Beta-Glycosidases from Hyperthermophiles as Biocatalysts
Sammanfattning: The thesis concerns glycosyl transfer with use of b-glycosidases, most of them from hyperthermophilic organisms. Both optimisation of the reaction conditions and protein engineering in terms of site-directed mutagenesis were used to improve b-glycosidase-catalysed reactions. The first part of the work deals with conversion of lactose into valuable products such as galacto-oligosaccharides (GOS) and alkyl-glycosides, the activity and selectivity of b-glycosidases in model reactions being investigated in the second part. b-glycosidases from the hyperthermophilic Archaea Sulfolobus solfataricus (LacS) and Pyrococcus furiosus (CelB) were shown to be very well suited for GOS synthesis from lactose. For both enzymes the maximum yield of GOS increased with increasing lactose concentration. In an optimally designed reaction involving LacS, a combined yield of 37% for the tri- and tetrasaccharides was attained and a maximum yield of 40% for CelB. Use of site-directed mutagenesis of CelB resulted in a further improvement in GOS yield. An exchange of phenylalanine for tyrosine (F426Y) increased the GOS yield (45%) compared with wild-type CelB (40%). Furthermore, a double mutant, M424K/F426Y, showed much better glycosyl transfer properties at low lactose concentrations than the wild-type enzyme did. At a lactose concentration of 10%, the GOS yield for the mutant was 40% as compared with 18% for the wild-type. In the synthesis of hexyl-b-glycoside from lactose in hexanol, these two b-glycosidases from hyperthermophiles showed higher activity than other b-glycosidases did, and produced high yields of both hexyl-b-galactoside (LacS: 41%, CelB: 63%) and hexyl-b-glucoside (LacS: 29%, CelB: 28%). Adding SDS to the reaction increased both the initial reaction rate of LacS and the hexyl-b-galactoside yield (from 41% to 51%). The competition between b-glucosidase-catalysed transglucosylation and hydrolysis was studied using wild-type CelB and active site mutants (M424K, F426Y, M424K/F426Y) in the conversion of pentyl-b-glucoside to hexyl-b-glucoside in hexanol as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction to this. Each of the mutants showed lower activity but higher selectivity than the wild-type enzyme. The largest increase in selectivity (2.6-fold) was attained by the F426Y mutant, for which the hexyl-b-glucoside yield increased from 56% to 69%. In contrast to the wild-type CelB, the F426Y mutant had a transferase activity as low as aw 0.29. Surprisingly, the selectivity of CelB towards transglucosylation increased as the water activity increased, up to aw 0.92. To investigate the influence of the water activity on the selectivity of b-glycosidases further five different enzymes (P. furiosus b-glucosidase, S. solfataricus b-galactosidase, Caldocellum saccharolyticum b-glucosidase, almond b-glucosidase and Escherichia coli b-galactosidase) were evaluated as transglycosylation catalysts in hexanol containing various amounts of water. For all the enzymes tested, the selectivity for alcohol increased with an increase in water activity. On the other hand, in a hexanol/water two-phase system, hydrolysis was by far the most dominant reaction, despite the total activity for all the enzymes increasing. It was also shown that b-glycosidases of differing origin differs markedly in their substrate specificity and in the extent to which the temperature influences their selectivity for the glycon part of the donor substrate.
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