Preparation and Characterization of Single-Enzyme Nanogels

2011 ◽  
pp. 119-130 ◽  
Author(s):  
Jun Ge ◽  
Ming Yan ◽  
Diannan Lu ◽  
Zhixia Liu ◽  
Zheng Liu
Keyword(s):  
Single Enzyme

scholarly journals Spurious regulatory connections dictate the expression-fitness landscape of translation termination factors

2020 ◽  
Author(s):  
Jean-Benoît Lalanne ◽  
Darren J. Parker ◽  
Gene-Wei Li
Keyword(s):  
Fitness Landscape ◽  
Stop Codon ◽  
Translation Termination ◽  
Mass Conservation ◽  
List Type ◽  
Master Regulators ◽  
Peptide Release ◽  
The Impact ◽  
Single Enzyme

AbstractDuring steady-state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in bacterium Bacillus subtilis. While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom-up models of expression-fitness landscapes will require near-exhaustive characterization of parts.HighlightsPrecision measurements enable multiscale expression-to-fitness mapping.RF depletion leads to imbalanced translation for co-transcribed gene pairs.Imbalanced translation induces unintended regulons to the detriment of cell fitness.Swapping a single stop codon rewires global susceptibility to RF perturbation.

scholarly journals EVIDENCE FOR THE INVOLVEMENT OF SERINE TRANSHYDROXYMETHYLASE IN SERINE AND GLYCINE INTERCONVERSIONS IN SALMONELLA TYPHIMURIUM

Genetics ◽  
1974 ◽  
Vol 77 (2) ◽  
pp. 185-198
Author(s):  
George V Stauffer ◽  
Jean E Brenchley
Keyword(s):  
Salmonella Typhimurium ◽  
Isolation And Characterization ◽  
Single Enzyme

ABSTRACT Salmonella typhimurium can normally use glycine as a serine source to support the growth of serine auxotrophs. This reaction was presumed to occur by the reversible activity of the cnzyme, serine transhydroxymethylase (E. C. 2. 1. 2. 1; L-serine: tetrahydrofolic-5, 10 transhydroxymethylase), which is responsible for glycine biosynthesis. However, this enzyme had not been demonstrated to be solely capable of synthesizing serine from glycine in vivo. The isolation and characterization of a mutant able to convert serine to glycine but unable to convert glycine to serine supports the conclusion that a single enzyme is involved in this reversible interconversion of serine and glycine. The mutation conferring this phenotype was mapped with other mutations affecting serine transhydroxymethylase (glyA) and assays demonstrated reduced activities of this enzyme in the mutant.

Purification and characterization of an extracellular exopolygalacturonase from Geotrichum lactis

1991 ◽  
Vol 37 (12) ◽  
pp. 974-977 ◽  
Author(s):  
C. Pardo ◽  
M. A. Lapeña ◽  
M. Gacto
Keyword(s):  
Molecular Weight ◽  
Galacturonic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Pectic Enzymes ◽  
Ammonium Sulphate Precipitation ◽  
Polygalacturonase Activity ◽  
Single Enzyme

Geotrichum lactis ATCC 48590 produced extracellular polygalacturonase (EC 3.2.1.67) in media containing pectate, pectin, or galacturonic acid as inducers. The synthesis of the enzyme was strongly repressed by glucose. The polygalacturonase was purified 80-fold by ammonium sulphate precipitation, Sephadex G-100 filtration, and DEAE Sephadex ion-exchange chromatography. Polyacrylamide gel electrophoresis with copolymerized substrate indicated that the isolated material was a single enzyme with polygalacturonase activity. The main product of enzyme action was galacturonic acid. The enzyme shows a molecular weight close to 53 000 by gel filtration and degrades preferentially de-esterified substrates. Km values for pectate and pectin (64% esterified) were 0.09 and 0.49 mg mL−1, respectively. The optimum pH for enzyme activity was 5.0, while the optimum temperature was 40 °C. The polygalacturonase was precipitated by concanavalin A – Sepharose, and treatment with endoglycosidase H reduced its precipitation by the lectin, suggesting that the enzyme is a glycoprotein. In addition to being found extracellularly, the polygalacturonase is also present in the periplasm of the cells. A different form of the polygalacturonase showing a lower molecular weight was located inside the cells. Key words: polygalacturonase, pectic enzymes, Geotrichum lactis.

Morphological Characterization of Mycobacteriophage R1

1974 ◽  
Vol 32 ◽  
pp. 254-255
Author(s):  
B. L. Soloff ◽  
T. A. Rado
Keyword(s):  
Carbon Source ◽  
Stationary Phase ◽  
Growth Phase ◽  
Minimal Medium ◽  
Morphological Characterization ◽  
Logarithmic Growth Phase ◽  
Complete Lysis ◽  
Lysogenic Culture ◽  
Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

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