WO2025213626A1 - 融合酶及其应用与组合物 - Google Patents

融合酶及其应用与组合物

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Publication number
WO2025213626A1
WO2025213626A1 PCT/CN2024/107944 CN2024107944W WO2025213626A1 WO 2025213626 A1 WO2025213626 A1 WO 2025213626A1 CN 2024107944 W CN2024107944 W CN 2024107944W WO 2025213626 A1 WO2025213626 A1 WO 2025213626A1
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pox
cat
fusion enzyme
composition
gel
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French (fr)
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黄鹏
陈欣
陈帅
林静
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Shenzhen University
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Shenzhen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/0301—Pyranose oxidase (1.1.3.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01006—Catalase (1.11.1.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the present invention relates to the field of biocatalysis technology, in particular to a fusion enzyme and its application and composition.
  • ROS reactive oxygen species
  • Glucose oxidase (GOx) in particular, has shown promise in controlling hyperglycemic environments.
  • H2O2 toxic hydrogen peroxide
  • CAT catalase
  • fusion enzymes have been applied in continuous flow biocatalysis, substrate channel optimization, protein ligation and other fields, highlighting the importance of fusion enzyme technology.
  • fusion enzymes specifically for tissue repair have not yet been developed.
  • the present invention provides a fusion enzyme and its application and composition.
  • the present invention uses genetic recombination technology to fuse pyranose oxidase (POx) and catalase (CAT) to express, and prepare a POx-CAT fusion enzyme that can simultaneously consume glucose and hydrogen peroxide and produce oxygen.
  • POx pyranose oxidase
  • CAT catalase
  • the present invention 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.
  • the fusion enzyme is CAT-ASGAGGSEGGGSEGGT-POx.
  • amino acid sequence of the pyranose oxidase POx is shown as SEQ ID No. 1
  • amino acid sequence of the catalase CAT is shown as SEQ ID No. 2.
  • the N-terminus of the fusion enzyme is connected to a hexahistidine tag.
  • the fusion enzyme is HHHHHH-CAT-ASGAGGSEGGGSEGGT-POx.
  • the second aspect of the present invention provides the use of the above-mentioned fusion enzyme in the preparation of diabetic wound repair products.
  • the third aspect of the present invention provides a composition comprising a gel, calcium carbonate nanoparticles encapsulated in the gel, and the above-mentioned fusion enzyme encapsulated in the calcium carbonate nanoparticles.
  • a fourth aspect of the present invention provides a method for preparing the above-mentioned composition, the preparation method comprising the following steps:
  • the fusion enzyme is encapsulated in the calcium carbonate nanoparticles by an in situ mineralization method to obtain the intermediate POx-CAT@CaCO 3 ;
  • Fibrinogen and thrombin are mixed with the intermediate POx-CAT@CaCO 3 to obtain the composition POx-CAT@CaCO 3 @Gel.
  • the fifth aspect of the present invention provides the use of the above composition in preparing a diabetic wound repair product.
  • the present invention discloses a fusion enzyme, its applications, and compositions.
  • the three major challenges to the diabetic wound microenvironment are hyperglycemia, high levels of reactive oxygen species, and hypoxia. Therefore, the core of the present invention is the design and synthesis of the fusion enzyme POx-CAT.
  • This fusion enzyme has dual functions, making it ideal for use in diabetic wound repair: it can regulate blood sugar levels and mitigate the effects of reactive oxygen species through the decomposition of H2O2 , while simultaneously correcting O2 deficiency at the wound site.
  • the present invention encapsulates this fusion enzyme within CaCO3 nanoparticles (resulting in the intermediate POx-CAT@ CaCO3 ), ensuring sustained release and in vivo stability of the fusion enzyme, thereby enhancing its lifespan and effectiveness.
  • the intermediate POx-CAT@ CaCO3 is incorporated into a biocompatible fibrin gel (resulting in the therapeutic agent), which 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.
  • the fusion enzyme provided by the present invention has very great clinical translation prospects in enhancing diabetic wound healing; the present invention not only demonstrates the potential application of the composition POx-CAT@ CaCO3 @Gel in diabetic wound treatment, but also opens up new research directions for future medical enzyme therapy.
  • FIG1 is a diagram showing the purification of the POx-CAT fusion enzyme prepared in an embodiment of the present invention.
  • FIG2 is a graph showing the glucose consumption capacity of the POx-CAT fusion enzyme prepared in an example of the present invention.
