WO2024098496A1 - Enzyme composition and use thereof - Google Patents

Enzyme composition and use thereof Download PDF

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WO2024098496A1
WO2024098496A1 PCT/CN2022/137790 CN2022137790W WO2024098496A1 WO 2024098496 A1 WO2024098496 A1 WO 2024098496A1 CN 2022137790 W CN2022137790 W CN 2022137790W WO 2024098496 A1 WO2024098496 A1 WO 2024098496A1
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glucose oxidase
glucanase
candida albicans
biofilm
enzyme
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PCT/CN2022/137790
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French (fr)
Chinese (zh)
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谭玉龙
蔺劝
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青岛农业大学
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of microorganisms, and in particular relates to an enzyme composition and application thereof.
  • Candida albicans is a common fungal pathogen that can parasitize on human skin and mucous membranes. When the body's immunity is low or affected by certain factors, Candida albicans can cause fungal infections in different parts of the body to varying degrees. Candida albicans will form extensive biofilms on the mucosal surface during infection. Biofilm is the main form of existence of Candida albicans, that is, fungal cells adhere to the contact surface, produce a large amount of extracellular matrix (mainly polysaccharides), and surround themselves to form a microbial aggregation structure. The extracellular matrix not only participates in the formation of the biofilm structure, but also protects the internal microorganisms from the harsh external environment, significantly improving their drug resistance.
  • extracellular matrix mainly polysaccharides
  • the present invention provides an enzyme composition, which consists of ⁇ -1,3-glucanase and glucose oxidase, wherein the mass ratio of ⁇ -1,3-glucanase to glucose oxidase is 5 to 80:30 to 500.
  • the mass ratio of ⁇ -1,3-glucanase to glucose oxidase is 10:250.
  • the present invention provides application of the enzyme composition in removing Candida albicans biofilm.
  • the present invention also provides the use of the above enzyme composition in the preparation of a preparation for removing Candida albicans biofilm.
  • the preparation is selected from the mixed enzyme solution of the above enzyme composition; in the mixed enzyme solution, the solvent includes but is not limited to water, PBS buffer, etc., which can disperse or dissolve ⁇ -1,3-glucanase and glucose oxidase therein and will not inhibit the activity of the enzyme.
  • the concentration of ⁇ -1,3-glucanase is preferably 5 to 80 ⁇ g/mL, and the concentration of glucose oxidase is preferably 30 to 500 ⁇ g/mL; preferably, the concentration of ⁇ -1,3-glucanase is preferably 10 ⁇ g/mL, and the concentration of glucose oxidase is preferably 250 ⁇ g/mL.
  • the present invention provides an enzyme preparation, which is a mixed enzyme solution formed by dispersing or dissolving ⁇ -1,3-glucanase and glucose oxidase in a solvent, wherein the concentration of ⁇ -1,3-glucanase is preferably 5-80 ⁇ g/mL, and the concentration of glucose oxidase is preferably 30-500 ⁇ g/mL; the solvent includes but is not limited to water, PBS buffer, etc., which can disperse or dissolve ⁇ -1,3-glucanase and glucose oxidase therein and will not inhibit enzyme activity.
  • the concentration of ⁇ -1,3-glucanase is preferably 10 ⁇ g/mL, and the concentration of glucose oxidase is preferably 250 ⁇ g/mL.
  • the enzyme composition and enzyme preparation can be used to kill Candida albicans, and can also be used to assist other active ingredients in killing Candida albicans.
  • the present invention provides the use of the enzyme composition and/or enzyme preparation in inhibiting Candida albicans; the use can be diagnostic or therapeutic, and can also be non-diagnostic or therapeutic.
  • the present invention provides a method for removing Candida albicans biofilm, the steps of which are as follows:
  • the Candida albicans and its biofilm are treated with the above-mentioned mixed enzyme solution at a treatment temperature of 30 to 37° C. for a treatment time of 12 to 24 hours to achieve the purpose of removing the biofilm.
  • the above treatment temperature is preferably 30°C.
  • the above treatment time is preferably 24 hours.
  • the above treatment method can be a treatment method such as coating, spraying, etc. that can make the mixed enzyme solution fully contact with the Candida albicans biofilm.
  • ⁇ -1,3-glucanase is proved to have the function of assisting glucose oxidase in increasing the production of hydrogen peroxide. Therefore, the present invention provides the use of ⁇ -1,3-glucanase in assisting glucose oxidase in increasing the production of hydrogen peroxide.
  • the present invention also provides the use of the enzyme composition and/or enzyme preparation in increasing the production of hydrogen peroxide.
  • the enzyme composition and/or enzyme preparation can increase the hydrogen peroxide production in the antibacterial system during the inhibition of Candida albicans.
  • the concentration of ⁇ -1,3-glucanase is selected from 10 ⁇ g/mL
  • the concentration of glucose oxidase is selected from 250 ⁇ g/mL.
  • the present invention establishes a multi-enzyme linkage cascade reaction system by combining ⁇ -1,3-glucanase and glucose oxidase, and destroys the biofilm matrix by degrading the matrix components, especially the polysaccharide components, so as to have a significant removal ability for the Candida albicans biofilm.
