A sphaerobacter polyphenylalanine degrading enzyme and its coding gene and application

Abstract

This invention discloses a PS degrading enzyme derived from the deep-sea spore-forming bacterium *Sporosarcina* sp. R9-12, its encoding gene, and its applications, belonging to the fields of microbial enzyme engineering and plastic pollution remediation technology. The amino acid sequence of the PS degrading enzyme is shown in SEQ ID NO: 1. It is a naturally occurring single-membrane anchoring protein belonging to class I dye decolorizing peroxidases. Its naturally occurring encoding gene sequence is shown in SEQ ID NO: 2, and the codon-optimized gene sequence is shown in SEQ ID NO: 3. This invention constructed a pET series vector-*Escherichia coli* BL21(DE3) heterologous expression system. Soluble purified enzymes were obtained by induction with 5-aminolevulinic acid and IPTG followed by nickel affinity chromatography. Under the synergistic effect of oxygen and hydrogen donors, this enzyme can directly degrade untreated polystyrene (PS) plastics and its water-soluble analogue 4-phenylbutyric acid, producing phenylacetic acid. Simultaneously, the enzyme is naturally displayed on the cell membrane surface of this strain, allowing for direct catalytic degradation by live bacteria. This invention possesses dual application value in both green bioremediation of PS plastics and biosynthesis of phenylacetic acid.

Description

Technical Field

[0001] This invention relates to the fields of deep-sea microbial resource mining, microbial enzyme engineering, biodegradation of polymer plastics and environmental bioremediation, and particularly to a polystyrene-degrading enzyme of Bacillus subtilis, its encoding gene and its application. Background Technology

[0002] Polystyrene (PS) is one of the world's five most widely used aromatic polymers. It is polymerized from styrene monomers, with its main molecular chain linked by high-energy C-C single bonds to form benzene rings. It exhibits extremely high chemical stability and surface hydrophobicity, allowing it to degrade over decades to centuries in natural environments. Global annual PS production is enormous, with widespread applications in electronic packaging, catering utensils, building insulation, and daily necessities. Waste PS, when broken down into microplastics and nanoplastics by sunlight and mechanical forces, not only damages the ecological structure of soil and water bodies but also adsorbs heavy metals and persistent organic pollutants, accumulating along the food chain and posing long-term health risks to various organisms and humans. This is a key global issue of "white pollution" that is currently being addressed.

[0003] Plastic pollution is becoming increasingly serious, with a large portion of the plastics produced and used each year ultimately ending up in the ocean. As early as 1977, there were reports of the microorganism *Alcaligenes* sp. 559 degrading styrene oligomers (Tsuchii et al., 1977). In 1978, researchers used a microbial community composed of *Pseudomonas*, *Bacillus*, *Micrococcus*, and *Nocardia* to degrade 1,3-diphenylbutane (the smallest molecular weight PS analog), identifying phenylacetic acid in the degradation products and speculating on the degradation pathway of 1,3-diphenylbutane. Researchers believed that monooxygenase and catalase play important roles in the degradation of PS plastics (Sielicki et al., 1978). In 1997, Japanese researchers reported that hydroquinone peroxidase derived from *Azotobacter beijerinckii* HM121 could degrade PS plastics in a two-phase system, but the amino acid sequence of this PS-degrading enzyme remained unknown (Nakamiya et al., 1997). In 2008, research found that the soil actinomycete *Rhodococcus ruber* (C208) could form a biofilm on the surface of PS plastic film to degrade PS and grow there (Mor and Sivan, 2008). In 2015, the mealworm (*Tenebrio molitor*) was reported to be able to consume expanded polystyrene (EPS) plastic, converting it into carbon dioxide and excreting it as excrement (Yang et al., 2015a). Studies found that the gut microbiota of the mealworm plays a crucial role in the degradation of PS. A microorganism, *Exiguobacterium* sp. strain YT2, capable of degrading PS, was isolated from the mealworm gut; this strain could degrade 7.4% of 2500 mg / L PS in 60 days (Yang et al., 2015b). Subsequently, it was reported that the black mealworm (*Tenebrio obscurus*) could also consume EPS, and a significant reduction in the molecular weight of EPS was observed (Peng et al., 2019).

