Estudos de poligalacturonases do fungo mutualista Leucoagaricus gongylophorus
Golfeto, Camilla Calemi
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Polygalacturonases (PGases) are enzymes which hydrolyze glycosidic linkages - 1,4 between pectic acid residues, and are produced by plants, fungi, bacteria and yeasts. The fungus L. gongylophorus, Atta sexdens ant mutualist, secretes enzymes with PGase activity. The project was developed in two approaches: study with recombinant and native PGase. The activity on polygalacturonic acid of native PGase in fungus culture medium was determined, and its purification from crude extract was performed by (NH4)2SO4 precipitation and molecular exclusion chromatography. PGase kinetic parameters (Vmax and KM), optimal pH and temperature were determined, and the enzyme was immobilized on magnetic particles, proved to be a good method to future work inhibitors search. A cDNA library was constructed from total RNA obtained from L. gongylophorus culture in induction medium. 816 clones from the library were sequenced, allowing the identification of enzymes sequences involved in the plant cell wall degradation, some already in cloning and expression in our laboratory. Using a deposited L. gongylophorus PGase (PGase-Lg) sequence (GenBank: ADV30326.1), primers were designed to amplify the PGase-Lg gene, with cleavage sites for EcoRI, HindIII and NotI enzymes, respecting the pETSUMO (for E. coli expression) and pPICZA (for P.pastoris expression) reading phase vectors. From the cDNA, the PGase-Lg ORF encoding was amplified by PCR and cloned in both vectors. The clones were confirmed by plasmid DNA extraction and PCR. E. coli expression experiments of pETSUMO-PGase-Lg construction showed a great expression of His.tag-SUMO.tag-Lg-PGase fusion protein in the insoluble form and many expression and solubility assays were inefficient in solubility of expressed fusion protein. The protein refolding was performed and this was obtained in soluble form but lacks PGase activity, showing that folding may not have been correctly or E. coli fusion protein expressed does not undergo post-translational modifications to have enzymatic activity. The rPGase-Lg expressed in P. pastoris showed PGase activity on polygalacturonic acid and electrophoresis analysis suggest more than one enzyme expression, with different glycosylation content.