Nutrient-degrading enzyme, engineering bacterial agent and application thereof in nutrient regulation of polymeric slow-release fertilizer

Abstract

This invention discloses a nutrient-degrading enzyme, engineered microbial agents, and their application in the nutrient regulation of polymeric slow-release fertilizers, belonging to the field of enzyme engineering technology. This invention utilizes a specific polymeric slow-release fertilizer-degrading enzyme modified by artificial intelligence, which not only retains the degradation activity of the source strain enzyme but also achieves efficient heterologous expression while promoting a dual improvement in stability and catalytic efficiency. The constructed composite engineered strain exhibits no antagonistic effect, enabling low-cost, large-scale production of the degrading enzyme. The composite engineered strain can achieve precise regulation of nutrient release from polymeric slow-release fertilizers. By adjusting the ratio of engineered strains producing peptidases and amidases, precise supply of nutrients to different stages throughout the growth cycle of different crops can be achieved, resulting in complete degradation of polymeric slow-release fertilizers, significantly improving crop yield and nutrient utilization, and showing significant application prospects in the development of precision agriculture.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, specifically to a nutrient-degrading enzyme, an engineered microbial agent, and their application in the nutrient regulation of polymeric slow-release fertilizers. Background Technology

[0002] Polymerized slow-release fertilizers, prepared through cross-linking polymerization reactions, achieve the effects of delaying nutrient release, reducing nutrient loss, and improving nutrient utilization. They are currently used in the cultivation and production of various crops. These fertilizers typically contain functional groups such as peptide bonds, amide bonds, amino groups, and hydroxyl groups. Their nutrient release rate mainly depends on the catalytic cleavage of these functional groups by degrading enzymes secreted by microorganisms in the environment. Therefore, the activity, stability, and substrate specificity of the degrading enzymes directly determine the application effect of polymerized slow-release fertilizers.

[0003] However, existing polymeric slow-release fertilizers still face many technical bottlenecks in practical applications. On the one hand, microbial resources capable of efficiently degrading polymeric slow-release fertilizers in the natural environment are scarce. The natural degrading enzymes they secrete suffer from low activity, poor thermal stability, and insufficient substrate binding efficiency, resulting in slow degradation rates and difficulty in matching the nutrient release cycle with the nutrient requirements of crops at different growth stages. On the other hand, traditional enzyme molecular modification technologies rely on empirical screening, which suffers from drawbacks such as high blindness, long screening cycles, and difficulty in simultaneously optimizing enzyme activity, stability, substrate specificity, and other multi-dimensional performance aspects, failing to meet the demands of large-scale agricultural production for highly efficient degrading enzymes. Simultaneously, existing heterologous expression systems for degrading enzymes suffer from low expression efficiency, poor stability of engineered strains, and high fermentation costs. Furthermore, single-function engineered strains are insufficient to achieve complete degradation of polymeric slow-release fertilizers, and the combined application of multiple strains easily leads to antagonistic effects, further limiting the large-scale application of degrading enzymes in agricultural production. Therefore, there is an urgent need to develop a technical system that can balance degradation efficiency, stability, low-cost large-scale production, and precise nutrient supply to promote the widespread application of polymeric slow-release fertilizers in precision agriculture. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a nutrient-degrading enzyme, an engineered microbial agent, and their application in the nutrient regulation of polymeric slow-release fertilizers.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a nutrient-degrading enzyme, said nutrient-degrading enzyme being a protein represented by at least one of the following (A1)-(A13):

[0007] (A1) pepE protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0008] (A2) lepB-1 protein, the amino acid sequence of which is shown in SEQ ID NO.2;

[0009] (A3) lepB-2 protein, the amino acid sequence of which is shown in SEQ ID NO.3;

[0010] (A4) amiABC protein, the amino acid sequence of which is shown in SEQ ID NO.4;

[0011] (A5) amiS protein, the amino acid sequence of which is shown in SEQ ID NO.5;

[0012] (A6) amiE protein, the amino acid sequence of which is shown in SEQ ID NO.6;

[0013] (A7) pepE-D100A protein, the amino acid sequence of which is shown in SEQ ID NO.13;

[0014] (A8) pepE-E135Q protein, the amino acid sequence of which is shown in SEQ ID NO.15;

[0015] (A9) pepE-D100A / E135Q protein, the amino acid sequence of which is shown in SEQ ID NO.17;

[0016] (A10)amiE-E80A protein, the amino acid sequence of which is shown in SEQ ID NO.19;

[0017] (A11)amiE-I135V protein, the amino acid sequence of which is shown in SEQ ID NO.21;

[0018] (A12)amiE-E80A / I135V protein, the amino acid sequence of which is shown in SEQ ID NO.23;

[0019] (A13) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of any of the proteins defined in (A1)-(A12).

[0020] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0021] Preferably, the nutrient-degrading enzyme is composed of pepE-D100A / E135Q protein and amiE-E80A / I135V protein in a mass ratio of 1:1.

[0022] This invention is derived from *Priestella megaterium* (… Priestia megaterium Nutrient-degrading enzymes pepE, lepB-1, lepB-2, amiABC, amiS, and amiE with high catalytic degradation activity for polymer slow-release fertilizers were isolated and purified from YC5 (CGMCC NO: 30135).

[0023] Further, the pepE and amiE proteins with excellent degradation activity were modified using artificial intelligence. The artificial intelligence modification model was selected from the enzyme molecule modification model constructed by deep learning in machine learning.

[0024] Preferably, the machine learning is selected from one or more of deep neural networks (DNN), convolutional neural networks (CNN), recurrent neural networks (RNN), and generative adversarial networks (GAN);

[0025] Preferably, the input data includes the amino acid sequence, three-dimensional structure, and substrate binding mode data of the nutrient-degrading enzyme;

[0026] Preferably, the three-dimensional structure is selected from one or more of the following: homology modeling, X-ray crystal diffraction, and AlphaFold prediction.

[0027] After artificial intelligence modification, three mutants of pepE protein (pepE-D100A, pepE-E135Q, and pepE-D100A / E135Q) and three mutants of amiE protein (amiE-E80A, amiE-I135V, and amiE-E80A / I135V) were obtained. The mutation modification significantly improved their degradation performance for polymeric slow-release fertilizers.

[0028] Furthermore, combining pepE-D100A / E135Q protein and amiE-E80A / I135V protein can synergistically improve the degradation performance of polymeric slow-release fertilizers.

