Efficient nylon-degrading enzyme mutant and application thereof
Patent Information
- Application Number
- CN202611269080.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-20
- Publication Date
- 2026-09-22
AI Technical Summary
然而，其催化活性仍有提升空间
本发明通过对天然尼龙酶NylC－E10与野生型NylCp2－TS进行序列比对和结构分析，揭示了NylC－E10高性能的分子基础；野生型NylCp2－TS的基础上，通过在四个位于同源四聚体亚基界面的氨基酸替换（A36D、G122D、Y130H、Q263E）或N端氨基酸截短，获得了系列的性能优异突变体；进一步通过分别优化界面替换组合和N端截短长度，发现双区域修饰存在协同作用；又以N端截短45个氨基酸的突变体为模板，引入Q263E单点突变，获得了最佳突变体NylC－E10pro，其活性是野生型的32.9倍，为目前报道的NylC型尼龙酶中活性最高的突变体之一，该最佳突变体的最适温度高达90°C，可在接近沸水条件下工作，显著提高反应速率和底物溶解度，且对多种形态尼龙底物（薄膜、颗粒、纤维）均有高效降解能力，在工业尼龙废弃物处理中具有广泛适用性；表明本发明提供的尼龙降解酶突变体在尼龙废弃物高温生物降解领域具有重大应用前景。
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of enzyme engineering and genetic engineering, specifically to a highly efficient nylon-degrading enzyme mutant and its applications. Background Technology
[0002] Polyamide (nylon) is widely used in textiles, automobiles, electronics, and other fields due to its excellent mechanical properties and chemical stability. However, nylon is extremely difficult to degrade in the natural environment, and large amounts of waste nylon cause serious "white pollution." Bio-enzymatic hydrolysis has become a research hotspot for nylon waste treatment due to its mild reaction conditions and environmental friendliness.
[0003] Nylon-degrading enzymes can catalyze the hydrolysis of amide bonds in nylon oligomers or polymers. Among them, the NylC type nylonase (EC 3.5.1.117) exhibits high hydrolytic activity against nylon-6 and its linear oligomers. Currently known nylon-degrading enzymes include those derived from... Arthrobacter sp. The wild-type NylCp2-TS of KI72, and the natural enzyme NylC-E10 obtained through sequence mining, exhibit 18.1 times the specific activity of the wild-type and a Tm of 97.9℃. However, there is still room for improvement in their catalytic activity. Furthermore, the high-performance molecular mechanism of NylC-E10 remains unclear, limiting further rational modification.
[0004] Therefore, there is an urgent need to design and construct mutants with superior catalytic activity and thermal stability through system structure analysis of NylC-E10 to meet the industrial requirements of high-temperature biodegradation of nylon waste. In view of this, this invention provides a highly efficient nylon-degrading enzyme mutant and its applications. Summary of the Invention
[0005] The technical problem to be solved by this invention is to provide a highly efficient nylon-degrading enzyme mutant and its applications. The aim is to obtain a highly efficient nylon-degrading enzyme mutant through systematic structural analysis and combinatorial modification based on wild-type NylCp2-TS or the natural nylon enzyme NylC-E10.
[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, a highly efficient nylon-degrading enzyme mutant, said mutant being a mutation in the amino acid sequence shown in SEQ ID NO: 1 involving any one or at least two of the following (a) to (l): (a) The A at position 36 mutates to D; (b) The G at position 122 mutates to D; (c) The Y at position 130 mutates to H; (d) The A at position 36 mutates into D; (e) The N-terminus is truncated by 5, 10, 15, 20, 25, 30, 35, 40, 45, 46 or 47 amino acids respectively.
[0007] Based on the above technical solution, the present invention can be further improved as follows.
[0008] The mutant is a mutation occurring in any one of the following (a) to (l) of the amino acid sequence shown in SEQ ID NO: 1: (a) The A at position 36 mutates to D, and the G at position 122 mutates to D; (b) The G at position 122 mutates to D, and the Q at position 263 mutates to E; (c) The Y at position 130 mutates to H, and the Q at position 263 mutates to E; (d) The A at position 36 mutates to D, the Y at position 130 mutates to H, and the Q at position 263 mutates to E; (e) The N-terminus is truncated by 40 amino acids; (f) The N-terminus is truncated by 45 amino acids; (g) The N-terminus is truncated by 46 amino acids; (h) The N-terminus is truncated by 47 amino acids; (i) The N-terminus is truncated by 45 amino acids, and the G at position 122 is mutated to D; (j) The N-terminus is truncated by 45 amino acids, and the Y at position 130 is mutated to H; (k) The N-terminus is truncated by 45 amino acids, and the Q at position 263 is mutated to E; (l) The N-terminus is truncated by 45 amino acids, and the G at position 122 is mutated to D, and the Y at position 130 is mutated to H.
