An amidase sp1 mutant and its use in hydrolyzing polyurethane alcoholysis products

Abstract

The present application relates to the fields of genetic engineering, enzyme engineering and plastic biocatalytic recovery, and in particular to an amidase SP1 mutant and its application in hydrolysis of polyurethane alcoholysis products. In view of the problems that the wild type SP1 has poor thermal stability and is difficult to continuously catalyze the hydrolysis of polyurethane alcoholysis products, the present application comprehensively uses means such as multi-strategy rational design, PNBC activity, thermal challenge and half-life grading screening, and obtains high-thermal-stability mutants such as G299M, G299M / M238L, G299M / Q327L and G299M / T389S. Among them, the 45 DEG C thermal half-life and melting temperature of G299M / Q327L are significantly improved, and it shows the ability of continuously and efficiently hydrolyzing TDA-DEG and improving the release amount of aromatic diamines in real TDI-based and MDI-based polyurethane alcoholysis products, and has important industrial application prospect.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering, enzyme engineering, and biocatalytic recycling of plastics, specifically to an amidase SP1 mutant and its application in the hydrolysis products of polyurethane. Background Technology

[0002] Polyurethane (PU or PUR) is an important polymer material formed by the polymerization of polyols, diisocyanates, and chain extenders. It is widely used in flexible foams, rigid foams, elastomers, coatings, adhesives, thermal insulation materials, and automotive interiors. Due to the stable urethane bonds, complex hard / soft segment structures, and thermosetting foam crosslinking networks in polyurethane materials, it is difficult to achieve high-value reuse of waste polyurethane materials through traditional mechanical recycling methods.

[0003] Chemical alcoholysis is one of the most promising treatment methods for polyurethane recycling. For TDI-based polyether polyurethane foam, the alcoholysis process can convert it into a polyol-enriched phase, a polymer dispersion phase, and a base phase containing urethane intermediates. The base phase may contain TDA-DEG, monourethane intermediates, TDA, and other aromatic urethane compounds. These low-molecular-weight urethane intermediates still require further hydrolysis to release high-value aromatic diamine monomers, such as 2,4-toluenediamine and 2,6-toluenediamine.

[0004] Amideases capable of hydrolyzing low molecular weight carbamate substrates offer a novel technological pathway for the chemical-enzymatic cascade recovery of polyurethane. Among them, SP1-type amidases can catalyze the hydrolysis of low molecular weight dicarbamate substrates in polyurethane alcoholysis products. However, the insufficient thermal stability of wild-type SP1 under medium- and high-temperature and long-term reaction conditions limits its application in practical enzymatic upgrading processes of polyurethane alcoholysis products.

[0005] In polyurethane chemical-enzymatic combined recovery systems, the performance of enzymes depends not only on their initial activity but also on their operational stability during prolonged reactions. The actual alcoholysis substrate has a complex composition, containing various carbamate intermediates and aromatic amine compounds, which makes wild / natural amidases prone to inactivation during extended reaction periods.

[0006] Therefore, developing the SP1 mutant with higher thermal stability and the ability to continuously catalyze the hydrolysis of polyurethane alcoholysis products such as TDA-DEG ​​is of great significance for improving polyurethane recovery efficiency and aromatic diamine monomer recovery rate. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to address the shortcomings of the existing technology, such as insufficient thermal stability of wild-type SP1 and limited ability to hydrolyze polyurethane alcoholysis products over a long period of time. The present invention provides an amidase SP1 mutant, its nucleic acid molecule, recombinant expression vector, recombinant host cell, enzyme preparation, and its application in the hydrolysis of polyurethane alcoholysis products.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides an amidase SP1 mutant, wherein the amino acid sequence of the amidase SP1 mutant is obtained by mutating G to M at position 299 of the amino acid sequence of wild-type SP1 (i.e., G299M).

[0010] The amino acid sequence of the wild-type SP1 is shown in SEQ ID NO.1.

[0011] Furthermore, the present invention also provides an amidase SP1 mutant, wherein the amino acid sequence of the amidase SP1 mutant is obtained by mutating G to M at position 299 and M to L at position 238 of the amino acid sequence of wild-type SP1 (i.e., G299M / M238L).

