Revision as of 03:01, 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

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

We characterised β-1,3-glucanase by cloning it into pVE vector. Moreover, an signal peptide were added.

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 following agarose gel electrophoresis (Figure 1).

Figure 1. Digestion and agarose gel electrophoresis of pVE-β-GA.

To assess the expression of our β-1,3-glucanase, Congo Red experiment was used.

Congo Red has a strong chromogenic reaction with β-1, 3-glucan, and 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. The results of activity detection of Congo Red hydrolytic ring (Figure 2) showed that compared to the control, there was a larger size of transparent hydrolytic circle caused by β-1, 3-glucanase expressed in E. coli with pVE-β-GA.

Figure 2. Expression of pVE-β-GA. Add 10 mg/ml Congo Red solution to LB medium containing β-1,3-glucan substrate (0.1 g/100 mL) at a ratio of 1:100. 100 μl supernatant obtained by centrifugation after ultrasonic crushing of E. coli with pVE-β-GA is added to the Oxford cup, and the pVE empty vector bacteria supernatant is used as the control. Stand at 37 ℃ for 24 hours.

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. The result indicates that β-1,3-glucanase has disruption effect on mature biofilm with concentration-dependent manner. Under the condition of using bacteria with pVE empty 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.

Figure 3. Effect of β-1,3-glucanase on biofilm in 96-well microplate. Biofilm formed in RPMI 1640 medium for 48 h. Mature biofilm was treated with RPMI 1640 medium, supernatant of bacteria with pVE empty vector or pVE-β-GA and standard β-1,3-glucanase in different concentrations (0.5, 1 and 2 μg/mL) for another 24 h. Values obtained are given as the percentage of biofilm. The experiment was performed three times in triplicate. *, P < 0.05 from mock control using Student’s t test. △, P < 0.05.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 274
Illegal NgoMIV site found at 483
Illegal NgoMIV site found at 622
1000
COMPATIBLE WITH RFC[1000]