CN115747180B - Enzyme composition and application thereof - Google Patents
Enzyme composition and application thereof Download PDFInfo
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- CN115747180B CN115747180B CN202211388414.7A CN202211388414A CN115747180B CN 115747180 B CN115747180 B CN 115747180B CN 202211388414 A CN202211388414 A CN 202211388414A CN 115747180 B CN115747180 B CN 115747180B
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- glucose oxidase
- glucanase
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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
Abstract
The invention discloses an enzyme composition and application thereof, and belongs to the technical field of microorganisms. The enzyme composition of the invention consists of beta-1, 3-glucanase and glucose oxidase; wherein the mass ratio of the beta-1, 3-glucanase to the glucose oxidase is 5-80:30-500. The invention establishes a multi-enzyme linked cascade reaction system by combining beta-1, 3-glucanase and glucose oxidase, and damages the biomembrane matrix by degrading matrix components, especially polysaccharide components, thereby having remarkable cleaning capacity on candida albicans biomembrane; meanwhile, the glucose oxidase can also generate hydrogen peroxide, and has a killing effect on candida albicans in the biofilm. On the basis, the invention provides a method for removing the candida albicans biofilm, which has high removal rate of the candida albicans biofilm, good sterilization effect and better application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an enzyme composition and application thereof.
Background
Candida albicans is a common fungal pathogen and can be parasitic on the skin and mucous membranes of a human body, and when the immunity of the human body is low or is influenced by certain factors, the candida albicans can cause fungal infections at different parts with different degrees. Candida albicans can form a broad biofilm on mucosal surfaces during infection. Biofilms are the predominant form of candida albicans, i.e., the microbial aggregate structure formed by fungal cells adhering to a contact surface, self-producing a large amount of extracellular matrix (primarily polysaccharides), surrounding itself. The extracellular matrix not only participates in the formation of a biological film structure, but also protects internal microorganisms from the influence of external severe environment, and obviously improves the drug resistance.
Disclosure of Invention
The invention provides an enzyme composition, which consists of beta-1, 3-glucanase and glucose oxidase; wherein the mass ratio of the beta-1, 3-glucanase to the glucose oxidase is 5-80:30-500. Preferably, the mass ratio of beta-1, 3-glucanase to glucose oxidase is 10:250.
The invention provides application of the enzyme composition in removing candida albicans biofilm.
The invention also provides application of the enzyme composition in preparation of a preparation for removing candida albicans biofilm. Preferably, the preparation is selected from the mixed enzyme solutions of the above enzyme compositions; in the mixed enzyme solution, the solvent includes, but is not limited to, water, PBS buffer, etc., which can disperse or dissolve the beta-1, 3-glucanase and glucose oxidase therein without inhibiting the enzyme activity. In the mixed enzyme solution, the concentration of beta-1, 3-glucanase is preferably 5-80 mug/mL, and the concentration of glucose oxidase is preferably 30-500 mug/mL; preferably, the concentration of the beta-1, 3-glucanase is preferably 10. Mu.g/mL and the concentration of the glucose oxidase is preferably 250. Mu.g/mL.
The invention provides an enzyme preparation, which is a mixed enzyme solution formed by dispersing or dissolving beta-1, 3-glucanase and glucose oxidase in a solvent, wherein the concentration of the beta-1, 3-glucanase is preferably 5-80 mug/mL, and the concentration of the glucose oxidase is preferably 30-500 mug/mL; the solvent includes, but is not limited to, water, PBS buffer, etc., capable of dispersing or dissolving the beta-1, 3-glucanase and glucose oxidase therein without inhibiting the enzyme activity. Preferably, the concentration of the beta-1, 3-glucanase is preferably 10. Mu.g/mL and the concentration of the glucose oxidase is preferably 250. Mu.g/mL.
The enzyme composition and enzyme preparation can be used for killing candida albicans, and can also be used for assisting other active ingredients in killing candida albicans. For this purpose, the invention provides the use of the above-mentioned enzyme composition and/or enzyme preparation for inhibiting candida albicans; the application may be either diagnostic or non-diagnostic.
The invention provides a candida albicans biofilm removal method, which comprises the following steps:
candida albicans and a biological film thereof are treated by the mixed enzyme liquid, the treatment temperature is 30-37 ℃ and the treatment time is 12-24 hours, so as to achieve the aim of removing the biological film.
The above-mentioned treatment temperature is preferably 30 ℃.
The treatment time is preferably 24 hours.
The treatment method may be a treatment method such as coating or spraying, which can sufficiently contact the mixed enzyme solution with the candida albicans biofilm.