[0012] Alternatively, the mutation at position 299 (G) can be changed to M, and at position 327 (Q) can be changed to L (i.e., G299M / Q327L).

[0013] Alternatively, the 299th position G is mutated to M, and the 389th position T is mutated to S (i.e., G299M / T389S).

[0014] The amino acid sequence of the wild-type SP1 is shown in SEQ ID NO.1.

[0015] Preferably, the amino acid sequence of the amidase SP1 mutant is obtained by mutating G to M at position 299 and Q to L at position 327 of the wild-type SP1 amino acid sequence.

[0016] The aforementioned amidase SP1 mutants were first screened using a combination of three strategies: PROSS / FireProt stability design, MutCompute local microenvironment prediction, and Yosshi and Disulfide by Design 2.0 disulfide bond engineering design, resulting in 25 single-substitution variants and 4 Cys-paired variants. Then, primary PNBC activity and thermostability screening yielded 7 beneficial single-substitution variants: A228L, I251L, M238L, G299M, Q327L, T334R, and T389S. These 7 single-substitution variants all maintained a certain level of PNBC hydrolytic activity while improving the 45 °C thermal half-life, providing multiple independently usable thermostability engineering elements for subsequent combined double mutants. Finally, the above 7 mutants were combined in pairs to construct double mutants. By examining the relative PNBC activity, 45 °C thermal challenge, and thermal half-life of the double mutants, 6 double mutants were obtained: A228L / G299M, A228L / T334R, A228L / T389S, G299M / M238L, G299M / Q327L, or G299M / T389S. All 6 candidate double mutants have long thermal lifetimes at 45 °C and TDA-DEG ​​hydrolysis capabilities, expanding the selection range of SP1 mutants that can be used for the hydrolysis of polyurethane alcoholysis products.

[0017] The aforementioned beneficial single-substitution variants and candidate double mutants all exhibit activity in hydrolyzing urethane bonds, and their thermal stability is improved.

[0018] The above-mentioned seven beneficial single-substitution variants, six candidate double mutants and their encoded nucleic acids, recombinant expression vectors, recombinant host cells and related enzymatic applications provide a more complete technical solution for selecting SP1 biocatalysts with suitable activity-stability balance under different substrate and process conditions.

[0019] Without altering the specified mutation sites and retaining the urethane bond hydrolysis activity, the present invention also includes proteins with added expression, purification, or immobilization tags at the N-terminus or C-terminus of the corresponding mutants, as well as functional variants that have at least 95%, 98%, or 99% identity with any of the above-mentioned beneficial single-substitution variants or double-mutant sequences, retain the specified mutation sites, and have the hydrolysis activity.

[0020] In a second aspect, the present invention provides an isolated nucleic acid molecule that encodes the amidase SP1 mutant described in the first aspect.

[0021] Thirdly, the present invention provides a recombinant expression vector containing the nucleic acid molecule described in the second aspect.

[0022] Fourthly, the present invention provides a recombinant host cell containing the recombinant expression vector described in the third aspect, or having the nucleic acid molecules described in the second aspect integrated into its genome.

[0023] Fifthly, the present invention provides an enzyme preparation containing the expression protein of the amidase SP1 mutant described in the first aspect.

[0024] The enzyme preparation may be in the form of, but is not limited to, purified enzyme, crude enzyme solution, or immobilized enzyme.

[0025] In some embodiments of the present invention, the enzyme preparation is in the form of a purified enzyme.

[0026] In a sixth aspect, the present invention provides the use of the amidase SP1 mutant described in the first aspect, or the nucleic acid molecule described in the second aspect, or the recombinant expression vector described in the third aspect, or the recombinant host cell described in the fourth aspect, or the enzyme preparation described in the fifth aspect in the hydrolysis of polyurethane alcoholysis products.

[0027] The polyurethane alcoholysis product is a TDI-based polyurethane alcoholysis product and / or an MDI-based polyurethane alcoholysis product.