  • glucose oxidase can also generate hydrogen peroxide, which has a significant killing effect on the Candida albicans inside the biofilm.
  • the anti-biofilm strategy of the present invention can not only overcome the limitations of a single enzyme, improve the antibacterial efficiency, but also is not easy to induce bacterial resistance.
  • Figure 1 shows the antibacterial effect of a single enzyme
  • Figure A shows different concentrations of ⁇ -1,3-glucanase
  • Figure B shows different concentrations of glucose oxidase
  • Figure 2 is a scanning electron micrograph of Candida albicans and its biofilm; wherein, Figure A is a blank control; Figure B is 10 ⁇ g/mL ⁇ -1,3-glucanase; Figure C is 250 ⁇ g/mL glucose oxidase; Figure D is 10 ⁇ g/mL ⁇ -1,3-glucanase + 250 ⁇ g/mL glucose oxidase;
  • Figure 3 is a fluorescence microscopy image of live/dead Candida albicans cells in the biofilm; wherein, Figure A is a blank control group; Figure B is 10 ⁇ g/mL ⁇ -1,3-glucanase; Figure C is 250 ⁇ g/mL glucose oxidase; and Figure D is 10 ⁇ g/mL ⁇ -1,3-glucanase + 250 ⁇ g/mL glucose oxidase.
  • ⁇ -1,3-glucanase solution was prepared with PBS as solvent, and the concentration gradient was set to 15.6 ⁇ g/mL, 31.2 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL, 1000 ⁇ g/mL.
  • Glucose oxidase solution was prepared with PBS as solvent, and the concentration gradient was set to 7.8 ⁇ g/mL, 15.6 ⁇ g/mL, 31.2 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL, 1000 ⁇ g/mL.
  • the wells treated with ⁇ -1,3-glucanase were all turbid, indicating that ⁇ -1,3-glucanase had no antibacterial effect.
  • the wells treated with 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL, and 1000 ⁇ g/mL glucose oxidase were all clear and transparent, so the MIC value of glucose oxidase was 125 ⁇ g/mL.
  • Candida albicans was activated and cultured, it was diluted with a culture medium so that the A value at 600 nm of the spectrophotometer was 0.01. 100 ⁇ L of the bacterial solution was added to a 96-well plate and cultured at 30° C. for 48 hours to form a biofilm.
  • the following groups were set up: (1) blank control; (2) 5 ⁇ g/mL ⁇ -1,3-glucanase solution; (3) 10 ⁇ g/mL ⁇ -1,3-glucanase solution; (4) 125 ⁇ g/mL glucose oxidase solution; (5) 250 ⁇ g/mL glucose oxidase solution; (6) mixed enzyme solution: 5 ⁇ g/mL ⁇ -1,3-glucanase + 125 ⁇ g/mL glucose oxidase; (7) mixed enzyme solution: 5 ⁇ g/mL ⁇ -1,3-glucanase + 250 ⁇ g/mL glucose oxidase; (8) mixed enzyme solution: 10 ⁇ g/mL ⁇ -1,3-glucanase + 125 ⁇ g/mL glucose oxidase; (9) mixed enzyme solution: 10 ⁇ g/mL ⁇ -1,3-glucanase + 250 ⁇ g/mL glucose oxidase.
  • the Candida albicans biofilm prepared in Example 2 was washed three times with PBS, divided into eight groups, and 100 ⁇ L of the above enzyme solution was added to each of the eight groups, and cultured at 30° C. for 24 h.
  • the removal rate of biofilm was determined by MTT staining.
  • the treated Candida albicans biofilm was washed 3 times with PBS and stained with 10 ⁇ L MTT solution (5 mg/mL) + 90 ⁇ L sterile PBS for 4 h.
  • the supernatant was removed and the cells were treated with 110 ⁇ L DMSO.
  • the A 490 value of the extract was measured with an ELISA instrument to determine the content of the biofilm.
  • the mixed enzyme solution has a very significant effect on the removal of Candida albicans biofilm, among which 10 ⁇ g/mL ⁇ -1,3-glucanase + 250 ⁇ g/mL glucose oxidase has the highest removal rate, which can reach about 80%.
  • the Candida albicans in the control group formed a dense three-dimensional biofilm structure (Figure A); after being treated with ⁇ -1,3-glucanase alone, the biofilm structure was looser than that of the blank control group ( Figure B). After being treated with glucose oxidase alone, the bacterial content was significantly reduced compared with the blank control group, which shows that glucose oxidase has a certain bactericidal effect, but there are still bacteria clumping (Figure C). However, after being treated with mixed enzymes, the structure of the Candida albicans biofilm collapsed, and the cell shedding was more serious (Figure D).
  • the culture system treated with glucose oxidase can produce more hydrogen peroxide, while the culture system treated with ⁇ -1,3-glucanase cannot actually increase the production of hydrogen peroxide.
  • the hydrogen peroxide content in the culture system can be significantly increased on the basis of glucose oxidase treatment alone. This shows that ⁇ -1,3-glucanase can assist glucose oxidase in increasing the production of hydrogen peroxide.

