Fusion protein based on adhesion promoting antibacterial peptide, enzyme immobilization system and preparation method and application thereof

Abstract

The invention discloses a fusion protein based on adhesion promoting antibacterial peptide, an enzyme immobilization system and a preparation method and application thereof. The fusion protein contains adhesion promoting antibacterial peptide and target protein, the adhesion promoting antibacterial peptide is selected from at least one of DS1, LCI and TA2, and the target protein is selected from at least one of green fluorescent protein eGFP and pullulanase pul. According to the fusion protein and enzyme immobilization system constructed by the invention, the target protein can be immobilized on immobilization carriers in different scenes, so that the target protein is recycled, and the fusion protein and enzyme immobilization system has the advantages of simplicity in operation, convenience, rapidness, high immobilization efficiency, good enzyme activity and stability and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme immobilization, and in particular relates to a fusion protein based on an adhesion-promoting antimicrobial peptide, an enzyme immobilization system, and a preparation method and application thereof. Background Art

[0002] Enzymes are mild catalysts with high selectivity in production practice. Compared with general chemical catalysts, they have many advantages such as mild reaction conditions, high catalytic efficiency, strong specificity, non-toxicity, and wide availability. They are currently widely used in the fields of food, pharmaceuticals, and chemical industry. At present, the catalytic technology of free enzymes is quite mature. However, in actual use, the use of free enzymes has significant limitations. They are unstable in environments such as high temperature, strong acid, strong base, high ionic strength, and organic solvents, and easily lose their catalytic activity. In addition, there are problems such as difficulty in separation and purification, and difficulty in recycling and reuse, which easily leads to product contamination and increased production costs. Enzyme immobilization technology can increase the stability of enzymes, overcome the shortcomings of free enzymes, and is conducive to improving the operational stability and reuse rate of enzymes, while reducing production costs, providing broad application prospects for enzymes.

[0003] Traditional enzyme immobilization methods can be categorized into encapsulation, adsorption, covalent bonding, and cross-linking. These immobilization methods immobilize soluble enzymes. Enzyme immobilization can be categorized into carrier-based immobilization and carrier-free immobilization, depending on the carrier used. Carrier-free immobilized enzymes do not require additional inactive substances and can be produced by direct cross-linking of cross-linked enzymes (CLEs), crystallized enzymes (CLECs), and aggregating enzymes (CLEAs). Cross-linked enzyme aggregates are the most mature and have been used to prepare a variety of enzymes, including hydrolases, proteases, and amidases. Cross-linked enzyme aggregates are formed by adding salts, organic solvents, or nonionic polymers to form protein aggregates, replacing the tedious crystallization process. This method is not only simple and cost-effective, but also exhibits excellent catalytic activity, storage stability, operational stability, and ease of recovery and recycling. However, there are still many key issues to be explored in cross-linked enzyme aggregate technology, including how to regulate the particle size of CLEAs without causing significant diffusion restrictions, how to adjust the activity, stability and selectivity of enzymes by changing aggregation conditions, how the microstructure of CLEAs changes under different conditions, and the development of various enzyme CLEAs and new cross-linkers.

[0004] Support-based immobilization methods rely on the physical interaction between the enzyme and the material surface; in particular, the enzyme's affinity for the material is crucial for immobilization, which requires the presence of specific active groups on the support. The interaction between the enzyme and the support must be considered when selecting materials. In selecting a support, considerations must be given to the shape and size, chemical stability, hydrophilicity, mechanical mobility, and type of functional groups, as well as the selection of different materials based on their intended application. Enzyme adsorption on the support surface is not a directional process and can result in unproductive enzyme-support interactions, leading to occlusion of the enzyme's active sites or reduced enzyme flexibility, resulting in significant loss of enzyme activity. Furthermore, excessively high packing density can result in a multilayered structure, resulting in enzyme waste and reduced activity.