In the present invention, the beta-1, 3-glucanase has been shown to have an effect of assisting glucose oxidase in increasing hydrogen peroxide production, and for this reason, the present invention provides the use of the beta-1, 3-glucanase in assisting glucose oxidase in increasing hydrogen peroxide production.
The invention also provides application of the enzyme composition and/or the enzyme preparation in improving the yield of hydrogen peroxide.
Preferably, the above-described enzyme composition and/or enzyme preparation is capable of increasing the hydrogen peroxide yield in a bacteriostatic system during its inhibition of candida albicans. The enzyme composition and/or enzyme preparation has a concentration of beta-1, 3-glucanase selected from 10 μg/mL and a concentration of glucose oxidase selected from 250 μg/mL.
The beneficial effects of the invention are as follows:
the invention establishes a multi-enzyme linked cascade reaction system by combining beta-1, 3-glucanase and glucose oxidase, and damages the biomembrane matrix by degrading matrix components, especially polysaccharide components, thereby having remarkable cleaning capacity on candida albicans biomembrane. Meanwhile, the glucose oxidase can also generate hydrogen peroxide, and has remarkable killing effect on candida albicans in the biofilm. In addition, the anti-biofilm strategy of the invention not only can overcome the limitation of single enzyme, improve the antibacterial efficiency, but also is not easy to induce bacterial drug resistance.
Drawings
FIG. 1 shows the bacteriostatic effect of single enzyme; wherein, the A graph shows beta-1, 3-glucanase with different concentrations; panel B shows glucose oxidase at various concentrations;
FIG. 2 is a scanning electron microscope image of Candida albicans and its biofilm; wherein, the A graph is blank control; panel B shows 10. Mu.g/mL beta-1, 3-glucanase; panel C shows 250. Mu.g/mL glucose oxidase; panel D shows 10. Mu.g/mL beta. -1, 3-glucanase+250. Mu.g/mL glucose oxidase;
FIG. 3 is a fluorescence microscope image of live/dead Candida albicans cells in a biofilm; wherein, the A graph is a blank control group; panel B shows 10. Mu.g/mL beta-1, 3-glucanase; panel C shows 250. Mu.g/mL glucose oxidase; panel D shows 10. Mu.g/mL beta. -1, 3-glucanase+250. Mu.g/mL glucose oxidase.
Detailed Description
Other terms used in the present invention, unless otherwise indicated, generally have meanings commonly understood by those of ordinary skill in the art. The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
Single enzyme antibacterial effect test:
beta-1, 3-glucanase solution is prepared by taking PBS as a solvent, and concentration gradients are set to be 15.6 mug/mL, 31.2 mug/mL, 62.5 mug/mL, 125 mug/mL, 250 mug/mL, 500 mug/mL and 1000 mug/mL respectively. A glucose oxidase solution was prepared using PBS as a solvent, and a concentration gradient was set at 7.8. Mu.g/mL, 15.6. Mu.g/mL, 31.2. Mu.g/mL, 62.5. Mu.g/mL, 125. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL, respectively.
100. Mu.L of the above beta-1, 3-glucanase solution, glucose oxidase solution and Candida albicans suspension (10) 6 CFU/mL) was added to the 96-well plate and incubated at 30 ℃ for 24h. The degree of turbidity of the bacterial solutions in the 96-well plates was observed, and the MIC value of the single enzyme was determined.
The test results are shown in FIG. 1:
all of the beta-1, 3-glucanase treated wells were cloudy, indicating that the beta-1, 3-glucanase had no bacteriostatic effect. Whereas wells treated with 125. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL of glucose oxidase were clear and transparent, so that the MIC value of glucose oxidase was 125. Mu.g/mL.
Example 2
Preparation of candida albicans biofilm:
candida albicans was subjected to activation culture and then diluted with a medium so that the A value of the culture medium was 0.01 at 600nm in a spectrophotometer. 100. Mu.L of the bacterial liquid was added to a 96-well plate, and the mixture was allowed to stand still at 30℃for 48 hours to form a biofilm.
Example 3
Removal of candida albicans biofilm:
the following groups are set: (1) a blank; (2) 5 μg/mL beta-1, 3-glucanase solution; (3) 10 μg/mL beta-1, 3-glucanase solution; (4) 125. Mu.g/mL glucose oxidase solution; (5) 250 μg/mL glucose oxidase solution; (6) mixing enzyme solution: 5 μg/mL β -1, 3-glucanase+125 μg/mL glucose oxidase; (7) mixing enzyme solution: 5 μg/mL β -1, 3-glucanase+250 μg/mL glucose oxidase; (8) mixing enzyme solution: 10 μg/mL β -1, 3-glucanase+125 μg/mL glucose oxidase; (9) mixing enzyme solution: 10 μg/mL β -1, 3-glucanase+250 μg/mL glucose oxidase.
