Revision as of 17:12, 21 October 2019

β-1,3-glucanase

β-1,3-glucanase protein coding region. β-1,3-glucanase can degrade biofilm.

1. Usage and Biology

β-1,3-glucan is one of the primary components in C. albicans biofilm EPS, which is important for Candida biofilm formation and resistance to stresses. The enzyme β-1,3-glucanase, form Cellulosimicrobium cellulans, can degrade β-1,3-glucan. Therefore, this year, we decided use β-1,3-glucanase to disrupt the Candida biofilm matrix and increase the effect of the antimicrobial drug.

2. Characterization

2.1 Validation of pVE-β-1,3-glucanase construction

To verify the construction of pVE-β-1,3-glucanase (pVE-β-GA) which we generated, the digestion by SalI/EcoRV was performed by a standard protocol followed by agarose gel electrophoresis (Figure 1).

2.2 Expression of pVE-β-1,3-glucanase

To assess the β-1,3-glucanase expression of our construct, Congo Red experiment was used. Congo Red has a strong red chromogenic reaction with β-1,3-glucan. In contrast, when β-1, 3-glucan is decomposed into reducing monosaccharides by β-1,3-glucanase, the hydrolyzed region forms a pale yellow transparent hydrolytic circle. Compared with control, there was a larger size of transparent hydrolytic circle caused by β-1,3-glucanase expressed in E. coli with pVE-β-GA, indicating the expression of β-1,3-glucanase (Figure 2).

2.3 Degradation effect of β-1,3-glucanase on biofilm

Crystal violet (CV) reduction method, which is commonly used for quantitative analysis of biofilm, was used to evaluate the antibiofilm activity of β-1,3-glucanase. Under the condition of using bacteria with pVE5523 vector(pVE vector) to exclude the influence of bacterial substances on the staining results, as Figure 3 shows, β-1,3-glucanase produced by our engineered bacteria has the effect of degrading biofilm. The supernatant of E. coli with pVE-β-GA diluted by one time is estimated to reach the effect of 0.5 μg/mL~2 μg/mL samples of standard β-1,3-glucanase. The result demonstrated that β-1,3-glucanase had disruption effect on mature biofilm with concentration-dependent manner(Figure 3).

3. Conclusion

Our engineered bacteria successfully characterized β-1,3-glucanase,moreover, it was effective in performing the function of degrading C. albicans biofilm.

Sequence and Features