[0005] In recent years, significant progress has been made in the development and application of site-specific protein immobilization. Notably, advances in organic chemistry and molecular biology have led to the development of some very powerful, efficient, and site-specific applications for anchoring proteins to supports. A prominent example is the carbohydrate-binding module (CBM), which is present in hydrolases such as glycosyl hydrolases and glycosyltransferases. CBMs can promote adhesion to their natural substrates (e.g., cellulose, chitin, starch, glycogen, inulin, and xylan), thereby improving hydrolysis efficiency. In addition to natural substrates, CBMs can also exhibit adhesion to synthetic polymers such as polyethylene terephthalate (PET). A comparison of eight CBMs from different sources revealed that a CBM derived from Bacillus anthracis exhibited superior adhesion to PET, with a dissociation constant for its interaction with PET (Kd = 25.4 μg / L). Similarly, Chen Kequan et al. (CN109880859 A) fixed lysine decarboxylase to the chitin surface via a chitin binding domain (ChBD), achieving high immobilization efficiency and better enzyme activity stability. Other site-specific protein immobilizations include six continuous histidine tags, biotin tags, and enzyme-active fusion proteins (such as the filamentous fungus Fusarium spp. serine esterase cutinase). Zhang Yu et al. (CN110423742A) co-expressed ferritin containing an Ecoil short peptide and an enzyme containing a Kcoil short peptide to achieve magnetic immobilization of the enzyme. This method requires the simultaneous expression of two elements and requires a magnetic material. The above site-specific immobilization methods all require specific carriers or corresponding proteins, which limits their scope of application. Summary of the Invention

[0006] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a fusion protein and enzyme immobilization system based on adhesion-promoting antimicrobial peptides. The fusion protein and enzyme immobilization system constructed by the present invention can fix the target protein on an immobilization carrier in different scenarios, thereby realizing the recycling of the target protein. It has the advantages of simple operation, convenience and speed, high immobilization efficiency, good enzyme activity and stability, etc.

[0007] The present invention also provides a preparation method and application of the fusion protein and enzyme immobilization system based on the adhesion-promoting antimicrobial peptide.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a fusion protein based on an adhesion-promoting antimicrobial peptide, which contains an adhesion-promoting antimicrobial peptide and a target protein, wherein the adhesion-promoting antimicrobial peptide is selected from at least one of DS1, LCI and TA2, and the target protein is selected from at least one of green fluorescent protein eGFP and pullulanase pul.

[0009] Wherein, the fusion protein is selected from at least one of DS1-eGFP, TA2-eGFP, eGFP-LCI, eGFP-TA2, DS1-pul, TA2-pul, pul-LCI and pul-TA2.

[0010] Wherein, the fusion protein is selected from at least one of DS1-pul, TA2-pul, pul-LCI and pul-TA2.

[0011] The method for constructing the fusion protein based on the adhesion-promoting antimicrobial peptide of the present invention comprises the following steps:

[0012] The adhesion-promoting antimicrobial peptide gene and the target gene are assembled and cloned into a plasmid, and the plasmid is transformed and extracted to obtain a fusion protein expression vector based on the adhesion-promoting antimicrobial peptide; the expression vector is introduced into a host bacterium, cultured, and the fusion protein is extracted; wherein the adhesion-promoting antimicrobial peptide gene is selected from at least one of the DS1 gene, the LCI gene, and the TA2 gene, and the target gene is selected from the eGFP gene (eGFP: green fluorescent protein) or the pul gene (pul: pullulanase),

[0013] Preferably, the sequence of the adhesion-promoting antimicrobial peptide gene is at least one selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0014] Wherein, the host bacteria is Escherichia coli or Bacillus subtilis.

[0015] The enzyme immobilization system based on adhesion-promoting antimicrobial peptides of the present invention contains the fusion protein.

[0016] Wherein, the enzyme immobilization system comprises an immobilization carrier and a fusion protein, and the fusion protein is immobilized on the immobilization carrier.

[0017] Wherein, the immobilization carrier is selected from at least one of PS, PP, PET, PMP, PCL, stainless steel and silicon wafer.

