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oai:gumushane-rdlopen.rawdatalibrary.net:gumushane/135362026-10-22T11:36:22Zopenairedrivercom_gumushane_10000col_gumushane_10001com_gumushane_10007col_gumushane_10180

Cloning and biochemical characterization of a novel GH1 β-glucosidase from thermophilic bacterium, Anoxybacillus flavithermus DSM 2641TAkgeyik, Aytul UzunAkyildiz, EsmaKaraoglu, HakanBeris, Fatih SabanAnoxybacillus flavithermus DSM 2641^TGH1Thermophilicβ-glucosidaseβ-glucosidases belong to the class of glycoside hydrolases (GHs) and can mediate the cleavage of β-1-4 glycosidic bonds in alkyl or aryl-β-D-glucosides, amino, cyanogenic glucosides, and oligo-or disaccharides. The gene encoding β-glucosidase of Anoxybacillus flavithermus DSM2641T was subcloned and expressed in E. coli BL21 Star(DE3) cells with pET100 Directional TOPO Expression Kit. The recombinant AFBGL was purified by a Ni-NTA affinity chromatography. The enzyme was 53 kDa and had 5.13 × 105 µmol/min/mg protein of Vmax and 422.3 µM of Km towards p-nitrophenyl β-D-glucoside as a main substrate. The optimum temperature and pH of the purified enzyme were 65°C and 6.8, respectively. We also determined kinetic parameters of AFBGL against the other artificial substrats, pNPC, pNPX, and oNPG. The enzyme had the Vmax and Km for 5.13 × 105 µmol/min/mg protein and 0.42 mM for pNPG, 0.007 µmol/min/mg protein and 0.19 mM for pNPC, 0.01 µmol/min/mg protein and 11.97 mM for pNPX, 0.06 µmol/min/mg protein and 0.134 mM for oNPG, respectively. AFBGL's substrate affinity was found to be more directed towards oNPG. Although 0.5 mM Mg2+ increases the enzyme's activity 1.6-fold, higher concentrations of Mg2+ and the presence of K+, Fe2+, Mn2+, Zn2+, Cu2+, and Al3+ decreases its activity. AFBGL retained 50% of the initial activity towards pNPG for 25 min and the activity was not detected after 225 min. According to amino acid motifs, AFBGL belongs to family 1 of glycosylhydrolase and 4/7 super family. So, AFBGL is a member of GH1, as the amino acid sequence contained a GH1 specific domain.National Institute of Science Communication and Policy Research2026Articlehttps://rdlopen.rawdatalibrary.net/r/gumushane/13536https://doi.org/10.56042/ijbb.v63i8.94110301-1208enIndian Journal of Biochemistry and Biophysicsinfo:eu-repo/semantics/openAccessinfo:eu-repo/semantics/articlehttps://gumushane-rdlopen.rawdatalibrary.net/tr/items/gumushane/13536