Fusion enzyme and use and composition thereof

Abstract

Provided are a fusion enzyme and the use and a composition thereof. The fusion enzyme is constructed by means of linking pyranose oxidase (POx) and catalase (CAT) via a peptide linker, and the peptide linker has an amino acid sequence of ASGAGGSEGGGSEGGT. The fusion enzyme is encapsulated in a calcium carbonate nanoparticle, and is integrated into a sprayable fibrin gel so as to prepare a POx-CAT@CaCO3@Gel composition. The composition has the triple functions of reducing the glucose level, scavenging excessive reactive oxygen, and increasing oxygen supply at a diabetic wound site. In-vivo experimental results show that the use of the POx-CAT@CaCO3@Gel composition significantly promotes the healing of wounds in mice with diabetes. The strategy combining the fusion enzyme with a medical gel provides a way for enhancing the healing of diabetic wounds, and can be used in the clinical treatment of diabetic wounds.

Description

Fusion enzymes, uses and compositions thereof TECHNICAL FIELD

[0001] The present application relates to the field of biocatalysis, in particular to fusion enzymes, uses and compositions thereof. BACKGROUND

[0002] Diabetes is difficult to heal due to the microenvironment damage of hyperglycemia, hypoxia and excessive active oxygen. Although there are many treatment methods, most of them cannot fully cope with these interrelated problems. Natural enzymes play a key role in various biological processes due to their excellent specificity, high biological activity, minimal side effects and limited multi-drug resistance. These characteristics make them increasingly used in biomaterials to treat cancer and promote wound healing. In particular, glucose oxidase (GOx) shows potential in controlling hyperglycemic environment. However, GOx also brings challenges such as oxygen depletion and accumulation of toxic hydrogen peroxide (H2O2) while converting glucose, which may exacerbate anaerobic conditions and disrupt the redox balance of the wound. And excess H2O2 may cause GOx inactivation. On the contrary, catalase (CAT) can catalyze the decomposition of H2O2 to generate oxygen, which helps wound healing by regulating ROS and hypoxic environment. However, there may be a lack of H2O2 in the wound area, and some CAT-based oxygen generation systems release toxic H2O2 at the initial stage of oxygen release. Therefore, solving these side effects is crucial to fully utilize the therapeutic potential of enzymes.

[0003] At present, chimeric enzymes can be designed by using genetic engineering methods, which effectively catalyze multi-step reactions by fusing two or more different enzymes end to end. The proximity of active sites promotes the transfer of intermediates to the next enzyme, increasing the overall yield of the desired product while significantly reducing the production of harmful intermediates. In addition, these artificially synthesized enzymes can exhibit optimized properties and functions to meet specific application requirements. Currently, several fusion enzymes have been applied in continuous flow biocatalysis, substrate channel optimization, protein ligation and other fields, highlighting the importance of fusion enzyme technology. However, fusion enzymes specifically for tissue repair have not been developed.

[0004] SUMMARY

[0005] In view of the deficiencies of the prior art described above, the present application provides fusion enzymes, uses and compositions thereof. The present application fuses and expresses pyranose oxidase (POx) and catalase (CAT) by genetic recombination technology to prepare POx-CAT fusion enzyme that can consume glucose and hydrogen peroxide simultaneously and produce oxygen.

[0006] The technical solutions of the present application are as follows:

[0007] In a first aspect, the present application provides a fusion enzyme, which is composed of pyranose oxidase POx and catalase CAT connected by a connecting peptide, and the amino acid sequence of the connecting peptide is ASGAGGSEGGGSEGGT.

[0008] Preferably, the fusion enzyme is CAT-ASGAGGSEGGGSEGGT-POx.

[0009] Preferably, the amino acid sequence of the pyranose oxidase POx is shown in SEQ ID No. 1, and the amino acid sequence of the catalase CAT is shown in SEQ ID No. 2.

[0010] Preferably, a hexahistidine tag is connected to the N-terminus of the fusion enzyme.

[0011] Preferably, the fusion enzyme is HHHHHH-CAT-ASGAGGSEGGGSEGGT-POx.

[0012] In a second aspect, the present application provides the use of the above-mentioned fusion enzyme in the preparation of a diabetic wound repair product.

[0013] In a third aspect, the present application provides a composition, which comprises a gel, calcium carbonate nanoparticles wrapped in the gel, and the above-mentioned fusion enzyme wrapped in the calcium carbonate nanoparticles.