[0018] Use of the adhesion-promoting antimicrobial peptide-based fusion protein of the present invention or the adhesion-promoting antimicrobial peptide-based enzyme immobilization system of claim 5 in enzyme-catalyzed reactions.

[0019] The present invention can utilize the antimicrobial peptide LCI and the antifungal peptide Tachystatin A2 (TA2) as adhesion-promoting peptides to specifically immobilize the fusion protein on the surface of immobilized carriers such as polystyrene (PS), polypropylene (PP), polyethylene terephthalate (PET), poly(4-methylpentene-1) (PMP), and polycaprolactone (PCL). Furthermore, the present invention can utilize the antimicrobial peptide Dermaseptin S1 (DS1) and the antimicrobial peptide LCI as adhesion-promoting peptides to specifically immobilize the fusion protein on stainless steel and silicon wafers. The above method allows for the selection of different immobilized carriers and, in turn, different types of adhesion-promoting peptides according to different application scenarios, allowing the enzyme or protein to be immobilized on the surface of the immobilized carrier to achieve enzyme immobilization and sustained catalytic reaction.

[0020] The present invention can immobilize the target protein on an immobilized carrier in different scenarios, thereby realizing the recycling of the target protein. It has the advantages of simple operation, convenience and rapidity, high immobilization efficiency, good enzyme activity and stability, etc.

[0021] This invention adopts the "two-site cooperative adhesion" theory. In the first binding domain, the antimicrobial peptide anchors to the carrier surface through electrostatic / hydrophobic interactions; in the second functional domain, the target protein maintains its native conformation and performs catalytic functions. At the same time, the natural preservative properties of the antimicrobial peptide are utilized to overcome the physical limitations of traditional carrier materials.

[0022] The combination of the specific adhesion-promoting antimicrobial peptide and the specific target protein of the present invention has a significant effect. For example, for the enzymatic activity of Pul on stainless steel, DS1-pul significantly exceeds that of other fusion proteins. The effects of other adhesion-promoting antimicrobial peptide and pul combinations on stainless steel are also significantly inferior to that of the present invention.

[0023] The present invention utilizes antimicrobial peptides as adhesion-enhancing modules to immobilize target proteins on immobilized carriers in different scenarios, thereby achieving the recycling of target proteins. It has the advantages of simple and convenient operation, high immobilization efficiency, and good enzyme activity and stability.

[0024] The core of the antimicrobial peptides DS1, LCI and TA2 and the connecting peptide used in the present invention lies in the fusion expression of the antimicrobial peptides and pullulanase, and the adhesion properties of the antimicrobial peptides are used to achieve the immobilization of the pullulanase in the fusion protein.

[0025] In the process of constructing the protein particle, the present invention uses four adhesion-promoting antimicrobial peptides and an N-short peptide tag fusion protein composed of an N-terminal short peptide tag separated by a rigid 17-amino acid helix and a C-terminal eGFP. All five EGFP-adhesion-promoting antimicrobial peptide fusion proteins (N or C terminus) showed green fluorescence on all studied synthetic polymer carriers (PS, PP, PET); while the negative control showed no fluorescence on any immobilized carrier material after washing. At the same time, in the pullulanase immobilization experiment, it was found that the activity of the immobilized fusion protein on PET, stainless steel and silicon wafers was significantly improved, up to 8.7 times the activity of the original enzyme. Therefore, these experimental results have preliminarily demonstrated that the antimicrobial peptides and connecting peptides selected by the present invention have obvious advantages in enhancing the immobilization effect of the fusion protein.