[0029] A second aspect of the present invention provides an engineered microbial agent, said engineered microbial agent being constructed by the following method:

[0030] The encoding genes of the above-mentioned nutrient-degrading enzymes were ligated into an expression vector to construct a recombinant expression vector; the recombinant expression vector was then transferred into a host cell to construct an engineered strain; the engineered strain was then fermented to obtain an engineered bacterial agent.

[0031] Preferably, the gene encoding the nutrient-degrading enzyme is any one of the nucleic acid molecules shown in (1)-(24) below:

[0032] (1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.7;

[0033] (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO.1;

[0034] (3) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 8;

[0035] (4) Nucleic acid molecules other than (3) that encode the amino acid sequence shown in SEQ ID NO.2;

[0036] (5) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.9;

[0037] (6) Nucleic acid molecules other than (5) that encode the amino acid sequence shown in SEQ ID NO.3;

[0038] (7) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.10;

[0039] (8) Nucleic acid molecules other than (7) that encode the amino acid sequence shown in SEQ ID NO.4;

[0040] (9) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.11;

[0041] (10) Nucleic acid molecules other than (9) that encode the amino acid sequence shown in SEQ ID NO. 5;

[0042] (11) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.12;

[0043] (12) Nucleic acid molecules other than (11) that encode the amino acid sequence shown in SEQ ID NO. 6;

[0044] (13) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.14;

[0045] (14) Nucleic acid molecules other than (13) that encode the amino acid sequence shown in SEQ ID NO. 13;

[0046] (15) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.16;

[0047] (16) Nucleic acid molecules other than (15) that encode the amino acid sequence shown in SEQ ID NO. 15;

[0048] (17) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.18;

[0049] (18) Nucleic acid molecules other than (17) that encode the amino acid sequence shown in SEQ ID NO. 17;

[0050] (19) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.20;

[0051] (20) Nucleic acid molecules other than (19) that encode the amino acid sequence shown in SEQ ID NO. 19;

[0052] (21) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.22;

[0053] (22) Nucleic acid molecules other than (21) that encode the amino acid sequence shown in SEQ ID NO.21;

[0054] (23) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.24;

[0055] (24) Nucleic acid molecules other than (23) that encode the amino acid sequence shown in SEQ ID NO.23.

[0056] Preferably, the expression vector is selected from one or more of plasmids, viscera, and artificial chromosomes;

[0057] More preferably, the expression vector is selected from one or more of the pET-28a expression vector, pET-32a expression vector, pGEX series expression vector, or pBAD series expression vector.

[0058] Preferably, the host cell is selected from one or more prokaryotic cells or eukaryotic cells;

[0059] Furthermore, the host cell is a bacterial cell or a fungal cell;

[0060] Preferably, the bacterial cells are selected from one or more of the genera *Escherichia coli*, *Bacillus*, *Staphylococcus aureus*, and *Lactobacillus*.

[0061] More preferably, the bacterial cells are selected from Escherichia coli;

[0062] Furthermore, the *Escherichia coli* is selected from one or more of BL21 (DE3), DH5α, or Rosetta (DE3);

[0063] Preferably, the fungal cells are selected from yeast.

[0064] Preferably, the fermentation conditions are: 37℃, aeration rate of 1.0 vvm, stirring speed of 200 rpm, and culture for 20-24 hours.

[0065] In some preferred embodiments of the present invention, the engineered bacterial agent is prepared by mixing the seed culture of an engineered strain expressing pepE-D100A / E135Q protein and the seed culture of an engineered strain expressing amiE-E80A / I135V protein in a volume ratio of (3-7):(3-7).

[0066] A third aspect of the present invention provides the application of the above-mentioned nutrient-degrading enzymes or engineered microbial agents in the nutrient regulation of polymeric slow-release fertilizers.

[0067] In the above applications, the polymeric slow-release fertilizer contains one or more of the following functional groups: peptide bond, amide bond, amino bond, and hydroxyl bond.

[0068] Furthermore, the polymeric slow-release fertilizer has any of the following structures:

[0069]

[0070] The beneficial effects of this invention are:

[0071] This invention utilizes a specific polymeric slow-release fertilizer-degrading enzyme modified by artificial intelligence. This enzyme not only retains the degradation activity of the original strain but also achieves efficient heterologous expression while simultaneously improving both stability and catalytic efficiency. The constructed composite engineered strain exhibits no antagonistic effect, enabling low-cost, large-scale production of the degradation enzyme.

[0072] The composite engineered strains can achieve precise control over the release of nutrients from polymeric slow-release fertilizers. By adjusting the ratio of engineered strains that produce peptidase and amidase, the nutrient requirements of different crops at different stages throughout their growth can be precisely supplied, achieving complete degradation of polymeric slow-release fertilizers and significantly improving crop yield and nutrient utilization. This demonstrates important application prospects in the development of precision agriculture. Attached Figure Description

[0073] Figure 1 Photographs of crude enzyme solutions containing pepE, lepB-1, lepB-2, amiABC, amiS, and amiE obtained in Example 1.

[0074] Figure 2 Example 1 shows the degradation rates of polymeric slow-release fertilizers by crude enzyme solutions of pepE, lepB-1, lepB-2, amiABC, amiS, and amiE. In the figure, a represents the degradation rate of polymeric slow-release fertilizers by the buffer control; b represents the degradation rate of polymeric slow-release fertilizers by pepE, lepB-1, and lepB-2; and c represents the degradation rate of slow-release fertilizers by amiABC, amiS, and amiE.

[0075] Figure 3The enzymatic performance of pepE-D100A / E135Q and amiE-E80A / I135V was evaluated. In the figure, a represents the enzyme activity of pepE-D100A / E135Q and amiE-E80A / I135V under different temperature conditions; b represents the enzyme activity of pepE-D100A / E135Q under different pH conditions; c represents the enzyme activity of amiE-E80A / I135V under different pH conditions; and d represents the catalytic efficiency of pepE-D100A / E135Q, amiE-E80A / I135V, and pepE-D100A / E135Q and amiE-E80A / I135V in a 1:1 mass ratio for degradation of slow-release fertilizer.

[0076] In the diagram, "pepE" represents pepE-D100A / E135Q, and "amiE" represents amiE-E80A / I135V.

[0077] Figure 4 ESI-MS results of polymeric slow-release fertilizers after degradation by pepE-D100A / E135Q and amiE-E80A / I135V compound enzyme solution.

