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- MakaleAçık Erişim2026Scopus SJRQ4Cloning and biochemical characterization of a novel GH1 β-glucosidase from thermophilic bacterium, Anoxybacillus flavithermus DSM 2641T
Akgeyik, Aytul Uzun; Akyildiz, Esma; Karaoglu, Hakan; Beris, Fatih Saban
β-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.
- MakaleYalnız künye2018Scopus SJRQ1New xylanolytic enzyme from Geobacillus galactosidasius BS61 from a geothermal resource in Turkey
Sari, Bilge; Faiz, Ozlem; Genc, Berna; Sisecioglu, Melda; Adiguzel, Ahmet; Adiguzel, Gulsah
In this study, isolation, conventional and molecular characterizations of ten thermophilic bacteria from Rize/Ayder were carried out. Xylanase from Geobacillus galactosidasius BS61 (GenBank number: KX447660) was purified by acetone precipitation, Diethylaminoethyl-cellulose and Sephadex G-100 chromatographies. The xylanase of G. galactosidasius BS61 in clarifying fruit juice was also investigated. Enzyme was purified 29.80-fold with 75.18% yield; and molecular weight was determined as 78.15 kDa. The optimum temperature of xylanase was 60 °C. The enzyme activity was maintained fully after 24 h and over 50% after 168 h at pH 4.0–10.0, while optimum pH was 7.0. Km and Vmax for beech wood xylan were measured as 3.18 mg mL−1, 123 U mg protein−1. In addition, Ca2+, Na+, Al3+, Zn2+, Cd2+, Mg2+, Ni2+, Cu2+ had decreasing effect on enzyme activity, while enzyme activity had been protected against anions, especially HSO3− and HPO4 2− stimulated enzyme activity. Xylanase applications (with 15 U/mL enzyme activity) in orange and pomegranate juices were increased; and the sugar and turbidity amounts were reduced 17.36% ± 1.18 and 30.52 ± 1.23, respectively. These results indicated that the xylanase of G. galactosidasius BS61 has biotechnological potential in juice clarification due to its stability against metal ions, chemicals and high pH-values.
AtıfScopus21WoS18OpenAlex22
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