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Functional Surface Display of Laccase in a Phenol-Inducible Bacterial Circuit for Bioremediation Purposes

ABSTRACT: 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.

SUBMITTER: Fathi-Roudsari M

PROVIDER: S-EPMC5889505 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Functional Surface Display of Laccase in a Phenol-Inducible Bacterial Circuit for Bioremediation Purposes

Fathi-Roudsari Mehrnoosh M Behmanesh Mehrdad M Salmanian Ali-Hatef AH Sadeghizadeh Majid M Khajeh Khosro K

Iranian biomedical journal 20171028 3

<h4>Background</h4>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 ph ...[more]

PMID: 29078699

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Project description:BackgroundCarbamate pesticides have been widely used in agricultural and forestry pest control. The large-scale use of carbamates has caused severe toxicity in various systems because of their toxic environmental residues. Carbaryl is a representative carbamate pesticide and hydrolase/carboxylesterase is the initial and critical enzyme for its degradation. Whole-cell biocatalysts have become a powerful tool for environmental bioremediation. Here, a whole cell biocatalyst was constructed by displaying a novel carboxylesterase/hydrolase on the surface of Escherichia coli cells for carbaryl bioremediation.ResultsThe carCby gene, encoding a protein with carbaryl hydrolysis activity was cloned and characterized. Subsequently, CarCby was displayed on the outer membrane of E. coli BL21(DE3) cells using the N-terminus of ice nucleation protein as an anchor. The surface localization of CarCby was confirmed by SDS-PAGE and fluorescence microscopy. The optimal temperature and pH of the engineered E. coli cells were 30 °C and 7.5, respectively, using pNPC4 as a substrate. The whole cell biocatalyst exhibited better stability and maintained approximately 8-fold higher specific enzymatic activity than purified CarCby when incubated at 30 °C for 120 h. In addition, ~ 100% and 50% of the original activity was retained when incubated with the whole cell biocatalyst at 4 ℃ and 30 °C for 35 days, respectively. However, the purified CarCby lost almost 100% of its activity when incubated at 30 °C for 134 h or 37 °C for 96 h, respectively. Finally, approximately 30 mg/L of carbaryl was hydrolyzed by 200 U of the engineered E. coli cells in 12 h.ConclusionsHere, a carbaryl hydrolase-containing surface-displayed system was first constructed, and the whole cell biocatalyst displayed better stability and maintained its catalytic activity. This surface-displayed strategy provides a new solution for the cost-efficient bioremediation of carbaryl and could also have the potential to be used to treat other carbamates in environmental bioremediation.

Dataset Information

Functional Surface Display of Laccase in a Phenol-Inducible Bacterial Circuit for Bioremediation Purposes

Publications

Functional Surface Display of Laccase in a Phenol-Inducible Bacterial Circuit for Bioremediation Purposes

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