[0009] The preferred amino acid sequence of the nylon-degrading enzyme mutant is shown in SEQ ID NO:2. This mutant is named NylC-E10pro.
[0010] Secondly, a nucleic acid molecule encodes the aforementioned highly efficient nylon-degrading enzyme mutant.
[0011] Furthermore, the nucleotide sequence of the nylon degrading enzyme mutant is shown in SEQ ID NO:3.
[0012] Thirdly, a recombinant expression vector containing the aforementioned nucleic acid molecule.
[0013] Fourthly, a recombinant strain, wherein the recombinant strain is obtained by introducing the nucleic acid molecule into a host cell, or by introducing the nucleic acid molecule into a host cell via the recombinant expression vector.
[0014] Fifthly, a method for preparing the protein of the highly efficient nylon-degrading enzyme mutant, wherein the seed culture of the recombinant strain is inoculated into a fermentation medium and cultured to express the protein of the highly efficient nylon-degrading enzyme mutant.
[0015] The sixth aspect concerns the application of the highly efficient nylon-degrading enzyme mutant, the nucleic acid molecule, the recombinant expression vector, or the recombinant strain in the degradation of nylon materials.
[0016] Furthermore, the nylon material includes at least one of nylon 6, nylon 66, nylon oligomers, nylon films, nylon particles, nylon fibers, and nylon-containing plastic waste.
[0017] In a seventh aspect, a method for degrading nylon material includes the following steps: reacting the highly efficient nylon-degrading enzyme mutant with a substrate containing nylon material in a degradation reaction; The degradation reaction conditions are: temperature 25-120℃, pH 4.0-10.0; preferably 30-100℃ and pH 6.0-9.0.
[0018] The beneficial effects of this invention are: This invention reveals the molecular basis for the high performance of NylC-E10 through sequence alignment and structural analysis of the natural nylonase NylC-E10 and wild-type NylCp2-TS. Based on wild-type NylCp2-TS, a series of high-performance mutants were obtained by replacing amino acids (A36D, G122D, Y130H, Q263E) or truncating the N-terminal amino acid at the interfaces of four homotetrameric subunits. Further optimization of the interface replacement combinations and the N-terminal truncation length revealed a synergistic effect of dual-region modification. A mutant with a 45-amino acid truncated N-terminus was used as a model. By introducing a single-point mutation of Q263E, the optimal mutant NylC-E10pro was obtained, with an activity 32.9 times that of the wild type. This is one of the most active mutants of NylC-type nylonases reported to date. The optimal temperature of this mutant is as high as 90°C, allowing it to operate under near-boiling water conditions. It significantly improves the reaction rate and substrate solubility, and has a high efficiency in degrading various forms of nylon substrates (films, particles, and fibers). It has wide applicability in the treatment of industrial nylon waste. This indicates that the nylon degrading enzyme mutant provided by this invention has significant application prospects in the field of high-temperature biodegradation of nylon waste. Attached Figure Description
[0019] Figure 1 A schematic diagram showing the structural differences between NylC-E10 and wild-type NylCp2-TS.
[0020] Figure 2The results show the activity and thermal stability analysis of mutants obtained by optimizing the interface sites and N-terminus of wild-type NylCp2-TS.
[0021] Figure 3 The results show the activity and thermal stability of mutants obtained by further modifying mutants based on N-terminal optimization.
[0022] Figure 4 The results are the optimal temperature analysis results for the dominant variant.
[0023] Figure 5 The optimal pH analysis results are for the dominant variant.
