scholarly journals Effect of Chitosan on Tissue Maceration and Enzyme Production by Erwinia carotovora in Potato

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 512A-512 ◽  
Author(s):  
M.V. Bhaskara Reddy ◽  
Alain Asselin ◽  
Joseph Arul
Keyword(s):  
Wide Spectrum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Erwinia Carotovora ◽  
Soft Rot ◽  
Reaction Products ◽  
Potato Tissue ◽  
Chitosan Concentration ◽  
Crude Extracts ◽  
Tissue Maceration

We have investigated the relationship between chitosan treatments and maceration of potato tissue by macerating enzymes secreted by Erwinia carotovora causal agent of soft rot of potato. Erwinia isolated from potato showing soft rot symptoms was used for inoculation. The bacteria secreted a wide spectrum of enzymes that degraded potato cell walls. Polygalacturonase (PG), pectate lyase (PL), pectinmethylesterase (PME), cellulase, xylanase, and protease showed the highest activity in potato tissue inoculated with the pathogen. Accordingly increased maceration and cell death were observed. On the other hand, in chitosan-treated tissue and challenged with the pathogen, significant decrease in enzymatic activity and tissue maceration were observed, more so with increasing chitosan concentration. This observation confirmed that chitosan interfered with multiplication and pathogenic powers of the bacteria, thereby improving cell texture and viability. Crude extracts obtained from treatments were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess pectinase activity. The electophoretic profiles showed significant lytic zone of pectin degradation in the control, which decreased with increase in chitosan concentrations. No lytic zone was observed at 8 mg·ml–1 chitosan concentration and was comparable to intact activity in untreated potato tissue. Pectic enzyme reaction products were analyzed to see the action pattern of pectinases in the crude extracts. Cellulose choromatographic profiles revealed monomers and dimers of polygalacturonic acid up to 6 mg·ml–1 chitosan concentrations. The results suggest that chitosan significantly inhibits bacterial growth and the production of macerating enzymes by the pathogen and thus chitosan can be a potential anti-bacterial agent.

scholarly journals Purification and Characterization of Chitosanase from Bacillus sp. Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides

2004 ◽  
Vol 70 (8) ◽  
pp. 4522-4531 ◽  
Author(s):  
Yeon Jin Choi ◽  
Eun Jung Kim ◽  
Zhe Piao ◽  
Young Chul Yun ◽  
Yong Chul Shin
Keyword(s):  
Liquid Chromatography ◽  
Glycoside Hydrolase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Glycoside Hydrolase Family ◽  
Reaction Products ◽  
Enzymatic Production ◽  
Chitosan Oligosaccharides ◽  
Hydrolase Family

ABSTRACT For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.

scholarly journals Simultaneous Ultrasound and Heat Enhance Functional Properties of Glycosylated Lactoferrin

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5774
Author(s):  
Zhipeng Li ◽  
Dexue Ma ◽  
Yiyang He ◽  
Siqi Guo ◽  
Fuguo Liu ◽  
...  
Keyword(s):  
Functional Properties ◽  
Maillard Reaction ◽  
Spatial Configuration ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Hydrophobicity ◽  
Orthogonal Experimental Design ◽  
Maillard Reaction Products ◽  
Reaction Products ◽  
Ultrasound Assisted

Protein-polysaccharide covalent complexes exhibit better physicochemical and functional properties than single protein or polysaccharide. To promote the formation of the covalent complex from lactoferrin (LF) and beet pectin (BP), we enhanced the Maillard reaction between LF and BP by using an ultrasound-assisted treatment and studied the structure and functional properties of the resulting product. The reaction conditions were optimized by an orthogonal experimental design, and the highest grafting degree of 55.36% was obtained by ultrasonic treatment at 300 W for 20 min and at LF concentration of 20 g/L and BP concentration of 9 g/L. The formation of LF-BP conjugates was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy. Ultrasound-assisted treatment can increase the surface hydrophobicity, browning index, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radicals scavenging activity of LF due to the changes in the spatial configuration and formation of Maillard reaction products. The thermal stability, antioxidant activity and emulsifying property of LF were significantly improved after combining with BP. These findings reveal the potential application of modified proteins by ultrasonic and heat treatment.