[0078] Figure 5 The results of antagonistic effect detection using the plate confrontation method are shown in the figure. "pepE" represents the engineered strain expressing pepE-D100A / E135Q, and "amiE" represents the engineered strain expressing amiE-E80A / I135V. Detailed Implementation

[0079] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0080] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0081] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0082] Priestella megaterium ( Priestia megaterium YC5 has the accession number CGMCC NO: 30135 and is recorded in patent CN 119162063B.

[0083] The "polymer slow-release fertilizer" used in this embodiment of the invention belongs to the slow-release nitrogen fertilizer type. The specific preparation method is as follows: 41.67 ml of formaldehyde solution and 70 ml of deionized water are added to a 250 ml three-necked round-bottom flask. The pH of the mixed solution is adjusted to 8.5 with 5% potassium hydroxide or 0.1% phosphoric acid solution. After the temperature of the heat-collecting magnetic stirrer reaches the corresponding 35 ℃, 65.5 g of urea is added and reacted for 1.5 h. Then, 6.25 g of ammonium dihydrogen phosphate is added and the reaction continues for 30 min. Then, 1.5% of polyphosphoric acid relative to the entire reaction system is added. When the reaction system becomes turbid and turns milky white, it is immediately removed and excess water is removed using a rotary evaporator. Then, it is completely dried in a vacuum drying oven at 40 ℃. After drying, the product is ground and passed through a 60-mesh sieve to obtain the polymer slow-release fertilizer.

[0084] Inorganic salt culture medium: anhydrous dipotassium hydrogen phosphate 4.58 g / L, potassium dihydrogen phosphate 1.00 g / L, ammonium chloride 2.50 g / L, anhydrous magnesium sulfate 0.20 g / L, manganese sulfate monohydrate 0.056 g / L, ferrous sulfate heptahydrate 0.01 g / L, anhydrous calcium chloride 0.022 g / L; pH 7.2-7.4.

[0085] Example 1: Screening of nutrient-degrading enzymes and investigation of their degradation effect on polymeric slow-release fertilizers

[0086] 1. Screening of nutrient-degrading enzymes:

[0087] Separately, *Priestella megaterium* ( Priestia megaterium YC5 was inoculated into inorganic salt medium and inorganic salt medium containing 10 wt% polymeric slow-release fertilizer, and cultured at 37 ℃ and 200 rpm for 48 h for fermentation culture. After the culture was completed, the bacterial suspension in the two media was collected, and the supernatant and bacterial cells were collected by centrifugation at 12000 r / min for 10 min. The bacterial cells were resuspended in pre-cooled Tris-HCl buffer at pH 7.5, and then 0.1 mol / L mild elution agent NaCl solution was added. After elution by shaking at 4 ℃ for 30 min, the mixture was centrifuged at 12000 r / min for 10 min, and the supernatant and bacterial cells were collected. The supernatant was combined with the supernatant in the original fermentation broth.

[0088] The collected bacterial cells were sent to a testing company for transcriptomics analysis, and the whole genome of strain YC5 was also sequenced to identify those involved in the degradation of *Priscilla megaterium* by polymer-release fertilizers. Priestia megaterium The upregulation of related degradation genes in YC5 was investigated. The results showed that: pepE , lepB-1 , lepB-2 , amiABC , amiSand amiE The gene was significantly upregulated, serving as a key gene for subsequent experiments and validation.

[0089] The amino acid sequence of pepE is shown in SEQ ID NO.1; the amino acid sequence of lepB-1 is shown in SEQ ID NO.2; the amino acid sequence of lepB-2 is shown in SEQ ID NO.3; the amino acid sequence of amiABC is shown in SEQ ID NO.4; the amino acid sequence of amiS is shown in SEQ ID NO.5; and the amino acid sequence of amiE is shown in SEQ ID NO.6.

[0090] pepE encoding gene pepE The nucleotide sequence is shown in SEQ ID NO.7; the encoding gene for lepB-1. lepB- 1 The nucleotide sequence is shown in SEQ ID NO.8; the gene encoding lepB-2. lepB-2 The nucleotide sequence is shown in SEQ ID NO. 9; the encoding gene for amiABC. amiABC The nucleotide sequence is shown in SEQ ID NO.10; the encoding gene for amiS. amiS The nucleotide sequence is shown in SEQ ID NO.11; the encoding gene for amiE. amiE The nucleotide sequence is shown in SEQ ID NO. 12.

[0091] 2. Evaluation of the degradation effect of polymeric slow-release fertilizer:

[0092] (1) Preparation of crude enzyme solution:

[0093] The coding genes for pepE, lepB-1, lepB-2, amiABC, amiS and amiE were respectively ligated into the pET-28a vector to construct recombinant expression vectors expressing the above six nutrient-degrading enzymes, namely pepE, lepB-1, lepB-2, amiABC, amiS and amiE.

[0094] The recombinant expression vectors were then transformed into Escherichia coli BL21(DE3) competent cells and plated on LB plates containing kanamycin (50 μg / mL) and cultured at 37°C for 12 h.

[0095] Single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm for 8 h. The inoculum was then transferred to 500 mL of LB medium at a 1% inoculation rate and cultured at 37°C until OD500 was reached. 600When the concentration of the enzyme was 0.6-0.8, IPTG was added to a final concentration of 0.5 mmol / L, and expression was induced at 28℃ for 12 h. The bacterial cells were collected by centrifugation, resuspended in PBS buffer (pH 7.5), and sonicated (300W, 3s operation, 5s interval, total 30 min). The supernatant was collected by centrifugation. Crude enzyme solutions of pepE, lepB-1, lepB-2, amiABC, amiS, and amiE were obtained. Figure 1 ).

[0096] (2) Degradation effect determination:

[0097] Take 0.2 g of polymerized slow-release fertilizer and place it in a 50 ml centrifuge tube. Add 10 ml of crude enzyme solution of pepE, lepB-1, lepB-2, amiABC, amiS, and amiE respectively, and replenish the entire system to 20 ml with Tris-HCl buffer at pH 7.5. Repeat each crude enzyme solution 18 times. At the same time, set up a control experiment with only 20 ml of Tris-HCl buffer at pH 7.5.