The Candida albicans biofilm prepared in example 2 was washed 3 times with PBS, divided into eight groups, 100. Mu.L of the above enzyme solutions were added, respectively, and incubated at 30℃for 24 hours.
The clearance of the biofilm was determined by MTT staining. The treated Candida albicans biofilm was washed 3 times with PBS and stained with 10. Mu.L MTT solution (5 mg/mL) +90. Mu.L sterile PBS for 4h. The supernatant was aspirated and the cells were treated with 110. Mu.L of DMSO. Measuring A of the extract by using an enzyme-labeled instrument 490 Values were measured to determine the biofilm content.
The test results are shown in table 1:
TABLE 1
| Group of | (1) | (2) | (3) | (4) | (5) | (6) | (7) | (8) | (9) |
| Biofilm content (%) | 100 | 91.09 | 82.25 | 49.45 | 35.96 | 28.93 | 23.15 | 23.77 | 19.24 |
As is clear from Table 1, the effect of the mixed enzyme solution on the removal of Candida albicans biofilm was very remarkable as compared with the beta-1, 3-glucanase and glucose oxidase alone, and the removal rate of 10. Mu.g/mL of beta-1, 3-glucanase+250. Mu.g/mL of glucose oxidase was the highest and could reach about 80%.
Example 4
Structural changes of biofilm:
1mL of Candida albicans suspension (10) 6 CFU/mL) and 1mL YPD medium were added to a 24-well plate, and the chip was placed on the bottom of the 24-well plate and cultured for 48 hours to form a biofilm. The supernatant was carefully removed and washed 3 times with PBS. To the formed biofilm, 10. Mu.g/mL of beta-1, 3-glucanase, 250. Mu.g/mL of glucose oxidase and 1mL of 10. Mu.g/mL of beta-1, 3-glucanase+250. Mu.g/mL of glucose oxidase were added, respectively, and the mixture was cultured for 24 hours. Washed 3 times with PBS. Then placing the chips in 2.5% glutaraldehyde solution for fixation for 4 hours, then sequentially placing the chips in 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol and 100% ethanol for dehydration and replacing the chips with 100% tertiary butanol, taking out the chips, freeze-drying the chips for 48 hours, spraying gold, and observing the influence of the enzyme solution on the structural change of candida albicans biological film by using a 10kv Scanning Electron Microscope (SEM).
The test results are shown in fig. 2:
as can be seen from fig. 2, candida albicans in the control group formed a dense three-dimensional structure of the biofilm (a panel); after treatment with beta-1, 3-glucanase alone, the biofilm structure was looser than in the blank (panel B). After being treated by glucose oxidase alone, the bacterial content is obviously reduced compared with a blank control group, which shows that the glucose oxidase has a certain bactericidal effect, but still has bacterial mass (C diagram). However, after the mixed enzyme treatment, the structure of candida albicans biofilm disintegrates, and the cell shedding is more serious (panel D).
Example 5
Fluorescent microscope observation:
1mL of Candida albicans suspension (10) 6 CFU/mL) and 1mL YPD medium were added to a 24-well plate and cultured for 48 hours to form a biofilm. The supernatant was carefully removed and washed 3 times with PBS. To the formed biofilm, 10. Mu.g/mL of beta-1, 3-glucanase, 250. Mu.g/mL of glucose oxidase and 10. Mu.g/mL of beta-1, 3-glucanase+250 were addedmu.g/mL glucose oxidase were incubated for 24h in 1mL each. Washed 3 times with PBS. By usingBaclight staining TM bacterial viability and counting kit treatment. 1 volume of DMAO and 2 volumes of EthD-III were mixed in a microcentrifuge tube, and then 8 volumes of 0.85% sodium chloride solution were added to give a 100 Xdye solution. Then incubated for 15min in the dark at room temperature. The biofilm was observed with a fluorescence microscope with FITC and Cy3 channels. When live/dead->When BacLight bacterial viability and the counting kit stain candida albicans, live cells show green fluorescence and damaged or dead cells show yellow-green or red fluorescence.