[0004] Dye-decolorizing peroxidases (EC 1.11.1.19, DyPs) are lignin-degrading enzymes, and PS (polysaccharide) shares some structural similarities with lignin. In 2022, researchers used two dye-decolorizing peroxidases, TcDyP from *Thermomonospora curvata* and AnaPX from *Anabaenasp. PCC 7120*, to degrade UV-pretreated PS. Gas chromatography-mass spectrometry (GC-MS) detected 23 degradation products (Du et al., 2022). Dye-decolorizing peroxidases (DyPs) are a unique family of peroxidases with heme as a cofactor, distinct from lignin peroxidases and manganese peroxidases. They possess high redox potentials, can oxidize non-phenolic aromatic compounds, and break high-molecular-weight C / C bonds, making them a core potential enzyme class for PS degradation. However, to date, no PS degrading enzymes with well-defined sequences that can directly degrade untreated PS plastics have been reported, and the process and mechanism of PS degradation by PS degrading enzymes are also unclear. Summary of the Invention

[0005] Therefore, the present invention aims to provide a strain of deep-sea spore-forming Micrococcus. Sporosarcoma A novel DyP-type polystyrene degrading enzyme (i.e., PS degrading enzyme) of sp. R9-12, with gene number gene0121 in the genome, was identified. Its physicochemical properties, enzymatic characteristics, and phylogenetic position were determined. A phylogenetic analysis of this protein was completed, and its homology and evolutionary specificity were elucidated through a phylogenetic tree. The natural coding gene, E. coli codon optimization gene, recombinant vector, and engineered bacteria were provided to achieve efficient heterologous expression of the soluble protein. The process of enzyme-catalyzed degradation of PS and 4-phenylbutyric acid was elucidated.

[0006] The *Dystrophus* species described in this invention are *Dystrophus* species (… Sporosarcoma sp.) R9-12 was deposited on August 7, 2019, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2019610. The *Spore-forming *Dystrophus* described in this invention has been disclosed in Chinese Invention Patent Application No. 2023107411371.

[0007] The PS-degrading enzyme described in this invention is derived from Bacillus subtilis (Bacillus subtilis). Sporosarcomasp.)R9-12 is a dye-decolorizing peroxidase (DyPs), belonging to Class I dye-decolorizing peroxidases (Class I DyPs). It is a naturally occurring functional protein anchored to the cell membrane of the strain and is the core rate-limiting enzyme for the initial depolymerization of PS.

[0008] To achieve the above objectives, the first aspect of the present invention provides a PS degrading enzyme of Bacillus subtilis, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] Furthermore, a phylogenetic tree was constructed by comparing the amino acid sequence of the Bacillus spores PS degrading enzyme with the sequence of the dye decolorizing peroxidase, and the Bacillus spores PS degrading enzyme belongs to Class I in the phylogenetic tree.

[0010] Furthermore, the amino acid sequence of the N-terminal transmembrane domain of the PS degrading enzyme is QMLKYTGAGAAGIAIGASGLGGI, and the amino acid sequence of the N-terminal intracellular domain is MSELTESKDDKFYDKKISRR.

[0011] The deoxynucleotide sequence of the natural encoding gene for the PS degrading enzyme of Bacillus spores is shown in SEQ ID NO: 2.

[0012] The second aspect of this invention provides a method for heterologous expression of a PS-degrading enzyme from *Escherichia coli*, specifically comprising: (1) Based on the amino acid sequence of the above-mentioned Bacillus spores PS degrading enzyme, the transmembrane domain and intracellular domain at the N-terminus were removed. After codon optimization, restriction sites NdeI and XhoI were added to the N-terminus and C-terminus of the optimized gene, respectively. Gene synthesis was carried out, and the synthesized gene fragment was ligated into the Escherichia coli pET series expression vector to construct the expression plasmid. The 6×His tag sequence of the pET series expression vector itself was used to add a 6×His tag to the C-terminus of the PS degrading enzyme. Then it was introduced into Escherichia coli BL21(DE3). Positive clones were obtained by kanamycin resistance screening. (2) Cultured in LB medium containing kanamycin resistance until OD 600 The concentration was 0.3-0.8. 5-aminolevulinic acid was added to promote the synthesis of heme cofactors, and the expression of the enzyme was induced by isopropyl-β-D-thiogalactoside. The PS degrading enzyme of Bacillus angiotensinus was obtained by separation and purification. (3) The separation and purification process is as follows: centrifugation to collect bacterial cells, ultrasonic disruption of cells, and separation and purification by nickel ion affinity chromatography to obtain the PS degrading enzyme of Bacillus angiotensinus.