scholarly journals Characterization of Proteins in the Outer Membrane Preparation of a Murine Pathogen, Helicobacter bilis

2001 ◽  
Vol 69 (5) ◽  
pp. 3502-3506 ◽  
Author(s):  
Zhongming Ge ◽  
Peter Doig ◽  
James G. Fox
Keyword(s):  
Outer Membrane ◽  
Wide Spectrum ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Membrane Preparation ◽  
Laboratory Animals ◽  
Helicobacter Bilis ◽  
H Pylori

ABSTRACT Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals. In this study, proteins present in the outer membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen. All four H. bilis strains had similar OMP protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins fromH. bilis and H. pylori have little cross-reactivity, except for their flagellins. Nine major immunogenic polypeptides were present in the H. bilis OMPs. By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is distinct from those of H. pylori.

scholarly journals Purification and Characterization of a Novel Bacteriocin Produced by Enterococcus faecalis Strain RJ-11

2003 ◽  
Vol 69 (10) ◽  
pp. 5746-5753 ◽  
Author(s):  
Yukio Yamamoto ◽  
Yoshikazu Togawa ◽  
Makoto Shimosaka ◽  
Mitsuo Okazaki
Keyword(s):  
Enterococcus Faecalis ◽  
Proteolytic Enzymes ◽  
Wide Spectrum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Culture Fluid ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Heat Stable ◽  
Alkaline Conditions

ABSTRACT Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.

Limited proteolysis of rabbit cardiac procathepsin D in a cell-free system

1986 ◽  
Vol 250 (4) ◽  
pp. C589-C596 ◽  
Author(s):  
A. M. Smarel ◽  
S. W. Worobec ◽  
A. G. Ferguson ◽  
R. S. Decker ◽  
M. Lesch
Keyword(s):  
Cathepsin B ◽  
Cathepsin D ◽  
Intracellular Transport ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Cysteine Proteases ◽  
Proteolytic Processing ◽  
Reaction Products ◽  
Free System

Rabbit cardiac cathepsin D is initially synthesized as an inactive, apparent molecular weight (Mr) 53,000, pI 6.6 precursor (procathepsin D) that is proteolytically processed during intracellular transport to produce the Mr 48,000 isoforms of active cathepsin D found in cardiac lysosomes. To examine potential proteases responsible for intracellular proteolytic processing, biosynthetically labeled procathepsin D was isolated from rabbit ventricular tissue perfused for 30 min with [35S]methionine. Procathepsin D was then incubated in vitro (40 degrees C, 1-240 min) with active cathepsin D, papain, and cathepsin B, either singly or sequentially, and the reaction products analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Incubation of 35S-labeled procathepsin D with active cathepsin D produced a single reaction product (Mr 51,000; pI 6.2). This limited proteolysis occurred at pH 3-5 and was inhibited by pepstatin. Incubation of 35S-labeled procathepsin D with papain or cathepsin B produced a major reaction product (Mr 48,000; pI 6.4) and a minor form (Mr 50,000; pI 6.0). These reactions occurred at pH 4-7 and were inhibited by leupeptin but not pepstatin. Only the Mr 48,000, pI 6.4 products of papain and cathepsin B-mediated proteolysis comigrated with the most basic isoform of active cathepsin D found in cardiac tissue. In addition, the Mr 51,000 intermediate produced by cathepsin D was susceptible to further limited proteolysis by cysteine proteases with resultant production of a Mr 48,000 product. Thus the intracellular proteolytic processing of rabbit cardiac procathepsin D does not result solely from autocatalysis but requires at least one other protease, possibly cathepsin B.

scholarly journals The location of arabinosyl:hydroxyproline transferase in the membrane system of potato tissue culture cells

1981 ◽  
Vol 195 (3) ◽  
pp. 661-667 ◽  
Author(s):  
R J Owens ◽  
D H Northcote
Keyword(s):  
Tissue Culture ◽  
Cell Walls ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chromatographic Behavior ◽  
Potato Tissue ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Discontinuous Sucrose Gradient ◽  
Potato Lectin

