scholarly journals Generalized enzymatic mechanism of catalysis by tetrameric l-asparaginases from mesophilic bacteria

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pawel Strzelczyk ◽  
Di Zhang ◽  
Marzena Dyba ◽  
Alexander Wlodawer ◽  
Jacek Lubkowski
Keyword(s):  
Structural Data ◽  
E Coli ◽  
Mesophilic Bacteria ◽  
Enzymatic Mechanism ◽  
Enzyme Intermediates ◽  
Mechanism Of Hydrolysis ◽  
Mechanism Of Catalysis ◽  
Structural Mass Spectrometry ◽  
Covalent Intermediate ◽  
Structural Mass

Abstract The mechanism of catalysis by the l-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of l-Asn by the type II l-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric l-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (l-Asp and l-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by l-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to l-Asn and l-Gln, we postulate that it is common for all these structurally related enzymes.

scholarly journals Аналіз мікробної контамінації туш свиней в процесі забою та первинної обробки

2017 ◽  
Vol 19 (77) ◽  
pp. 194-199
Author(s):  
V.B. Kusturov ◽  
V.V. Kasyanchuk ◽  
A.M. Bergievich
Keyword(s):  
Microbiological Analysis ◽  
E Coli ◽  
Mesophilic Bacteria ◽  
Microbiological Criteria ◽  
Important Pathway ◽  
Control Procedures ◽  
Permissible Levels ◽  
Standard Values ◽  
Animal Feeding ◽  
High Level

The article presents the results of studies on the еxploring of microbial contamination of pigs' carcasses in the pork slaughter and primary processing with microorganisms such as general mesophilic bacteria, Enterobacteriaceae coliforms and E.coli. The carcass surfaces were examined in six technological operations: after bleeding (1) after scalding and removing bristles and hair (2) after singeing and polishing (3); аfter the nutration (4); аt the final point after a veterinary examination (5); аfter cooling down to a temperature of 4–5 °C (6). Sampling swabs was carried out during 2015–2016 years, 530 samples were selected from 260 carcasses accordance with ISO Standard 17604 Microbiology of food and animal feeding stuffs-Carcass sampling for microbiological analysis. Swabs sampling from carcass sites taken from thigh and outer and inner surfaces of the chest and abdominal wall. It was found that the high level of contamination of surfaces carcasses with general mesophilic bacteria, Enterobacteriaceae. They were exceeded after bleeding the microbiological criteria an average of 2.2–2.4 Log CFU/cm2 and 2.5-2.7 Log CFU/cm2, respectively. Each subsequent technological operation reduced the level of contamination of carcass surfaces: the amount of general mesophilic bacteria, Enterobacteriaceae after scalding, removing bristles and hair on the carcass surface, significantly decreased compared to levels that were after bleeding, but were above the standard values by an average of 0.7 Log CFU/Cm2 and by 0.35 Log CFU/cm2, respectively. The number of coliform forms and E. coli on the carcass surface after singeing and polishing was less than after bleeding by an average of 1.8 Log CFU/cm2 and 1.23 Log CFU/cm2, respectively. The intestinal tract is also the an important pathway for contamination of pigs' carcasses. Enterobacteriaceae and E. coli testing demonstrate the effectiveness of slaughter process control procedures and is the indicator for fecal contamination. After cooling of the carcasses, on their surfaces the amount of microorganisms studied was within the permissible levels.

Prevalence of antibiotic-resistant Escherichia coli isolated from urban and agricultural streams in Canterbury, New Zealand

2019 ◽  
Vol 366 (8) ◽  
Author(s):  
Sophie Van Hamelsveld ◽  
Muyiwa E Adewale ◽  
Brigitta Kurenbach ◽  
William Godsoe ◽  
Jon S Harding ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
New Zealand ◽  
Pathogenic Bacteria ◽  
Freshwater Ecosystems ◽  
Urban Setting ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Mesophilic Bacteria ◽  
Resistant Strains ◽  
Population Counts