The test results are shown in fig. 3:
as can be seen from FIG. 3, untreated Candida albicans showed almost all fluorescence as green fluorescence and large and dense area (panel A). Candida albicans treated with beta-1, 3-glucanase was also almost green light, but had a more loose structure than the blank (panel B). After glucose oxidase treatment, red and yellow fluorescence account for the majority of the graph, indicating that glucose oxidase has killing ability against candida albicans, but yellow fluorescence is large in area and compact in structure, indicating that glucose oxidase can kill surface candida albicans, but biofilm can still protect internal candida albicans from killing (graph C). After the mixed enzyme treatment, the red and yellow fluorescence is very strong, and the phenomena of sparse and dispersion of bacterial load are accompanied, which indicates that the structure of a biological film is destroyed and candida albicans is killed in a large amount (D graph). The above results indicate that the mixed enzyme treatment more easily lyses the biofilm and kills candida albicans cells hidden in the biofilm.
Example 6
Hydrogen peroxide production effect:
100. Mu.L of Candida albicans suspension (10 6 CFU/mL) and 100 μl YPD medium were added to 96-well plates and cultured for 48h to form biofilms. Carefully remove the supernatant, useThe PBS was washed 3 times. To the formed biofilm, 10. Mu.g/mL of beta-1, 3-glucanase, 250. Mu.g/mL of glucose oxidase and 10. Mu.g/mL of beta-1, 3-glucanase+250. Mu.g/mL of glucose oxidase were added, and the mixture was cultured for 24 hours. Washed 3 times with PBS. The hydrogen peroxide content in each culture system was determined using a hydrogen peroxide kit.
The measurement results are shown in Table 2:
TABLE 2
| Group of | H 2 O 2 (μmol/mL) |
| Blank control | 0.0626 |
| 10. Mu.g/mL beta-1, 3-glucanase | 0.0534 |
| Glucose oxidase 250. Mu.g/mL | 0.1855 |
| 10 μg/mL beta-1, 3-glucanase+250 μg/mL glucose oxidase | 0.5613 |
As can be seen from Table 2, the glucose oxidase-treated culture system produced more hydrogen peroxide than the blank, whereas the beta-1, 3-glucanase-treated culture system did not actually increase the hydrogen peroxide yield. However, after the mixed enzyme treatment, the hydrogen peroxide content in the culture system can be obviously improved on the basis of the independent treatment of glucose oxidase. This suggests that beta-1, 3-glucanase can assist glucose oxidase in increasing hydrogen peroxide production.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (6)
1. An enzyme composition comprising beta-1, 3-glucanase and glucose oxidase; wherein the mass ratio of the beta-1, 3-glucanase to the glucose oxidase is 5-10:125-250.
2. The enzyme composition according to claim 1, characterized in that the mass ratio of beta-1, 3-glucanase to glucose oxidase is 10:250.
3. Use of the enzyme composition of claim 1 for the preparation of a formulation for removing biofilm from candida albicans.
4. An enzyme preparation, characterized in that it is a mixed enzyme solution formed by dispersing or dissolving the enzyme composition of claim 1 in a solvent; wherein the concentration of the beta-1, 3-glucanase is 5-10 mug/mL, and the concentration of the glucose oxidase is 125-250 mug/mL.
5. The enzyme preparation according to claim 4, wherein the concentration of the beta-1, 3-glucanase is 10 μg/mL and the concentration of the glucose oxidase is 250 μg/mL.
6. Use of the enzyme composition of claim 1 and/or the enzyme preparation of claim 4 for inhibiting candida albicans for non-diagnostic purposes.
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| WO2022079315A1 (en) * | 2020-10-16 | 2022-04-21 | Onelife S.A. | Parapharmaceutical or pharmaceutical composition administrable to a living being, preferably a human being, comprising at least one enzyme for the treatment and/or prevention of bacterial infections involving biofilm formation |
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- 2022-11-08 CN CN202211388414.7A patent/CN115747180B/en active Active
Patent Citations (6)
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| WO2015091772A1 (en) * | 2013-12-19 | 2015-06-25 | Ludwig-Maximilians-Universität München | Method of determining the degradation of cellulosic materials |
| CN108866036A (en) * | 2018-07-24 | 2018-11-23 | 浙江大学 | A kind of cascade enzyme reaction microballoon and preparation method thereof with antibacterial functions |
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| WO2022079315A1 (en) * | 2020-10-16 | 2022-04-21 | Onelife S.A. | Parapharmaceutical or pharmaceutical composition administrable to a living being, preferably a human being, comprising at least one enzyme for the treatment and/or prevention of bacterial infections involving biofilm formation |
| CN113209031A (en) * | 2021-04-30 | 2021-08-06 | 青岛农业大学 | Double-targeting composite nano system loaded with amphotericin B and beta-1, 3-glucanase, preparation method and application thereof |
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