[0098] The above system was incubated in a constant-temperature shaker at 37 ℃ and 200 rpm. Samples were taken on day 30, with three replicates per sample. Undegraded polymeric slow-release fertilizer was collected and dried in a vacuum drying oven at 40 ℃. The degradation rate of the polymeric slow-release fertilizer was determined by the mass difference method. The calculation formula is as follows:

[0099] Degradation rate of polymeric slow-release fertilizer (%) = [(0.2 - undegraded polymeric slow-release fertilizer) / 0.2] × 100%.

[0100] The results are as follows Figure 2 As shown, the results indicate that the six nutrient-degrading enzymes, pepE, lepB-1, lepB-2, amiABC, amiS, and amiE, all exhibit good degradation effects on polymeric slow-release fertilizers; pepE and amiE are the two nutrient-degrading enzymes with the best degradation effects.

[0101] Example 2: Artificial Intelligence Modification of Nutrient-Degrading Enzymes

[0102] To further improve the performance of nutrient-degrading enzymes, pepE and amiE were modified using artificial intelligence, as detailed below:

[0103] 1. AI Transformation of PEPE:

[0104] (1) Mutation site prediction:

[0105] The amino acid sequence of pepE (SEQ ID NO.1), the three-dimensional structure predicted by AlphaFold2, and the molecular docking mode data of polymeric slow-release fertilizer substrate and enzyme were input into a deep neural network (DNN) model, where the molecular docking mode data of polymeric slow-release fertilizer substrate and enzyme were calculated by AutoDock Vina.

[0106] With the goals of optimizing enzyme active site, improving thermal stability, and enhancing substrate binding efficiency, a DNN model was trained to output the optimal mutation site and type, and three mutants, D100A, E135Q, and D100A / E135Q, were obtained through screening.

[0107] (2) Construction of mutant vectors:

[0108] Using a site-directed mutagenesis kit, mutation primers were designed targeting amino acid positions 100 and 135 of SEQ ID NO.1 to perform site-directed mutagenesis on the recombinant expression vector pET-28a-pep, constructing mutant vectors pET-28a-pep-D100A, pET-28a-pep-E135Q, and pET-28a-pep-D100A / E135Q;

[0109] The mutant vector was sequenced to ensure that the mutation site was correct and the amino acid sequence consistency was ≥99%.

[0110] (3) Heterologous expression and purification of enzymes:

[0111] The recombinant vector pET-28a-pep and the mutant vectors pET-28a-pep-D100A, pET-28a-pep-E135Q, and pET-28a-pep-D100A / E135Q were transformed into Escherichia coli BL21(DE3) competent cells, respectively, and plated on LB plates containing kanamycin (50 μg / mL) and cultured at 37°C for 12 h.

[0112] Pick a single colony and inoculate it into 5 mL of LB medium (containing kanamycin). Incubate at 37°C and 200 rpm for 8 h. Transfer the inoculum to 500 mL of LB medium at a 1% inoculation rate and incubate at 37°C until OD500. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5 mmol / L and induce expression at 28℃ for 12 h;

[0113] Collect bacterial cells by centrifugation, resuspend in PBS buffer (pH 7.5), sonicate (300W power, 3s operation, 5s interval, total duration 30 min), and collect the supernatant by centrifugation.

[0114] The supernatant was purified by Ni-NTA affinity chromatography, equilibrated with PBS buffer containing 20 mmol / L imidazole, eluted with PBS buffer containing 300 mmol / L imidazole, the elution peak was collected, and after dialysis and desalting, the protein concentration was determined by the BCA method; pepE enzyme solution and mutant enzyme solution were prepared.

[0115] (4) Enzyme activity detection:

[0116] The polymeric slow-release fertilizer was dissolved in PBS buffer (pH 7.5) to prepare a substrate solution of 10 mg / mL;

[0117] Add 0.5 mL of substrate solution, 0.1 mL of enzyme solution, and 0.4 mL of PBS buffer. Incubate at 37°C for 30 min. Terminate the reaction by adding 0.5 mL of 10% trichloroacetic acid.

[0118] The content of free amino acids in the reaction product was detected by HPLC, the enzyme activity was calculated, and the specific activity was calculated.

[0119] The results showed that the specific activity of the pepE enzyme solution was 125 U / mg, while the specific activities of the mutants D100A, E135Q, and D100A / E135Q were 158 U / mg, 165 U / mg, and 182 U / mg, respectively. The results indicate that mutating amino acids 100 and 135 of the pepE amino acid sequence, individually or simultaneously, can further enhance the enzyme activity.

[0120] Three mutants were obtained through artificial intelligence modification of the pepE enzyme: pepE-D100A, pepE-E135Q, and pepE-D100A / E135Q; among which:

[0121] The amino acid sequence of pepE-D100A is shown in SEQ ID NO.13, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.14;

[0122] The amino acid sequence of pepE-E135Q is shown in SEQ ID NO.15, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.16.

[0123] The amino acid sequence of pepE-D100A / E135Q is shown in SEQ ID NO.17, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.18.

[0124] 2. AI Transformation of amiE:

[0125] (1) Mutation site prediction:

[0126] The amino acid sequence of amiE (SEQ ID NO.6), the three-dimensional structure predicted by AlphaFold2, and the molecular docking mode data of polymeric slow-release fertilizer substrate and enzyme were input into a deep neural network (DNN) model, where the molecular docking mode data of polymeric slow-release fertilizer substrate and enzyme were calculated by AutoDock Vina.

[0127] With the goals of optimizing enzyme active sites, improving thermal stability, and enhancing substrate binding efficiency, a DNN model was trained to output the optimal mutation sites and types, and three mutants, E80A, I135V, and E80A / I135V, were obtained through screening.

[0128] (2) Construction of mutant vectors:

[0129] Using a site-directed mutagenesis kit, mutation primers were designed targeting amino acid sites 80 and 135 of SEQ ID NO.6 to perform site-directed mutagenesis on the recombinant expression vector pET-28a-ami, constructing mutant vectors pET-28a-ami-E80A, pET-28a-ami-I135V, and pET-28a-ami-E80A / I135V;

[0130] The mutant vector was sequenced to ensure that the mutation site was correct and the amino acid sequence consistency was ≥99%.

[0131] (3) Heterologous expression and purification of enzymes:

[0132] The recombinant vector pET-28a-ami and the mutant vectors pET-28a-ami-E80A, pET-28a-ami-I135V, and pET-28a-ami-E80A / I135V were transformed into Escherichia coli BL21(DE3) competent cells, respectively, and plated on LB plates containing kanamycin (50 μg / mL) and cultured at 37°C for 12 h.

