P45: Molecular cloning and enzymatic characterization of two highly thermostable β-glucanase isozymes from Thermotoga neapolitana

β-Glucanases (EC 3.2.1.4) are members of Glycoside Hydrolase (GH) family 12, which are known to hydrolyze β-(1,4)-glycosidic linkages of cereal β-glucans or celluloses. Commonly, cereal β-glucans are composed of β-(1,4)-cellotriosyl and/or β-(1,4)-cellotetraosyl alternatively repeating units connected via β-(1,3)-glycosidic linkages.In the present study, two β-glucanase isozymes (TnβGn-A and B) genes were cloned from a hyperthermophile, Thermotoga neapolitana DSM4359. TnβGn-A has an open reading frame of 292 amino acids (about 34.4 kDa), while TnβGn-B gene encodes 274 amino acids (about 32.8 kDa). TnβGn-A shares about 48 % amino acids sequence identity with TnβGn-B. Recombinant TnβGn-A and B constitutively expressed in E. coli were simply purified to homogeneity via heat treatment at 60 °C for 30 min followed by an Ni-NTA affinity chormatography. TnβGn-A shows the highest acitivity at 100 °C and pH 7.5, while TnβGn-B has its optimual condition at 110 °C and pH 7.0 respectively. Both β-glucanases can hydrolyze the internal β-(1,4)-linkages of barley and oat β-glucans to release glucose (G), cellobiose (βG4G), and glucotriose-A (βG3G4G) as major end products. In order to understand their hydrolysis patterns in detail, a variety of β-glucooligosaccharides were treated and the resulting products were analyzed by high performance anion exchange chromatography (HPAEC) and thin layer chromatography (TLC). Interestingly, TnβGn-A and B share very similar hydrolysis patterns against various β-glucooligosaccharides containing mixed β-(1,4)- and β-(1,3)-linkages, except laminaribiose (βG3G), cellotetraose (βG4G4G4G), glucotriose-B (βG4G3G), glucotetraose-A (βG3G4G4G), glucotetraose-B (βG4G4G3G), and glucotetraose-C (βG4G3G4G).