The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 μmol min-1 mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.
- MeSH
- asparaginasa izolace a purifikace metabolismus MeSH
- glutamin metabolismus MeSH
- glutaminasa metabolismus MeSH
- kinetika MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- poločas MeSH
- Pyrobaculum enzymologie genetika MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Increasing trend of antibiotic resistance and expression of Extended Spectrum Beta Lactamases (ESBLs) are serious threats for public health as they render the treatment ineffective. Present study was designed to elucidate the antibiotic-susceptibility patterns of ESBL and non-ESBL producing E. coli and K. pneumoniae causing urinary tract infections so that the ineffective antibiotics could be removed from the line of treatment. The bacterial isolates obtained from the urine of patients visiting a tertiary health care facility were cultured for strain identification using API20E. Antimicrobial susceptibility and ESBL detection were done by Kirby-bauer diffusion technique. Almost 53.4 % isolates of E. coli and 24.5 % isolates of K. pneumoniae were found to be ESBL producers. The ESBL producing bacteria were found to be more resistant towards various antibiotics. The most effective drugs against E. coli ESBL isolates were imipenem (99.54 %), ampicillin-sulbactam (97.48 %), piperacillin-tazobactam (96.86 %), fosfomycin (94.51 %), amikacin (92.26 %) and nitrofurantoin (90.68 %). The most effective drugs against K. pneumoniae ESBL isolates were imipenem (97.62 %), piperacillin-tazobactam (95.35 %), ampicillin-sulbactam (90.48 %) and amikacin (88.37 %). The antibiotics having the highest resistance, particularly by the ESBL producers were amoxicillin clavulanic acid, sulphamethoxalzole/ trimethoprim, cefuroxime, cefpirome, ceftriaxone and ciprofloxacin. Most of the isolates showed multi drug resistance (MDR). High frequency of ESBL producing E. coli and K. pneumoniae were observed as compared to previous data. Penicillins, cephalosporins and some representatives of fluoroquinolones were least effective against the common UTIs and are recommended to be removed from the line of treatment.
- Publikační typ
- časopisecké články MeSH
