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In silico analysis, expression and purification of a new laccase from Chitinophaga sp. CB10

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Bárbara Bonfá Buzzo
Total Authors: 1
Document type: Master's Dissertation
Press: Jaboticabal. 2019-03-26.
Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Agrárias e Veterinárias. Jaboticabal
Defense date:
Advisor: Eliana Gertrudes de Macedo Lemos

Enzymes are biological catalysts that increasingly arouse industry interest in applying them in their processes. Among all the enzymes, laccases are a class of enzymes belonging to the family of Multicopper oxidases, with high industrial potential because they act on several substrates, thus having a diversity of applications. Between all laccases, bacterial are greater interest for industrial processes than fungi, once bacteria have a greater tolerance to extreme environments than these, besides presenting a time of reproduction and cloning faster. Therefore, a bacterial laccase was prospected in the genome of Chitinophaga sp. CB10 from the genomic database belonging to the Laboratory of Biochemistry of Microorganisms and Plants (LBMP), based on orthologous genes and search for HMM ("Hidden Markov Model") followed by the search for laccases using as motif the standard L3 (HLHGH which is a specific signature of laccases. This prospection revealed a ORF CB10_180.4889 which was cloned and subjected to expression, extraction and purification analyzes. The protein was included in inclusion bodies and it was only possible to extract it in its soluble fraction using denaturing agents, such as urea and guanidine. After extraction, the denatured protein was subjected to purification with concomitantly renaturation, which proved to be efficient according to the dye degradation screening tests. From the in silico analyzes, it was observed that the enzyme CB10_180.4889 has three domains of copper (cupredoxin) and belongs to the class of CopAs, characteristic only of bacterial laccases with greater resistance to copper. Considering the possible biotechnological application, the enzyme presents potential for the degradation of dye. (AU)

FAPESP's process: 17/09421-0 - Mining of metagenomes to search for multifunctional enzymes lacases
Grantee:Bárbara Bonfá Buzzo
Support type: Scholarships in Brazil - Master