[0024] Figure 6 The results are the optimal substrate analysis results for the dominant variant. Detailed Implementation
[0025] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0026] Example 1. Sequence alignment and structural analysis, and mutant design strategies: 1.1 Sequence Alignment and Structural Analysis: The amino acid sequences of natural nylonase NylC-E10 and wild-type NylCp2-TS (whose sequence is shown in SEQ ID NO:1) were compared. The results showed that NylC-E10 differed from wild-type in the following ways: (1) four amino acid substitutions: A36D, G122D, Y130H, and Q263E; (2) 25 amino acids were missing at the N-terminus. Using the three-dimensional structure of the homotetramer of wild-type NylCp2-TS (which can be obtained based on known crystal structures or homology modeling), the structure was located. It was found that the four substitutions were all located at the subunit-to-subunit contact interface, and the nonpolar or uncharged residues (alanine, glycine, tyrosine, glutamine) were changed to charged residues (aspartic acid, histidine, glutamic acid); the missing 25 N-terminal residues were located in the ring region of the tetramer ( Figure 1 ).
[0027] 1.2 Mutant Design Strategy: Based on sequence alignment of NylC-E10 with the wild type, four interface substitutions and N-terminal truncated regions were identified. Mutants containing only the four substitutions (WT-4, A36D / G122D / Y130H / Q263E) and only the N-terminal truncated region were constructed. All single, double, and three-site interface combination mutants were constructed. Interface substitutions were introduced using amino acids with the optimal truncated length as templates, and the optimal mutant was screened, as detailed below: ① Construct mutants WT-4 (A36D / G122D / Y130H / Q263E) containing only four substitutions and mutants containing only N-terminal truncation. Systematic construction of N-terminal truncated mutants of different lengths was used to screen for highly active combinations, such as WT-TN5, WT-TN10, WT-TN15, WT-TN20, WT-TN25, WT-TN30, WT-TN35, WT-TN40, WT-TN45, WT-TN46, and WT-TN47, to determine the optimal amino acid truncation length.
[0028] ② The system constructs all single, double, and three-point interface combination mutants for screening highly active combinations, such as A36D, G122D, Y130H, Q263E, A36D / G122D, A36D / Y130H, A36D / Q263E, G122D / Y130H, G122D / Q263E, Y130H / Q263E, A36D / G122D / Y130H, A36D / G122D / Q263E, G122D / Y130H / Q263E, and A36D / Y130H / Q263E.
[0029] ③ Using amino acids of optimal truncated length as templates, interfacial substitutions are introduced to screen for the best mutants, such as WT-TN45-G122D, WT-TN45-Y130H, WT-TN45-Q263E, WT-TN45-G122D / Y130H, WT-TN45-G122D / Q263E, WT-TN45-Y130H / Q263E, and WT-TN45-G122D / Y130H / / Q263E.
[0030] 2. Construction of mutants and protein expression and purification.
[0031] 2.1 Mutant Construction: Using the encoding gene of wild-type NylCp2-TS (its sequence is shown in SEQ ID NO:4) as a template, mutants were constructed according to the mutation combinations listed in Table 1 using a rapid site-directed mutagenesis kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). All mutants were verified by DNA sequencing.
[0032] Table 1 As shown in Table 1, the system constructed all single, double, and three-point interface combination mutants for screening highly active combinations, specifically as follows: A36D, G122D, Y130H, Q263E, A36D / G122D, A36D / Y130H, A36D / Q263E, G122D / Y130H, G122D / Q263E, Y130H / Q263E, A36D / G122D / Y130H, A36D / G122D / Q263E, G122D / Y130H / Q263E, A36D / Y130H / Q263E.
[0033] As shown in Table 1, N-terminal truncated mutants of different lengths were constructed to screen for highly active combinations, specifically: WT-TN5, WT-TN10, WT-TN15, WT-TN20, WT-TN25, WT-TN30, WT-TN35, WT-TN40, WT-TN45, WT-TN46, and WT-TN47.
[0034] As shown in Table 1, the optimal mutants were screened by introducing interfacial replacements using the amino acid WT-TN45 with the optimal truncated length as a template. The specific mutants are as follows: WT-TN45-G122D, WT-TN45-Y130H, WT-TN45-Q263E, WT-TN45-G122D / Y130H, WT-TN45-G122D / Q263E, WT-TN45-Y130H / Q263E, and WT-TN45-G122D / Y130H / / Q263E.
