Criação de uma metodologia para termoestabilização de proteínas via mutagenese in silico e aplicação em uma enzima da classe das endoglucanases

Abstract

The search for more sustainable industrial production has made enzymes an interesting target for application in several industries, such as the production of biofuels. Besides being more sustainable than traditional chemical reactions, enzymes are also capable of catalyzing novel reactions, that can't be done using convetional chemistry. However, one of the issues with its use in industrial settings is it's sensitivity to enviromental conditions, with most industrial processes being conducted at conditions that are adverse to the enzyme function. One way to address this problem is through protein engineering, which modifies aminoacids in the protein sequence in order to improve specific characteristics of it's structure and function. In the present work, an in silico methodology was developed to predict point mutations with high probability of increasing protein thermostability. The methodology starts by "cleaning" the protein through the PyMol software, followed by selecting the residues to be mutated using the YASARA software. Site-saturation mutagenesis is then applied to the selected residues through the FRESCO libraries, using the FoldX and Rosetta_ddG softwares. The residues which are predicted to be stable are then sumitted to backrub simulations and sequence tolerance approaches via the Rosetta package. Remaining stabilizing mutations are then submitted to 100 nanoseconds of molecular dynamics simulations, and the results of RMSF, hydrogen bond and radius of gyration analysis of each mutant are compared to the native protein. To test the performance of the method, a celulose degrading enzyme of the endoglucanase class, Xaccel5A was used as a model. Two mutations were selected, and the native protein and its mutations were expressed and tested in vitro in the 40ºC, 50ºC and 60ºC temperature range through the DNS assay. The results showed the ability of the methodology to produce a reduced mutant library, with high stabilization capacity, in a short time period. Of the two tested mutations, one showed incresed performance compared to the native proteins in all temperatures. However, further studies are necessary in order to better characterize the enzyme kinetics of the mutations, as well as increasing the tested temperature range.