scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Characterization of a Novel Lytic Protein Encoded by the Bacillus cereus E33L GeneampDas a Bacillus anthracis Antimicrobial Protein

2012 ◽  
Vol 78 (8) ◽  
pp. 3025-3027 ◽  
Author(s):  
Feliza A. Bourguet ◽  
Brian E. Souza ◽  
Angela K. Hinz ◽  
Matthew A. Coleman ◽  
Paul J. Jackson
Keyword(s):  
Bacillus Cereus ◽  
Bacillus Anthracis ◽  
Antimicrobial Protein ◽  
Cell Wall Biosynthesis ◽  
Bacterial Genomes ◽  
Content Type ◽  
Highly Active ◽  
Low Concentrations

ABSTRACTLytic proteins encoded by bacterial genomes have been implicated in cell wall biosynthesis and recycling. TheBacillus cereusE33LampDgene encodes a putativeN-acetylmuramoyl-l-alanine amidase. This gene, expressedin vitro, produced a very stable, highly active lytic protein. Very low concentrations rapidly and efficiently lyse vegetativeBacillus anthraciscells.

scholarly journals A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins

2015 ◽  
Vol 82 (4) ◽  
pp. 1196-1204 ◽  
Author(s):  
Jonas Wick ◽  
Daniel Heine ◽  
Gerald Lackner ◽  
Mathias Misiek ◽  
James Tauber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Basis ◽  
Carrier Protein ◽  
Fungal Enzymes ◽  
Content Type ◽  
Acceptor Substrate ◽  
Armillaria Mellea ◽  
Product Assembly

ABSTRACTThe basidiomycetous tree pathogenArmillaria mellea(honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemicalin vitrocharacterization of five flavin-dependentA. melleaenzymes (ArmH1 to ArmH5) that were heterologously produced inEscherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

scholarly journals Characterization of a Mutant Escherichia coli Heat-Labile Toxin, LT(R192G/L211A), as a Safe and Effective Oral Adjuvant

2011 ◽  
Vol 18 (4) ◽  
pp. 546-551 ◽  
Author(s):  
Elizabeth B. Norton ◽  
Louise B. Lawson ◽  
Lucy C. Freytag ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Oral Vaccines ◽  
Content Type ◽  
Intracellular Degradation ◽  
Heat Labile ◽  
Increased Sensitivity

ABSTRACTDespite the fact that the adjuvant properties of the heat-labile enterotoxins ofEscherichia coli(LT) andVibrio cholerae(CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies.In vitroandin vivodata suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects.

scholarly journals High-Level Formation of Active Pseudomonas cepaciaLipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in Escherichia coli and Rapid In Vitro Refolding

1999 ◽  
Vol 65 (2) ◽  
pp. 787-794 ◽  
Author(s):  
Dinh Thi Quyen ◽  
Claudia Schmidt-Dannert ◽  
Rolf D. Schmid
Keyword(s):  
Escherichia Coli ◽  
Burkholderia Cepacia ◽  
Specific Activity ◽  
Gc Content ◽  
Pseudomonas Cepacia ◽  
Membrane Anchor ◽  
E Coli ◽  
Highly Active ◽  
High Level

ABSTRACT The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λPRL promoter. However, no overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5′ region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5′ region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E. coli. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed inE. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.

scholarly journals Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

2013 ◽  
Vol 79 (6) ◽  
pp. 1948-1955 ◽  
Author(s):  
Nicholas M. Thomson ◽  
Azusa Saika ◽  
Kazunori Ushimaru ◽  
Smith Sangiambut ◽  
Takeharu Tsuge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Chaperones ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Efficient Production ◽  
Type I ◽  
Content Type ◽  
Molecular Weights ◽  
Polyhydroxyalkanoate Synthase ◽  
Potential Use

ABSTRACTThe type I polyhydroxyalkanoate synthase fromCupriavidus necatorwas heterologously expressed inEscherichia coliwith simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter−1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter−1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.

scholarly journals Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics

2016 ◽  
Vol 60 (11) ◽  
pp. 6867-6871 ◽  
Author(s):  
Peng Cui ◽  
Hongxia Niu ◽  
Wanliang Shi ◽  
Shuo Zhang ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Enhancement Effect ◽  
Bacterial Cells ◽  
Sulfa Drugs ◽  
Content Type ◽  
Bacterial Membranes ◽  
Highly Active ◽  
High Concentrations

ABSTRACTPersisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active againstEscherichia colipersisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect onE. colipersisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugsin vitro. The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

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