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Abstract

Provided are an enzyme composition and use thereof. The enzyme composition consists of β-1,3-glucanase and glucose oxidase, wherein the mass ratio of the β-1,3-glucanase to the glucose oxidase is (5-80):(30-500). The enzyme composition has a significant clearing ability against a Candida albicans biofilm. Meanwhile, the glucose oxidase can generate hydrogen peroxide, thereby having a killing effect on Candida albicans in the biofilm.

Description

一种酶组合物及其应用An enzyme composition and its application 技术领域Technical Field
本发明属于微生物技术领域，具体涉及一种酶组合物及其应用。The invention belongs to the technical field of microorganisms, and in particular relates to an enzyme composition and application thereof.
背景技术Background technique
白色念珠菌是常见的真菌病原体，可寄生于人体的皮肤和粘膜，当机体免疫力低下或受某些因素影响时，白色念珠菌便可引起不同程度不同部位的真菌感染。白色念珠菌在感染期间会在粘膜表面形成广泛的生物被膜。生物被膜是白色念珠菌主要存在形式，即真菌细胞粘附于接触表面，自身产生大量胞外基质(主要为多糖)，将其自身包绕其中而形成的微生物聚集结构。胞外基质不仅参与生物被膜结构形成，而且保护内部的微生物免受外界恶劣环境影响，显著提高其耐药性。Candida albicans is a common fungal pathogen that can parasitize on human skin and mucous membranes. When the body's immunity is low or affected by certain factors, Candida albicans can cause fungal infections in different parts of the body to varying degrees. Candida albicans will form extensive biofilms on the mucosal surface during infection. Biofilm is the main form of existence of Candida albicans, that is, fungal cells adhere to the contact surface, produce a large amount of extracellular matrix (mainly polysaccharides), and surround themselves to form a microbial aggregation structure. The extracellular matrix not only participates in the formation of the biofilm structure, but also protects the internal microorganisms from the harsh external environment, significantly improving their drug resistance.
发明内容Summary of the invention
本发明提供了一种酶组合物，由β-1,3-葡聚糖酶和葡糖氧化酶组成；其中，β-1,3-葡聚糖酶和葡糖氧化酶的质量比为5～80:30～500。优选地，β-1,3-葡聚糖酶和葡糖氧化酶的质量比为10:250。The present invention provides an enzyme composition, which consists of β-1,3-glucanase and glucose oxidase, wherein the mass ratio of β-1,3-glucanase to glucose oxidase is 5 to 80:30 to 500. Preferably, the mass ratio of β-1,3-glucanase to glucose oxidase is 10:250.
本发明提供了上述酶组合物在清除白色念珠菌生物被膜中的应用。The present invention provides application of the enzyme composition in removing Candida albicans biofilm.
本发明还提供了上述酶组合物在制备清除白色念珠菌生物被膜的制剂中的应用。优选地，所述制剂选自上述酶组合物的混合酶液；所述混合酶液中，溶剂包括但不限于水、PBS缓冲液等能够将β-1,3-葡聚糖酶和葡糖氧化酶分散或溶解在其中，且不会抑制酶活性的溶剂。在所述混合酶液中，β-1,3-葡聚糖酶的浓度优选为5～80μg/mL，葡糖氧化酶的浓度优选为30～500μg/mL；优选地，所述β-1,3-葡聚糖酶的浓度优选为10μg/mL，葡糖氧化酶的浓度优选为250μg/mL。The present invention also provides the use of the above enzyme composition in the preparation of a preparation for removing Candida albicans biofilm. Preferably, the preparation is selected from the mixed enzyme solution of the above enzyme composition; in the mixed enzyme solution, the solvent includes but is not limited to water, PBS buffer, etc., which can disperse or dissolve β-1,3-glucanase and glucose oxidase therein and will not inhibit the activity of the enzyme. In the mixed enzyme solution, the concentration of β-1,3-glucanase is preferably 5 to 80 μg/mL, and the concentration of glucose oxidase is preferably 30 to 500 μg/mL; preferably, the concentration of β-1,3-glucanase is preferably 10 μg/mL, and the concentration of glucose oxidase is preferably 250 μg/mL.
本发明提供了一种酶制剂，是将β-1,3-葡聚糖酶和葡糖氧化酶分散或溶解于溶剂中所形成的混合酶液，其中，β-1,3-葡聚糖酶的浓度优选为5～80μg/mL，葡糖氧化酶的浓度优选为30～500μg/mL；所述溶剂包括但不限于水、PBS缓冲液等能够将β-1,3-葡聚糖酶和葡糖氧化酶分散或溶解在其中，且不会抑制酶活性的溶剂。优选地，所述β-1,3-葡聚糖酶的浓度优选为10μg/mL，葡糖氧化酶的浓度优选为250μg/mL。The present invention provides an enzyme preparation, which is a mixed enzyme solution formed by dispersing or dissolving β-1,3-glucanase and glucose oxidase in a solvent, wherein the concentration of β-1,3-glucanase is preferably 5-80 μg/mL, and the concentration of glucose oxidase is preferably 30-500 μg/mL; the solvent includes but is not limited to water, PBS buffer, etc., which can disperse or dissolve β-1,3-glucanase and glucose oxidase therein and will not inhibit enzyme activity. Preferably, the concentration of β-1,3-glucanase is preferably 10 μg/mL, and the concentration of glucose oxidase is preferably 250 μg/mL.