[0014] In a fourth aspect, the present application provides a preparation method of the above-mentioned composition, which comprises the following steps:

[0015] The fusion enzyme is wrapped in the calcium carbonate nanoparticles by in-situ mineralization to obtain an intermediate POx-CAT@CaCO3;

[0016] Fibrinogen and thrombin are mixed with the intermediate POx-CAT@CaCO3 to obtain the composition POx-CAT@CaCO3@Gel.

[0017] In a fifth aspect, the present application provides the use of the above-mentioned composition in the preparation of a diabetic wound repair product. Beneficial effects:

[0018] The present application discloses a fusion enzyme, its application and a composition. The three major challenges of the microenvironment of diabetic wounds are high blood sugar, high active oxygen and low oxygen. Therefore, the core of the present application is to design and synthesize a fusion enzyme POx-CAT. This fusion enzyme has a dual function, which is suitable for application in diabetic wound repair: it can regulate blood sugar level, reduce the influence of active oxygen by decomposing H2O2, and correct the O2 deficiency at the wound site.

[0019] And, for better application, in the present application, the fusion enzyme is encapsulated in CaCO3 nanoparticles (intermediate POx-CAT@CaCO3) to ensure the sustained release and in vivo stability of the fusion enzyme, thereby enhancing its life and effectiveness. And, the integration of intermediate POx-CAT@CaCO3 into biocompatible fibrin gel (obtaining therapeutic agent) not only facilitates application, but also ensures that the therapeutic agent is localized to the wound site, providing a controlled healing environment for the wound.

[0020] In summary, the fusion enzyme provided by the present application, and the application strategy of its combination with medical gel, has very great clinical transformation prospects in enhancing the healing of diabetic wounds; the present application not only demonstrates the potential application of composition POx-CAT@CaCO3@Gel in the treatment of diabetic wounds, but also opens up a new research direction for future medical enzyme therapy. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a purification diagram of the POx-CAT fusion enzyme prepared in the embodiment of the present application.

[0022] Figure 2 is a diagram of the ability of the POx-CAT fusion enzyme prepared in the embodiment of the present application to consume glucose.

[0023] Figure 3 is a diagram of the ability of the POx-CAT fusion enzyme prepared in the embodiment of the present application to consume glucose to produce hydrogen peroxide.

[0024] Figure 4 is a diagram of the ability of the POx-CAT fusion enzyme prepared in the embodiment of the present application to consume hydrogen peroxide.

[0025] Figure 5 is a diagram of the ability of the POx-CAT fusion enzyme prepared in the embodiment of the present application to produce oxygen.

[0026] Figure 6 is a DLS diagram of POx-CAT@CaCO3 prepared in the embodiment of the present application.

[0027] Figure 7 is a TEM diagram of POx-CAT@CaCO3 prepared in the embodiment of the present application.

[0028] Figure 8 is a SEM diagram of POx-CAT@CaCO3@Gel prepared in the embodiment of the present application.

[0029] Figure 9 is a diagram of the POx-CAT@CaCO3@Gel prepared in the embodiment of the present application to promote the healing of diabetic wounds.

[0030] Figure 10 is a diagram of hematoxylin and eosin (H&E) staining of wound tissue on the 7th day and the 14th day of diabetic wound repair using POx-CAT@CaCO3@Gel prepared in the embodiment of the present application. DETAILED DESCRIPTION

[0031] The present application provides a fusion enzyme and its application and composition. To make the purpose, technical solution and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0032] In the development of fusion enzymes, a series of technical challenges are faced, mainly including: finding natural enzymes with medical potential; when different enzymes are fused together, ensuring the correct folding of the fusion enzyme and maintaining the inherent function and activity of each enzyme; and ensuring the stability and solubility of the fusion enzyme to ensure isolation and purification. These challenges make the whole process of developing multifunctional fusion enzymes from design to application full of challenges.

[0033] Based on this, the present application provides a POx-CAT fusion enzyme, which is composed of pyranose oxidase POx and catalase CAT connected by a connecting peptide, and the amino acid sequence of the connecting peptide is ASGAGGSEGGGSEGGT.