[0026] The present invention selected these antimicrobial peptides and connecting peptides from among numerous candidate sequences, among which the TA2 peptide can be referenced as follows: J Biol Chem. 1999 Sep 10; 274(37): 26172-8; the LCI peptide can be referenced as follows: Biochemistry. 2011 May 10; 50(18): 3621-7; and the DS1 peptide can be referenced as follows: J Biol Chem. 2002 Dec 20; 277(51): 49332-40), based on their good performance shown in preliminary experiments, including efficient adhesion and immobilization effects.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] The present invention provides a method for constructing a fusion protein expression vector based on an adhesion-promoting antimicrobial peptide, for example, by expressing a fusion of an antimicrobial peptide and pullulanase, and utilizing the adhesion properties of the antimicrobial peptide to immobilize the pullulanase in the fusion protein. The present invention enables the immobilization of target proteins on immobilized carriers in different scenarios, thereby enabling the recycling of target proteins. It has the advantages of simple operation, convenience and rapidity, high immobilization efficiency, and good enzyme activity and stability. For example, on a stainless steel surface, the C-terminal fusion activity of Pul and DS1 was significantly increased by 8.7 times compared to WT Pul, and was significantly higher than other combinations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The SDS-PAGE electrophoresis detection of Example 1; wherein: 1: eGFP; 2: DS1-eGFP; 3: TA2-eGFP; 4: eGFP-LCI; 5: eGFP-TA2;

[0030] Figure 2 The immobilization results of each fusion protein in Example 3 on the immobilization carrier PS;

[0031] Figure 3 The immobilization results of each fusion protein in Example 3 on the immobilization carrier PP;

[0032] Figure 4 The immobilization results of each fusion protein in Example 3 on the immobilization carrier PET;

[0033] Figure 5 The immobilization results of each fusion protein in Example 3 on the immobilization carrier stainless steel;

[0034] Figure 6 The immobilization results of each fusion protein in Example 3 on the immobilization carrier silicon wafer;

[0035] Figure 7 The results of the study on the reusability of Pul-LCI immobilized on the immobilization carrier PS in Example 7 are as follows;

[0036] Figure 8 These are the results of a study on the reusability of Pul-LCI immobilized on the immobilization carrier PP in Example 7. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0038] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Experimental methods without specific conditions specified in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0039] Example 1

[0040] Construction of green fluorescent protein plasmids containing adhesion-promoting antimicrobial peptides

[0041] 1. Materials

[0042] 1.1. Strains and vectors

[0043] The Escherichia coli (E. coli) strain TOP 10 used for cloning and the expression strain E. coli BL21 (DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0044] Adhesion-promoting antimicrobial peptide genes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; their amino acid sequences are shown in Table 1. The sequences of the four synthesized adhesion-promoting antimicrobial peptide genes are shown in Table 2. The N-peptide-tagged fusion protein consists of an N-terminal peptide tag separated by a rigid 17-amino acid helix (17H, AEAAAKEAAAKEAAAKA) and a C-terminal eGFP. The C-peptide-tagged fusion protein, on the other hand, consists of an N-terminal eGFP separated by a rigid linker peptide 17H. The resulting fusion genes or proteins, with corresponding abbreviations, are shown in Table 3.

[0045] Table 1 Adhesion-promoting peptide information

[0046]

[0047] Table 2 Base sequences of four synthetic adhesion-promoting antimicrobial peptide genes

[0048]

[0049]

[0050] Table 3 Fusion genes and fusion proteins

[0051]

[0052] 1.2 Culture medium

[0053] LB medium: 0.5% yeast powder, 1% peptone, 1% NaCl; solid LB medium is supplemented with 2% agar; when screening transformants of the recombinant plasmid, kanamycin is added to a final concentration of 50 μg / mL.

[0054] The E. coli expression medium used was TB medium containing 1.2% peptone, 2.4% yeast powder, 0.4% (v / v) glycerol, 0.231% KH2PO4 and 1.254% K2HPO4.

[0055] 2. Gene amplification and vector construction

[0056] Based on the synthetic gene sequences of four adhesion-promoting antimicrobial peptides with linkers (see Table 2) and the EGFP gene sequence of the vector pEGFP-N1, primers of the corresponding genes in Table 4 were used for amplification to obtain Ds1-linker, TA2-linker, linker–LCI, linker-TA2 and EGFP genes, respectively.