  • FIG3 is a graph showing the ability of the POx-CAT fusion enzyme prepared in an embodiment of the present invention to consume glucose and produce hydrogen peroxide.
  • FIG4 is a graph showing the ability of the POx-CAT fusion enzyme prepared in an example of the present invention to consume hydrogen peroxide.
  • FIG5 is a graph showing the oxygen production capability of the POx-CAT fusion enzyme prepared in an embodiment of the present invention.
  • Figure 6 is a DLS graph of POx-CAT@ CaCO3 prepared in an example of the present invention.
  • Figure 7 is a TEM image of POx-CAT@ CaCO3 prepared in an embodiment of the present invention.
  • FIG8 is a SEM image of POx-CAT@CaCO 3 @Gel prepared in an example of the present invention.
  • FIG9 is a diagram showing that POx-CAT@CaCO 3 @Gel prepared in an embodiment of the present invention promotes diabetic wound healing.
  • FIG10 is a hematoxylin and eosin (H&E) staining image of wound tissue on the 7th and 14th days in the validation of diabetic wound repair using POx-CAT@CaCO 3 @Gel prepared in an example of the present invention.
  • H&E hematoxylin and eosin
  • the present invention provides a fusion enzyme and its application and composition. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the present invention.
  • fusion enzymes presents a series of technical challenges, including: identifying natural enzymes with medical potential; ensuring the correct folding of the fusion enzymes while maintaining the inherent function and activity of each enzyme when fusing them together; and ensuring the stability and solubility of the fusion enzymes to ensure isolation and purification.
  • the embodiment of the present invention 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.
  • the POx-CAT fusion enzyme combines the approximately 69.3 kDa POx enzyme and the approximately 84.1 kDa CAT enzyme. These two enzymes are linked by a 16-amino acid peptide (ASGAGGSEGGGSEGGT). In addition, a six-amino acid tag (His6) is added to the N-terminus of the fusion enzyme to facilitate its purification.
  • the POx-CAT fusion enzyme prepared in Example 1 was expressed and purified.
  • the POx-CAT gene was codon-optimized for expression in Escherichia coli, then amplified by PCR and cloned into the pET28a vector.
  • the pET28a vector provides an N-terminal His6 tag to facilitate protein purification.
  • 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 bipyrrolidine chloride (BV), 1 mM TCEP, and 0.5 mM PMSF.
  • the cells were lysed using an ultrahigh pressure homogenizer and centrifuged at 20,000 rpm for 50 minutes to obtain a clarified lysate.
  • the supernatant was treated with immobilized metal affinity chromatography (IMAC) using a HisTrapTM HP column (Cytiva, USA).
  • the supernatant was treated with immobilized metal affinity chromatography (IMAC) and purified using a HisTrapTM HP column.
  • the purified protein was subjected to size exclusion chromatography on a Superdex 200 16/600 column and further purified using an AKTA Pure FPLC system.
  • the purified POx-CAT showed a single elution peak at approximately 51 mL, indicating its homogeneity.
  • SDS-PAGE analysis under reducing conditions revealed a clear band at approximately 156 kDa, confirming the purity of the protein (see Figure 1).
  • the present examples evaluated the ability of the POx-CAT fusion enzyme prepared in Example 1 to consume glucose and simultaneously degrade H2O2 .
  • the glucose consumption abilities of the POx and POx-CAT fusion enzymes were compared, and the results showed that their glucose consumption rates were similar (see Figure 2). Their abilities to produce H2O2 in glucose-containing media were further compared.
  • the results showed that, compared with POx, the POx-CAT fusion enzyme produced significantly less H2O2 at high glucose concentrations ( see Figure 3). This suggests that the POx-CAT fusion enzyme, while catalyzing the production of H2O2 from glucose, effectively accelerates H2O2 degradation .
  • the present invention also evaluated the activity of CAT in the POx-CAT fusion enzyme and found that the POx-CAT fusion enzyme could effectively degrade H 2 O 2 and simultaneously produce O 2 in a concentration-dependent manner (see Figures 4 and 5). These results demonstrate the activity of the POx-CAT fusion enzyme.
  • the design and purification process well maintains the activity of POx and CAT while reducing the generation of harmful byproduct H 2 O 2 .
  • the POx-CAT fusion enzyme prepared in Example 1 was encapsulated in calcium carbonate (CaCO 3 ) nanoparticles by an in situ mineralization method as follows: CaCl 2 (0.2 mL, 1 M) was added to a POx-CAT solution (1 mL, 2 mg/mL) in Tris buffer (3.6 mL, 1 mM) under constant stirring. Na 2 CO 3 (0.2 mL, 1 M) was then quickly added to the mixture at 22°C. The mixture was stirred on 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 supernatants were collected.
