scholarly journals Single-molecule mechanochemical characterization of E. coli pol III core catalytic activity

2017 ◽  
Vol 26 (7) ◽  
pp. 1413-1426 ◽  
Author(s):  
M. Nabuan Naufer ◽  
David A. Murison ◽  
Ioulia Rouzina ◽  
Penny J. Beuning ◽  
Mark C. Williams
Keyword(s):  
Catalytic Activity ◽  
Single Molecule ◽  
E Coli ◽  
Pol Iii

scholarly journals Single-Molecule Characterization of E. Coli Pol III Core Catalytic Activity

2017 ◽  
Vol 112 (3) ◽  
pp. 514a
Author(s):  
M. Nabuan Naufer ◽  
David A. Murison ◽  
Ioulia Rouzina ◽  
Penny J. Beuning ◽  
Mark C. Williams
Keyword(s):  
Catalytic Activity ◽  
Single Molecule ◽  
E Coli ◽  
Pol Iii

scholarly journals In situ genotyping of a pooled strain library after characterizing complex phenotypes

2017 ◽  
Author(s):  
Michael J. Lawson ◽  
Daniel Camsund ◽  
Jimmy Larsson ◽  
Özden Baltekin ◽  
David Fange ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Detailed Characterization ◽  
E Coli ◽  
Complex Phenotypes

So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strain’s genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

scholarly journals Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

2020 ◽  
Author(s):  
Martin Rieu ◽  
Jessica Valle-Orero ◽  
Bertrand Ducos ◽  
Jean-François Allemand ◽  
Vincent Croquette
Keyword(s):  
Single Molecule ◽  
Functional Characterization ◽  
Nucleic Acid Binding ◽  
Single Molecule Force Spectroscopy ◽  
E Coli ◽  
Natural Function ◽  
Protein Nucleic Acid ◽  
Free Quantification

ABSTRACTFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.

scholarly journals Characterization of a new bifunctional endo-1,4-β-xylanase/esterase found in the rumen metagenome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gabriella Cavazzini Pavarina ◽  
Eliana Gertrudes de Macedo Lemos ◽  
Natália Sarmanho Monteiro Lima ◽  
João Martins Pizauro
Keyword(s):  
Single Molecule ◽  
Production Efficiency ◽  
Animal Feed ◽  
Metagenomic Data ◽  
Synergistic Activity ◽  
E Coli ◽  
Biomass Processing ◽  
Nellore Cattle ◽  
Rumen Metagenome

AbstractMetagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-β-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s−1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-β-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.

scholarly journals Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Martin Rieu ◽  
Jessica Valle-Orero ◽  
Bertrand Ducos ◽  
Jean-François Allemand ◽  
Vincent Croquette
Keyword(s):  
Single Molecule ◽  
Functional Characterization ◽  
Nucleic Acid Binding ◽  
Single Molecule Force Spectroscopy ◽  
E Coli ◽  
Natural Function ◽  
Protein Nucleic Acid ◽  
Free Quantification

AbstractFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

Effect of alteration of translation error rate on enzyme microheterogeneity as assessed by variation in single molecule electrophoretic mobility and catalytic activity

2009 ◽  
Vol 87 (3) ◽  
pp. 517-529 ◽  
Author(s):  
Ellert R. Nichols ◽  
Elnaz Shadabi ◽  
Douglas B. Craig
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Single Molecule ◽  
Hydrodynamic Radius ◽  
No Reduction ◽  
E Coli ◽  
Translation Error ◽  
Enzyme Molecules ◽  
Catalytic Heterogeneity

The role of translation error for Escherichia coli individual β-galactosidase molecule catalytic and electrophoretic heterogeneity was investigated using CE-LIF. An E. coli rpsL mutant with a hyperaccurate translation phenotype produced enzyme molecules that exhibited significantly less catalytic heterogeneity but no reduction of electrophoretic heterogeneity. Enzyme expressed with streptomycin-induced translation error had increased thermolability, lower activity, and no significant change to catalytic or electrophoretic heterogeneity. Modeling of the electrophoretic behaviour of β-galactosidase suggested that variation of the hydrodynamic radius may be the most significant contributor to electrophoretic heterogeneity.

Characterization of two plasmid-encoded cefotaximases found in clinical Escherichia coli isolates: CTX-M-65 and a novel enzyme, CTX-M-87

2009 ◽  
Vol 58 (6) ◽  
pp. 811-815 ◽  
Author(s):  
Jun Yin ◽  
Jun Cheng ◽  
Zhen Sun ◽  
Ying Ye ◽  
Yu-Feng Gao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Hydrolytic Activity ◽  
E Coli ◽  
High Affinity ◽  
Extended Spectrum ◽  
M 14

Three clinical strains of Escherichia coli (p168, p517 and p667) were collected in 2006 from three hospitals in Anhui Province (China). PCR and DNA sequencing revealed that E. coli p168 carried a novel extended-spectrum β-lactamase (ESBL), which was designated CTX-M-87. The extended-spectrum β-lactamase which was carried by E. coli p517 and E. coli p667 was previously named CTX-M-65. The deduced amino acid sequence of CTX-M-87, with pI 9.1, differed from that of CTX-M-14 by the substitutions Ala77→Val and Pro167→Leu. Like CTX-M-14, CTX-M-87 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and had high affinity for cefuroxime and cefotaxime. These data show that mutations at position 167 in CTX-M do not always affect catalytic activity and substrate preference.

Characterization of a new esterase for enantioselective resolution of (R, S)-methyl mandelate

2019 ◽  
Vol 9 (9) ◽  
pp. 1112-1119
Author(s):  
Haiwei Xie ◽  
Yongzhi Chen ◽  
Dun Deng
Keyword(s):  
Catalytic Activity ◽  
High Activity ◽  
Optical Purity ◽  
E Coli ◽  
Enantioselective Resolution ◽  
Esterase Gene ◽  
Biotechnology Application

A new esterase gene (EST35) was cloned from Dactylosporangium sp. BB08 and expressed in E. coli BL21 (DE3). Optimum catalytic activity of EST35 was at 30 C and it could be activated at 0 °C. EST35 remained high activity in 20% (V/V) cyclohexane, hexane, heptane, methanol and DMSO. Interestingly the enzyme exhibits good enantioselectivity towards (R, S)-methyl mandelate leaving with an optical purity of 97% (R)-methyl mandelate and make EST35 a promising enzyme for biotechnology application.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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