Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana

The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.