Biochemical and structural analysis of thermostable esterases

Biocatalysts play an important role in modern biotechnology because of their specificity, selectivity, efficiency and sustainability. One of the industrially most exploited and important groups of biocatalysts are the esterases. These enzymes catalyze, in the presence of water, the hydrolysis of an ester-bond resulting in the formation of an alcohol and a carboxylic acid. The use of enzymes in industrial processes also has its restrictions. Many processes are operated at elevated temperatures or in the presence of organic solvents. These conditions are detrimental to most enzymes and therefore there is a growing demand for enzymes with an improved stability. In this regard, there is a special interest from industry in enzymes of thermophilic origin since these enzymes generally display a high intrinsic thermal and chemical stability. This thesis describes the results of biochemical and structural analyses of thermostable esterases. Bioinformatics was used to identify new ester-hydrolyzing enzymes in the genomes of the hyperthermophilic bacterium Thermotoga maritima and the hyperthermophilic archaeon Archaeoglobus fulgidus. These potential esterases were cloned and heterologously expressed in Escherichia coli. Different types of ester hydrolyzing enzymes were found, including carboxylesterases, an acetyl esterase and a lipase. The biochemical properties of these enzymes were studied in detail. In addition, crystallization trials were performed, resulting in the three-dimensional structures of several of these enzymes. New structural features were revealed, such as the combination of an esterase domain with an immunoglobulin-like domain. The information obtained in this study provides fundamental knowledge, which may act as a basis for modern methods of enzyme engineering, with the aim to improve the applicability of these enzymes.