[0013] Furthermore, the gene sequence of *Bacillus spp.* PS degrading enzyme after removing the N-terminal transmembrane domain and the N-terminal intracellular domain and optimizing the codons is shown in SEQ ID NO: 3.

[0014] The third aspect of the present invention provides the use of the above-mentioned Bacillus spores PS degrading enzyme in the preparation of products for degrading polystyrene plastics or 4-phenylbutyric acid.

[0015] PS plastic is a high molecular weight compound characterized by high crystallinity, poor water solubility, and difficulty in degradation. 4-Phenylacetic acid (4-Phenylacetic butyric acid) is a water-soluble small-molecule analog of PS plastic and can be used as a substrate for PS degrading enzymes. This facilitates the analysis of degradation intermediates, final products, and the catalytic degradation conditions of the enzyme.

[0016] Furthermore, the degradation is carried out in the presence of oxygen, and a hydrogen donor is added to the reaction system; the reaction system for the degradation is a 50-300 mM phosphate buffer solution or a Tris-HCl buffer solution.

[0017] Furthermore, the hydrogen donor is selected from at least one of ascorbic acid, panthenol, pantothenic acid, histidine, histamine, cytochrome c, vitamin B1, vitamin B6, tetramethylhydroquinone, and FMNH2.

[0018] The aforementioned PS-degrading enzyme of *Bacillus subtilis* is a single-membrane protein, naturally exhibited in *Bacillus subtilis* (…). Sporosarcina sp. On the cell membrane surface of R9-12, through the propagation and culture of spore-forming Micrococcus ( Sporosarcoma sp. R9-12 can obtain bacterial cells with the PS-degrading enzyme of Bacillus spores on the cell membrane surface, which can be used to catalyze the degradation of 4-phenylbutyric acid or PS plastic to produce phenylacetic acid.

[0019] The present invention also provides a microbial agent containing Bacillus subtilis (Bacillus subtilis). Sporosarcoma sp.) R9-12 bacterial cells, the cell membrane surface of which naturally displays the above-mentioned PS degrading enzyme, the bacterial agent can be used to catalyze the degradation of 4-phenylbutyric acid or PS plastic to produce phenylacetic acid.

[0020] The PS-degrading enzyme of *Bacillus spp.* described in this invention can be obtained through heterologous expression or through fermentation culture of *Bacillus spp.* Sporosarcina sp. (R9-12 can be obtained directly.) This invention is the first reported PS degrading enzyme with a clearly defined sequence that can directly degrade untreated PS plastic. Its degradation product, phenylacetic acid, is an important industrial product with promising applications in environmental pollution control and biochemical production.

[0021] The beneficial effects of this invention are: This invention provides an amino acid residue sequence of a *Bacillus spp.* PS degrading enzyme, the primary sequence of which possesses high novelty; the *Bacillus spp.* PS degrading enzyme can degrade PS plastic to produce phenylacetic acid, and simultaneously possesses the functions of green degradation of PS plastic pollutants and production of industrial-grade phenylacetic acid; the *Bacillus spp.* PS degrading enzyme is exhibited in *Bacillus spp.* (…). Sporosarcoma The single transmembrane protein on the cell membrane of sp.) R9-12 can therefore be used in the application of *Spore-forming Micrococcus* (sp.) Sporosarcoma The bacterial cells of sp.)R9-12 catalytically degrade PS plastics; only fermentation culture of Bacillus spp. (Spp.) is required. Sporosarcoma Large quantities of Bacillus spores PS degrading enzyme can be obtained by using sp.)R9-12, which is simple and convenient to obtain. At the same time, large quantities of purified Bacillus spores PS degrading enzyme can also be obtained through heterologous expression. Attached Figure Description

[0022] Figure 1 It is a spore-forming Micrococcus ( Sporosarcoma Phylogenetic tree of sp.)R9-12 PS degrading enzyme and previously reported dye decolorizing peroxidase.

[0023] Figure 2 It is a heterologous expression of spore-forming Micrococcus in Escherichia coli ( Sporosarcoma SDS-PAGE electrophoresis image of R9-12 PS degrading enzyme after isolation and purification. Lane M represents protein molecular weight standards, and lane 1 represents isolated and purified Bacillus subtilis (sp.) Sporosarcoma sp.)R9-12 PS degrading enzyme.

[0024] Figure 3 Heterologous expression of spore-forming Micrococcus ( Sporosarcoma The apparent color of the isolated and purified R9-12 PS degrading enzyme is red (a) and the ultraviolet-visible spectrophotometric scan (b).