[0028] The TDI-based polyurethane alcoholysis product includes TDA-DEG ​​and its monocarbamate intermediate; the MDI-based polyurethane alcoholysis product includes MDA-DEG ​​and its monocarbamate intermediate.

[0029] Wherein, TDA-DEG ​​and MDA-DEG ​​are N-aryl dicarboxylate esters; the monocarboxylate intermediates of TDA-DEG ​​and MDA-DEG ​​are N-aryl monocarboxylate esters.

[0030] Specifically, the TDA-DEG ​​is 2,4-TDA-DEG ​​and / or 2,6-TDA-DEG; the MDA-DEG ​​is 2,4-MDA-DEG ​​and / or 4,4-MDA-DEG.

[0031] In a seventh aspect, the present invention provides a method for the chemical-enzymatic cascade recovery of polyurethane alcoholysis products, wherein polyurethane material is chemically alcoholyzed to obtain alcoholysis products, the amidase SP1 mutant described in the first aspect is added to the alcoholysis products or their separated components to carry out a hydrolysis reaction, and the generated aromatic diamine is recovered.

[0032] The hydrolysis reaction is carried out under the following conditions: pH 6.0~8.0, 25~40 ℃ for 6~48 h.

[0033] In some embodiments of the present invention, the hydrolysis reaction is carried out under the following conditions: pH 68.0 and 37-40 °C for 6-24 h.

[0034] In some embodiments of the present invention, the reaction system is: 50 mM pH 8.0 phosphate buffer, approximately 5 wt% alcoholysis product or its isolated fraction, and 0.2 mg / mL amidase SP1 mutant.

[0035] Beneficial effects:

[0036] (1) This invention employs three strategies for screening: PROSS / FireProt stability design, MutCompute local microenvironment prediction, and Yosshi and Disulfide by Design 2.0 disulfide bond engineering design. Combined with PNBC activity, thermal stability, and thermal half-life classification screening, a type of amidase SP1 mutant G299M, or G299M / M238L, or G299M / Q327L, or G299M / T389S that can be used for the hydrolysis of polyurethane alcoholysis products, has the activity of hydrolyzing urethane bonds, and has improved thermal stability is obtained.

[0037] (2) The SP1 mutant of the amidase of the present invention has a significantly prolonged thermal half-life at 45°C, which enables it to operate continuously at higher temperatures in industrial applications and has good prospects for industrial application. Among them, the G299M / Q327L mutant has a thermal half-life of 25 h at 45 °C, which is about 47 times that of WT, and the melting temperature is increased by about 6.7 °C. It can show the most sustained substrate consumption ability in long-term reactions.

[0038] (3) The mutant G299M / Q327L of this invention exhibits significantly better substrate consumption capacity and product release than the wild type in both real TDI-based and MDI-based polyurethane alcoholysis products, demonstrating good tolerance and adaptability to complex industrial substrate systems. Specifically, in real TDI-based polyurethane alcoholysis products, G299M / Q327L produces approximately 28.82 mM of total TDA in 24 h, while WT produces approximately 19.01 mM; in real MDI-based polyurethane alcoholysis products, it releases approximately 15.6 mM of MDA in 24 h, while WT releases approximately 8.2 mM. Attached Figure Description

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0040] Figure 1 The graph shows the half-life and relative PNBC activity of the SP1 double mutant at 45 °C.

[0041] Figure 2 The figure shows the hydrolysis results of the SP1 double mutant on the TDA-DEG ​​substrate.

[0042] Figure 3 The results are from repeated feed-and-batch hydrolysis of TDA-DEG ​​for wild-type SP1 and double mutant G299M / Q327L.

[0043] Figure 4 The enzymatic hydrolysis effects of wild-type SP1 and double mutant G299M / Q327L on MDI-based polyurethane alcoholysis products were evaluated. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0046] Example 1: SP1 amidase mutant site design

[0047] This embodiment employs a multi-strategy synergistic rational design approach, using wild-type SP1 amidase as a template. Its amino acid sequence is shown in SEQ ID NO.1, and its encoding nucleotide sequence is shown in SEQ ID NO.2, to screen for potential thermostable engineering sites.