[0133] Pick a single colony and inoculate it into 5 mL of LB medium containing 50 μg / mL kanamycin. Incubate at 37°C and 200 rpm for 8 h. Transfer the inoculum to 500 mL of LB medium at a 1% inoculation rate and incubate at 37°C until OD500. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5 mmol / L and induce expression at 28℃ for 12 h;

[0134] Collect bacterial cells by centrifugation, resuspend in PBS buffer (pH 7.5), sonicate (300W power, 3s operation, 5s interval, total duration 30 min), and collect the supernatant by centrifugation.

[0135] The supernatant was purified by Ni-NTA affinity chromatography, equilibrated with PBS buffer containing 20 mmol / L imidazole, eluted with PBS buffer containing 300 mmol / L imidazole, the elution peak was collected, and after dialysis and desalting, the protein concentration was determined by the BCA method; amiE enzyme solution and mutant enzyme solution were prepared.

[0136] (4) Enzyme activity detection:

[0137] The polymeric slow-release fertilizer was dissolved in PBS buffer (pH 7.5) to prepare a substrate solution of 10 mg / mL;

[0138] Add 0.5 mL of substrate solution, 0.1 mL of enzyme solution, and 0.4 mL of PBS buffer. Incubate at 37°C for 30 min. Terminate the reaction by adding 0.5 mL of 10% trichloroacetic acid.

[0139] The content of free amino acids in the reaction product was detected by HPLC, the enzyme activity was calculated, and the specific activity was calculated.

[0140] The results showed that the specific activity of the amiE enzyme solution was 105 U / mg, while the specific activities of the mutants E80A, I135V, and E80A / I135V were 125 U / mg, 131 U / mg, and 146 U / mg, respectively. This indicates that mutating amino acids 80 and 135 of the amiE amino acid sequence, individually or simultaneously, can further enhance the enzyme activity.

[0141] Three mutants were obtained by artificial intelligence modification of the amiE enzyme: amiE-E80A, amiE-I135V, and amiE-E80A / I135V; among which:

[0142] The amino acid sequence of amiE-E80A is shown in SEQ ID NO.19, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.20;

[0143] The amino acid sequence of amiE-I135V is shown in SEQ ID NO.21, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.22;

[0144] The amino acid sequence of amiE-E80A / I135V is shown in SEQ ID NO.23, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.24.

[0145] Example 3: Enzymatic performance evaluation of pepE-D100A / E135Q and amiE-E80A / I135V:

[0146] 1. Investigation of catalytic reaction temperature:

[0147] The polymerized slow-release fertilizer was dissolved in PBS buffer (pH 7.5) to prepare a substrate solution of 10 mg / mL. 0.5 mL of the substrate solution, 0.1 mL each of pepE-D100A / E135Q and amiE-E80A / I135V enzyme solutions, and 0.4 mL of PBS buffer were added and reacted at 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 30 min, respectively. The reaction was then terminated by adding 0.5 mL of 10% trichloroacetic acid. An equal volume of pH 7.5 PBS buffer without enzyme solution was used as a blank control.

[0148] The content of free amino acids in the reaction product was determined by HPLC, and the enzyme activity was calculated. Enzyme activity (U / mg) = (Free amino acid content in the sample group - Content in the blank control group) / (Enzyme dosage × Reaction time);

[0149] 1 U is defined as the ability of each milligram of enzyme to catalyze the production of 1 μg of free amino acids within 30 min;

[0150] The initial enzyme activity was determined at 37 °C and pH 7.5, and this enzyme activity was used as the baseline 100%.

[0151] The results are as follows Figure 3 As shown in Figure a, the results indicate that after 30 min of reaction, the pepE-D100A / E135Q and amiE-E80A / I135V enzymes exhibited the highest enzyme activity at 50 ℃ and 55 ℃, respectively.

[0152] 2. pH assessment of the catalytic reaction:

[0153] Polymerized slow-release fertilizers were dissolved in PBS buffer at pH values ​​of 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, and 10.5 to prepare substrate solutions of 10 mg / mL. 0.5 mL of the substrate solution, 0.1 mL each of pepE-D100A / E135Q and amiE-E80A / I135V enzyme solutions, and 0.4 mL of PBS buffer were added and reacted at 37°C for 30 min. The reaction was then terminated by adding 0.5 mL of 10% trichloroacetic acid.

[0154] The content of free amino acids in the reaction product was detected by HPLC, and the enzyme activity was calculated according to the above method.

[0155] The results are as follows Figure 3 As shown in Figures b and c, the results indicate that the optimal reaction pH for the pepE-D100A / E135Q and amiE-E80A / I135V enzymes is 7.5 and 8.5, respectively.

[0156] 3. Investigation on the degradation effect of pepE-D100A / E135Q and amiE-E80A / I135V composites on polymeric slow-release fertilizer:

[0157] The polymeric slow-release fertilizer was dissolved in PBS buffer (pH 7.5) to prepare a substrate solution of 10 mg / mL.

[0158] Take 0.5 mL of substrate solution, 0.1 mL of pepE-D100A / E135Q enzyme solution, and 0.4 mL of PBS buffer, and mix them to form reaction system 1 (pepE).

[0159] Take 0.5 mL of substrate solution, 0.1 mL of amiE-E80A / I135V enzyme solution, and 0.4 mL of PBS buffer, and mix them to form reaction system 2 (amiE).

[0160] Take 0.5 mL of substrate solution, 0.1 mL of pepE-D100A / E135Q and amiE-E80A / I135V complex enzyme solution at a mass ratio of 1:1, and 0.4 mL of PBS buffer, and mix them to form reaction system 3 (pepE + amiE (1:1)).

[0161] A control (CK) was prepared using the same volume of pH 7.5 PBS buffer.

[0162] Reaction system 1, reaction system 2, reaction system 3 and control were reacted at 37°C; samples were taken on days 5, 10, 15, 20, 25 and 30.