[0035] 2.2 Protein Expression and Purification: The coding genes of each mutant were cloned into the pET-28a(+) expression vector (NdeI / XhoI site) (purchased from Beijing Qingke Biotechnology Co., Ltd.), and transformed into *E. coli* BL21(DE3) competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.). Single colonies were picked and inoculated into LB medium containing 50 µg / mL kanamycin, and cultured at 37°C with shaking until OD500. 600Approximately 0.6–0.8 mg of the solution was added with 0.1 mM IPTG, and expression was induced at 18°C for 20 hours. The bacterial cells were collected by centrifugation, sonicated, and then the target protein was purified using nickel affinity chromatography. Purification was performed using buffer A (20 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5), buffer B (50 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5), and buffer C (500 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5). A 50 mM Tris-HCl buffer at pH 7.5 was used for desalting. Purity was verified by SDS-PAGE, and protein concentration was determined using the BCA method.
[0036] 3. Nylon degrading enzyme activity assay.
[0037] Nylon 6 (PA6) membranes (0.2 mm thick, Goodfellow) were used as substrates. The reaction system consisted of 50 mM Tris-HCl buffer (pH 7.5), with an enzyme (the mutant constructed above) to substrate ratio of 0.04 mM enzyme / g PA6. The reaction was carried out at 60 °C for 24 hours. High-performance liquid chromatography (HPLC) was used for quantitative detection of the hydrolysis products. The mobile phase was acetonitrile and water (3:1 v / v), the column was a C18 column (150 Å, 5 µm), and the detection wavelength was 220 nm.
[0038] The results are as follows Figure 2 and 3 The results showed that WT-4 activity was 1.2 times that of wild type, and WT-TN25 activity was 4.8 times. The interface double mutants G122D / Q263E and Y130H / Q263E showed activities increased by 31.4 times and 30.9 times, respectively. The interface triple mutant A36D / Y130H / Q263E showed an activity increase of 26.8 times. Among the N-terminal truncated mutants, WT-TN45 showed the highest activity, 28.4 times that of wild type (1.6 times that of NylC-E10). After introducing Q263E using WT-TN45 as a template, the activity of NylC-E10pro was 32.9 times that of wild type.
[0039] 4. Thermal stability test The melting temperature of the enzyme was determined using differential scanning calorimetry (DSC) or quantitative real-time PCR (SYPRO Orange dye method). The purified enzyme solution (1 mg / mL) was heated from 30 °C to 110 °C at a rate of 1 °C / min, and the thermal denaturation curve was recorded.
[0040] The results are as follows Figure 2 and 3The results showed that the wild-type Tm was 77.1℃. WT-4 Tm was 60.9℃ (a decrease of 16.2℃). WT-TN25 Tm was 77.9℃. The Tm of the interface double mutants G122D / Q263E and Y130H / Q263E decreased to 45.1℃ and 45.6℃, respectively. The interface triple mutant A36D / Y130H / Q263E had a Tm of 82.2℃ (an increase of 5.1℃). WT-TN45 had a Tm of 83.9℃ (an increase of 6.8℃). NylC-E10pro had a Tm of 89.6℃ (an increase of 12.5℃).
[0041] 5. Systematic enzymatic characterization of ylC-E10pro.
[0042] 5.1 Optimal Temperature: Within the temperature range of 25–95°C, using PA6 membrane as a substrate, the reaction was carried out for 2 hours, and the amount of product generated was determined. The optimal temperature for the wild type was 70°C; the optimal temperature for NylC-E10pro was increased to 90°C; it still maintained high activity at 95°C. Figure 4 ).
[0043] 5.2 Optimal pH: The reaction was carried out at 70°C for 2 hours within a pH range of 4–10. The optimal pH for both the wild-type and NylC-E10pro was 7.5. At pH 5, NylC-E10pro retained 64.0% of its maximum activity (wild-type 54.0%); at pH 10, it retained 37.4% (wild-type 29.6%). Figure 5 ).
[0044] 5.3 Optimal substrate: Using 0.2 mm membranes, 0.5 mm membranes, and PA6 particles as substrates, the reaction was carried out at 70 °C for 24 hours. The activity of the 0.2 mm membrane was considered 100%. Results showed that when the substrate was increased from a 0.2 mm membrane to a 0.5 mm membrane, the wild-type activity decreased by 35.4%, while NylC-E10pro only decreased by 3.7%; when particles were used instead of membranes, the wild-type activity decreased by 59.5%, and NylC-E10pro by 17.9%. Figure 6 ).