[0048] Using the predicted 3D structure of SP1 as a design template, three strategies were comprehensively employed: PROSS / FireProt stability design, MutCompute local microenvironment prediction, and Yosshi and Disulfide by Design 2.0 disulfide bond engineering design. Specifically, PROSS / FireProt was used to screen single-amino acid substitutions that were beneficial to protein stability in terms of sequence conservation and structural energy; MutCompute was used to predict substitutions with potential stabilizing effects based on the local 3D microenvironment of residues; and disulfide bond engineering was used to screen Cys residue combinations with suitable spatial geometry that could potentially improve the rigidity of the protein's local skeleton through intramolecular disulfide bond constraints.

[0049] To preserve the catalytic function of SP1 as much as possible, when integrating candidate variants, the conserved Lys-Ser-Ser catalytic triplet, the conserved GGSSGG motif, and their adjacent potential substrate processing regions are treated as functional protection regions, and no direct mutations are performed on these regions.

[0050] PROSS / FireProt yielded 16 single substitution candidates; MutCompute yielded 9 single substitution candidates; and disulfide bond engineering yielded 4 Cys pair candidate variants, involving 7 Cys substitutions that needed to be introduced. Thus, a total of 32 design amino acid substitutions were obtained, which were organized into 29 primary candidate engineered variants for actual construction and screening, including 25 single substitution variants and 4 Cys pair variants.

[0051] Table 1. Candidate engineering variants obtained from screening three design strategies.

[0052]

[0053] Example 2: Construction and protein expression of the candidate engineered variant—SP1 mutant

[0054] 1. Construction of a single substitution variant

[0055] The wild-type SP1 encoding gene was cloned into the pET29a vector to obtain the recombinant expression plasmid pET29a-SP1. Using pET29a-SP1 as a template, the 25 single-substitution variants described in Example 1 were constructed using the whole-plasmid PCR site-directed mutagenesis method.

[0056] Each 50 μL PCR reaction system contained 50 ng of template plasmid, 0.5 μmol / L each of forward and reverse primers, 25 μL of PrimeSTAR Max premix (2×), and nuclease-free water to a final volume of 50 μL. The PCR reaction program was as follows: 98 °C pre-denaturation for 3 min; 30 cycles, including 98 °C denaturation for 10 s, 55 °C annealing for 15 s, and 72 °C extension for 5 min; and a final extension at 72 °C for 10 min.

[0057] The PCR product was added to 20 U Dpn I and digested at 37 °C for 2 h to remove the methylated parental template plasmid. The digested product was then transformed into E. coli DH5α competent cells, positive clones were picked, and the mutation sites were confirmed by colony PCR and DNA sequencing to obtain 25 single-mutant recombinant plasmids.

[0058] 2. Construction of Cys Paired Variants

[0059] The disulfide bond engineering candidates were constructed into four paired variants according to the designed Cys pairing relationships: A67C / L70C, A157C / C184, R247C / E280C, and P351C / V410C.

[0060] For A67C / L70C, R247C / E280C, and P351C / V410C, a stepwise site-directed mutagenesis method was used for construction. First, using pET29a-SP1 as a template, following the construction process in step 1, Cys substitutions were introduced at the first site (A67C, R247C, P351C) of each paired variant. After sequencing verification, recombinant plasmids with the first site mutation were obtained. Using these recombinant plasmids as templates, Cys substitutions were further introduced at the second site of each paired variant. Finally, DNA sequencing confirmed the correct presence of both target substitutions, yielding double-mutant recombinant plasmids for each paired variant.

[0061] Since the 184th residue of wild-type SP1 is itself a cysteine ​​residue, when constructing the A157C / C184 variant, it is only necessary to introduce an A157C substitution into pET29a-SP1. The Cys157 introduced into the resulting protein and the natural Cys184 constitute the expected Cys pair constraint. The construction process is the same as step 1.

[0062] The single Cys intermediate plasmids used in the above construction process are only used as intermediates for the construction of paired variants and are not used as independent first-level engineering variants for screening.