[0025] Figure 4 Heterologous expression of spore-forming Micrococcus ( Sporosarcoma HPLC detection results of PS plastic film degradation by sp.)R9-12 PS degrading enzyme.

[0026] Figure 5 Heterologous expression of spore-forming Micrococcus ( Sporosarcoma HPLC detection results of 4-phenylbutyric acid degradation by sp.)R9-12 PS degrading enzyme, (a) is the global chromatogram, and (b) is a magnified view of (a).

[0027] Figure 6 It is a spore-forming Micrococcus ( SporosarcomaHPLC detection results of PS plastic degradation by sp.)R9-12 bacterial cells.

[0028] Figure 7 It is a spore-forming Micrococcus ( Sporosarcoma HPLC detection results of 4-phenylbutyric acid degradation by sp.)R9-12 bacterial cells. Detailed Implementation

[0029] To explain in detail the technical content, structural features, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0030] The experimental methods used in the following examples are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0032] Bacillus subtilis ( Sporosarcoma Construction of the phylogenetic tree of the R9-12 PS degrading enzyme system. Bacillus subtilis ( Sporosarcoma The entire genome of sp.)R9-12 has been sequenced. The gene encoding the PS degrading enzyme is designated gene0121 in the genome. A phylogenetic tree of dye decolorizing peroxidase (gene0121) and previously reported different types of peroxidases was constructed using MEGA software. Figure 1 Based on primary structural homology, they can be divided into classes A, B, C, and D. Then, based on the relationship between protein and function, they are further divided into classes P, I, V, and D. Bacillus spores R9-12 gene0121 belongs to class I. Class I DyPs primarily function as iron chelate enzymes, derived from... Escherichia coli EfeB belongs to class I. efeB yes efeUOB Part of the operon, it is a component encoding the iron transport system and has the highest homology with Bacillus subtilis R9-12 gene0121. Bacillus subtilis According to reports, Dyp, Bacillus subtilis BsDyP (Class I) is a homolog of EfeB, exhibiting dye decolorization peroxidase activity, and possesses high redox potential, substrate heterogeneity, and good thermal stability. Example 2

[0033] Bacillus subtilis ( Sporosarcoma sp.)R9-12 PS degrading enzyme heterologously expressed, isolated and purified in E. coli According to the present invention, the spore-forming diatomaceous earth ( Sporosarcoma The amino acid residue sequence of sp.)R9-12 PS degrading enzyme was extracted, and the N-terminal intracellular and transmembrane domains were removed. The DNA sequence of dye decolorizing peroxidase (gene0121) was codon-optimized and the entire gene synthesized. Using plasmid PET-30a (C-terminal His-tag) as the starting vector, the gene0121 protein expression vector was constructed. The restriction enzyme sites selected were NdeI and XhoI. The 6×His tag sequence of the pET-30a expression vector itself was used in *Bacillus subtilis* (sp.) Sporosarcoma The C-terminus of sp.)R9-12 PS degradative enzyme is tagged with a 6×His tag to facilitate the purification of the target protein. This codon-optimized Spore-forming Micrococcus (sp.) Sporosarcoma The gene sequence of sp.)R9-12 PS degrading enzyme is shown in SEQ ID NO: 3. The gene was heterologously expressed in *Escherichia coli* using plasmid pET-30a. Sporosarcoma sp.)R9-12 PS degrading enzyme.

[0034] Containing Spore-forming Micrococcus ( Sporosarcoma The gene sequence of sp.)R9-12 PS degrading enzyme was transformed into Escherichia coli BL21(DE3) using the vector pET-30a-gene0121 and cultured in LB medium containing kanamycin resistance until OD. 600 When the OD value is between 0.3 and 0.8, this embodiment selects OD. 600 When the concentration reaches 0.6, proceed to the next induction step. During induction, add 5-aminolevulinic acid to a final concentration of 2 mM and IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.5 mM. Shake well and incubate at 16°C and 180 rpm for 16-18 hours to induce expression of the target protein.

