Sürdürülebilir Kalkınma Amaçları
Yayınlar
- MakaleAçık Erişim2026Cloning 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.
- MakaleAçık Erişim2015Distribution of β-Lactamase Genes Among Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated From Patients in Turkey
Iraz, Meryem; Düzgün, Azer Ozad; Sandalli, Cemal; Doymaz, Mehmet Ziya; Akkoyunlu, Yasemin; Saral, Aysegul; Peleg, Anton Y.; Özgümüs, Osman Birol; Beris, Fatih Saban; Karaoglu, Hakan
Background: The emergence of carbapenem-resistant Klebsiella pneumoniae poses a serious problem to antibiotic management. We investigated the beta-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey. Methods: Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of p-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)- FOR analysis. Results: All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to FOR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like beta-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M-1-like (62%) beta-lactamases. Sequence analysis of bla(TEM) genes identified a bla(TEM-166) with an amino acid change at position 53 (Arg53Gly) from bla(TEM-1b), the first report of a mutation in this region. REP-FOR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. Conclusions: Combinations of beta-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other beta-lactamases, such as TEM, SHV, and CTX-M beta-lactamases.