上述酶组合物和酶制剂可被用于杀灭白色念珠菌，也可被用于辅助其它活性成分杀灭白色念珠菌。为此，本发明提供了上述酶组合物和/或酶制剂在抑制白色念珠菌中的应用；该应用可以是诊疗性的，也可以是非诊疗性的。The enzyme composition and enzyme preparation can be used to kill Candida albicans, and can also be used to assist other active ingredients in killing Candida albicans. To this end, the present invention provides the use of the enzyme composition and/or enzyme preparation in inhibiting Candida albicans; the use can be diagnostic or therapeutic, and can also be non-diagnostic or therapeutic.
本发明提供了一种白色念珠菌生物被膜清除方法，步骤如下：The present invention provides a method for removing Candida albicans biofilm, the steps of which are as follows:
将白色念珠菌及其生物被膜利用上述混合酶液进行处理，处理温度为30～37℃，处理时间为12～24h，以达到生物被膜清除的目的。The Candida albicans and its biofilm are treated with the above-mentioned mixed enzyme solution at a treatment temperature of 30 to 37° C. for a treatment time of 12 to 24 hours to achieve the purpose of removing the biofilm.
上述处理温度优选为30℃。The above treatment temperature is preferably 30°C.
上述处理时间优选为24h。The above treatment time is preferably 24 hours.
上述处理方式可以是涂覆、喷洒等能够使混合酶液与白色念珠菌生物被膜充分接触的处理方式。The above treatment method can be a treatment method such as coating, spraying, etc. that can make the mixed enzyme solution fully contact with the Candida albicans biofilm.
在本发明中，β-1,3-葡聚糖酶被证明具有辅助葡糖氧化酶提高过氧化氢产量的作用，为此，本发明提供了β-1,3-葡聚糖酶在辅助葡糖氧化酶提高过氧化氢产量中的应用。In the present invention, β-1,3-glucanase is proved to have the function of assisting glucose oxidase in increasing the production of hydrogen peroxide. Therefore, the present invention provides the use of β-1,3-glucanase in assisting glucose oxidase in increasing the production of hydrogen peroxide.
本发明还提供了上述酶组合物和/或酶制剂在提高过氧化氢产量中的应用。The present invention also provides the use of the enzyme composition and/or enzyme preparation in increasing the production of hydrogen peroxide.
优选地，上述酶组合物和/或酶制剂能够在其抑制白色念珠菌过程中提高抑菌体系中的过氧化氢产量。所述酶组合物和/或酶制剂中，β-1,3-葡聚糖酶的浓度选自10μg/mL，葡糖氧化酶的浓度选自250μg/mL。Preferably, the enzyme composition and/or enzyme preparation can increase the hydrogen peroxide production in the antibacterial system during the inhibition of Candida albicans. In the enzyme composition and/or enzyme preparation, the concentration of β-1,3-glucanase is selected from 10 μg/mL, and the concentration of glucose oxidase is selected from 250 μg/mL.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明通过联合使用β-1,3-葡聚糖酶与葡糖氧化酶，建立一个多酶联动的级联反应系统，并通过降解基质成分，尤其是多糖成分，破坏生物膜基质，从而对白色念珠菌生物被膜具有显著的清除能力。同时，葡糖氧化酶还能够生成过氧化氢，对生物被膜内部的白色念珠菌具有显著的杀灭作用。此外，本发明的抗生物被膜策略，不仅能够克服单一酶的限制，提高抗菌效率而且不易诱发细菌耐药性。The present invention establishes a multi-enzyme linkage cascade reaction system by combining β-1,3-glucanase and glucose oxidase, and destroys the biofilm matrix by degrading the matrix components, especially the polysaccharide components, so as to have a significant removal ability for the Candida albicans biofilm. At the same time, glucose oxidase can also generate hydrogen peroxide, which has a significant killing effect on the Candida albicans inside the biofilm. In addition, the anti-biofilm strategy of the present invention can not only overcome the limitations of a single enzyme, improve the antibacterial efficiency, but also is not easy to induce bacterial resistance.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为单酶抑菌效果；其中，A图为不同浓度β-1,3-葡聚糖酶；B图为不同浓度葡糖氧化酶；Figure 1 shows the antibacterial effect of a single enzyme; Figure A shows different concentrations of β-1,3-glucanase; Figure B shows different concentrations of glucose oxidase;
图2为白色念珠菌及其生物被膜的扫描电镜图；其中，A图为空白对照；B图为10μg/mLβ-1,3-葡聚糖酶；C图为250μg/mL葡糖氧化酶；D图为10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶；Figure 2 is a scanning electron micrograph of Candida albicans and its biofilm; wherein, Figure A is a blank control; Figure B is 10 μg/mL β-1,3-glucanase; Figure C is 250 μg/mL glucose oxidase; Figure D is 10 μg/mL β-1,3-glucanase + 250 μg/mL glucose oxidase;
图3为生物被膜中活/死白色念珠菌细胞的荧光显微镜图；其中，A图为空白对照组；B图为10μg/mLβ-1,3-葡聚糖酶；C图为250μg/mL葡糖氧化酶；D图为10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶。Figure 3 is a fluorescence microscopy image of live/dead Candida albicans cells in the biofilm; wherein, Figure A is a blank control group; Figure B is 10 μg/mL β-1,3-glucanase; Figure C is 250 μg/mL glucose oxidase; and Figure D is 10 μg/mL β-1,3-glucanase + 250 μg/mL glucose oxidase.
