The synthesis of glycosidic bonds is of high commercial value, because the produced compounds can be used for a wide range of applications. Oligosaccharides, for example, have great potential in the food industry, not only as essential nutrients that stimulate the immune system but also as low-caloric and non-cariogenic sweeteners. In turn, glycosylation of a non-carbohydrate acceptor, resulting in a glycoside or a glycoconjugate, can drastically influence both the physicochemical and biological properties of that molecule. Attaching a glycosyl group to a vitamin, for example, can improve its stability, solubility and bio-availability.As carbohydrates can be branched and connected in many different ways due the presence of multiple hydroxyl groups, their potential structural diversity is enormous. Consequently, chemical synthesis of glycosidic molecules is a very challenging task that requires the use of protecting and activating groups, resulting in multi-step synthetic routes with a low overall yield. Furthermore, chemical synthesis also makes use of toxic catalysts such as heavy metals, which limits its application in large-scale processes. Enzymatic glycosylation methods are therefore preferred since they result in higher yields and are more regiospecific than the chemical methods. Many enzymes can be applied for the production of glycosides. We have selected sucrose phosphorylase (SP) for glycosylation reactions because it can transfer a glucosyl moiety from an inexpensive donor substrate -simple table sugar- to a wide variety of acceptor molecules. Unfortunately, the thermostability of this enzyme is too low for industrial applications, which need to be operated at 60 °C or higher to avoid microbial contamination. Consequently, the goal of this PhD thesis is to increase the thermostability of sucrose phosphorylase. First, the most promising SP enzymes, i.e. from L. mesenteroides (LmSP) and B. adolescentis (BaSP) were recombinantly expressed and thoroughly characterized. The characterization of BaSP has revealed that this enzyme exhibits a relatively high temperature optimum (58 °C) and a remarkable stability at 60 °C. In contrast, LmSP has an optimal temperature of only 42 °C and loses all of its activity after 5 minutes incubation at 60 °C. The intriguing difference in thermostability of these two SP enzymes has been examined in more detail. Based on sequence alignment and mutational analysis, two amino acid substitutions have been identified that have a rigidifying effect on the enzyme’s structure.Several strategies have then been successfully applied to increase the thermostability of SP from B. adolescentis. Engineering of the enzyme by (semi-)rational mutagenesis has resulted in five mutants that are about 40 % more stable than the wild-type enzyme. These beneficial mutations could potentially be combined to obtain a stable biocatalyst at 60 °C. However, immobilization of the enzyme, either by covalent attachment to a carrier or by cross-linking, was found to be a more efficient technique, as it generates a biocatalyst that is stable for at least 2 weeks at 60 °C and can be used for more than one reaction cycle. Furthermore, the temperature optimum of the immobilized enzyme was found to be increased by as much as 17 °C, in the case of the cross-linked enzyme. For the first time, production of αG1P has become possible at elevated temperatures, which serves as proof of concept for the production of other glycosylated compounds with SP under industrial conditions.
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