Cloning and biochemical characterization of a novel GH1 (3-glucosidase from thermophilic bacterium,<i> Anoxybacillus</i><i> flavithermus</i> DSM 2641<SUP>T</SUP>
INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS, cilt.63, sa.8, ss.869-880, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 63 Sayı: 8
- Basım Tarihi: 2026
- Doi Numarası: 10.56042/ijbb.v63i8.9411
- Dergi Adı: INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, Chemical Abstracts Core, Directory of Open Access Journals, Academic Search Ultimate (EBSCO)
- Sayfa Sayıları: ss.869-880
- Recep Tayyip Erdoğan Üniversitesi Adresli: Evet
Özet
(3-glucosidases belong to the class of glycoside hydrolases (GHs) and can mediate the cleavage of (3-1-4 glycosidic bonds in alkyl or aryl-(3-D-glucosides, amino, cyanogenic glucosides, and oligo- or disaccharides. The gene encoding (3-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 & times; 105 & micro;mol/min/mg protein of Vmax and 422.3 & micro;M of Km towards p-nitrophenyl (3-D-glucoside as a main substrate. The optimum temperature and pH of the purified enzyme were 65 degrees 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 & times; 105 & micro;mol/min/mg protein and 0.42 mM for pNPG, 0.007 & micro;mol/min/mg protein and 0.19 mM for pNPC, 0.01 & micro;mol/min/mg protein and 11.97 mM for pNPX, 0.06 & micro;mol/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.