具体实施方式Detailed ways
本发明中所使用的其它术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。下面结合具体实施例，并参照数据进一步详细的描述本发明。以下实施例只是为了 举例说明本发明，而非以任何方式限制本发明的范围。Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.
实施例1Example 1
单酶抑菌效果测试：Single enzyme antibacterial effect test:
以PBS为溶剂配制β-1,3-葡聚糖酶溶液，设置浓度梯度，分别为15.6μg/mL、31.2μg/mL、62.5μg/mL、125μg/mL、250μg/mL、500μg/mL、1000μg/mL。以PBS为溶剂配制葡糖氧化酶溶液，设置浓度梯度，分别为7.8μg/mL、15.6μg/mL、31.2μg/mL、62.5μg/mL、125μg/mL、250μg/mL、500μg/mL、1000μg/mL。β-1,3-glucanase solution was prepared with PBS as solvent, and the concentration gradient was set to 15.6μg/mL, 31.2μg/mL, 62.5μg/mL, 125μg/mL, 250μg/mL, 500μg/mL, 1000μg/mL. Glucose oxidase solution was prepared with PBS as solvent, and the concentration gradient was set to 7.8μg/mL, 15.6μg/mL, 31.2μg/mL, 62.5μg/mL, 125μg/mL, 250μg/mL, 500μg/mL, 1000μg/mL.
分别取100μL上述β-1,3-葡聚糖酶溶液、葡糖氧化酶溶液，和白色念珠菌悬浮液(10 6CFU/mL)加入96孔板中，在30℃下孵育24h。观察96孔板中菌液浑浊程度，并确定单酶的MIC值。 100 μL of the above β-1,3-glucanase solution, glucose oxidase solution, and Candida albicans suspension (10 6 CFU/mL) were added to a 96-well plate and incubated at 30° C. for 24 h. The turbidity of the bacterial solution in the 96-well plate was observed, and the MIC value of the single enzyme was determined.
试验结果如图1所示：The test results are shown in Figure 1:
经β-1,3-葡聚糖酶处理的孔全部是浑浊状态，这表明，β-1,3-葡聚糖酶没有抑菌效果。而经125μg/mL、250μg/mL、500μg/mL、1000μg/mL葡糖氧化酶处理的孔都是澄清透明的，因此葡糖氧化酶的MIC值为125μg/mL。The wells treated with β-1,3-glucanase were all turbid, indicating that β-1,3-glucanase had no antibacterial effect. However, the wells treated with 125μg/mL, 250μg/mL, 500μg/mL, and 1000μg/mL glucose oxidase were all clear and transparent, so the MIC value of glucose oxidase was 125μg/mL.
实施例2Example 2
白色念珠菌生物被膜的制备：Preparation of Candida albicans biofilm:
将白色念珠菌活化培养后用培养基进行稀释，使其在分光光度计600nm下的A值为0.01。将100μL菌液加入96孔板中，在30℃下静置培养48h，使之形成生物被膜。After the Candida albicans was activated and cultured, it was diluted with a culture medium so that the A value at 600 nm of the spectrophotometer was 0.01. 100 μL of the bacterial solution was added to a 96-well plate and cultured at 30° C. for 48 hours to form a biofilm.
实施例3Example 3
白色念珠菌生物被膜的清除：Elimination of Candida albicans biofilm:
设置如下组别：(1)空白对照；(2)5μg/mLβ-1,3-葡聚糖酶溶液；(3)10μg/mLβ-1,3-葡聚糖酶溶液；(4)125μg/mL葡糖氧化酶溶液；(5)250μg/mL葡糖氧化酶溶液；(6)混合酶液：5μg/mLβ-1,3-葡聚糖酶+125μg/mL葡糖氧化酶；(7)混合酶液：5μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶；(8)混合酶液：10μg/mLβ-1,3-葡聚糖酶+125μg/mL葡糖氧化酶；(9)混合酶液：10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶。The following groups were set up: (1) blank control; (2) 5 μg/mL β-1,3-glucanase solution; (3) 10 μg/mL β-1,3-glucanase solution; (4) 125 μg/mL glucose oxidase solution; (5) 250 μg/mL glucose oxidase solution; (6) mixed enzyme solution: 5 μg/mL β-1,3-glucanase + 125 μg/mL glucose oxidase; (7) mixed enzyme solution: 5 μg/mL β-1,3-glucanase + 250 μg/mL glucose oxidase; (8) mixed enzyme solution: 10 μg/mL β-1,3-glucanase + 125 μg/mL glucose oxidase; (9) mixed enzyme solution: 10 μg/mL β-1,3-glucanase + 250 μg/mL glucose oxidase.