Abstract Baseline studies are needed to identify environmental reservoirs of non-pathogenic but associating microbiota or pathogenic bacteria that are resistant to antibiotics and to inform safe use of freshwater ecosystems in urban and agricultural settings. Mesophilic bacteria and Escherichia coli were quantified and isolated from water and sediments of two rivers, one in an urban and one in an agricultural area near Christchurch, New Zealand. Resistance of E. coli to one or more of nine different antibiotics was determined. Additionally, selected strains were tested for conjugative transfer of resistances. Despite having similar concentrations of mesophilic bacteria and E. coli, the rivers differed in numbers of antibiotic-resistant E. coli isolates. Fully antibiotic-susceptible and -resistant strains coexist in the two freshwater ecosystems. This study was the first phase of antibiotic resistance profiling in an urban setting and an intensifying dairy agroecosystem. Antibiotic-resistant E. coli may pose different ingestion and contact risks than do susceptible E. coli. This difference cannot be seen in population counts alone. This is an important finding for human health assessments of freshwater systems, particularly where recreational uses occur downstream.

The interaction between the natural metalloendopeptidase inhibitor BJ46a and its target toxin jararhagin analyzed by structural mass spectrometry and molecular modeling

2020 ◽  
Vol 221 ◽  
pp. 103761
Author(s):  
Viviane A. Bastos ◽  
Francisco Gomes-Neto ◽  
Surza Lucia G. Rocha ◽  
André Teixeira-Ferreira ◽  
Jonas Perales ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Modeling ◽  
Structural Mass Spectrometry ◽  
Structural Mass

The growing role of structural mass spectrometry in the discovery and development of therapeutic antibodies

The Analyst ◽  
2018 ◽  
Vol 143 (11) ◽  
pp. 2459-2468 ◽  
Author(s):  
Yuwei Tian ◽  
Brandon T. Ruotolo
Keyword(s):  
Mass Spectrometry ◽  
State Of The Art ◽  
Therapeutic Antibodies ◽  
Critical Importance ◽  
Current State ◽  
Measurement Science ◽  
Structural Mass Spectrometry ◽  
Structural Mass

The comprehensive structural characterization of therapeutic antibodies is of critical importance for the successful discovery and development of such biopharmaceuticals, yet poses many challenges to modern measurement science. Here, we review the current state-of-the-art mass spectrometry technologies focusing on the characterization of antibody-based therapeutics.

scholarly journals Structural mass spectrometry of tissue extracts to distinguish cancerous and non-cancerous breast diseases

2014 ◽  
Vol 10 (11) ◽  
pp. 2827-2837 ◽  
Author(s):  
Kelly M. Hines ◽  
Billy R. Ballard ◽  
Dana R. Marshall ◽  
John A. McLean
Keyword(s):  
Mass Spectrometry ◽  
Breast Tissue ◽  
Breast Diseases ◽  
Molecular Signatures ◽  
Human Breast ◽  
Human Breast Tissue ◽  
Tissue Extracts ◽  
Ms Analysis ◽  
Structural Mass Spectrometry ◽  
Structural Mass

UPLC-IM-MS/MS analysis of human breast tissue extracts distinguished cancerous and non-cancerous breast diseases by characteristic molecular signatures.

scholarly journals Structural Mass Spectrometry: Rapid Methods for Separation and Analysis of Peptide Natural Products

2012 ◽  
Vol 75 (1) ◽  
pp. 48-53 ◽  
Author(s):  
Cody R. Goodwin ◽  
Larissa S. Fenn ◽  
Dagmara K. Derewacz ◽  
Brian O. Bachmann ◽  
John A. McLean
Keyword(s):  
Mass Spectrometry ◽  
Natural Products ◽  
Rapid Methods ◽  
Structural Mass Spectrometry ◽  
Structural Mass

scholarly journals Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975

1999 ◽  
Vol 341 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Donna D. SONG ◽  
Nicholas A. JACQUES
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Streptococcus Salivarius ◽  
Acetobacter Diazotrophicus ◽  
E Coli ◽  
Ping Pong ◽  
Mechanism Of Catalysis ◽  
Enzyme Intermediate

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum activity at pH 6.0 and 37 °C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than for the two other enzymes.

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