[0034] Example 1: Design of POx-CAT fusion enzyme

[0035] The POx-CAT fusion enzyme combines a POx enzyme of about 69.3 kDa and a CAT enzyme of about 84.1 kDa. The two enzymes are connected by a peptide segment (ASGAGGSEGGGSEGGT) composed of 16 amino acids. In addition, a six-group amino acid (His6) tag is added to the N-terminus of the fusion enzyme to facilitate its purification process.

[0036] Example 2: Expression and purification of POx-CAT fusion enzyme

[0037] In the present embodiment, the POx-CAT fusion enzyme prepared in Example 1 was expressed and purified. The POx-CAT gene was codon-optimized for expression in E. coli, followed by PCR amplification and cloning into the pET28a vector. The pET28a vector provides a N-terminal His6 tag for protein purification. When the optical density at 600 nm (OD600) of the bacterial culture reached 0.8, protein expression was induced using 0.8 mM isopropyl β-D-l-thiogalactopyranoside (IPTG). After induction, the culture was incubated at 16 °C for 20 hours, and the cells were harvested by centrifugation at 8,000 rpm for 30 minutes. The cell pellet was resuspended in a buffer containing Tris-HCl (0.03 M, pH 7.6), 500 mM NaCl, 5 mM imidazole, 0.1 mM biliverdin chloride (BV), 1 mM TCEP, and 0.5 mM PMSF, and cell lysis was performed using an ultra-high pressure homogenizer. The clarified lysate was obtained after centrifugation at 20,000 rpm for 50 minutes. The supernatant was treated by immobilized metal affinity chromatography (IMAC) using a HisTrap™ HP column (Cytiva, USA). The supernatant was purified by immobilized metal affinity chromatography (IMAC) using a HisTrap™ HP column. The purified protein was further purified by size exclusion chromatography using a Superdex 200 16 / 600 column using an AKTA Pure FPLC system. The purification of POx-CAT showed a single elution peak at approximately 51 mL, indicating its homogeneity. SDS-PAGE analysis under reducing conditions showed a clear band at approximately 156 kDa, confirming the purity of the protein (see Figure 1).

[0038] Example 3 Enzymatic activity characterization of POx-CAT fusion enzyme

[0039] The present embodiment evaluated the ability of the POx-CAT fusion enzyme prepared in Example 1 to consume glucose and simultaneously degrade H2O2. The ability of POx and POx-CAT fusion enzyme to consume glucose was compared, and the results showed similar glucose consumption rates for both (see Figure 2). Further comparison of their ability to produce H2O2 in a glucose-containing medium was performed. The results showed that the amount of H2O2 produced by POx-CAT fusion enzyme was significantly lower at high glucose concentrations compared to POx (see Figure 3). This indicates that the POx-CAT fusion enzyme effectively accelerates the degradation of H2O2 while catalyzing the production of H2O2 from glucose.

[0040] The activity of CAT in the POx-CAT fusion enzyme was also evaluated in the embodiments of the present application, and it was found that the POx-CAT fusion enzyme could effectively degrade H2O2 and simultaneously produce O2 in a concentration-dependent manner (see FIGS. 4, 5). These results demonstrate that the design and purification process of the POx-CAT fusion enzyme well maintained the activities of POx and CAT, while reducing the generation of the harmful byproduct H2O2.

[0041] Example 4 Preparation of composition POx-CAT@CaCO3@Gel

[0042] In the embodiments of the present application, the POx-CAT fusion enzyme prepared in Example 1 was encapsulated in calcium carbonate (CaCO3) nanoparticles by in-situ mineralization, as follows: CaCl2(0.2 mL, 1 M) was added to Tris buffer (3.6 mL, 1 mM) in the POx-CAT solution (1 mL, 2 mg / mL) under constant stirring. Subsequently, Na2CO3(0.2 mL, 1 M) was rapidly added to the mixture at 22°C. The mixture was stirred in a magnetic stirrer for 40 seconds and incubated for 15 minutes. The precipitate was separated by centrifugation at 12,000 rpm for 5 minutes, then washed twice, and all the supernatant was collected. The protein concentration was determined using an ultramicro spectrophotometer, and the POx-CAT@CaCO3 particles were observed using a hydrodynamic size analyzer and a transmission electron microscope (TEM). The loading capacity of this method was 20%, and the encapsulation efficiency was 55%. Morphologically, POx-CAT@CaCO3 was monodisperse spherical, with an average diameter of about 200 nm (see FIGS. 6-7).