采用PBS洗涤实施例2所制备的白色念珠菌生物被膜3次，分为八组，分别加入100μL上述酶液，在30℃下培养24h。The Candida albicans biofilm prepared in Example 2 was washed three times with PBS, divided into eight groups, and 100 μL of the above enzyme solution was added to each of the eight groups, and cultured at 30° C. for 24 h.
采用MTT染色法测定生物被膜的清除率。将处理后的白色念珠菌生物被膜，用PBS洗涤3次，采用10μL MTT溶液(5mg/mL)+90μL无菌PBS染色4h。吸去上清液，用110μL DMSO处理细胞。用酶标仪测定提取液的A 490值，以测定生物被膜的含量。 The removal rate of biofilm was determined by MTT staining. The treated Candida albicans biofilm was washed 3 times with PBS and stained with 10 μL MTT solution (5 mg/mL) + 90 μL sterile PBS for 4 h. The supernatant was removed and the cells were treated with 110 μL DMSO. The A 490 value of the extract was measured with an ELISA instrument to determine the content of the biofilm.
试验结果如表1所示：The test results are shown in Table 1:
表1Table 1
组别Group (1)(1) (2)(2) (3)(3) (4)(4) (5)(5) (6)(6) (7)(7) (8)(8) (9)(9)
生物膜含量(％)Biofilm content (%) 100100 91.0991.09 82.2582.25 49.4549.45 35.9635.96 28.9328.93 23.1523.15 23.7723.77 19.2419.24
由表1可知，与单独的β-1,3-葡聚糖酶和葡糖氧化酶相比，混合酶液对白色念珠菌生物被膜的清除效果非常显著，其中，10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶的清除率最高，能达到80％左右。As shown in Table 1, compared with the single β-1,3-glucanase and glucose oxidase, the mixed enzyme solution has a very significant effect on the removal of Candida albicans biofilm, among which 10μg/mL β-1,3-glucanase + 250μg/mL glucose oxidase has the highest removal rate, which can reach about 80%.
实施例4Example 4
生物被膜结构变化：Changes in biofilm structure:
将1mL白色念珠菌悬浮液(10 6CFU/mL)和1mL YPD培养基加入到24孔板中，并将芯片放入24孔板底部，培养48h形成生物被膜。小心取出上清液，用PBS冲洗3次。在形成的生物被膜中分别加入10μg/mLβ-1,3-葡聚糖酶、250μg/mL葡糖氧化酶、10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶各1mL，培养24h。采用PBS洗涤3次。然后置于2.5％戊二醛溶液中固定4h，然后依次置于30％乙醇、50％乙醇、70％乙醇、80％乙醇、90％乙醇、100％乙醇中脱水并用100％叔丁醇置换，将芯片取出，冷冻干燥48h，喷金，采用10kv扫描电子显微镜(SEM)观察上述酶液对白色念珠菌生物被膜结构变化的影响。 1mL of Candida albicans suspension (10 6 CFU/mL) and 1mL of YPD medium were added to a 24-well plate, and the chip was placed at the bottom of the 24-well plate and cultured for 48 hours to form a biofilm. The supernatant was carefully removed and rinsed with PBS three times. 10μg/mL β-1,3-glucanase, 250μg/mL glucose oxidase, and 10μg/mL β-1,3-glucanase + 250μg/mL glucose oxidase were added to the formed biofilm, and cultured for 24 hours. Washed with PBS three times. Then placed in 2.5% glutaraldehyde solution for 4 hours, and then placed in 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol for dehydration and replaced with 100% tert-butanol. The chip was taken out, freeze-dried for 48 hours, sprayed with gold, and the effects of the above enzyme solutions on the structural changes of Candida albicans biofilm were observed using a 10kv scanning electron microscope (SEM).
测试结果如图2所示：The test results are shown in Figure 2:
由图2可知，对照组中的白色念珠菌形成了致密的生物膜三维结构(A图)；经β-1,3-葡聚糖酶单独处理后，与空白对照组相比，生物被膜结构较松散(B图)。经葡糖氧化酶单独处理后，与空白对照组相比，菌含量明显减少，这说明葡糖氧化酶具有一定的杀菌作用，但是仍旧有菌抱团(C图)。但经混合酶处理后，白色念珠菌生物被膜的结构发生崩解，而且，细胞脱落的情况更加严重(D图)。As shown in Figure 2, the Candida albicans in the control group formed a dense three-dimensional biofilm structure (Figure A); after being treated with β-1,3-glucanase alone, the biofilm structure was looser than that of the blank control group (Figure B). After being treated with glucose oxidase alone, the bacterial content was significantly reduced compared with the blank control group, which shows that glucose oxidase has a certain bactericidal effect, but there are still bacteria clumping (Figure C). However, after being treated with mixed enzymes, the structure of the Candida albicans biofilm collapsed, and the cell shedding was more serious (Figure D).