[0057] Using pET-30a(+) as the base plasmid, Ds1-linker-EGFP, TA2-linker-EGFP, EGFP-linker-LCI, and EGFP-linker-TA2 were assembled and cloned into pET-30a(+) using the Gibson assembly method after double digestion with NdeⅠ and XhoⅠ. The clones were then transformed into E. coli TOP 10 competent cells. Positive transformants were identified by colony PCR, restriction enzyme digestion, and sequencing analysis to obtain four vectors: pET30a-Ds1-linker-eGFP, pET30a-TA2-linker-eGFP, pET30a-eGFP-linker-LCI, and pET30a-eGFP-linker-TA2. A control plasmid containing only the EGFP-linker was also constructed.

[0058] Table 4 Primer sequences

[0059]

[0060]

[0061] Note: The underlined fragments are homologous fragments

[0062] Example 2

[0063] Inducible expression of fusion protein in recombinant Escherichia coli

[0064] The four vectors and one control plasmid constructed in Example 1 were transformed into E. coli BL21 (DE3) competent cells, and a single colony was picked and inoculated into 5 mL of LB medium. 50 μg / mL of kanamycin was added, and the cells were cultured at 37° C. overnight to obtain a seed solution.

[0065] The next day, the seed solution was inoculated into 100 mL of fresh TB medium and cultured at 37°C for approximately 2 hours, until the OD600 reached 0.5-0.6. 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture medium and incubated at 20°C for 24 hours to induce expression. The cells were then collected by centrifugation at 12,000 rpm and 4°C for 10 minutes and washed twice with buffer to remove any residual culture medium.

[0066] The collected cells were resuspended in 20 mL of buffer (50 mM Tris-HCl, pH 8.0), disrupted by ultrasound, and centrifuged (10,000 × g, 4°C, 20 min). The supernatant was collected for later use.

[0067] Purification was performed using a Ni affinity chromatography column, which was equilibrated to the baseline with an equilibrium buffer. The sample was loaded onto the Ni affinity chromatography column at a flow rate of 1 mL / min through a constant flow pump, and gradient eluted with an imidazole elution solution. The elution peak was collected, dialyzed overnight, and concentrated 10 times for later use.

[0068] The results were detected by SDS-PAGE electrophoresis. Figure 1 ; Figure 1 The results showed that four fusion proteins, namely DS1-eGFP, TA2-eGFP, TA2-eGFP and eGFP-TA2, were obtained by the above method.

[0069] Example 3

[0070] eGFP immobilization system

[0071] The immobilized carrier materials PP, PS, PET, stainless steel and silicon wafers were cut into 1×1 cm sizes, and the purified fusion proteins DS1-eGFP, TA2-eGFP, eGFP-LCI, eGFP-TA2 and the control group eGFP in Example 2 were applied to the above different immobilized carrier materials respectively.

[0072] Take 50 μL protein solution and carrier mix, room temperature for 15 minutes. Then, remove the protein solution, rinse the material with 10mL of Tris-HCl buffer (50mM, pH 8.0), and transfer it to a 25-well square culture dish containing 2ml Tris-HCl buffer (50mM, pH 8.0). Incubate the sample (2 minutes), remove the buffer, and then add new buffer (three times). Take out the sample, rinse with 10ml ddH2O, and dry with a nitrogen stream. Determine the binding between the eGFP-adhesion-promoting antimicrobial peptide tag fusion protein by confocal fluorescence microscopy (TCS SP8). Excite the sample with a laser intensity of 488nm and 10%. Detect with a PMT2 detector (excitation light 500-565nm).

[0073] The binding of eGFP-adhesion-promoting antimicrobial peptide tag fusion protein to the five immobilized carrier materials was observed by confocal fluorescence microscopy; as a negative control, EGFP-linker was also coated on the surface to determine nonspecific binding.