[0043] In the embodiments of the present application, a fibrin gel was prepared using a mixture of fibrinogen and thrombin. By mixing a specific volume of a fibrinogen solution (containing 50 mg / mL of fibrinogen and 200 μg of POx-CAT@CaCO3) with an equal volume of a thrombin solution (containing 500 IU / mL of thrombin), the composition POx-CAT@CaCO3@Gel was generated. The morphology of the composite gel was observed using a scanning electron microscope (SEM) (see FIG. 8).

[0044] Example 5 Application of composition POx-CAT@CaCO3@Gel in diabetic wound repair

[0045] To verify the effect of the composition POx-CAT@CaCO3@Gel prepared in Example 4 in the repair of diabetic wounds, the present embodiment selected 6-8 week old male C57BL / 6 mice to establish a type 1 diabetes model. In the wound healing study, a sterile biopsy punch with a diameter of 10 mm was used to make skin wounds on the back of the mice. Subsequently, the diabetic mice were divided into four experimental groups, each with 8 mice, and received different treatments: a diabetic control group (DC group without treatment), a Gel group, a POx-CAT group, and a normal mouse wound group (NC group) as a control. To study the effectiveness of wound healing, the present embodiment monitored the progress of the wounds and took photos of the wounds on day 0, day 3, day 6, day 8, and day 11. The wound healing rate was quantified using the following formula: Wound area percentage = (initial wound area - wound area on a specific day) / initial wound area x 100%. The results showed that on day 11, the POx-CAT group showed the most significant improvement in promoting wound healing, while scars could still be seen in the other groups, especially the DC group. The curve of the wound healing rate further showed that the mice treated with POx-CAT@CaCO3@Gel healed significantly faster, with almost complete closure of the wound compared to the other groups, especially the DC group, exhibiting a healing rate comparable to the NC group under non-diabetic conditions. The results of H&E staining showed that on day 7, obvious tissue defects appeared in all groups except the NC group and the POx-CAT group. On day 14, the group treated with POx-CAT@CaCO3@Gel showed significant progress in wound healing, with the appearance of continuous epidermis, the formation of new blood vessels, and even the regeneration of hair follicles. See Figures 9 and 10. The newly formed epithelial tissue in the POx-CAT@CaCO3@Gel treatment group was very similar in structural characteristics to normal skin tissue. This study confirmed that the POx-CAT fusion enzyme designed and synthesized by the present embodiment, by mineralization and combination with medical fibrin hydrogel, showed significant effects in promoting the repair of diabetic wounds.

[0046] It should be understood that the application is not limited to the above examples, and can be improved or modified by those of ordinary skill in the art based on the above description, and all such improvements and modifications shall fall within the scope of the appended claims of the present application.

Claims

1. A fusion enzyme, characterized in that The fusion enzyme is composed of pyranose oxidase POx and catalase CAT connected by a connecting peptide, and the amino acid sequence of the connecting peptide is ASGAGGSEGGGSEGGT.

2. The fusion enzyme according to claim 1, characterized in that The fusion enzyme is CAT-ASGAGGSEGGGSEGGT-POx.

3. The fusion enzyme according to claim 1, characterized in that The amino acid sequence of the pyranose oxidase POx is shown in SEQ ID No. 1, and the amino acid sequence of the catalase CAT is shown in SEQ ID No.

2.

4. The fusion enzyme according to claim 1, characterized in that The N-terminus of the fusion enzyme is connected with a hexahistidine tag.

5. The fusion enzyme according to claim 4, characterized in that The fusion enzyme is HHHHHH-CAT-ASGAGGSEGGGSEGGT-POx.

6. Use of the fusion enzyme according to any one of claims 1 to 5 in preparing a diabetic wound repair product.

7. A composition, characterized in that The composition comprises gel, calcium carbonate nanoparticles encapsulated in the gel, and the fusion enzyme according to any one of claims 1 to 5 encapsulated in the calcium carbonate nanoparticles.

8. A method for preparing the composition according to claim 7, characterized in that: The preparation method comprises the following steps: The fusion enzyme according to any one of claims 1 to 5 is encapsulated in the calcium carbonate nanoparticles by an in situ mineralization method to obtain the intermediate POx-CAT@CaCO3; Fibrinogen and thrombin are mixed with the intermediate POx-CAT@CaCO3 to obtain the composition POx-CAT@CaCO3@Gel.

9. Use of the composition according to claim 7 in preparing a diabetic wound repair product.

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