实施例5Example 5
荧光显微镜观察：Fluorescence microscopy observation:
将1mL白色念珠菌悬浮液(10 6CFU/mL)和1mL YPD培养基加入到24孔板中，培养48h形成生物被膜。小心取出上清液，用PBS冲洗3次。在形成的生物被膜中分别加入10μg/mLβ-1,3-葡聚糖酶、250μg/mL葡糖氧化酶、10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶各1mL，培养24h。采用PBS洗涤3次。采用
Figure PCTCN2022137790-appb-000001
染色 TM细菌活力和计数试剂盒处理。将1个体积DMAO和2个体积EthD-III混合在微离心管中，然后加入8个体积0.85％氯化钠溶液，得到100×染料溶液。然后在室温黑暗中孵育15min。用带有FITC和Cy3通道 的荧光显微镜观察生物被膜。当用活/死
Figure PCTCN2022137790-appb-000002
细菌活力和计数试剂盒染色白色念珠菌时，活细胞显示绿色荧光，受损或死细胞显示黄绿色或红色荧光。 Add 1 mL of Candida albicans suspension (10 6 CFU/mL) and 1 mL of YPD medium to a 24-well plate and culture for 48 hours to form a biofilm. Carefully remove the supernatant and rinse three times with PBS. Add 1 mL each of 10 μg/mL β-1,3-glucanase, 250 μg/mL glucose oxidase, and 10 μg/mL β-1,3-glucanase + 250 μg/mL glucose oxidase to the formed biofilm and culture for 24 hours. Wash three times with PBS.
Figure PCTCN2022137790-appb-000001
StainTM Bacterial Viability and Counting Kit. Mix 1 volume of DMAO and 2 volumes of EthD-III in a microcentrifuge tube, then add 8 volumes of 0.85% sodium chloride solution to make a 100× dye solution. Then incubate for 15 min at room temperature in the dark. Observe the biofilm using a fluorescence microscope with FITC and Cy3 channels. When using Live/Dead
Figure PCTCN2022137790-appb-000002
When the Bacterial Viability and Counting Kit stains Candida albicans, live cells show green fluorescence, while damaged or dead cells show yellow-green or red fluorescence.
测试结果如图3所示：The test results are shown in Figure 3:
由图3可知，未经处理的白色念珠菌，几乎所有荧光都是绿色荧光且面积大而致密(A图)。经β-1,3-葡聚糖酶处理的白色念珠菌，也几乎都是绿光，但与空白组相比结构更加松散(B图)。经葡糖氧化酶处理后，红色和黄色荧光占图的大部分，这表明葡糖氧化酶对白色念珠菌具有杀灭能力，但是黄色荧光面积大且结构致密，这表明葡糖氧化酶虽可杀死表面白色念珠菌，但生物被膜仍能保护内部白色念珠菌免于灭亡(C图)。经混合酶处理后，红色和黄色荧光非常强烈，伴随由菌量稀疏、分散等现象，这表明，生物被膜的结构被破坏，白色念珠菌被大量杀死(D图)。上述结果表明，混合酶处理更容易使生物被膜裂解，并杀死藏于生物被膜中的白色念珠菌细胞。As shown in Figure 3, almost all fluorescence of untreated Candida albicans is green fluorescence with a large and dense area (Figure A). Candida albicans treated with β-1,3-glucanase also emits almost all green light, but the structure is looser than that of the blank group (Figure B). After treatment with glucose oxidase, red and yellow fluorescence occupy most of the figure, indicating that glucose oxidase has the ability to kill Candida albicans, but the yellow fluorescence area is large and the structure is dense, indicating that although glucose oxidase can kill Candida albicans on the surface, the biofilm can still protect the internal Candida albicans from extinction (Figure C). After treatment with mixed enzymes, red and yellow fluorescence are very strong, accompanied by sparse and dispersed bacteria, indicating that the structure of the biofilm is destroyed and Candida albicans is killed in large quantities (Figure D). The above results show that mixed enzyme treatment makes it easier to lyse the biofilm and kill Candida albicans cells hidden in the biofilm.
实施例6Example 6
过氧化氢产量影响：Hydrogen peroxide production impact:
将100μL白色念珠菌悬浮液(10 6CFU/mL)和100μL YPD培养基加入到96孔板中，培养48h形成生物被膜。小心取出上清液，用PBS冲洗3次。在形成的生物被膜中分别加入10μg/mLβ-1,3-葡聚糖酶、250μg/mL葡糖氧化酶、10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶各100μL，培养24h。采用PBS洗涤3次。利用过氧化氢试剂盒测定各培养体系中的过氧化氢含量。 100 μL of Candida albicans suspension (10 6 CFU/mL) and 100 μL of YPD medium were added to a 96-well plate and cultured for 48 hours to form a biofilm. The supernatant was carefully removed and rinsed three times with PBS. 100 μL of 10 μg/mL β-1,3-glucanase, 250 μg/mL glucose oxidase, and 10 μg/mL β-1,3-glucanase + 250 μg/mL glucose oxidase were added to the formed biofilm and cultured for 24 hours. Washed with PBS three times. The hydrogen peroxide content in each culture system was determined using a hydrogen peroxide kit.
