS utilizes an endonuclease mechanism to convert potentially existing cyclic and linear oligomers into linear dimers. Furthermore, this invention uses calcium hydroxide solution to adjust the pH of the solution system. Calcium hydroxide does not affect enzyme activity, and the calcium ions in it can react with sulfate ions to form calcium sulfate, which can be separated from aminocaproic acid after filtration. This avoids the use of expensive ion exchange resins to purify aminocaproic acid, achieving low-cost separation and purification of products obtained from high substrate concentrations. The processing method is more economical and feasible. Moreover, excess calcium ions in the system are removed by forming calcium oxalate precipitate with oxalic acid solution, resulting in a neutral filtrate with a pH of 7-8. High-purity aminocaproic acid monomers can be obtained directly through concentration and recrystallization.
[0008] In conjunction with the first aspect, prior to the acidic pretreatment, the polycaprolactam waste to be treated is cleaned, dried, and then crushed into granules or filaments for later use.
[0009] Pre-crushing the polycaprolactam waste into granules or filaments can increase the contact area between sulfuric acid and water molecules and PA6, thereby improving the degradation rate and ensuring degradation efficiency.
[0010] In conjunction with the first aspect, the volume concentration of the sulfuric acid aqueous solution is 20% to 30%.
[0011] The sulfuric acid aqueous solution used in this invention has a volume concentration of 20% to 30%, preferably 25%. This concentration range ensures that PA6 molecules undergo initial degradation, improving the accessibility of hydrolytic enzymes to PA6, while avoiding increased post-processing difficulty due to excessively high sulfate ion concentration.
[0012] In conjunction with the first aspect, the mass ratio of the polycaprolactam waste to the volume ratio of the sulfuric acid aqueous solution is 1:1 to 1.5.
[0013] Preferably, the mass ratio of the polycaprolactam waste to the volume ratio of the sulfuric acid aqueous solution is 1:1.25. Based on a sulfuric acid aqueous solution volume concentration of 25%, this means that only 0.3125 mL of concentrated sulfuric acid is needed to degrade 1 g of PA6 waste.
[0014] In conjunction with the first aspect, the reaction temperature in the acidic pretreatment step is 90~120℃.
[0015] Preferably, the reaction temperature in the acidic pretreatment step is 90~120℃.
[0016] In conjunction with the first aspect, the hydrolytic enzyme is a free enzyme or an immobilized enzyme; When the hydrolytic enzyme is an immobilized enzyme, the enzyme-catalyzed depolymerization step specifically involves adding hydrolytic enzymes NylB-DNY and NylC to the slurry. k -TS was stirred at 40~55℃ for 40~60h for catalytic depolymerization, and then filtered to obtain an enzymatic hydrolysate containing aminocaproic acid.
[0017] The immobilized enzyme obtained from filtration can be recycled for the enzyme-catalyzed depolymerization step.
[0018] In conjunction with the first aspect, the immobilized enzyme includes at least one of carrier covalently immobilized enzyme, carrier adsorption immobilized enzyme, cross-linked enzyme polymer immobilized enzyme, and cross-linked enzyme crystal immobilized enzyme.
[0019] In conjunction with the first aspect, in the enzyme-catalyzed depolymerization step, the substrate concentration in the slurry is 25-75 g / L, and the enzyme concentration after adding the hydrolytic enzyme to the slurry is 0.5-1 g / L. This range of substrate and enzyme concentrations is sufficient to completely degrade the aminocaproic acid polymer into aminocaproic acid monomers.
[0020] Preferably, the substrate concentration in the slurry can be 45~75 g / L, and the enzyme concentration after adding hydrolytic enzyme to the slurry can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, or any value between any two of these values.
[0021] Preferably, the hydrolytic enzyme used in this invention contains NylB-DNY and NylC. k - A crude enzyme solution of TS protein, which can be prepared by the following method: This will express NylB-DNY and NylC. k After culturing the -TS strain in the medium, the seed culture was transferred to ZYM medium containing ampicillin for further shaking culture. The culture was then diluted and the OD was tested. 600 Once the culture medium reaches 0.6-0.8, centrifuge the culture medium, discard the supernatant, and collect the bacterial precipitate. Resuspend the bacterial precipitate in water, and then use a high-pressure homogenizer to break up the bacterial suspension to obtain the final product.
[0022] In practical applications, NylB-DNY and NylC can be used. k -TS crude enzyme solution, purified enzyme or immobilized enzyme are used for enzymatic hydrolysis.
