Screening of strains and recombinant enzymes from Thermus thermophilus for their use in disaccharide synthesis

Abstract

Abstract Cell extracts from ten different strains of Thermus thermophilus have been screened for glycosidase activity. Among these, the sequenced strain HB27 hydrolyzed a wide variety of glycosides and increased six fold its β-glycosidase activity when grown with cellobiose in nutrient-limited media. We selected five genes encoding (putative) glycosidases (TTP0042, TTP0072, TTP0220, TTC0107 and TTP0222) from the genome of this strain, and the corresponding recombinant enzymes were overexpressed and purified. Several transglycosylation reactions using cellobiose-induced HB27 cell extracts and the purified recombinant enzymes were assayed. Biochemical properties and biosynthetic capabilities of the HB27 cell extracts and the TTP0042 enzyme were very similar, suggesting that this enzyme was responsible for most of the β-glycosidase activity detected in the HB27 strain. This was confirmed through the isolation and analysis of a null mutant of its encoding gene. With both, HB27 cell extracts and purified TTP0042 recombinant enzyme, we finally achieved high yields conditions for disaccharide production by transglycosylation with low amounts of self-condensed donor when high concentrations of a 1:5 donor:acceptor molar ratio was used.