[0074] Figures 2 to 6The results showed that the negative control showed no fluorescence on any immobilized carrier material after washing; while all five EGFP-adhesion-promoting antimicrobial peptide fusion proteins (N or C terminus) showed green fluorescence on all studied synthetic polymer carriers (PS, PP, PET); further, by observing the intensity of fluorescence, DS1 and TA2 fusion proteins showed the strongest binding on stainless steel and silicon wafers, and similar results were obtained on the silicon wafer surface where C-terminus fused LCI and TA2 showed the strongest binding.

[0075] Example 4

[0076] Construction of recombinant Bacillus subtilis

[0077] 1. Materials

[0078] 1.1. Strains and vectors

[0079] Escherichia coli (E. coli) strain TOP 10 used for cloning was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0080] Plasmid pDG1730 was purchased from BGSC ( www.bgsc.org ), and its sequence is available in NCBI (accession number: EF473728.1 );

[0081] Geobacillus thermocatenulatus (Geobacillus thermocatenulatus GSMZ730) was purchased from DSM Culture Collection, Germany;

[0082] Bacillus subtilis WB800 was used as the expression strain, and the four genes containing the adhesion-promoting peptide and the rigid 17-amino acid helical gene sequences were the same as those in Example 1.

[0083] 1.2 Culture medium

[0084] LB medium: 0.5% yeast powder, 1% peptone, 1% NaCl, and 2% agar added to solid LB medium;

[0085] M1 medium: 0.5% peptone, 0.3% yeast extract, and 2% agar added to solid LB medium; when screening transformants of the recombinant plasmid, add ampicillin to a final concentration of 50 μg / mL to Escherichia coli, and add spectinomycin to a final concentration of 100 μg / mL to Bacillus subtilis.

[0086] Bacillus subtilis fermentation medium: contains peptone 2%, yeast powder 1%, KH2PO4 0.09%, K2HPO4 0.118%, NH4Cl 0.5%, NaCl 0.5%, MgSO4·7H2O 0.102%, and CaCl2·2H2O 0.013%.

[0087] 1.3. Pulase activity assay

[0088] The recombinant pullulanase activity was measured using the DNS termination method using pullulan solution as the substrate. Pullulanase activity is defined as the amount of enzyme required to decompose pullulan per minute under the selected conditions to produce reducing sugars with a reducing power equivalent to 1 μmol of glucose, expressed as 1 U.

[0089] 2. Methods

[0090] 2.1 Genome Extraction

[0091] Geobacillus thermocatenulatus GSMZ730 was cultured in M1 medium at 60°C overnight, and Bacillus subtilis WB800 was cultured in LB medium at 37°C overnight. The genomes of the two strains were extracted according to the instructions of the Omega Bacterial Genome Extraction Kit.

[0092] 2.2 Gene amplification and vector construction

[0093] The promoter and signal peptide were amplified according to the P43 gene (promoter) fragment sequence (accession number: EF473728.1) and the signal peptide NprE gene fragment sequence (accession number: JQ302263.1) in NCBI. The genome of WB800 was used as a template and the primers of the corresponding genes in Table 4 were used for amplification. After the amplified products were recovered by agarose gel electrophoresis, the P43 gene fragment and the NprE gene fragment were connected by overlapping PCR to obtain the fusion PCR product P43-Signal NprE ; as the basic plasmid, double-digested with BamHI and HindⅢ, and assembled with Gibson method to form P43-Signal NprEAssemble and clone into pDG1730 to obtain pDG1730-P43. Based on the pullulanase (pul) gene sequence in NCBI (accession number: KY613502), the genome of GSMZ730 was used as a template and primers of the corresponding genes in Table 3 were used for amplification. At the same time, based on the synthetic 4 adhesion-promoting peptide gene sequences with linkers, 4 genes, Ds1-linker, TA2-linker, linker-LCI and linker-TA2, were amplified respectively. Using plasmid pDG1730-P43 as the starting plasmid, double enzyme digestion with HindⅢ and EcoRI was used, and Ds1-linker-pul, TA2-linker-pul, pul-linker-LCI, and pul-linker-TA2 were assembled and cloned into pDG1730-P43 using the Gibson assembly method and transformed into E. coli TOP 10 competent cells were cultured and positive transformants were identified by colony PCR, restriction enzyme digestion, and sequencing analysis to obtain pDG1730P43-Ds1-linker-pul, pDG1730P43-TA2-linker-pul, pDG1730P43-pul-linker-LCI, and pDG1730P43-pul-linker-TA2. A control plasmid containing only pul-linker was also constructed.