  • CaCO 3 calcium carbonate
  • the protein concentration was determined using an ultramicrospectrophotometer, and the POx-CAT@CaCO 3 particles were observed using a hydrodynamic size analyzer and transmission electron microscopy (TEM). This method achieved a loading capacity of 20% and an encapsulation efficiency of 55%. Morphologically, POx-CAT@CaCO 3 is monodispersed and spherical with an average diameter of about 200 nm (see Figures 6-7).
  • a fibrin gel was prepared using a mixture of fibrinogen and thrombin.
  • a specific volume of fibrinogen solution (containing 50 mg/mL of fibrinogen and 200 ⁇ g of POx-CAT@CaCO 3 ) was mixed with an equal volume of thrombin solution (containing 500 IU/mL of thrombin) to produce the POx-CAT@CaCO 3 @Gel composite.
  • the morphology of the composite gel was observed using a scanning electron microscope (SEM) (see Figure 8 ).
  • a type 1 diabetes model was established in male C57BL/6 mice aged 6-8 weeks using a sterile biopsy punch with a diameter of 10 mm. The diabetic mice were then divided into four experimental groups of 8 mice each, each receiving different treatments: a diabetic control group (untreated DC group), a Gel group, a POx-CAT group, and a normal mouse wound group (NC group) as a control.
  • a diabetic control group untreated DC group
  • a Gel group a POx-CAT group
  • NC group normal mouse wound group
  • the present invention monitored wound progression and photographed the wounds on days 0, 3, 6, 8, and 11.
  • the results showed that on day 11, the POx-CAT group showed the most significant improvement in promoting wound healing, while scarring was still visible in the other groups, particularly the DC group.
  • the wound healing rate curve further showed that the wound healing speed of mice treated with POx-CAT@CaCO 3 @Gel was significantly accelerated. Compared with other groups, especially the DC group, the wounds were almost completely closed, showing a healing speed comparable to that of the NC group under non-diabetic conditions.