测定结果如表2所示：The measurement results are shown in Table 2:
表2Table 2
组别Group H 2O 2(μmol/mL) H 2 O 2 (μmol/mL)
空白对照Blank control 0.06260.0626
10μg/mLβ-1,3-葡聚糖酶10 μg/mL β-1,3-glucanase 0.05340.0534
250μg/mL葡糖氧化酶250μg/mL glucose oxidase 0.18550.1855
10μg/mLβ-1,3-葡聚糖酶+250μg/mL葡糖氧化酶10μg/mL β-1,3-glucanase + 250μg/mL glucose oxidase 0.56130.5613
由表2可知，与空白对照组相比，经葡糖氧化酶处理的培养体系能够产生较多的过氧化氢，而经β-1,3-葡聚糖酶处理的培养体系并不能实际提高过氧化氢的产量。但是，经混合酶处理后，培养体系中的过氧化氢含量，又能在葡糖氧化酶单独处理的基础上得到显著提高。这说明，β-1,3-葡聚糖酶能够协助葡糖氧化酶提高过氧化氢的产量。As shown in Table 2, compared with the blank control group, the culture system treated with glucose oxidase can produce more hydrogen peroxide, while the culture system treated with β-1,3-glucanase cannot actually increase the production of hydrogen peroxide. However, after the mixed enzyme treatment, the hydrogen peroxide content in the culture system can be significantly increased on the basis of glucose oxidase treatment alone. This shows that β-1,3-glucanase can assist glucose oxidase in increasing the production of hydrogen peroxide.
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施 例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims (10)

  1. 一种酶组合物，其特征在于，由β-1,3-葡聚糖酶和葡糖氧化酶组成；其中，β-1,3-葡聚糖酶和葡糖氧化酶的质量比为5～80:30～500；优选地，β-1,3-葡聚糖酶和葡糖氧化酶的质量比为10:250。An enzyme composition, characterized in that it consists of β-1,3-glucanase and glucose oxidase; wherein the mass ratio of β-1,3-glucanase to glucose oxidase is 5-80:30-500; preferably, the mass ratio of β-1,3-glucanase to glucose oxidase is 10:250.
  2. 权利要求1所述酶组合物在清除白色念珠菌生物被膜中的应用。Use of the enzyme composition according to claim 1 in removing Candida albicans biofilm.
  3. 权利要求1所述酶组合物在制备清除白色念珠菌生物被膜的制剂中的应用。Use of the enzyme composition according to claim 1 in the preparation of a preparation for removing Candida albicans biofilm.
  4. 一种酶制剂，其特征在于，是将权利要求1所述酶组合物分散或溶解于溶剂中所形成的混合酶液；其中，所述β-1,3-葡聚糖酶的浓度为5～80μg/mL，所述葡糖氧化酶的浓度为30～500μg/mL。An enzyme preparation, characterized in that it is a mixed enzyme solution formed by dispersing or dissolving the enzyme composition according to claim 1 in a solvent; wherein the concentration of the β-1,3-glucanase is 5 to 80 μg/mL, and the concentration of the glucose oxidase is 30 to 500 μg/mL.
  5. 根据权利要求4所述的酶制剂，其特征在于，所述β-1,3-葡聚糖酶的浓度为10μg/mL，所述葡糖氧化酶的浓度为250μg/mL。The enzyme preparation according to claim 4, characterized in that the concentration of the β-1,3-glucanase is 10 μg/mL and the concentration of the glucose oxidase is 250 μg/mL.
  6. 权利要求1所述酶组合物和/或权利要求4所述酶制剂在非诊疗目的的抑制白色念珠菌中的应用。Use of the enzyme composition according to claim 1 and/or the enzyme preparation according to claim 4 in inhibiting Candida albicans for non-diagnostic purposes.
  7. 一种白色念珠菌生物被膜清除方法，其特征在于，步骤如下：A method for removing Candida albicans biofilm, characterized in that the steps are as follows:
    将白色念珠菌及其生物被膜利用权利要求4所述酶制剂进行处理，处理温度为30～37℃，处理时间为12～24h，以达到生物被膜清除的目的。The Candida albicans and its biofilm are treated with the enzyme preparation according to claim 4 at a treatment temperature of 30 to 37° C. for a treatment time of 12 to 24 hours to achieve the purpose of removing the biofilm.
  8. β-1,3-葡聚糖酶在辅助葡糖氧化酶提高过氧化氢产量中的应用。Application of β-1,3-glucanase in assisting glucose oxidase to increase hydrogen peroxide production.
  9. 权利要求1所述酶组合物和/或权利要求4所述酶制剂在提高过氧化氢产量中的应用。Use of the enzyme composition according to claim 1 and/or the enzyme preparation according to claim 4 in increasing the production of hydrogen peroxide.
  10. 根据权利要求9所述的应用，其特征在于，所述酶组合物和/或酶制剂是在其抑制白色念珠菌过程中提高抑菌体系中的过氧化氢产量。The use according to claim 9 is characterized in that the enzyme composition and/or enzyme preparation is used to increase the hydrogen peroxide production in the antibacterial system during the process of inhibiting Candida albicans.
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