[0063] 3. Expression and purification of recombinant proteins

[0064] The 29 recombinant plasmids correctly sequenced in steps 1 and 2, along with the wild-type pET29a-SP1 plasmid, were transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD500. 600 =0.6~0.8, add IPTG to a final concentration of 0.5 mM, and induce expression at 16 ℃ for 18 h. After induction, collect bacterial cells by centrifugation at 4 ℃, resuspend the cells in pre-cooled lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0), sonicate, centrifuge at 12,000 rpm for 30 min at 4 ℃, collect the supernatant, and purify by gradient elution using a Ni-NTA affinity chromatography column to obtain the purified recombinant protein, which is stored at -80 ℃.

[0065] Example 3: Screening for PNBC activity and thermal stability of SP1 mutants

[0066] 1. PNBC hydrolysis activity assay

[0067] Wild-type SP1 and mutant proteins purified in Example 2 were diluted to a concentration of 0.2 mg / mL with phosphate buffer (50 mM pH 8.0). The amidase activity was determined using 4-nitrophenylcarbamate (PNBC) as a model substrate. A 1000 μL reaction system contained: 970 μL phosphate buffer (50 mM pH 8.0), 20 μL PNBC (10 mM) (PNBC dissolved in DMSO, final concentration ≤2%), and 10 μL enzyme solution (0.2 mg / mL). The reaction was carried out at 37 °C for 10 min, and the rate of increase in absorbance within the linear reaction range was recorded by monitoring the absorbance change at 405 nm. The p-nitrophenol formation rate was calculated based on the p-nitrophenol standard curve. The specific activity of PNBC in wild-type SP1 (WT) was 2.7358 U / mg, and its relative activity was defined as 100%.

[0068] 2. Thermal stability evaluation

[0069] The purified wild-type SP1 and mutant protein samples were uniformly diluted to 0.2 mg / mL and incubated at 45 °C. Samples were taken at different time points (10 min, 20 min, 30 min, 40 min, 60 min, 90 min, 120 min, 150 min, 180 min, 240 min, and 300 min), immediately cooled in an ice bath for 5 min, and then briefly centrifuged at 4 °C. The supernatant was used to determine the residual activity of PNBC hydrolysis. The inactivation curve of residual activity over time was plotted with the initial activity (0 min) as 100%. The thermal inactivation half-life T was calculated by nonlinear fitting according to the first-order inactivation kinetic equation ln(A / A0)=-kt. 1 / 2 =ln2 / k.

[0070] Table 2. Half-life at 45 °C and relative PNBC activity of 29 mutants

[0071]

[0072] The screening results showed that the A228L, I251L, M238L, G299M, Q327L, T334R, and T389S single mutants maintained certain PNBC activity while increasing their thermal half-life at 45 °C. Among them, the G299M single mutant had a half-life of 234.2 min (3.903 h), approximately 7.2 times longer than the WT half-life of 32.7 min (0.545 h), while maintaining 134.4% of the relative PNBC activity. The amino acid sequences of the A228L, I251L, M238L, G299M, Q327L, T334R, and T389S single mutants obtained from the screening are shown in SEQ ID NO. 3–9, respectively.

[0073] Example 4: Construction and Screening of SP1 Double Mutants

[0074] Seven single mutants (A228L, I251L, M238L, G299M, Q327L, T334R, and T389S) that performed well in Example 3 were selected and combined in pairs to construct 21 double mutants. Taking the A228L / I251L double mutant as an example, primers for introducing the I251L mutation were designed and synthesized based on the SP1 gene sequence (as shown in SEQ ID NO.2). Using the A228L single mutant recombinant plasmid pET29a-SP1-A228L constructed in step 1 of Example 2 as a template, PCR amplification was performed with the corresponding primers. The amplified PCR product was digested and transformed with enzymes, and verified by sequencing. The plasmid was extracted using a plasmid extraction kit to obtain the A228L / I251L double mutant recombinant plasmid. The recombinant proteins were expressed and purified according to the recombinant protein expression and purification process in Example 2, and the relative activity of PNBC was examined according to the process in Example 3. The specific combinations of the 21 double mutants and the corresponding relative PNBC activity results are shown in Table 3.

[0075] Table 3. Specific combinations of 21 double mutants and their corresponding relative PNBC activities.

