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| Author(s): |
Fabiana Maria de Almeida
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2003-04-22 |
| Examining board members: |
Hector Francisco Terenzi;
Maria Celia Bertolini;
Wilson Roberto Navega Lodi;
Maria de Lourdes Teixeira de Moraes Polizeli;
Aline Maria da Silva
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| Advisor: | Hector Francisco Terenzi |
| Abstract | |
The coding sequence of the neutral trehalase gene of Neurospora crassa (Genbank AF 044218) was cloned in the expression vector pET21b in fusion with a sequence encoding six histidine residues (histidine tail or His tag) at the carboxy terminus. The construct pET21b-NcTreB was then used to transform Escherichia coli BL21(DE3) and the recombinant protein was obtained upon induction with 1mM IPTG at 25ºC. After three hours, a significative induction a polypeptide of the expected size for the neutral trehalase of N. crassa (~84kDa) was observed. Cells from the induced cultures were submitted to lysis and the resulting extracts divided into soluble and insoluble fractions by centrifugation. The soluble fraction of IPTG (Isopropyl-β-D-thiogalactopyranoside) induced cultures was used for measurements of the activity of the recombinant protein. The recombinant neutral trehalase exhibited a distinct behavior of the endogenous E. coli trehalase, being stimulated by 10mM calcium and inhibited by EDTA. The recombinant protein was also used for immunizing rabbits, in order to obtain polyclonal antibodies. These antibodies were then used in to follow neutral trehalase protein levels along the life cycle of N. crassa by immunoblotting. We observed that neutral trehalase levels increased during exponential growth and dropped at the stationary phase. The results suggested that the mobilization of trehalose observed at the onset of germination was due to a pre-existent enzyme. The recombinant trehalase, purified by affinity chromatography on Ni-NTA agarose, exhibited a specific activity of about 80-140mU/mg protein. Maximum of activity was observed at pH 7.0, and at 30ºC. Molecular mass of the recombinant enzyme, estimated by gel filtration, was approximately 81kDa. The enzyme was absolutely specific for trehalose, and exhibited a half-life of 2.5 min at 40ºC. The presence of calcium partially protected the enzyme from thermal inactivation. The recombinant enzyme was activated by calcium and manganese, and inhibited by ATP, copper, silver, aluminum and cobalt. The observed KM was of 42mM, with a Vmax of 30.6nmol of glucose per min. The effect of phosphorylation on the recombinant trehalase was tested by incubating with recombinant cyclic AMP-dependent kinase (PKA). ln these conditions only 0.13% of the protein molecules were phosphorylated, resulting in about 30% of activation. The results from this study demonstrated that the neutral trehalase from N. crassa, expressed in E. coli, exhibited kinetic and biochemical characteristics typical of other described fungal neutral trehalases. The analysis of the expression of the N. crassa neutral trehalase suggested that the enzyme appears to be regulated by post-translational modification, with the possible involvement of the cAMP signaling pathway. Taken together our results and data from literature suggest that the in vivo activation of the neutral trehalase is a complex phenomenon, which may involve interactions between the enzyme and other proteins. Perhaps a sequential phosphorylation with the participation of other protein kinases may be required for full activation of the enzyme. (AU) | |