Abstract
Laccases (EC:1.10.3.2) are enzymes of the multi-copper oxidase family capable of degrading a wide range of substrates using oxygen as the final electron acceptor and producing water as a by-product of the reaction. Owing to this characteristic, they are promising tools in the field of bioremediation. However, the application of laccases in large-scale procedures is hindered by the low thermostability of many of these enzymes, a property whose molecular determinants remain to be fully characterised. Therefore, the aim of this project was to analyse thermostable and non-thermostable laccases to elucidate their physicochemical differences and identify key residues contributing to thermostability. To achieve this, multiple-sequence alignments of thermostable and non-thermostable laccases were constructed and analysed through statistical tests and the Kullback-Leibler divergence. The analysis revealed a high degree of global similarity between the two groups of enzymes, suggesting that thermostability is mostly determined by local conservation patterns. Using the Kullback-Leibler divergence and Bacillus subtilis CotA structure as a model, several discriminating positions were identified and structurally interpreted, revealing conservation patterns with a potential to be used in the targeted rational design of laccases to increase their thermostability.
Keywords: bacterial laccase, thermostability, bioremediation, rational engineering, multiple-sequence alignment (MSA)