[0076]

[0077] First, based on the standard that the relative PNBC activity is not less than 50% of WT, a total of 13 SP1 double mutants were obtained: A228L / I251L, A228L / G299M, A228L / T334R, A228L / Q327L, A228L / T389S, I251L / G299M, I251L / M238L, I251L / T389S, G299M / M238L, G299M / Q327L, G299M / T389S, M238L / Q327L, and M238L / T389S.

[0078] Secondly, the above 13 SP1 double mutants were incubated at 45 °C for 10 h, and screened based on the criterion of still having detectable residual activity. Nine SP1 double mutants were retained: A228L / I251L, A228L / G299M, A228L / T334R, A228L / Q327L, A228L / T389S, I251L / T389S, G299M / M238L, G299M / Q327L, and G299M / T389S.

[0079] Finally, the half-life of the above nine SP1 double mutants at 45 °C was determined, and the results were recorded at t 1 / 2 Based on the standard of >5 h, a total of 6 candidates were ultimately retained: A228L / G299M, A228L / T334R, A228L / T389S, G299M / M238L, G299M / Q327L, and G299M / T389S.

[0080] like Figure 1 As shown, the combinations of A228L / T334R, A228L / T389S, G299M / M238L, G299M / Q327L, and G299M / T389S achieved significantly longer thermal lifetimes while the model substrate activity decreased to approximately 56.3%–68.8%. Among them, the SP1 double mutant G299M / Q327L had a 45℃ half-life of 25 h, which was about 47 times longer than WT, demonstrating a balance between activity and stability.

[0081] The amino acid sequences of the above SP1 double mutants A228L / G299M, A228L / T334R, A228L / T389SG, G299M / M238L, G299M / Q327L, and G299M / T389S are shown in SEQ ID NO.10~15, respectively.

[0082] Example 5: Hydrolysis of TDA-DEG ​​by SP1 double mutant

[0083] Using TDA-DEG ​​as a substrate, the hydrolytic catalytic activity of wild-type SP1 (WT) and SP1 double mutants (A228L / G299M, A228L / T334R, A228L / T389SG, 299M / M238L, G299M / Q327L, G299M / T389S) was compared. The total reaction volume was 5 mL, specifically composed of 20 mM TDA-DEG, 50 mM pH 8.0 phosphate buffer, and 0.05 mg / mL enzyme protein. The reaction was carried out at 37℃–40℃ for 24 h. During the reaction, 100 μL samples were taken periodically, and the samples were immediately added to 400 μL of pre-cooled acetonitrile to terminate the reaction and precipitate the protein. After the sample was filtered through a 0.22 μm filter membrane, the residual concentration of TDA-DEG, the accumulation of monocarbamate intermediates, and the amount of target product TDA (2,4-toluenediamine) were determined by HPLC.

[0084] The results are as follows Figure 2 As shown, in the initial stage of the reaction (0–3 h), both wild-type SP1 and SP1 double mutants rapidly catalyzed the hydrolysis of TDA-DEG, and the TDA-DEG ​​concentration decreased rapidly. However, with the extension of reaction time, the hydrolytic ability of wild-type SP1 gradually decreased. In contrast, some SP1 enzymes with introduced double mutants exhibited excellent stability and hydrolytic ability, such as G299M / Q327L, A228L / G299M, G299M / T389S, and A228L / T389SG. In particular, the G299M / Q327L double mutant maintained a more sustained and robust TDA-DEG ​​hydrolytic ability throughout the entire 24-h reaction period, and reduced the residual TDA-DEG ​​concentration to a minimum of approximately 5.0 mM at 24 h. This result demonstrates that through reasonable site combination design, not only can the thermostability problem of wild-type enzymes be effectively solved, but their practical application potential for long-term catalytic hydrolysis of aromatic carbamates under complex substrate environments can also be significantly improved.

