Citation: LIMA, Priscila da Silva. Clonagem, expressão heteróloga e caracterização molecular de um gene de lacase de Pycnoporus sanguineus. 2009. 74 f., il. Dissertação (Mestrado em Biologia Molecular)-Universidade de Brasília, Brasília, 2009.
Lacases are enzymes with potential application in a set of industrial processes. Some authors described their use in bioremediation of phenolic compounds of pharmaceutical and dyes industries and in the removal of lignin of the cellulose pulp. In this study a laccase from Pycnoporus sanguineus previously described by Garcia and co-workers 2006), was expressed in the metylhothrofic yeast Pichia pastoris, aiming their production for biotechnological purposes. The laccase cDNA was obtained by RT-PCR and cloned using the vector PGEM-T. Escherichia coli harbouring the construction were selected and the clone called PGLac 1 was used for further experiments (subcloning and sequencing). Two different vectors were used for P. pastoris transformation, pPIC9 e pPICZ-A. The laccase activity was detected only for P. pastoris harbouring the construction 2 (pPIC9/laccase cDNA). The heterologous laccase presents identity with lacases from P. sanguineus, P. cinnabarinus (97%) and Trametes sp. (79%). Moreover, the predicted protein has 502 amino acids, 3 cupric-oxidase conserveddomains, molecular mass of 58,8 KDa and isoelectric point of 5,7. The molecular modelling revelead a globular protein with a active site containing copper atoms, coordinated by histidin and cistein residues.
Description: Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2009.