Cloning, expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13.
A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co(2+) affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 °C and pH of 8.3: kcat of 4.8 × 10(5) s(-1) and kcat/Km of 5.6 × 10(7) M(-1) × s(-1). This activity was potently inhibited by acetazolamide which showed a KI of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO3 biomineralization processes.
a Department of Biology , Faculty of Art and Science, Molecular Biology Research Laboratories, Recep Tayyip Erdogan University , Rize , Turkey .
Ayşenur
A
Eminoğlu
b Neurofarba Department, Sezione Di Scienze Farmaceutice E Nutraceutiche, University of Florence , Sesto Fiorentino (Firenze) , Italy .
Daniela
D
Vullo
c Department of Medical Biology , Faculty of Medicine, Selcuk University , Konya , Turkey .
Aycan
A
Aşık
d Bulancak School of Applied Sciences, Giresun University , Giresun , Turkey , and.
Dilşat Nigar
DN
Çolak
b Neurofarba Department, Sezione Di Scienze Farmaceutice E Nutraceutiche, University of Florence , Sesto Fiorentino (Firenze) , Italy .
Claudiu T
CT
Supuran
e Department of Biology , Faculty of Science, Karadeniz Technical University , Trabzon , Turkey.
Sabriye
S
Çanakçı
e Department of Biology , Faculty of Science, Karadeniz Technical University , Trabzon , Turkey.
Ali
A
Osman Beldüz
D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000145:classification / Q000201:enzymology*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology
D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D002256:Carbonic Anhydrases
Enterobacter sp.; kinetics; β-Carbonic anhydrase