Quantitative Turbidimetric Assay of Enzymatic Gram-Negative Bacteria Lysis

Левашов, П. А.; Sedov, Sergey Alekseevich; Shipovskov, Stepan; Belogurova, N. G.; Levashov, Andrey V.

doi:10.1021/ac902978u

Cited by 45 publications

(27 citation statements)

References 25 publications

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“…This fact may be explained by the presence of hydrophobic regions on the protein surface, which leads to the nonproductive sorption of the enzyme on substrate cells as the ionic strength increases. In the case of M34EM, the fall in the lysis rate with increas ing ionic strength of buffer is insignificant, and the picture is on the whole similar to that observed earlier for chicken egg lysozyme [7,9].…”

Section: Resultssupporting

confidence: 84%

“…Bacteriolytic activity of protein factors in fractions after the chromatographic separation was determined by the turbidimetric method at 650 nm at 37°C [7,9,11] using E. coli, M. luteus, and B. subtilis cells as sub strates. The activity is expressed in conventional units (c.u.)…”

Section: Resultsmentioning

confidence: 99%

“…The sources of bacteriolytic enzymes are animals, plants, fungi, microorganisms, and bacteriophages [1][2][3][4][5][6]. Today there are a great many publications devoted to bacteriolytic enzymes [1,[4][5][6][7], which is explained by their wide application in genetic engineering, medicine, and various branches of industry. The recent outbreak of infectious diseases in Europe has shown the urgency of developing alternative methods of diagnosis of, and therapy for, diseases of various etiology, in particular, diseases caused by gram negative microorganisms [8].…”

Section: Introductionmentioning

confidence: 99%

“…This substantially restricts the possibilities for studying the 1 properties of enzymes able to lyse bacterial cells. In recent years, novel approaches to the description of these complex systems have been developed [7,[9][10][11], which opens up further opportunities for the detection and characterization of these enzymes in living sys tems, in particular, the blood plasma of animals. Detailed investigations of the properties of bacteri olytic factors of animals are of current interest for understanding various aspects of the functioning of the immune system and the design of novel drugs.…”

Section: Introductionmentioning

confidence: 99%

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Bacteriolytic enzymes from the blood plasma of the sheep

et al. 2012

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In the present work the studies ofbacteriolytic factors from sheep blood plasma have been performed. Three novel enzymes have been identified and characterized. Two of them have a molecular weight 15 +/- 2 kDa and able to lyse the gram-negative Escherichia coli bacteria. The third enzyme has a molecular weight 34 +/- 4 kDa and is able to lyse both gram-negative Escherichia coli and gram-positive Micrococcus luteus bacteria. The bacteriolytic reactions have been studied for all three enzymes; particularly, pH-optima have been identified with respect to the substrate. To identify the enzymes trypsinolysis and consequent MALDI-TOF mass spectrometry studies were performed. The results were compared to data from publicly available databases, such as Swiss-Prot, NCBI, MSDB.

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bacteriolytic enzymes from the blood plasma of the sheep

et al. 2012

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“…Detection of bacterial lysis can be achieved by turbidimetric method (Gorin, Wang, & Papapavlou, 1971;Lee & Yang, 2002;Levashov, Sedov, Shipovskov, Belogurova, & Levashov, 2010;Shuga, 1952;Zhao, Zhang, & Yang, 2002), agar diffusion method (Modeer & Soder, 1971;Qin & Peng, 1998), fluorometric method (Hannig, Spitzmüller, & Hannig, 2009;Helal & Melzig, 2008), Resonance scattering spectra method (Jiang & Huang, 2007), and agarose rocket electrophoresis method (Chen, Wang, Zhou, Zhong, & Liu, 1988). However, it should be noted that bacterial lysis is a complex reaction that is mediated by both bacterial enzymes and lysozyme.…”

Section: Introductionmentioning

confidence: 99%

Quantitative determination of chitinolytic activity of lysozyme using half-deacetylated chitosan as a substrate

Zhang

Wang

Zhang

et al. 2011

Carbohydrate Polymers

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The bacteriolytic activity of native and covalently immobilized lysozyme against Gram‐positive and Gram‐negative bacteria is differentially affected by charged amino acids and glycine

et al. 2019

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The emergence of new antibiotic‐resistant bacterial strains means it is increasingly important to find alternatives to traditional antibiotics, such as bacteriolytic enzymes. The bacteriolytic enzyme lysozyme is widely used in medicine as an antimicrobial agent, and covalent immobilization of lysozyme can expand its range of possible applications. However, information on the effect of such immobilized preparations on whole bacterial cells is quite limited. Here, we demonstrate the differential effects of glycine and charged (basic and acidic) amino acids on the enzymatic lysis of Gram‐positive and Gram‐negative bacteria by soluble and immobilized lysozyme. Glycine and basic amino acids (histidine, lysine, and arginine) significantly increase the rate of lysis of Gram‐negative Escherichia coli cells in the presence of soluble lysozyme, but they do not substantially affect the rate of enzymatic lysis of Gram‐positive Micrococcus luteus . Glutamate and aspartate significantly enhance enzymatic lysis of both E. coli and M. luteus . When using immobilized lysozyme, the effects of amino acids on the rate of cell lysis are significantly reduced. For immobilized lysozyme, the presence of an external diffusion mode on cell lysis kinetics at bacterial concentrations below 4 × 10 8 colony‐forming units·mL −1 was shown. The broadening of the pH optimum of lysozyme activity after immobilization has been demonstrated for both Gram‐positive and Gram‐negative bacteria. The Michaelis constant ( K m ) values of immobilized lysozyme were increased by 1.5‐fold for E. coli cell lysis and 4.6‐fold for M. luteus cell lysis compared to soluble enzyme. A greater understanding of the effect of amino acids on the activity of native and immobilized lysozyme is important for both the development of new materials for medical purposes and elucidating the interaction of lysozyme with bacterial cells. Of particular interest is our finding that lysozyme activity against Gram‐negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations.

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Quantitative Turbidimetric Assay of Enzymatic Gram-Negative Bacteria Lysis

Cited by 45 publications

References 25 publications

Bacteriolytic enzymes from the blood plasma of the sheep

Bacteriolytic enzymes from the blood plasma of the sheep

Quantitative determination of chitinolytic activity of lysozyme using half-deacetylated chitosan as a substrate

The bacteriolytic activity of native and covalently immobilized lysozyme against Gram‐positive and Gram‐negative bacteria is differentially affected by charged amino acids and glycine

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