Functional surface display of laccase in a phenol-inducible bacterial circuit for bioremediation purposes

Background: Phenolic compounds, which are produced routinely by industrial and urban activities, possess dangers to live organisms and environment. Laccases are oxidoreductase enzymes with the ability of remediating a wide variety of phenolic compounds to more benign molecules. The purpose of the present research is surface display of a laccase enzyme with adhesin involved in diffuse adhesion [AIDA-I] autotransporter system on the surface of Escherichia coli cells for bioremediation of phenolic compounds

Methods: The expression of laccase was regulated by a phenol-responsive promoter [a 54 promoter]. The constitutively-expressed CapR transcription activator was able to induce laccase expression in the presence of phenolic compounds

Results: Western blot analysis showed the expression and correct transfer of the enzyme to the outer membrane of E. coli cells in the presence of phenol. Activity assay confirmed the correct folding of the enzyme after translocation through the autotransporter system. HPLC analysis of residual phenol in culture medium showed a significant reduction of phenol concentration in the presence of cells displaying laccase on the surface

Conclusion: Our findings confirm that autodisplay enables functional surface display of laccase for direct substrate-enzyme availability by overcoming membrane hindrance

Overexpression of full-length core protein of hepatitis C virus by Escherichia coli cultivated in stirred tank fermentor

The mature core protein of the Hepatitis C virus [HCVC173] carrying pelB as a signal peptide [PelB::core] was overexpressed in Escherichia coli as 18% and 23.3% of the host's total protein, in flask and fermentor cultivation, respectively. A final specific yield of 25 +/- 1 mg HCVC173/g dry cell weight and an overall productivity of 51 +/- 1 mg HCVC173/l/h were obtained in the stirred-tank fermentor. The recombinant PelB::core protein was overexpressed as the inclusion body [IB] form, higher than the expected level when compared to the HCVC173, which was also showed by the analysis of secondary structure of mRNAs and calculation of the Codon Adaptation Index of the gene. The results showed that the combined effects of protein fusion and the signal sequence significantly enhanced the production of recombinant mature HCVC173 in E. coli. Therefore, the fusion form of the mature HCV core protein and the conditions defined in this study provide an alternative strategy for HCVC173 production in high cell density culture of E. coli