[0163] The enzyme's degradation efficiency for polymeric slow-release fertilizer was characterized by measuring the amount of nitrogen released from the solution. The solution was removed and digested with 5 ml of sulfuric acid. The digested solution was then transferred to a sample inlet tube, and the nitrogen content in the solution was determined using the Kjeldahl method. Simultaneously, the nitrogen content in the substrate solution was determined using the same method. The formula for calculating the enzyme's degradation efficiency for polymeric slow-release fertilizer is as follows:

[0164] Enzyme catalytic efficiency for degradation of polymeric slow-release fertilizer (%) = (Nitrogen content in solution / Nitrogen content in substrate solution) × 100%

[0165] The results are as follows Figure 3As shown in Figure d, the results indicate that the pepE-D100A / E135Q and amiE-E80A / I135V composite enzyme solutions exhibit significant degradation catalytic efficiency for polymeric slow-release fertilizer, achieving a nitrogen release of 89.60% within 30 days. In contrast, pepE-D100A / E135Q enzyme solution alone achieved a nitrogen release of 51.83% within 30 days, while amiE-E80A / I135V enzyme solution alone achieved a nitrogen release of 63.07% within 30 days. The control group achieved a nitrogen release of 25.98% within 30 days.

[0166] Therefore, compared with using pepE-D100A / E135Q and amiE-E80A / I135V alone, combining pepE-D100A / E135Q and amiE-E80A / I135V can synergistically improve the degradation catalytic efficiency of polymeric slow-release fertilizer, achieving a synergistic effect of 1+1>2.

[0167] The degradation effect of the polymer slow-release fertilizer after degradation by pepE-D100A / E135Q and amiE-E80A / I135V composite enzyme solutions was characterized by ESI-MS. The results are as follows: Figure 4 As shown: The process of nutrient release from polymeric slow-release fertilizer is the gradual breaking of bonds in the polymer chains to form smaller molecular chains, which further release the nutrients needed by the crop. The five substances indicated by the arrows in the figure are amino nitrogen products of different chain lengths produced by enzymatic hydrolysis. The corresponding m / z values ​​and products are as follows:

[0168] m / z 117: Tetramethylenepentaurea, intermediate product;

[0169] m / z 274: Hexamethylene heptaurea, the longest molecular weight structure in this polymeric slow-release fertilizer;

[0170] m / z 294: Trimethylenetetraurea, intermediate product;

[0171] m / z 361: Dimethylenetriurea, which degrades into low molecular weight products;

[0172] m / z 406: Tetramethylenepentaurea, intermediate product;

[0173] The results showed that the pepE-D100A / E135Q and amiE-E80A / I135V complex enzymes can specifically break peptide bonds and amide bonds in polymerized slow-release fertilizers, thereby gradually generating nitrogen forms that can be utilized by crops.

[0174] Example 4: Construction and fermentation performance testing of engineered strains expressing pepE-D100A / E135Q and amiE-E80A / I135V

[0175] 1. Construction of engineered strains:

[0176] The gene encoding pepE-D100A / E135Q (SEQ ID NO.18) was ligated into the pET-28a vector to construct the recombinant expression vector pET-28a-pepE-D100A / E135Q;

[0177] The recombinant expression vector pET-28a-pepE-D100A / E135Q was transformed into Escherichia coli BL21(DE3) competent cells to construct engineered strain A (pepE) expressing pepE-D100A / E135Q.

[0178] The coding gene of amiE-E80A / I135V (SEQ ID NO.24) was ligated into the pET-28a vector to construct the recombinant expression vector pET-28a-amiE-E80A / I135V;

[0179] The recombinant expression vector pET-28a-amiE-E80A / I135V was transformed into Escherichia coli BL21(DE3) competent cells to construct engineered strain B (amiE) expressing amiE-E80A / I135V.

[0180] Antagonistic effects were detected using the plate confrontation method. Engineered strain A and engineered strain B were inoculated at both ends of an LB plate, respectively, and incubated at 37°C for 24 h. The presence of inhibition zones between the two strains was then observed.

[0181] The results are as follows Figure 5 As shown, no inhibition zone appeared in the plate confrontation experiment, proving that there is no antagonistic effect between engineered strain A and engineered strain B, and they can be used in combination.

[0182] Engineered strain A and engineered strain B were inoculated into LB medium containing kanamycin and cultured at 37°C and 200 rpm for 12 h to obtain seed culture.

[0183] The seed culture of engineered strain A and the seed culture of engineered strain B were mixed at a volume ratio of 1:1 to obtain a composite engineered strain.

[0184] 2. Fermentation performance testing:

[0185] The composite engineered strain was inoculated into LB medium at pH 7.5 at a concentration of 5%, and fermented at 37°C, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm.

[0186] Effective viable cell count: After 24 h of fermentation culture, the fermentation broth was serially diluted, spread on LB plates, and incubated at 37℃ for 12 h. The colony count (CFU / mL) was then counted.

[0187] Degrading enzyme yield: Fermentation broth cultured for 24 h was collected by centrifugation, and enzyme activity was detected according to the method in Example 2. The total enzyme yield (U / mg) was calculated.

[0188] Stability testing: The pH of the LB medium used for fermentation was adjusted to 6.5, 7.5, 8.5, and 10.0, and the fermentation temperature was adjusted to 30℃, 35℃, and 40℃; after 24 hours of cultivation, the viable cell count and enzyme yield were measured.

[0189] The results showed that after incubation at 37 ℃ and pH 7.5 for 24 h, the effective viable bacterial count reached 9.6 × 10⁻⁶. 7 The total yield of the degrading enzyme was 680 U / mg, with a concentration of CFU / mL.

[0190] The composite engineered strains can grow normally within a pH range of 6.5-10.0 and a temperature range of 30-40℃, with a viable count ≥8×10⁻⁶. 6 The enzyme yield is ≥550 U / mL, with CFU / mL, and good stability.

[0191] Example 5: Application of composite engineered strains in crops at different growth stages

[0192] To verify the application potential of the double-mutant composite engineered strain in crops with different growth cycles, short-cycle crops (leeks), medium-cycle crops (corn), and long-cycle crops (pears) were selected for the experiment. The corresponding indicators were measured at the time of crop harvest to explore the nitrogen utilization efficiency of the polymer slow-release fertilizer.

[0193] In planting trials of leeks, corn, and pears, nitrogen was entirely provided by polymeric slow-release fertilizer, phosphorus was provided by phosphorus in the polymeric slow-release fertilizer and the remainder was supplemented by superphosphate, and potassium was entirely provided by potassium sulfate, thus achieving correspondingly different nitrogen, phosphorus, and potassium nutrient application rates.