[0035] The bacterial cells were collected by centrifugation, the cells were disrupted by sonication, and the *Dystrophus spores* were obtained by nickel ion affinity chromatography. Sporosarcoma sp.)R9-12 PS degrading enzyme, SDS-PAGE showed that its molecular weight was approximately 45 kDa. Figure 2 The purified protein (gene0121) appears red. Figure 3 (a)), UV scanning showed a characteristic absorption peak of heme at 407 nm ( Figure 3 (b)). Example 3

[0036] Bacillus subtilis ( Sporosarcoma sp.)R9-12 PS degrading enzyme degrades PS plastics The spore-forming Micrococcus prepared using Example 2 ( Sporosarcomasp.)R9-12 PS degrading enzyme degrades PS plastics. The catalytic system (300 μL) consisted of six 2×2 cm PS plastic membranes as substrate, flavin reductase FRE (Uniprot No.: P0AEN1) (1 μM), dye decolorizing peroxidase gene0121 (4 μM), NADH solution (100 μM), FMN solution (100 μM), 0.8% dimethyl sulfoxide (DMSO), and 50 mM phosphate (pH 7.0) to a final volume of 300 μL. The control group did not contain dye decolorizing peroxidase gene0121. The reaction was carried out at 30 ℃ and 180 rpm for three days. Afterwards, the solution was centrifuged and filtered through a 0.22 μm filter. HPLC analysis was performed using an Agilent 1260 HPLC system with a COSMOSIL 5C18-AR-II 4.6 ID×250 mm column, 55% methanol, 45% phosphate, an injection volume of 30 μL, and a column temperature of 30 ℃. The experimental group showed a detection time of 4.9 seconds. The chromatographic peak of phenylacetic acid was detected in the control group, while no chromatographic peak of phenylacetic acid was detected in the control group. Figure 4 This indicates that the enzyme can directly degrade untreated PS plastic film to produce phenylacetic acid. Example 4

[0037] Bacillus subtilis ( Sporosarcoma sp.)R9-12 PS degrading enzyme degrades small molecule analogues of PS plastics, such as 4-phenylbutyric acid (4PBA). The spore-forming Micrococcus prepared using Example 2 ( Sporosarcoma R9-12 PS degradative enzyme (sp.) degraded 4-phenylbutyric acid. The catalytic system (300 μL) consisted of: substrate 4PBA (1 mM), flavin reductase (Uniprot No.: P0AEN1) (1 μM), dye decolorizing peroxidase gene0121 (4 μM), NADH solution (100 μM), FMN solution (100 μM), and 50 mM phosphate (pH 7.0), brought to a final volume of 300 μL. The control group did not contain dye decolorizing peroxidase gene0121. The reaction was carried out at 30℃ and 180 rpm for two days. After two days, the solution was centrifuged and filtered through a 0.22 μm filter membrane, and detected by high-performance liquid chromatography (HPLC). HPLC conditions: Agilent 1260 HPLC system, COSMOSIL 5C18-AR-II 4.6 ID×250 mm column, column temperature 30℃, 60% chromatographic methanol, 40% phosphate, injection volume 5 μL.

[0038] The results showed that chromatographic peaks of 3-hydroxy-4-phenylbutyric acid (4.5 min) and phenylacetic acid (4.9 min) were detected in the experimental group, while these two peaks were not detected in the control group. Figure 5This indicates that the enzyme catalyzes the conversion of 4-phenylbutyric acid to 3-hydroxy-4-phenylbutyric acid, and then further degrades it to phenylacetic acid, thus verifying the catalytic pathway of the enzyme. Example 5

[0039] Using Bacillus subtilis ( Sporosarcoma sp.)R9-12 bacterial cell degradation of PS plastic Using Bacillus subtilis ( Sporosarcoma PS-degrading enzymes displayed on the cell membrane surface of sp.)R9-12 degrade PS plastics. First, *Sacchariformis* (sp.) R9-12... Sporosarcoma sp.)R9-12 was fermented in 100 mL LB medium for 48 h. After centrifugation, the cells were washed three times with 50 mM phosphate buffer (pH 7.0) and resuspended in 50 mL of 50 mM phosphate buffer. 100 mg of PS plastic film was added, and the mixture was hydrolyzed at 30 °C for 72 h in a shaker at 150 rpm. After the reaction, the plastic film was removed, and the cells were removed by centrifugation. The degradation products were determined by HPLC. The results showed that *Bacillus occulta* (S.)R9-12 was fermented. Sporosarcoma sp.) R9-12 bacterial cells can degrade PS plastic to produce phenylacetic acid and a small amount of 4-hydroxyphenylacetic acid (sp.) Figure 6 ). Example 6

[0040] Using Bacillus subtilis ( Sporosarcoma (sp.)R9-12 bacterial cells degrade 4-phenylbutyric acid.