[0023] In conjunction with the first aspect, the solvent used for recrystallization is selected from at least one of methanol, ethanol, and isopropanol.
[0024] In conjunction with the first aspect, the polycaprolactam waste includes textile fabrics, engineering plastics, fiber products, or film and pipe products containing polycaprolactam.
[0025] Preferably, the enzymatic hydrolysis process of the present invention can also be used to degrade other types of waste containing polycaprolactam.
[0026] Compared with existing technologies, the high-efficiency enzymatic hydrolysis process for polycaprolactam provided by this invention has the following advantages: (1) This invention addresses the challenge of directly and gently degrading PA6. First, a rapid chemical acid hydrolysis method is used to pretreat PA6, depolymerizing it into aminocaproic acid and aminocaproic acid polymers, significantly improving the substrate's accessibility to the enzyme. Then, a specific hydrolytic enzyme is used to further convert the intermediate obtained from the acidic pretreatment into high-purity aminocaproic acid monomers. This invention combines chemical and biological methods, effectively overcoming the limitations of a single biological method and significantly improving degradation efficiency.
[0027] (2) The present invention further optimizes the acidic pretreatment conditions. The substrate slurry with a concentration of up to 1000 g / L can be treated with a low concentration of sulfuric acid solution (volume concentration of 20%~30%). The oligomers suitable for enzymatic hydrolysis can be obtained at a temperature below 130°C and within 5 hours, so that the total yield of the final enzymatically hydrolyzed aminocaproic acid monomer remains above 93%, and can reach up to 95%.
[0028] (3) The efficient enzymatic hydrolysis process provided by the present invention can directly use the slurry containing oligomers obtained in the chemical depolymerization step as the substrate in the enzyme-catalyzed depolymerization step, and can also directly use unpurified crude enzyme solution for enzymatic hydrolysis reaction. This "crude substrate + crude enzyme solution" method avoids the cumbersome and costly purification steps, greatly simplifies the operation process, and reduces the overall processing cost. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
[0030] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the meaning consistent with their meaning in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined.
[0031] Unless otherwise specified, all raw materials, reagents, and equipment used in this invention are conventional commercially available reagents and equipment. The enzymatic hydrolysis process provided by this invention is illustrated below through specific examples.
[0032] The enzymatic hydrolysis process provided by the present invention will be described below through specific embodiments.
[0033] The NylB-DNY sequence used in this invention (as shown in SEQ ID NO.1) is derived from the NylB homolog of Arthrobacter sp. K172. After modification by Yasuyuki Kawashima et al., NylB-DNY (G181D / H266N / D370Y) was obtained. The vector is pET28a and it is expressed in Escherichia coli BL21(DE3).
[0034] NylC k The -TS sequence (as shown in SEQ ID NO.2) is derived from basophilic Kocuria sp. and modified by Gregg T. Beckham et al. to obtain NylC. k The -TS (S111G / A137L) mutant, expressed in Escherichia coli BL21(DE3) using the pET28a vector.
[0035] The NylB-SCY sequence (as shown in SEQ ID NO.3) is derived from the NylB homolog of Arthrobacter sp. K172. After modification by Gregg T. Beckham et al., NylB-SCY (R187S / F264C / D370Y) was obtained and expressed in Escherichia coli BL21(DE3) using the pET28a vector.
[0036] In the enzyme-catalyzed depolymerization steps of the following embodiments and comparative examples of the present invention, crude enzyme solution (free enzyme) of hydrolytic enzyme is used for enzymatic hydrolysis. The crude enzyme solution is obtained as follows: The expression NylB-DNY or NylB-SCY will be related to NylC. k The -TS mutant strain was cultured in 5 mL of sterile LB medium at 37 °C and 220 rpm for 12 h. 1 mL of the above seed culture was transferred to 100 mL of ZYM medium supplemented with 100 μg / mL ampicillin and cultured for another 24 h with shaking at 21 °C and 160 rpm. At this point, the bacterial culture was diluted 10-fold, and the OD... 600 The culture medium was centrifuged at 4 °C and 10,000 rpm for 10 min. The supernatant was discarded, and the bacterial pellet was collected. The bacterial pellet was resuspended in 10 mL of water, and the bacterial suspension was homogenized using a high-pressure homogenizer to obtain crude enzyme solutions containing the target enzyme protein.