[0194] 1. Test method:

[0195] (1) Leeks (791 Leeks)

[0196] The experiment was set up with five treatment groups, each with 10 crops, as follows:

[0197] Blank control group: No polymeric slow-release fertilizer or engineered microbial agent expressing nutrient-degrading enzymes was applied.

[0198] Polymerized slow-release fertilizer group: Only polymerized slow-release fertilizer was applied, at a rate of 15-8-10 kg / hm² before sowing. 2 The nutrient ratio is applied to polymeric slow-release fertilizers and other fertilizers; the nutrient ratio refers to the ratio of nitrogen (as N) - phosphorus (as P2O5) - potassium (as K2O).

[0199] Polymer slow-release fertilizer + pET-28a-pep group: Apply at 15-8-10 kg / hm before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was adjusted accordingly. After the chives emerged, the engineered microbial agent expressing pepE-D100A / E135Q was applied near the chive roots using the "furrow application followed by soil covering" method to ensure full contact between the engineered microorganisms and the polymeric slow-release fertilizer. The effective viable count in the engineered microbial agent was 8 × 10⁶. 3 CFU / g, the amount of engineered microbial agent applied is 8% of the weight of polymeric slow-release fertilizer.

[0200] Polymer slow-release fertilizer + pET-28a-ami group: Apply at 15-8-10 kg / hm² before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was adjusted accordingly. After the chives emerged, the engineered microbial agent expressing amiE-E80A / I135V was applied near the chive roots using the "furrow application followed by soil covering" method to ensure full contact between the engineered microorganisms and the polymeric slow-release fertilizer. The effective viable count in the engineered microbial agent was 8 × 10⁶. 3 CFU / g, the amount of engineered microbial agent applied is 8% of the weight of polymeric slow-release fertilizer.

[0201] Polymer slow-release fertilizer + pET-28a-pep / ami (7:3) group: Apply at 15-8-10 kg / hm before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was adjusted accordingly. After the chives emerged, a compound engineered microbial agent expressing pepE-D100A / E135Q and amiE-E80A / I135V was applied near the chive roots using a "furrow application followed by soil covering" method to ensure full contact between the engineered bacteria and the polymeric slow-release fertilizer. The compound engineered microbial agent was obtained by mixing the seed liquid of engineered bacteria expressing pepE-D100A / E135Q and the seed liquid of engineered bacteria expressing amiE-E80A / I135V at a volume ratio of 7:3, followed by fermentation. The effective viable count in the compound engineered microbial agent was 8 × 10⁻⁶. 3 The CFU / g compound engineered microbial agent is applied at a rate of 8% of the weight of the polymer slow-release fertilizer.

[0202] Other management practices followed standard farmer practices. The experiment lasted 28 days, and relevant indicators were measured when the chives were harvested.

[0203] (2) Corn (Denghai 652)

[0204] The experiment was set up with five treatment groups, each covering 1 mu (approximately 0.16 acres):

[0205] Blank control group: No polymeric slow-release fertilizer or engineered microbial agent expressing nutrient-degrading enzymes was applied.

[0206] Polymerized slow-release fertilizer group: Only polymerized slow-release fertilizer was applied before sowing at a rate of 250-90-60 kg / hm². 2 The nutrient ratio is applied to polymeric slow-release fertilizers and other fertilizers; the nutrient ratio refers to the ratio of nitrogen (as N) - phosphorus (as P2O5) - potassium (as K2O).

[0207] Polymer slow-release fertilizer + pET-28a-pep group: Apply 250-90-60 kg / hm² before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was adjusted accordingly. After the corn seedlings emerged, the engineered microbial agent expressing pepE-D100A / E135Q was applied near the corn roots using the "furrow application followed by soil covering" method to ensure full contact between the engineered microorganisms and the polymeric slow-release fertilizer. The effective viable count in the engineered microbial agent was 8 × 10⁶. 4 CFU / g, the amount of engineered microbial agent applied is 5% of the weight of polymeric slow-release fertilizer.

[0208] Polymer slow-release fertilizer + pET-28a-ami group: Apply at 250-90-60 kg / hm² before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was adjusted accordingly. After the corn seedlings emerged, the engineered microbial agent expressing amiE-E80A / I135V was applied near the corn roots using the "furrow application followed by soil covering" method to ensure full contact between the engineered microorganisms and the polymeric slow-release fertilizer. The effective viable count in the engineered microbial agent was 8 × 10⁶. 4 CFU / g, the amount of engineered microbial agent applied is 5% of the weight of polymeric slow-release fertilizer.

[0209] Polymer slow-release fertilizer + pET-28a-pep / ami (1:1) group: Apply at 250-90-60 kg / hm before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was adjusted accordingly. After the corn seedlings emerged, a compound engineered microbial agent expressing pepE-D100A / E135Q and amiE-E80A / I135V was applied near the roots of the leeks using a "furrow application followed by soil covering" method to ensure full contact between the engineered bacteria and the polymeric slow-release fertilizer. The compound engineered microbial agent was obtained by mixing the seed liquid of engineered bacteria expressing pepE-D100A / E135Q and the seed liquid of engineered bacteria expressing amiE-E80A / I135V in a 1:1 volume ratio, followed by fermentation. The effective viable count in the compound engineered microbial agent was 8 × 10⁻⁶. 4 CFU / g, the amount of compound engineered microbial agent applied is 5% of the weight of polymeric slow-release fertilizer.

[0210] Other management practices followed standard farmer practices. The experiment lasted 120 days, and relevant indicators were measured at the time of corn harvest.

[0211] (3) Pear (Laiyang Chi Pear)

[0212] The experiment was set up with five treatment groups, each with 10 trees, as follows:

[0213] Blank control group: No polymeric slow-release fertilizer or engineered microbial agent expressing nutrient-degrading enzymes was applied.

[0214] Polymerized slow-release fertilizer group: Only polymerized slow-release fertilizer was applied before sowing at a rate of 300-225-375 kg / hm². 2 The nutrient ratio is applied to polymeric slow-release fertilizers and other fertilizers; the nutrient ratio refers to the ratio of nitrogen (as N) - phosphorus (as P2O5) - potassium (as K2O).

[0215] Polymer slow-release fertilizer + pET-28a-pep group: Apply 300-225-375 kg / hm² before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was applied. During the pear tree budding stage, an engineered microbial agent expressing pepE-D100A / E135Q was inoculated near the root system to ensure sufficient contact between the engineered microorganisms and the polymeric slow-release fertilizer. The effective viable count in the engineered microbial agent was 8 × 10⁶. 6 CFU / g, the amount of engineered microbial agent applied is 3% of the weight of polymeric slow-release fertilizer.