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Abstract

提供一种融合酶及其应用与组合物，所述融合酶由吡喃糖氧化酶POx和过氧化氢酶CAT通过连接肽连接构成，所述连接肽的氨基酸序列为ASGAGGSEGGGSEGGT。将此融合酶包裹在碳酸钙纳米颗粒中，并整合入可喷洒的纤维蛋白凝胶中，制成组合物POx-CAT@CaCO3@Gel。该组合物具有三重功能：在糖尿病伤口部位降低葡萄糖水平、清除过量活性氧，增加氧气供应。体内实验结果显示，组合物POx-CAT@CaCO3@Gel的使用显著促进了糖尿病小鼠伤口的愈合。这种结合融合酶和医用凝胶的策略，为加强糖尿病伤口愈合提供了一个途径，可用于临床上糖尿病伤口的处理。

Description

融合酶及其应用与组合物 技术领域
本发明涉及生物催化技术领域，尤其涉及融合酶及其应用与组合物。
背景技术
糖尿病因高血糖、缺氧和活性氧过多等微环境损害而导致伤口愈合困难。虽有众多治疗手段，但多数未能全面应对这些相互关联的问题。天然酶因其出色的特异性、高生物活性、最小副作用及有限的多药耐受性，在多种生物过程中扮演关键角色。这些特性使得它们日益被用于生物材料中，以治疗癌症和促进伤口愈合。特别地，葡萄糖氧化酶(Glucose oxidase,GOx)在控制高血糖环境方面显示出潜力。然而，GOx在转化葡萄糖的同时，也带来了氧气耗尽和有毒的过氧化氢(H2O2)积累等挑战，可能加重厌氧状态，扰乱伤口的氧化还原平衡。且过量H2O2可能导致GOx失活。相反，过氧化氢酶(Catalase,CAT)能催化分解H2O2，生成氧气，通过调节ROS和缺氧环境，有助于伤口愈合。然而，伤口区域可能存在H2O2不足，以及某些基于CAT的氧气生成系统在释放氧气初期释放有毒H2O2的问题。因此，解决这些副作用对于充分利用酶的治疗潜力至关重要。
当下，利用基因工程手段可以设计嵌合酶，通过将两个或多个不同酶的端到端融合，有效催化多步反应。活性位点的接近促进中间体向下一酶的转移，增加了所需产物的总产率，同时大幅减少有害中间体的产生。此外，这些人工合成的酶可展示优化的特性和功能，适应特定应用需求。目前，已有若干融合酶在连续流生物催化、底物通道优化、蛋白质连接等领域的应用，突显了融合酶技术的重要性。然而，尚未开发专门用于组织修复的融合酶。
发明内容
鉴于上述现有技术的不足，本发明提供了融合酶及其应用与组合物，本发明通过基因重组技术，将吡喃糖氧化酶(Pyranose Oxidase,POx)和过氧化氢酶(Catalase,CAT)融合表达，制备出能同时消耗葡萄糖和过氧化氢，且产生氧气的POx-CAT融合酶。
本发明的技术方案如下：
本发明的第一方面，提供一种融合酶，所述融合酶由吡喃糖氧化酶POx和过氧化氢酶CAT通过连接肽连接构成，所述连接肽的氨基酸序列为ASGAGGSEGGGSEGGT。
优选的，所述融合酶为CAT-ASGAGGSEGGGSEGGT-POx。
优选的，所述吡喃糖氧化酶POx的氨基酸序列如SEQ ID No.1所示，所述过氧化氢酶CAT的氨基酸序列如SEQ ID No.2所示。
优选的，所述融合酶的N端连接有六组氨酸标签。
优选的，所述融合酶为HHHHHH-CAT-ASGAGGSEGGGSEGGT-POx。
本发明的第二方面，提供上述融合酶在制备糖尿病伤口修复产品中的应用。
本发明的第三方面，提供一种组合物，所述组合物包括凝胶、包裹于所述凝胶内的碳酸钙纳米颗粒以及包裹于所述碳酸钙纳米颗粒内的上述的融合酶。
本发明的第四方面，提供上述组合物的制备方法，所述制备方法包括以下步骤：
采用原位矿化的方法将融合酶包裹在所述碳酸钙纳米颗粒内，得到中间体POx-CAT@CaCO3；
将纤维蛋白原、凝血酶与所述中间体POx-CAT@CaCO3混合，得到所述组合物POx-CAT@CaCO3@Gel。
本发明的第五方面，提供上述组合物在制备糖尿病伤口修复产品中的应用。
有益效果：
本发明公开了融合酶及其应用与组合物。糖尿病伤口微环境的三大挑战是高血糖、高活性氧和低氧，因此，本发明的核心首先是设计合成了融合酶POx-CAT，这种融合酶具有双重功能，在糖尿病伤口修复中的应用恰到好处：既能调节血糖水平，又能通过H2O2分解减轻活性氧的影响，同时纠正伤口位置的O2不足。
并且，为了更好的应用，在本发明中，将这种融合酶封装在CaCO3纳米颗粒中(得到中间体POx-CAT@CaCO3)，确保了融合酶的持续释放和体内稳定性，从而增强了其寿命和有效性。并且，将中间体POx-CAT@CaCO3整合到生物相容的纤维蛋白凝胶中(得到治疗剂)，这不仅便于应用，而且确保治疗剂局限于伤口部位，为伤口提供了一个受控的愈合环境。