[0041] Using Bacillus subtilis ( Sporosarcoma PS-degrading enzymes displayed on the cell membrane surface of sp.)R9-12 degrade 4-phenylbutyric acid. First, *Sacchariformis sp.* (sp.)R9-12 degrades 4-phenylbutyric acid. Sporosarcoma sp.)R9-12 was fermented in 100 mL LB medium for 48 h. After centrifugation, the cells were washed three times with 50 mM phosphate buffer (pH 7.0) and resuspended in 50 mL of 50 mM phosphate buffer. 50 mg of 4-phenylbutyric acid was added, and the mixture was hydrolyzed at 30 °C for 48 h in a shaker at 150 rpm. After the reaction, the cells were removed by centrifugation, and the degradation products were determined by HPLC. HPLC analysis showed that *Bacillus occulta* (S.)R9-12 was a viable biodegradable bacterial culture. Sporosarcoma sp.) R9-12 cells can catalyze the initial conversion of 4-phenylbutyric acid to 3-hydroxy-4-phenylbutyric acid, followed by further degradation to phenylacetic acid (sp.) Figure 7 ).

[0042] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A PS-degrading enzyme for Bacillus subtilis, characterized in that, The amino acid sequence of the PS degrading enzyme is shown in SEQ ID NO:

1.

2. The Bacillus sporeans PS-degrading enzyme according to claim 1, characterized in that, The amino acid sequence of the N-terminal transmembrane domain of the PS degrading enzyme is QMLKYTGAGAAGIAIGASGLGGI, and the amino acid sequence of the N-terminal intracellular domain is MSELTESKDDKFYDKKISRR.

3. The gene encoding the PS-degrading enzyme of Bacillus subtilis as described in claim 1, characterized in that, The deoxynucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

4. The encoding gene according to claim 3, characterized in that, The sequence of the encoding gene is an optimized E. coli codon sequence, and its nucleotide sequence is shown in SEQ ID NO:

3.

5. An expression vector or genetically engineered bacteria containing the coding gene of claim 3 or the codon-optimized gene of claim 4.

6. A method for heterologously expressing the PS-degrading enzyme of Bacillus angiosporins as described in claim 1 or 2 in Escherichia coli, characterized in that, Includes the following steps: (1) According to the amino acid sequence of the PS degrading enzyme of Bacillus angiosporins as described in claim 1 or 2, the transmembrane domain and intracellular domain of the N-terminus are removed, and after codon optimization, restriction sites NdeI and XhoI are added to the N-terminus and C-terminus of the optimized gene, respectively, and the gene is synthesized. The synthesized gene fragment is ligated into the pET series expression vector of Escherichia coli to construct the expression plasmid. The 6×His tag sequence of the pET series expression vector itself is used to add a 6×His tag to the C-terminus of the PS degrading enzyme. Then it is introduced into Escherichia coli BL21(DE3), and positive clones are obtained by kanamycin resistance screening. (2) Cultured in LB medium containing kanamycin resistance until OD 600 The expression of the enzyme was induced by adding 5-aminolevulinic acid and isopropyl-β-D-thiogalactoside to a concentration of 0.3-0.8, and the enzyme was obtained by separation and purification. (3) The separation and purification process is as follows: centrifugation to collect bacterial cells, ultrasonic disruption of cells, and separation and purification by nickel ion affinity chromatography to obtain the PS degrading enzyme of Bacillus angiotensinus.

7. The use of the Bacillus spp. PS degrading enzyme according to claim 1 in the preparation of products for degrading polystyrene plastics or 4-phenylbutyric acid.

8. The application according to claim 7, characterized in that, The degradation is carried out in the presence of oxygen, and a hydrogen donor is added to the reaction system; The degradation reaction system is a 50-300 mM phosphate buffer solution or a Tris-HCl buffer solution.

9. The application according to claim 8, characterized in that, The hydrogen donor is selected from at least one of ascorbic acid, panthenol, pantothenic acid, histidine, histamine, cytochrome c, vitamin B1, vitamin B6, tetramethylhydroquinone, and FMNH2.

10. A microbial agent, characterized in that, Contains spore-forming Micrococcus ( Sporosarcina sp.) R9-12 bacterial cells, the preservation number of the Bacillus spores R9-12 is CCTCC NO: M2019610, and its cell membrane surface displays the PS degrading enzyme as described in claim 1.

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