[0216] Polymer slow-release fertilizer + pET-28a-ami group: Apply at 300-225-375 kg / hm² before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was adjusted accordingly. During the pear tree budding stage, an engineered microbial agent expressing amiE-E80A / I135V was inoculated near the roots to ensure sufficient contact between the engineered microorganisms and the polymeric slow-release fertilizer. The effective viable count in the engineered microbial agent was 8 × 10⁶. 6 CFU / g, the amount of engineered microbial agent applied is 3% of the weight of polymeric slow-release fertilizer.

[0217] Polymer slow-release fertilizer + pET-28a-pep / ami (3:7) group: Apply 300-225-375 kg / hm² before sowing. 2 The nutrient ratio of polymeric slow-release fertilizer and other fertilizers was applied. During the pear tree budding period, a compound engineered microbial agent expressing pepE-D100A / E135Q and amiE-E80A / I135V was inoculated near the root system to ensure sufficient contact between the engineered bacteria and the polymeric slow-release fertilizer. The compound engineered microbial agent was obtained by mixing the seed liquid of engineered bacteria expressing pepE-D100A / E135Q and the seed liquid of engineered bacteria expressing amiE-E80A / I135V at a volume ratio of 3:7, followed by fermentation. The effective viable count in the compound engineered microbial agent was 8 × 10⁻⁶. 6 CFU / g, the amount of compound engineered microbial agent applied is 3% of the weight of polymeric slow-release fertilizer.

[0218] Other management practices followed standard farmer practices. The experiment lasted for one year, with relevant indicators measured at the pear harvest.

[0219] 2. Test Results:

[0220] The results are shown in Table 1.

[0221] Table 1: Application and effects of composite engineered strains on crops at different growth stages

[0222]

[0223] The method for determining "nitrogen use efficiency" in the table is as follows:

[0224] Yield was immediately determined by weighing at harvest. Fresh plants were then brought back to the laboratory, dried, weighed, ground, and sieved through a 100-mesh sieve for later use. The cumulative nitrogen uptake of the crop was determined using the Kjeldahl method. Simultaneously, nitrogen use efficiency was calculated using the following formula:

[0225] Nitrogen utilization efficiency (%) = [(Y i ×C N1 )-(Y0×C N2 )] / F N × 100%

[0226] Note: "Y i “C” N1 “Y0”, “C” N2 “F” N "These represent the yield of the fertilized crop, the nitrogen content of the fertilized crop, the yield of the unfertilized crop, the nitrogen content of the unfertilized crop, and the nitrogen content in the fertilizer, respectively."

[0227] The results showed that the double-mutant composite engineered strains could synergistically improve the nitrogen use efficiency in polymeric slow-release fertilizers, thereby increasing crop yield.

[0228] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A nutrient-degrading enzyme, characterized in that, The nutrient-degrading enzyme is at least one of pepE protein, pepE protein mutant, amiE protein, and amiE protein mutant; The amino acid sequence of the pepE protein is shown in SEQ ID NO.1; The pepE protein mutants are pepE-D100A protein, pepE-E135Q protein, or pepE-D100A / E135Q protein; the amino acid sequence of pepE-D100A protein is shown in SEQ ID NO.13, the amino acid sequence of pepE-E135Q protein is shown in SEQ ID NO.15, and the amino acid sequence of pepE-D100A / E135Q protein is shown in SEQ ID NO.

17. The amino acid sequence of the amiE protein is shown in SEQ ID NO.6; The amiE protein mutants are amiE-E80A protein, amiE-I135V protein, or amiE-E80A / I135V protein; the amino acid sequence of amiE-E80A protein is shown in SEQ ID NO.19, the amino acid sequence of amiE-I135V protein is shown in SEQ ID NO.21, and the amino acid sequence of amiE-E80A / I135V protein is shown in SEQ ID NO.

23.

2. The nutrient-degrading enzyme according to claim 1, characterized in that, The nutrient-degrading enzyme is composed of pepE-D100A / E135Q protein and amiE-E80A / I135V protein in a mass ratio of 1:

1.

3. An engineered microbial agent, characterized in that, The engineered microbial agent is constructed by the following method: The gene encoding the nutrient-degrading enzyme in claim 1 is ligated into an expression vector to construct a recombinant expression vector; the recombinant expression vector is then transferred into a host cell to construct an engineered strain; the engineered strain is fermented to obtain an engineered bacterial agent. The gene encoding the pepE protein is the nucleic acid molecule shown in SEQ ID NO.7, and the gene encoding the pepE protein mutant is the nucleic acid molecule shown in SEQ ID NO.14, SEQ ID NO.16 or SEQ ID NO.18; The gene encoding the amiE protein is the nucleic acid molecule shown in SEQ ID NO.12, and the gene encoding the amiE protein mutant is the nucleic acid molecule shown in SEQ ID NO.20, SEQ ID NO.22 or SEQ ID NO.

24.

4. The engineered microbial agent according to claim 3, characterized in that, The expression vector is selected from one or more of plasmids, viscera, and artificial chromosomes.

5. The engineered microbial agent according to claim 3, characterized in that, The host cell is a bacterial cell or a fungal cell; the bacterial cell is selected from one or more of the genera *Escherichia coli*, *Bacillus*, *Staphylococcus aureus*, and *Lactobacillus*; the fungal cell is selected from yeast.

6. The engineered microbial agent according to claim 3, characterized in that, The engineered bacterial agent is prepared by mixing the seed culture of an engineered strain expressing pepE-D100A / E135Q protein and the seed culture of an engineered strain expressing amiE-E80A / I135V protein in a volume ratio of (3-7):(3-7).

7. The application of the nutrient-degrading enzyme according to claim 1 or 2 or the engineered microbial agent according to any one of claims 3-6 in the nutrient regulation of polymeric slow-release fertilizer, characterized in that, The nutrient regulation is achieved by applying the degradation enzyme or engineered microbial agent to degrade the polymer slow-release fertilizer, thereby improving nutrient utilization.

8. The application according to claim 7, characterized in that, The polymeric slow-release fertilizer contains amide bonds.

9. The application according to claim 8, characterized in that, The polymeric slow-release fertilizer has any of the following structures: 。

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