综上，本发明提供的融合酶，以及其与医用凝胶结合的应用策略，在增强糖尿病伤口愈合方面具有非常大的临床转化前景；本发明不仅展示了组合物POx-CAT@CaCO3@Gel在糖尿病伤口治疗中的潜在应用，也为未来的医用酶疗法开辟了新的研究方向。
附图说明
图1是本发明实施例制备的POx-CAT融合酶的纯化图。
图2是本发明实施例制备的POx-CAT融合酶消耗葡萄糖的能力图。
图3为本发明实施例制备的POx-CAT融合酶消耗葡萄糖产生过氧化氢的能力图。
图4为本发明实施例制备的POx-CAT融合酶消耗过氧化氢的能力图。
图5为本发明实施例制备的POx-CAT融合酶产生氧气的能力图。
图6为本发明实施例制备的POx-CAT@CaCO3的DLS图。
图7为本发明实施例制备的POx-CAT@CaCO3的TEM图。
图8为本发明实施例制备的POx-CAT@CaCO3@Gel的SEM图。
图9为本发明实施例制备的POx-CAT@CaCO3@Gel促进糖尿病伤口愈合图。
图10为本发明实施例制备的POx-CAT@CaCO3@Gel验证糖尿病伤口修复中第7天和第14天伤口组织的苏木精和伊红(H&E)染色图。
具体实施方式
本发明提供了融合酶及其应用与组合物，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
在开发融合酶的过程中，面临一系列技术挑战，主要包括：找到具有医用潜力的天然酶；将不同的酶融合在一起时，要确保融合酶的正确折叠，并保持每个酶的固有功能和活性；以及确保融合酶的稳定性和可溶性，保证能够分离纯化。这些挑战使得从设计到应用的整个多功能融合酶开发过程充满挑战。
基于此，本发明实施例提供了一种POx-CAT融合酶，所述融合酶由吡喃糖氧化酶POx和过氧化氢酶CAT通过连接肽连接构成，所述连接肽的氨基酸序列为 ASGAGGSEGGGSEGGT。
实施例1POx-CAT融合酶的设计
POx-CAT融合酶结合了大约69.3kDa的POx酶和大约84.1kDa的CAT酶。这两种酶通过一个由16个氨基酸组成的肽段(ASGAGGSEGGGSEGGT)连接。此外，融合酶的N端添加了六组氨基酸(His6)标签，以促进其纯化过程。
实施例2POx-CAT融合酶的表达和纯化
本发明实施例中，对实施例1制备的POx-CAT融合酶进行表达和纯化。POx-CAT基因经过密码子优化以便于在大肠杆菌中表达，随后通过PCR扩增并克隆到pET28a载体中。pET28a载体提供了N端His6标签以便于蛋白纯化。当细菌培养物的光密度600nm(OD600)达到0.8时，使用0.8mM异丙基β-D-1-硫代半乳糖苷(IPTG)诱导蛋白表达。诱导后，将培养物在16℃下孵育20小时，通过8,000rpm离心30分钟收获细胞，细胞沉淀物重悬于含有Tris-HCl(0.03M，pH 7.6)，500mM NaCl，5mM咪唑，0.1mM双吡咯绿胆碱氯化物(BV)，1mM TCEP和0.5mM PMSF的缓冲液中，使用超高压均质机进行细胞裂解，并经20,000rpm离心50分钟后得到澄清的裂解液。上清液通过固定金属亲和层析(IMAC)处理，使用HisTrapTM HP柱(Cytiva,USA)。上清液经过固定金属亲和层析(IMAC)处理，使用HisTrapTM HP柱进行纯化。纯化后的蛋白通过Superdex 200 16/600柱进行尺寸排阻层析，使用AKTA Pure FPLC系统进行进一步纯化。POx-CAT的纯化表现为一个单一的洗脱峰，位于大约51mL处，显示其均一性。还原条件下的SDS-PAGE分析显示了大约156kDa的清晰条带，证实了蛋白的纯度(见图1)。
实施例3POx-CAT融合酶的酶活表征
本发明实施例评估了实施例1制备的POx-CAT融合酶在消耗葡萄糖和同时降解H2O2方面的能力。比较了POx和POx-CAT融合酶消耗葡萄糖的能力，结果显示两者的葡萄糖消耗率相似(见图2)。进一步比较了它们在含葡萄糖介质中产生H2O2的能力。结果表明，与POx相比，POx-CAT融合酶在高葡萄糖浓度下产生的H2O2量明显较低(见图3)。这说明POx-CAT融合酶在催化葡萄糖生成H2O2的同时，有效加速了H2O2的降解。
本发明实施例还评估了POx-CAT融合酶中CAT的活性，发现POx-CAT融合酶能依赖浓度地有效降解H2O2并同时产生O2(见图4,5)。这些结果证明了POx-CAT融合酶的 设计及纯化过程很好地保持了POx和CAT的活性，同时减少了有害副产物H2O2的生成。
实施例4组合物POx-CAT@CaCO3@Gel的制备