[0085] Example 6: Application of SP1 double mutant in fed-batch reaction

[0086] The G299M / Q327L double mutant was selected, and repeated fed-batch hydrolysis experiments were conducted using TDA-DEG ​​as a substrate to evaluate its application potential in continuous or repeated batch reactions. The total reaction volume was 5 mL, with 50 mM pH 8.0 phosphate buffer as the reaction buffer. In each round of the reaction, a final concentration of 5 mM TDA-DEG ​​and 0.2 mg / mL enzyme protein (wild-type or double mutant) were added, and the reaction was carried out at 37 ℃ for 6 h. After each round, 100 μL of sample was taken for HPLC detection of TDA production. Fresh TDA-DEG ​​was then added to the remaining reaction solution to a final concentration of 5 mM, without adding additional enzyme protein, and the next round of the reaction was immediately initiated. This process was repeated for multiple rounds of continuous fed-batch reactions.

[0087] The amount of TDA generated at the end of each reaction round was determined by HPLC, and the amount of newly generated TDA in each round and the cumulative amount of TDA generated were calculated. The results are as follows: Figure 3 As shown, wild-type SP1 (WT) can catalyze the hydrolysis of TDA-DEG ​​to TDA normally in the early stages of the reaction, but the amount of newly generated TDA per cycle decreases significantly with increasing cycle number. In contrast, the double mutant G299M / Q327L maintains a high TDA generation capacity in multiple consecutive feed reactions, with a cumulative TDA generation significantly higher than that of wild-type SP1. This indicates that the G299M / Q327L double mutant has good operational stability and can achieve multiple batches of continuous hydrolysis without the need for intermediate enzyme addition, demonstrating its potential to replace wild-type enzymes in polyurethane chemical-enzymatic cascade recovery processes.

[0088] Example 7: Enzymatic upgrading treatment of real polyurethane alcoholysis products by G299M / Q327L

[0089] TDI-based polyether polyurethane foam was pulverized and dried. 40 g of diethylene glycol was weighed and added to a four-necked flask equipped with a mechanical stirrer, thermometer, condenser, and inert gas inlet. The mixture was heated to 200 °C under nitrogen protection. 1 wt% dibutyltin dilaurate was added as an alcoholysis catalyst, based on the mass of the polyurethane. 40 g of the pulverized and dried TDI-based polyether polyurethane foam was slowly added while stirring at 300 rpm. After the polyurethane material was completely added and fully dispersed, the mixture was kept at 200 °C with stirring for 2 h. Heating was then stopped and the mixture was cooled to room temperature to obtain the TDI-based polyurethane chemical alcoholysis product.

[0090] The alcoholysis products were allowed to stand until phase separation was complete, yielding a polyol-enriched upper phase, a SAN / polymer dispersion intermediate phase, and a carbamate-containing substrate phase. The carbamate-containing substrate phase was collected as the substrate for subsequent enzymatic upgrading. HPLC analysis showed that the carbamate-containing substrate phase mainly contained TDA-DEG, the monocarbamate intermediate TAC, and a small amount of TDA.

[0091] Wild-type SP1 and G299M / Q327L groups were set up separately. The total volume of the enzymatic digestion reaction in each group was 2 mL, containing 50 mM phosphate-buffered saline (PBS, pH 8.0), approximately 5 wt% of the above-mentioned carbamate intermediate substrate, and wild-type SP1 or G299M / Q327L protein at a final concentration of 0.2 mg / mL. The volume was then brought up to 2 mL with PBS. Each reaction system was incubated at 40 °C for 24 h.

[0092] 200 μL samples were taken at 6 h, 12 h, and 24 h of reaction, respectively, and mixed with an equal volume of acetonitrile to terminate the enzymatic reaction and precipitate the protein. The samples were centrifuged at 12,000 × g for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. HPLC analysis was performed using a C18 column with an acetonitrile / water (60:40, v / v) mobile phase at a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, and an injection volume of 20 μL. The amounts of 2,4-TDA and 2,6-TDA generated were quantified using the external standard method.

[0093] Table 4. Generation amounts of 2,4-TDA and 2,6-TDA at different time points

[0094]

[0095] The results are shown in Table 4. With prolonged reaction time, the amount of TDA produced gradually increased. Compared to WT, G299M / Q327L produced higher concentrations of 2,4-TDA and 2,6-TDA during the longer reaction time. At 24 h, the total TDA produced by G299M / Q327L was approximately 28.82 mM, while the total TDA produced by WT was approximately 19.01 mM, indicating that its improved thermal stability translates into a higher release capacity of aromatic diamines in the actual polyurethane alcoholysis products.

