scholarly journals Biochemical Characterization of TEM-92 Extended-Spectrum β-Lactamase, a Protein Differing from TEM-52 in the Signal Peptide

2002 ◽  
Vol 46 (12) ◽  
pp. 3981-3983 ◽  
Author(s):  
Mariagrazia Perilli ◽  
Bernardetta Segatore ◽  
Maria Rosaria De Massis ◽  
Laura Pagani ◽  
Francesco Luzzaro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Signal Peptide ◽  
Proteus Mirabilis ◽  
Biochemical Characterization ◽  
Extended Spectrum ◽  
Penicillanic Acid ◽  
Hydrolysis Of

ABSTRACT A bla TEM-92 gene was cloned from a Proteus mirabilis isolate and expressed in Escherichia coli. Production of the enzyme caused reduction of susceptibility to penicillins and narrow- to expanded-spectrum cephalosporins but not to moxalactam and cephamycins. Determination of kinetic parameters with the purified enzyme revealed hydrolysis of expanded-spectrum cephalosporins, while cephamycins, moxalactam, and aztreonam were very poorly or not hydrolyzed. Clavulanate and penicillanic acid sulfones acylated TEM-92 slowly, and deacylation occurred at measurable rates.

scholarly journals Overproduction and Biochemical Characterization of the Chryseobacterium meningosepticum BlaB Metallo-β-Lactamase

2002 ◽  
Vol 46 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Sandrine Vessillier ◽  
Jean-Denis Docquier ◽  
Sandrine Rival ◽  
Jean-Marie Frere ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Kinetic Parameters ◽  
Culture Supernatant ◽  
Biochemical Characterization ◽  
Expression System ◽  
T7 Promoter ◽  
Hydrolysis Of

ABSTRACT The BlaB metallo-β-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k cat/Km ratios of >106 M−1 s−1) toward most penam and carbapenem compounds, with the exception of the 6-α-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

scholarly journals Biochemical Characterization of Laboratory Mutants of Extended-Spectrum β-Lactamase TEM-60

2004 ◽  
Vol 48 (9) ◽  
pp. 3579-3582 ◽  
Author(s):  
Bibiana Caporale ◽  
Nicola Franceschini ◽  
Mariagrazia Perilli ◽  
Bernardetta Segatore ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Extended Spectrum

ABSTRACT Three mutants of the extended-spectrum β-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

scholarly journals Characterization of a New Extended-Spectrum β-Lactamase (TEM-87) Isolated in Proteus mirabilis during an Italian Survey

2002 ◽  
Vol 46 (3) ◽  
pp. 925-928 ◽  
Author(s):  
Mariagrazia Perilli ◽  
Bernardetta Segatore ◽  
Maria Rosaria De Massis ◽  
Nicola Franceschini ◽  
Ciro Bianchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Analysis ◽  
Proteus Mirabilis ◽  
Host Susceptibility ◽  
Italian Survey ◽  
Italian Hospital ◽  
Extended Spectrum ◽  
Spectrum Activity

ABSTRACT A new natural TEM derivative, named TEM-87, was identified in a Proteus mirabilis isolate from an Italian hospital. Compared to TEM-1, TEM-87 contains the following mutations: E104K, R164C, and M182T. Kinetic analysis of TEM-87 revealed extended-spectrum activity against oxyimino cephalosporins (preferentially ceftazidime) and aztreonam. Expression of bla TEM-87 in Escherichia coli decreased the host susceptibility to these drugs.

Characterization of the extended-spectrum b-lactamases and determination of the virulence factors of uropathogenic Escherichia coli strains isolated from children

2012 ◽  
Vol 124 (15-16) ◽  
pp. 504-515 ◽  
Author(s):  
Branka Bedenić ◽  
Jasmina Vraneš ◽  
Sabine Hofmann-Thiel ◽  
Marija Tonkić ◽  
Anita Novak ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Uropathogenic Escherichia Coli ◽  
Extended Spectrum

scholarly journals Purification and Biochemical Characterization of IMP-13 Metallo-β-Lactamase

2010 ◽  
Vol 55 (1) ◽  
pp. 399-401 ◽  
Author(s):  
Gisela Santella ◽  
Jean-Denis Docquier ◽  
Gabriel Gutkind ◽  
Gian Maria Rossolini ◽  
Marcela Radice
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Chromatography Analysis ◽  
Marked Preference

ABSTRACTThe IMP-13 metallo-β-lactamase was overproduced inEscherichia coliBL21(DE3) and purified by chromatography. Analysis of kinetic parameters revealed some notable differences with other IMP-type enzymes, noteworthily a higher catalytic efficiency toward ticarcillin and piperacillin and a marked preference for imipenem over meropenem.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Isolation and characterization of pathogenic Escherichia coli bacteriophages from chicken and beef offal

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Celosia Lukman ◽  
Christopher Yonathan ◽  
Stella Magdalena ◽  
Diana Elizabeth Waturangi
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
High Efficiency ◽  
Multiplicity Of Infection ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Pathogenic Escherichia Coli ◽  
Family Based

Abstract Objective This study was conducted to isolate and characterize lytic bacteriophages for pathogenic Escherichia coli from chicken and beef offal, and analyze their capability as biocontrol for several foodborne pathogens. Methods done in this research are bacteriophage isolation, purification, titer determination, application, determination of host range and minimum multiplicity of infection (miMOI), and bacteriophage morphology. Results Six bacteriophages successfully isolated from chicken and beef offal using EPEC and EHEC as host strain. Bacteriophage titers observed between 109 and 1010 PFU mL−1. CS EPEC and BL EHEC bacteriophage showed high efficiency in reduction of EPEC or EHEC contamination in meat about 99.20% and 99.04%. The lowest miMOI was 0.01 showed by CS EPEC bacteriophage. CI EPEC and BL EPEC bacteriophage suspected as Myoviridae family based on its micrograph from Transmission Electron Microscopy (TEM). Refers to their activity, bacteriophages isolated in this study have a great potential to be used as biocontrol against several foodborne pathogens.

Sign in / Sign up

Export Citation Format

Share Document