Incubation of a particulate preparation from potato tissue culture cells with UDP-beta-L-[1-3H] arabinose yielded a glycoprotein fraction containing labelled material with the characteristics of hydroxyproline arabinosides. The sugar-protein linkage was resistant to hot alkaline hydrolysis, and the hydrolytic products showed similar electrophoretic and chromatographic behavior to authentic hydroxyproline-arabinosides prepared from potato tissue culture cell walls. Incorporation of arabinose into glycoprotein was stimulated by the addition of de-arabinosylated potato lectin. The product of the incubation co-migrated with native potato lectin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The subcellular distribution of the arabinosyl-transferase was investigated by fractionating potato tissue culture membranes on a discontinuous sucrose gradient in the presence or absence of Mg2+. Under both fractionation conditions the highest specific activity of the enzyme was found in the Golgi-enriched fraction. The results are discussed in relation to the synthesis of the hydroxy-proline-rich glycoprotein component of plant cell walls.

scholarly journals Involvement of N-acylhomoserine Lactones Throughout Plant Infection by Erwinia carotovora subsp. atroseptica (Pectobacterium atrosepticum)

2004 ◽  
Vol 17 (11) ◽  
pp. 1269-1278 ◽  
Author(s):  
Bruno Smadja ◽  
Xavier Latour ◽  
Denis Faure ◽  
Sylvie Chevalier ◽  
Yves Dessaux ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Erwinia Carotovora ◽  
Soft Rot ◽  
Type I ◽  
Blackleg Disease ◽  
Second Stage ◽  
Plant Infection ◽  
Two Phases ◽  
Tissue Maceration

Erwinia carotovora subsp. atroseptica is responsible for potato blackleg disease in the field and tuber soft rot during crop storage. The process leading to the disease occurs in two phases: a primary invasion step followed by a maceration step. Bacteria-to-bacteria communication is associated with a quorum-sensing (QS) process based on the production of N-acylhomoserine lactones (HSL). The role of HSL throughout plant infection was analyzed. To this purpose, HSL produced by a specific E. carotovora subsp. atroseptica wild-type strain, which was particularly virulent on potato, were identified. A derivative of this strain that expressed an HSL lactonase gene and produced low amounts of HSL was generated. The comparison of these strains allowed the evaluation of the role of HSL and QS in disease establishment and development. Bacterial growth and motility; activity of proteins secreted by type I, II, and III systems; and hypersensitive and maceration reactions were evaluated. Results indicated that HSL production and QS regulate only those traits involved in the second stage of the host plant infection (i.e., tissue maceration) and hypersensitive response in nonhost tobacco plants. Therefore, the use of QS quenching strategies for biological control in E. carotovora subsp. atroseptica cannot prevent initial infection and multiplication of this pathogen.

scholarly journals An Exo-Polygalacturonase Pgc4 Regulates Aerial Hyphal Growth and Virulence in Fusarium oxysporum f. sp. cubense race 4

2020 ◽  
Vol 21 (16) ◽  
pp. 5886
Author(s):  
Zhangyong Dong ◽  
Mei Luo ◽  
Zhenzhong Wang
Keyword(s):  
Fusarium Oxysporum ◽  
Molecular Mechanisms ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Hyphal Growth ◽  
Its Sequence Analysis ◽  
Tissue Maceration ◽  
Race 4

Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 kDa based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis. We further performed its sequence analysis and biochemical characterization. The two pgc4 genes encoding Pgc4 from Foc4 and Foc1 were 1434 bp in length and encoded 477 amino acids with differences, due to some nucleotide differences between the two. The Km and Vmax values of Pgc4 purified from Foc4 were determined to be 0.45 mg/mL and 105.26 Units·mg·protein−1 ·min−1, respectively. The recombinant proteins, r-Foc1-Pgc4 and r-Foc4-Pgc4, were expressed and purified from Pichia pastoris and showed optimal Pgc4 activity at 55 °C and pH 4.0; both could induce tissue maceration and necrosis in the “Guangfen-1” and “Baxi” varieties of banana but to a different extent. Phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Therefore, our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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