Thinker-ShenZhen  ChDeHome

Our project is to improve chitinase's antifungal ability to treat fungal skin diseases. By adding chitin-binding domains to both ends of the original chitinase through DNA synthesis, the binding ability of chitinase to chitin is greatly enhanced. We hope that chitinase can degrade chitin in fungal cell walls more efficiently, destroy cell structures to kill fungi, and treat fungal skin diseases.

Colloidal chitin was chosen to measure the binding capacity of chitinase due to the weak water solubility of chitin. We used chitin, concentrated hydrochloric acid, and ethanol to prepare 2% alternate chitin, which was stored at 4°C until use. The 2μmol/L chitinase of the experimental group and the control group, 1mg/mL colloidal chitin and other fungal agents were fully mixed with 50mmol/L phosphate buffer solution (pH=8), and then rotated and mixed at 4°C for 1h. The concentration of supernatant protein was measured by the BCA method. The chitin-binding ability chart of the experimental and control groups was obtained by sorting out the experimental data.

We used α-chitin, colloidal chitin, Malassezia chitin, and Candida albicans chitin.

Malassezia usually infects areas with well-developed sebaceous glands and uses oil to grow. Growing too fast can interfere with normal cell metabolism, causing itching and dandruff in the infected area.

Candida albicans can infect the oral cavity, esophagus, vagina, and other areas. It is very common, such as thrush and candida vulvovaginitis.

Experimental group: modified chitinase

Control group: unmodified chitinase

It can be seen that the binding ability of our modified chitinase to chitin is greatly enhanced, whether it is colloidal chitin or chitin in the fungal cell wall, verifying that the chitin-binding domain we added to the nitrogen and carbon ends is effective and can better bind to chitin.

In 0.3mL of 50mmol/mL phosphate buffer solution (pH=8), we used 0.1mL chitinase to hydrolyze 0.2mL 2% chitin to produce n-acetylglucosamine. -5 dinitrosalicylate reacts with it at 45°C to form a brown red compound 3-amino-5-nitrosalicylate, which has a characteristic absorption peak at λ=540nm. After 1h, the reaction is terminated by adding DNS, and the reaction is heated at 100°C for 10min. The absorbance value was measured at λ=540nm to characterize chitinase activity.

Experimental group: modified chitinase reacted with chitin control group: unmodified chitinase reacted with chitin

As can be seen from the above figure, the degradation ability of the modified chitinase is also greatly enhanced compared with that before, and the improvement in the degradation of pure fungal chitin is extremely significant.

Malassezia was selected as the test strain for the antifungal experiment. It was transferred to a PDA medium for activation, and cultured at 25°C for 48h, then the bacterial plate was knocked out with a hole punch, and inoculated into the center of the new petri dish with PDA medium. The purified chitinase of the experimental group and the control group were added respectively, and the inhibition was observed after 48 hours of culture.

Experimental group: modified chitinase inhibited fungi; control group: unmodified chitinase inhibited fungi

It can be seen that the modified chitinase has about three times higher antifungal activity than before, which meets our needs for efficient antifungal inhibition and treatment of fungal skin diseases.

The optimal pH and temperature of the modified chitinase were also obtained. The optimal pH value is about 7, and our chitinase can work to the maximum extent under neutral conditions, without adding weak acid or weak base, which means that there is no corrosive effect on the skin. The optimal temperature of chitinase was about 36℃, which was similar to the normal temperature of the human body, indicating that our chitinase could work to the maximum extent under the normal temperature of the human body. In general, our chitinase is highly applicable, not demanding for environmental conditions, and can be used under normal physiological conditions without side effects on the skin.

Considering the issue of biosafety, in order to release a large amount of chitinase and prevent gene leakage and pollution, our E.coli BL21 cannot directly act on human skin, so we chose to design a lysis system to destroy the cell structure of E.coli in the factory, release the produced chitinase and purify it. To make highly purified enzyme products for the treatment of fungal skin diseases.

Our cleavage system included the L-arabinose operon, BBa_B0034 RBS, SRRz gene, and BBa_B0015 terminator. The cleavage gene was introduced into BL21, and a certain concentration of Arabinose was added to initiate the cleavage process. Porous structures are formed on the cell membrane so that the R and RZ gene products can reach the cell wall; Transglycosidase gene R generates a water-soluble transglycosylase to decompose peptidoglycan in E. coli cell walls; The RZ gene generates a peptide chain endonuclease that cleaves connections between peptidoglycan oligosaccharides and cross-links between peptidoglycan and the outer membrane of the cell wall. Finally, the cell wall of E.coli BL21 is decomposed and a large amount of chitinase is released. After the purification process and removal of endotoxin and other impurities in the product, we obtain high-purity chitinase, and then make it into drugs to treat skin diseases.

Cracking System

We cultivated the engineered strain BL21 in LB medium at 37 ° c and 220 RPM, when the OD value was about 0.3, different concentrations of arabinose were added, and three sets of parallel experiments were repeated, the cultures were incubated under the same conditions, and OD600 was measured at 0.5h, 1h, 1.5h, 2h, 3h, and 4h, respectively. The Control group was the BL21 engineering strain group without arabinose.

The above experimental results showed that when the concentration of arabinose reached 10^-6 mol/L and above, the OD value was almost 0, which means that most of the strains were induced to lysis and died, and the intracellular chitinase was released, which proved that our lysis system could work normally.

Comparison

10^-6 mol/L vs Control

10^-5 mol/L vs Control

In general, in order to verify the success of our project, we obtained the data of the binding ability, degradation ability, optimal pH value and temperature of the modified chitinase, and the starting condition of the lysis system, which successfully verified the feasibility of our project at the theoretical and practical levels.

1. https://zh.wikipedia.org/zh-sg/%E7%99%BD%E8%89%B2%E5%BF%B5%E7%8F%A0%E8%8F%8C#%E7%94%9F%E7%89%A9%E8%96%84%E8%86%9C