[0037] Example 1 This embodiment provides a highly efficient enzymatic hydrolysis process for polycaprolactam, the specific steps of which are as follows: Acidic pretreatment: The polycaprolactam waste to be treated was washed, dried and crushed into filaments. 8g was added to 10mL of 25% sulfuric acid aqueous solution and stirred at 120℃ and 400rpm for 3h. After the reaction was completed, it was cooled to room temperature, the pH was adjusted to 7.2 with calcium hydroxide solution, and the volume was adjusted to 80mL with water to obtain a slurry containing aminocaproic acid and aminocaproic acid polymer.
[0038] Enzymatic depolymerization: Water was added to the slurry obtained from acid pretreatment to achieve a substrate concentration of 40 g / L, and hydrolytic enzymes NylB-DNY and NylC were added respectively. k The crude enzyme solution of -TS was used to achieve an enzyme concentration of 0.5 g / L. The mixture was stirred at 48℃ and 160 rpm for 48 h to carry out catalytic depolymerization. During the reaction, the pH of the system was monitored and maintained at 7~7.5 to obtain an enzymatic hydrolysate containing aminocaproic acid, which was then inactivated by heating.
[0039] Product post-processing: The pH of the obtained enzymatic hydrolysate was adjusted to 9.5 with calcium hydroxide solution, and calcium sulfate precipitate was precipitated. The calcium sulfate was removed by filtration. Oxalic acid solution was added dropwise to the obtained filtrate to adjust the pH to 7.5. During this process, calcium oxalate precipitate was formed. The calcium oxalate was removed by filtration again. The obtained filtrate was evaporated to remove water, and then recrystallized in ethanol to obtain 0.0672 mol of pure aminocaproic acid monomer. Its purity was tested to be >98%, and the yield was calculated to be 95.05%.
[0040] Yield (%) = Actual amount of aminocaproic acid obtained / Theoretical amount of aminocaproic acid to be obtained × 100%, where the theoretical amount of aminocaproic acid to be obtained is 0.0707 mol.
[0041] The yields of the following examples and comparative examples were calculated according to the above formula.
[0042] Example 2 This embodiment provides a highly efficient enzymatic hydrolysis process for polycaprolactam, the specific steps of which are as follows: Acidic pretreatment: The polycaprolactam waste to be treated was washed, dried and crushed into filaments. 8g was added to 12mL of 20% sulfuric acid aqueous solution and stirred at 90℃ and 400rpm for 5h. After the reaction was completed, it was cooled to room temperature, the pH was adjusted to 7.1 with calcium hydroxide solution, and the volume was adjusted to 80mL with water to obtain a slurry containing aminocaproic acid and aminocaproic acid polymer.
[0043] Enzymatic depolymerization: Water was added to the slurry obtained from acid pretreatment to achieve a substrate concentration of 30 g / L, and hydrolytic enzymes NylB-DNY and NylC were added respectively. k The crude enzyme solution of -TS was used to achieve an enzyme concentration of 0.7 g / L. The mixture was stirred at 40℃ and 160 rpm for 60 h to carry out catalytic depolymerization. During the reaction, the pH of the system was monitored and maintained at 7~7.5 to obtain an enzymatic hydrolysate containing aminocaproic acid, which was then inactivated by heating.
[0044] Product post-processing: The pH of the obtained enzymatic hydrolysate was adjusted to 9.4 with calcium hydroxide solution, precipitating calcium sulfate. The calcium sulfate was removed by filtration. Oxalic acid solution was added dropwise to the obtained filtrate to adjust the pH to 7.2. During this process, calcium oxalate precipitate was formed. The calcium oxalate was removed by filtration again. The obtained filtrate was evaporated to remove water, and then recrystallized in methanol to obtain 0.0669 mol of pure aminocaproic acid monomer. Its purity was tested to be >97.35%, and the yield was calculated to be 94.63%.
[0045] Example 3 This embodiment provides a highly efficient enzymatic hydrolysis process for polycaprolactam, the specific steps of which are as follows: Acidic pretreatment: The polycaprolactam waste to be treated was washed, dried and crushed into filaments. 8g was added to 8mL of 30% sulfuric acid aqueous solution and stirred at 130℃ and 400rpm for 2h. After the reaction was completed, it was cooled to room temperature, the pH was adjusted to 7.4 with calcium hydroxide solution, and the volume was adjusted to 80mL with water to obtain a slurry containing aminocaproic acid and aminocaproic acid polymer.
[0046] Enzymatic depolymerization: Water was added to the slurry obtained from acid pretreatment to achieve a substrate concentration of 70 g / L, and hydrolytic enzymes NylB-DNY and NylC were added respectively. k The crude enzyme solution of -TS was used to achieve an enzyme concentration of 0.8 g / L. The mixture was stirred at 55℃ and 160 rpm for 40 h to carry out catalytic depolymerization. During the reaction, the pH of the system was monitored and maintained at 7~7.5 to obtain an enzymatic hydrolysate containing aminocaproic acid, which was then inactivated by heating.