[0096] Example 8: Enzymatic upgrading treatment of real MDI polyurethane alcoholysis products by G299M / Q327L

[0097] MDI-based polyether polyurethane foam was chemically alcoholyzed according to the alcoholysis steps in Example 7 to obtain a homogeneous alcoholysis product. The homogeneous alcoholysis product contained polyols and urethane-containing intermediates, among other substances.

[0098] The homogeneous alcoholysis product was used as the substrate and diluted with phosphate buffer to a final concentration of approximately 5 wt%. WT and G299M / Q327L solutions with a final concentration of 0.2 mg / mL were then added to initiate the enzyme-catalyzed reaction. The reaction system was incubated at 37–40 °C, and the amount of MDA generated was detected by HPLC.

[0099] The results are as follows Figure 4 As shown, WT can release about 8.2 mM MDA within 24 h, while G299M / Q327L can release about 15.6 mM MDA, which is about twice that of wild type.

[0100] In summary, this invention yielded seven SP1 single mutants with improved thermal stability: A228L, I251L, M238L, G299M, Q327L, T334R, and T389S; and six double mutants with a good activity-stability balance: A228L / G299M, A228L / T334R, A228L / T389S, G299M / M238L, G299M / Q327L, and G299M / T389S. Among these, G299M / Q327L exhibited excellent long-term aromatic diamine release ability in the alcoholysis products of real TDI-based and MDI-based polyurethanes.

[0101] The above embodiments are only used to illustrate the technical concept and preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, without departing from the technical concept of the present invention, conventional adjustments can be made to the expression form, tag sequence, recombinant vector, host cell, enzyme preparation form, and reaction process conditions of the mutant according to actual application needs.

Claims

1. An amidase SP1 mutant, characterized in that, The amino acid sequence of the amidase SP1 mutant is obtained by mutating G to M at position 299 of the wild-type amidase SP1, as shown in SEQ ID NO.

1.

2. An amidase SP1 mutant, characterized in that, The amino acid sequence of the amidase SP1 mutant is obtained by mutating G at position 299 to M and Q at position 327 to L in the wild-type amidase SP1 shown in SEQ ID NO.

1. Alternatively, it can be obtained by mutating G at position 299 to M and simultaneously mutating A at position 228 to L; Alternatively, it can be obtained by mutating G at position 299 to M and simultaneously mutating T at position 389 to S.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the amidase SP1 mutant as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 3.

5. A recombinant host cell, characterized in that, It contains the recombinant expression vector of claim 4, or the nucleic acid molecule of claim 3 is integrated into its genome.

6. An enzyme preparation, characterized in that, The expressed protein contains the amidase SP1 mutant as described in claim 1 or claim 2.

7. The use of the amidase SP1 mutant of claim 1, or the amidase SP1 mutant of claim 2, or the recombinant expression vector of claim 4, or the recombinant host cell of claim 5, or the enzyme preparation of claim 6 in the hydrolysis of polyurethane alcoholysis products.

8. The application according to claim 7, characterized in that, The polyurethane alcoholysis product is a TDI-based polyurethane alcoholysis product and / or an MDI-based polyurethane alcoholysis product. The TDI-based polyurethane alcoholysis product includes TDA-DEG ​​and its monocarbamate intermediate; the MDI-based polyurethane alcoholysis product includes MDA-DEG ​​and its monocarbamate intermediate.

9. A method for the chemical-enzymatic cascade recovery of polyurethane alcoholysis products, characterized in that, The polyurethane material is chemically alcoholyzed to obtain the alcoholysis product. The amidase SP1 mutant according to any one of claims 1 to 2 is added to the alcoholysis product or its separated components to carry out a hydrolysis reaction, and the generated aromatic diamine is recovered.

10. The method according to claim 9, characterized in that, The hydrolysis reaction is carried out under the following conditions: pH 6.0~8.0, 25~40 ℃ for 6~48 h.

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