[0045] In summary, this invention, through sequence alignment and structural analysis of the natural nylonase NylC-E10 and wild-type NylCp2-TS, revealed the molecular basis for the high performance of NylC-E10. Based on wild-type NylCp2-TS, a series of high-performance mutants were obtained by replacing amino acids (A36D, G122D, Y130H, Q263E) or truncating the N-terminal amino acid at the interfaces of four homotetrameric subunits. Further optimization of the interface replacement combinations and the N-terminal truncation length revealed a synergistic effect of dual-region modification. A mutant with a 45-amino acid truncated N-terminus was also identified. Using a template, a single-point mutation of Q263E was introduced to obtain the optimal mutant NylC-E10pro, whose activity is 32.9 times that of the wild type. This is one of the most active mutants of NylC-type nylonases reported to date. The optimal temperature of this mutant is as high as 90°C, and it can operate under near-boiling water conditions, significantly improving the reaction rate and substrate solubility. It also has a high efficiency in degrading various forms of nylon substrates (films, particles, and fibers), and has wide applicability in the treatment of industrial nylon waste. This indicates that the nylon degrading enzyme mutant provided by this invention has significant application prospects in the field of high-temperature biodegradation of nylon waste.
[0046] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. A highly efficient nylon-degrading enzyme mutant, characterized in that, The mutant is a mutation occurring in the amino acid sequence shown in SEQ ID NO: 1, involving any one or at least a combination of the following (a) to (l): (a) The A at position 36 mutates to D; (b) The G at position 122 mutates to D; (c) The Y at position 130 mutates to H; (d) The A at position 36 mutates into D; (e) The N-terminus is truncated by 5, 10, 15, 20, 25, 30, 35, 40, 45, 46 or 47 amino acids respectively.
2. The highly efficient nylon-degrading enzyme mutant according to claim 1, characterized in that, The mutant is a mutation occurring in any one of the following (a) to (l) of the amino acid sequence shown in SEQ ID NO: 1: (a) The A at position 36 mutates to D, and the G at position 122 mutates to D; (b) The G at position 122 mutates to D, and the Q at position 263 mutates to E; (c) The Y at position 130 mutates to H, and the Q at position 263 mutates to E; (d) The A at position 36 mutates to D, the Y at position 130 mutates to H, and the Q at position 263 mutates to E; (e) The N-terminus is truncated by 40 amino acids; (f) The N-terminus is truncated by 45 amino acids; (g) The N-terminus is truncated by 46 amino acids; (h) The N-terminus is truncated by 47 amino acids; (i) The N-terminus is truncated by 45 amino acids, and the G at position 122 is mutated to D; (j) The N-terminus is truncated by 45 amino acids, and the Y at position 130 is mutated to H; (k) The N-terminus is truncated by 45 amino acids, and the Q at position 263 is mutated to E; (l) The N-terminus is truncated by 45 amino acids, and the G at position 122 is mutated to D, and the Y at position 130 is mutated to H.
3. A nucleic acid molecule, characterized in that, Encodes the highly efficient nylon-degrading enzyme mutant of claim 1 or 2.
4. A nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nylon degrading enzyme mutant is shown in SEQ ID NO:
3.
5. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 3 or 4.
6. A recombinant bacterial strain, characterized in that, The recombinant strain is obtained by introducing the nucleic acid molecule of claim 3 or 4 into a host cell, or by introducing the nucleic acid molecule of claim 3 or 4 into a host cell using the recombinant expression vector of claim 5.
7. A method for preparing the protein of the highly efficient nylon-degrading enzyme mutant according to claim 1 or 2, characterized in that, The seed culture of the recombinant strain according to claim 6 is inoculated into a fermentation medium and cultured to express the protein of the highly efficient nylon-degrading enzyme mutant.
8. The application of the highly efficient nylon-degrading enzyme mutant of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the recombinant expression vector of claim 5, or the recombinant strain of claim 6 in the degradation of nylon materials.
9. The application according to claim 8, characterized in that, The nylon material includes at least one of nylon 6, nylon 66, nylon oligomers, nylon films, nylon particles, nylon fibers, and nylon-containing plastic waste.
10. A method for degrading nylon material, characterized in that, The process includes the following steps: reacting the highly efficient nylon-degrading enzyme mutant of claim 1 or 2 with a substrate containing nylon material to perform a degradation reaction; The degradation reaction conditions are: temperature 25-120℃, pH 4.0-10.0.