[0047] Product post-processing: The pH of the obtained enzymatic hydrolysate was adjusted to 9.7 with calcium hydroxide solution, precipitating calcium sulfate. The calcium sulfate was removed by filtration. Oxalic acid solution was added dropwise to the filtrate to adjust the pH to 7.5, during which calcium oxalate precipitate was formed. The calcium oxalate was removed by filtration again. The filtrate was then evaporated by rotary evaporation to remove water, and then recrystallized in isopropanol to obtain 0.0663 mol of pure aminocaproic acid monomer. Its purity was tested to be >97.64%, and the yield was calculated to be 93.78%.
[0048] Comparative Example 1 This comparative example provides a highly efficient enzymatic hydrolysis process for polycaprolactam. The specific steps are similar to those in Example 1, except that NylB-DNY and NylC are used in the enzymatic depolymerization step of Example 1. k - The crude enzyme solution of TS was replaced with equal amounts of NylB-SCY and NylC. k The crude enzyme solution of -TS is prepared, and the remaining steps are the same as in Example 1, so they will not be repeated here.
[0049] Finally, 0.049 mol of high-purity aminocaproic acid monomer was obtained, with a purity of >80.25% and a calculated yield of 69.31%.
[0050] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A highly efficient enzymatic hydrolysis process for polycaprolactam, characterized in that, The process includes acidic pretreatment, enzyme-catalyzed depolymerization, and product post-treatment, with the specific steps as follows: Acidic pretreatment: The polycaprolactam waste to be treated is added to a sulfuric acid aqueous solution and stirred at 90~130℃ for 2~5h. After the reaction is completed, it is cooled to room temperature and the pH is adjusted to 7~7.5 with calcium hydroxide solution to obtain a slurry containing aminocaproic acid and aminocaproic acid polymer. Enzymatic depolymerization: Hydrolytic enzymes NylB-DNY and NylC were added to the slurry. k -TS was stirred at 40~55℃ for 40~60h to carry out catalytic depolymerization, and an enzymatic hydrolysate containing aminocaproic acid was obtained. Product post-processing: The pH of the enzymatic hydrolysate was adjusted to 9-10 with calcium hydroxide solution, and calcium sulfate was removed by filtration. The pH of the filtrate was adjusted to 7-8 with oxalic acid solution, and calcium oxalate was removed by filtration again. The filtrate was concentrated and recrystallized to obtain the high-quality aminocaproic acid monomer.
2. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 1, characterized in that, Before the acidic pretreatment, the polycaprolactam waste to be treated is cleaned, dried, and then crushed into granules or filaments for later use.
3. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 1, characterized in that, The volume concentration of the sulfuric acid aqueous solution is 20% to 30%.
4. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 3, characterized in that, The mass ratio of the polycaprolactam waste to the volume ratio of the sulfuric acid aqueous solution is 1:1 to 1.
5.
5. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 1, characterized in that, The reaction temperature in the acidic pretreatment step is 90~120℃.
6. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 1, characterized in that, The hydrolytic enzyme is a free enzyme or an immobilized enzyme; When the hydrolytic enzyme is an immobilized enzyme, the enzyme-catalyzed depolymerization step specifically involves adding hydrolytic enzymes NylB-DNY and NylC to the slurry. k -TS was stirred at 40~55℃ for 40~60h for catalytic depolymerization, and then filtered to obtain an enzymatic hydrolysate containing aminocaproic acid.
7. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 6, characterized in that, The immobilized enzyme includes at least one of the following: carrier covalently immobilized enzyme, carrier adsorbed immobilized enzyme, cross-linked enzyme polymer immobilized enzyme, and cross-linked enzyme crystal immobilized enzyme.
8. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 1, characterized in that, In the enzyme-catalyzed depolymerization step, the substrate concentration in the slurry is 25~75 g / L, and the enzyme concentration after adding hydrolytic enzyme to the slurry is 0.5~1 g / L.
9. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 1, characterized in that, The solvent used for recrystallization is selected from at least one of methanol, ethanol, and isopropanol.
10. The high-efficiency enzymatic hydrolysis process for polycaprolactam as described in claim 1, characterized in that, The polycaprolactam waste includes textile fabrics, engineering plastics, fiber products, or film and pipe products containing polycaprolactam.