本发明实施例中，通过原位矿化的方法将实施例1制备的POx-CAT融合酶包裹在碳酸钙(CaCO3)纳米颗粒中，方法如下：在不断搅拌的条件下，将CaCl2(0.2mL,1M)添加到POx-CAT溶液(1mL,2mg/mL)中的Tris缓冲液(3.6mL,1mM)中。随后在22℃快速加入Na2CO3(0.2mL,1M)到混合物中。混合物在磁力搅拌器中搅拌40秒，并孵育15分钟。通过12,000rpm离心5分钟分离沉淀物，然后洗涤两次，收集所有上清液。使用超微分光光度计测定蛋白质浓度，使用流体动力学尺寸仪和透射电子显微镜(TEM)观察POx-CAT@CaCO3颗粒。该方法的负载能力为20％，封装效率为55％。形态上，POx-CAT@CaCO3呈单分散的球形，平均直径约200nm(见图6-7)。
本发明实施例使用纤维蛋白原和凝血酶的混合物制备纤维蛋白凝胶。通过将特定体积的纤维蛋白原溶液(含有50mg/mL的纤维蛋白原和200μg的POx-CAT@CaCO3)与等体积的凝血酶溶液(含有500IU/mL的凝血酶)混合，生成了组合物POx-CAT@CaCO3@Gel。使用扫描电子显微镜(SEM)观察复合凝胶的形态(见图8)。
实施例5组合物POx-CAT@CaCO3@Gel在糖尿病伤口修复中的应用
为了验证实施例4制备的组合物POx-CAT@CaCO3@Gel在糖尿病伤口修复中的效果，本发明实施例选取了6-8周龄的雄性C57BL/6小鼠，建立了1型糖尿病模型。在伤口愈合研究中，使用直径10毫米的无菌活检冲孔器，在小鼠的背部制造了皮肤伤口。随后，将糖尿病小鼠分为四个实验组，每组8只，分别接受不同的治疗：糖尿病对照组(未治疗的DC组)、Gel组、POx-CAT组，以及正常小鼠伤口组(NC组)作为对照。为了研究伤口愈合的有效性，本发明实施例监测了伤口的进展情况，并在第0天、第3天、第6天、第8天和第11天拍摄了伤口的照片。使用以下公式量化了伤口愈合率：伤口面积百分比＝(初始伤口面积-特定日的伤口面积)/初始伤口面积×100％。结果显示，在第11天时，POx-CAT组在促进伤口愈合方面表现出最显著的改善，而其他组特别是DC组中仍然可以看到疤痕。伤口愈合率的曲线进一步表明，使用POx-CAT@CaCO3@Gel治疗的小鼠伤口愈合速度明显加快，与其他组特别是DC组相比，伤口几乎完全闭合，展现出与非糖尿病条件下的NC组相当的愈合速度。H&E染色的结果显示，在第7天，除了NC组和POx-CAT 组外，所有组都出现了明显的组织缺损。而在第14天，使用POx-CAT@CaCO3@Gel治疗的组展示出了显著的伤口愈合进展，呈现出连续性表皮的出现、新血管的形成，甚至有毛囊的重生。见图9和图10。POx-CAT@CaCO3@Gel治疗组新形成的上皮组织，在结构特征上与正常皮肤组织非常相似。这一研究证实了本发明实施例设计合成的POx-CAT融合酶，通过矿化并结合医用纤维蛋白水凝胶，显示出在促进糖尿病伤口修复方面的明显效果。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (9)

  1. 一种融合酶，其特征在于，所述融合酶由吡喃糖氧化酶POx和过氧化氢酶CAT通过连接肽连接构成，所述连接肽的氨基酸序列为ASGAGGSEGGGSEGGT。
  2. 根据权利要求1所述的融合酶，其特征在于，所述融合酶为CAT-ASGAGGSEGGGSEGGT-POx。
  3. 根据权利要求1所述的融合酶，其特征在于，所述吡喃糖氧化酶POx的氨基酸序列如SEQ ID No.1所示，所述过氧化氢酶CAT的氨基酸序列如SEQ ID No.2所示。
  4. 根据权利要求1所述的融合酶，其特征在于，所述融合酶的N端连接有六组氨酸标签。
  5. 根据权利要求4所述的融合酶，其特征在于，所述融合酶为HHHHHH-CAT-ASGAGGSEGGGSEGGT-POx。
  6. 一种权利要求1-5任一项所述的融合酶在制备糖尿病伤口修复产品中的应用。
  7. 一种组合物，其特征在于，所述组合物包括凝胶、包裹于所述凝胶内的碳酸钙纳米颗粒以及包裹于所述碳酸钙纳米颗粒内的权利要求1-5任一项所述的融合酶。
  8. 一种权利要求7所述的组合物的制备方法，其特征在于，所述制备方法包括以下步骤：
    采用原位矿化的方法将权利要求1-5任一项所述的融合酶包裹在所述碳酸钙纳米颗粒内，得到中间体POx-CAT@CaCO3；
    将纤维蛋白原、凝血酶与所述中间体POx-CAT@CaCO3混合，得到所述组合物POx-CAT@CaCO3@Gel。
  9. 一种权利要求7所述的组合物在制备糖尿病伤口修复产品中的应用。
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