Part:BBa_K3150002
ScLac
laccase from Streptomyces cyaneus with higher expression level for further use since the wide-used CotA has a problem on its low heterologous expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1328
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 716
Illegal NgoMIV site found at 1085
Illegal NgoMIV site found at 1244
Illegal NgoMIV site found at 1636 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 786
Illegal BsaI.rc site found at 145
Usage and Biology
We find a new laccase ScLac from Streptomyces cyanobacteriae with higher expression level for further use since the wide-used CotA has a problem on its low heterologous expression. ScLac showed a higher heterologous expression than CotA in E. Coli, which could reach 104 mg/L while CotA was only 20 mg/L.
References Ece S , Lambertz C , Fischer R , et al. Heterologous expression of aStreptomyces cyaneuslaccase for biomass modification applications[J]. AMB Express, 2017, 7(1):86.
SDS-Page Verification
SDS-PAGE experiments were performed to verify the expression of the enzyme protein. ScLac-, ScLac+ represent no induction and after induction by IPTG. By reviewing the literature, we found that ScLac has a protein size 70.9 kDa. After comparison with the images, we found that all proteins were successfully expressed under the induction of IPTG.
Decolorization Ability Test
We divided the scLac into two groups: one group was induced by IPTG, which was labeled as ScLac +, while the other group without IPTG was labeled as ScLac -. We detect the absorbance at regular intervals, and after statistical analysis, we get the results of Fig.1. From the analysis in Figure 1, we can see that the degradation speed of ScLac group is faster than that of control group, the decolorization effect increases significantly after 12h, and the absorbance has decreased to 0.4 after 48h, which is significantly different from the control group.
At the same time of the absorbance detection of the supernatant, we also retained the bacteria and observed them. It can be seen from Fig. 2 that in IPTG induction group, the color of bacteria is purple, which is closer to the color of dye. However, in the control group without IPTG, the color of bacteria was yellow, which was more similar to the color of the mixture of bacteria and pigment, so it was speculated that its adsorption on dye was very weak. According to the color observation of the supernatant in Figure 3, after 48 hours, the color of the supernatant under the catalysis of scLac decreased obviously.
Actual sewage test
We also carried out an actual sewage test. The wastewater from the printing and dyeing factory was added with the same concentration of dye RR as the previous experiment for RR degradation test. In order to ensure the consistency of the experimental method, we have done the dye degradation test of the sewage group and the experimental group at the same time, and recorded the light absorption value of the dye in 24 hours, 48 hours and 72 hours. The results are as figure 4. In the experimental group, we repeated the experiment according to the method of functional test. In this result, scLac showed good degradation effect on RR. It can be seen that the dye had been degraded by naked eyes at 48 hours.
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