[0094] 2.3. Transformation of Bacillus subtilis WB800 with recombinant plasmid

[0095] The four recombinant plasmids and the control plasmid constructed in 2.2 were transformed into Bacillus subtilis. pDG1730 uses the amylase gene as a homology arm, and under antibiotic selection pressure, it integrates the foreign gene into the Bacillus subtilis genome. Competent Bacillus subtilis cells were prepared according to the classic starvation method described by Anagnostopoulos and Spizizen in 1961. Recombinants were selected with spectinomycin, and positive clones were verified by colony PCR to obtain recombinant Bacillus subtilis.

[0096] Example 5

[0097] Secretion and expression of fusion protein using recombinant Bacillus subtilis

[0098] The recombinant Bacillus subtilis strain verified correctly in Example 4 was fermented. Specifically, a single clone was selected and inoculated into 5 mL of LB liquid medium containing Spc resistance, shaken at 37°C and 200 rpm overnight, and then transferred to expression medium at a ratio of 1:100 the next day. Fermentation was continued at 30°C and 200 rpm for 48 hours. The enzyme activity of the fermentation supernatant of the recombinant Bacillus subtilis strain and the fermentation supernatant of the control strain (wild-type Bacillus subtilis WB800) was measured and normalized.

[0099] Example 6

[0100] Pullulanase immobilization system

[0101] The five active recombinant pullulanases from the fermentation broth supernatant of Example 5, where WT was the supernatant of the pull-linker vector, were diluted with citrate buffer (pH 6.0) to prepare activity-normalized samples. 50 μL of each sample was applied to 1×1 cm immobilization support materials (PP, PS, PET, stainless steel, and silicon wafers) and incubated at room temperature for 15 minutes. After removing the supernatant, the samples were washed three times with 200 μL of citrate buffer (pH 6.0) and plated in a 24-well plate. Pullulan was added to determine activity. This was repeated four times for each enzyme solution, and the average activity (U) was calculated. The results are shown in Table 5.

[0102] Table 5 Pullulanase activity determination results

[0103]

[0104]

[0105] The thermostable type I pullulanase gene (pul) from Bacillus thermophilus DSMZ730 has a 2154bp open reading frame encoding 718 amino acids with a molecular weight of approximately 80.37kDa. Four adhesion-promoting peptides were fused to pul and expressed using the Bacillus subtilis secretion expression system. After 48 hours of fermentation, enzyme activity was detected in the fermentation supernatant. The average activity of the wild-type and fusion enzymes was approximately 28.5 U / mL, indicating that the addition of adhesion-promoting peptides had minimal effect on pullulanase.

[0106] After the enzyme activity of the five groups of fermentation supernatants was homogenized, they were immobilized on the surfaces of five different materials and subsequently washed, and the activity was determined using a pullulan substrate. The coating of PS and PP resulted in high background activity. On PET, a significant increase in the activity of the adhesion-promoting peptide tag fusion protein was observed (up to 3.7 times). The N-terminal fusion of Pul and TA2 increased the activity on PET. On the stainless steel surface, the C-terminal fusion activity of Pul and DS1 was significantly increased by 8.7 times compared to WT Pul, and was significantly higher than other combinations. Compared with WT Pul, a significant increase in the activity of the immobilized fusion protein was observed on the silicon wafer (up to 6.21 times).

[0107] Example 7

[0108] Reusability study

[0109] The reusability of pul-LCI and pul-TA2 was studied on PS and PP. The steps are as follows:

[0110] The pul-LCI and pul-TA2 supernatants (see Example 6) were adsorbed on PS and PP, followed by three washes with 200 μl of citrate buffer (pH 6.0) in a 24-well plate, followed by treatment with 0.01% Tween 20 for 2 minutes, and finally the activity was determined by adding pullulan in citrate buffer. After each reaction, 200 μl of citrate buffer (pH 6.0) was added to each well. Subsequently, the liquid was removed from each well by pipetting, and an additional washing step was performed by adding 200 μl of citrate buffer (pH 6.0) for three washes, followed by treatment with 0.01% Tween 20 for 2 minutes. The average of the four repetitions was calculated and compared with the starting value, and the residual activity after each cycle was calculated. The reusability of the eight cycles was determined, and the results are shown in Figure 2. Figure 7 、 Figure 8 .

[0111] The results showed that after one use of Pul-LCI fixed on PS and PP materials, the residual activity on PS and PP dropped to 86% and 85%, respectively; after repeated use for 8 times, the enzyme activity still remained above 50% of the initial enzyme activity.

[0112] In summary, the adhesion-promoting antimicrobial peptide tag of the present invention can be used to specifically fix the fusion protein on polystyrene (PS), polypropylene (PP), polyethylene terephthalate (PET), stainless steel, silicon wafers and even gold surfaces, achieving one-step fixation and purification, and fixing the crude enzyme on carriers in different scenarios. The operation is simple, convenient and fast, the immobilization efficiency is high, and the enzyme activity and stability are good.

Claims

1. A fusion protein based on an adhesion-promoting antimicrobial peptide, characterized in that: The fusion protein contains an adhesion-promoting antimicrobial peptide and a target protein, wherein the adhesion-promoting antimicrobial peptide is selected from at least one of DS1, LCI and TA2, and the target protein is selected from at least one of green fluorescent protein eGFP and pullulanase pul.

2. The fusion protein based on adhesion-promoting antimicrobial peptide according to claim 1, characterized in that The fusion protein is preferably selected from at least one of DS1-eGFP, TA2-eGFP, eGFP-LCI, eGFP-TA2, DS1-pul, TA2-pul, pul-LCI and pul-TA2.

3. The fusion protein based on adhesion-promoting antimicrobial peptide according to claim 1, characterized in that The fusion protein is selected from at least one of DS1-pul, TA2-pul, pul-LCI and pul-TA2.

4. A method for constructing a fusion protein based on an adhesion-promoting antimicrobial peptide according to claim 1, characterized in that: The following steps are involved: The adhesion-promoting antimicrobial peptide gene and the target gene are assembled and cloned into a plasmid, and the plasmid is transformed and extracted to obtain a fusion protein expression vector based on the adhesion-promoting antimicrobial peptide; the expression vector is introduced into a host bacterium, cultured and the fusion protein is extracted; wherein the adhesion-promoting antimicrobial peptide gene is selected from at least one of the DS1 gene, the LCI gene and the TA2 gene, and the target gene is selected from the eGFP gene or the pul gene.

5. The preparation method according to claim 4, characterized in that The sequence of the adhesion-promoting antimicrobial peptide gene is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

6. The preparation method according to claim 4, characterized in that The host bacteria is preferably Escherichia coli or Bacillus subtilis.

7. An enzyme immobilization system based on adhesion-promoting antimicrobial peptides, characterized in that: It contains the fusion protein according to any one of claims 1 to 4.

8. The enzyme immobilization system based on adhesion-promoting antimicrobial peptide according to claim 7, characterized in that: The enzyme immobilization system comprises an immobilization carrier and a fusion protein, wherein the fusion protein is immobilized on the immobilization carrier.

9. The enzyme immobilization system based on adhesion-promoting antimicrobial peptide according to claim 8, characterized in that: The immobilization carrier is selected from at least one of PS, PP, PET, PMP, PCL, stainless steel and silicon wafer.

10. Use of the adhesion-promoting antimicrobial peptide-based fusion protein according to claim 1 or the adhesion-promoting antimicrobial peptide-based enzyme immobilization system according to claim 5 in an enzyme-catalyzed reaction.

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