scholarly journals Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

2004 ◽  
Vol 70 (12) ◽  
pp. 7311-7320 ◽  
Author(s):  
Judit Marokházi ◽  
Katalin Lengyel ◽  
Szilvia Pekár ◽  
Gabriella Felföldi ◽  
András Patthy ◽  
...  
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secondary Phase ◽  
Activity Measurement ◽  
Intracellular Enzyme ◽  
Thimet Oligopeptidase ◽  
Proteolytic Activities ◽  
Reduced Activity ◽  
Phase Variants

ABSTRACT Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (∼74, ∼55, ∼54, and ∼37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.

Use of proteolytic enzymes as an additional tool for trypanosomatid identification

Parasitology ◽  
2004 ◽  
Vol 130 (1) ◽  
pp. 79-88 ◽  
Author(s):  
A. L. S. SANTOS ◽  
C. M. ABREU ◽  
C. S. ALVIANO ◽  
R. M. A. SOARES
Keyword(s):  
Proteinase Inhibitors ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Pattern ◽  
Extracellular Medium ◽  
Additional Tool ◽  
Potential Marker ◽  
Sds Page ◽  
Proteolytic Activities

The expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants. We have comparatively analysed the proteolytic profiles of 19 monoxenous trypanosomatids (Herpetomonas anglusteri,H. samuelpessoai,H. mariadeanei,H. roitmani,H. muscarum ingenoplastis,H. muscarum muscarum,H. megaseliae,H. dendoderi,Herpetomoassp.,Crithidia oncopelti,C. deanei,C. acanthocephali,C. harmosa,C. fasciculata,C. guilhermei,C. luciliae,Blastocrithidia culicis,Leptomonas samueliandLept. seymouri) and 4 heteroxenous flagellates (Phytomonas serpens,P. mcgheei,Trypanosoma cruziandLeishmania amazonensis) byin situdetection of enzyme activities on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE ) containing co-polymerized gelatine as substrate, in association with specific proteinase inhibitors. All 23 trypanosomatids expressed at least 1 acidic proteolytic enzyme. In addition, a characteristic and specific pattern of cell-associated metallo and/or cysteine proteinases was observed, except for the similar profiles detected in 2Herpetomonas(H. anglusteriandH. samuelpessoai) and 3Crithidia(C. fasciculata,C. guilhermeiandC. luciliae) species. However, these flagellates released distinct secretory proteinase profiles into the extracellular medium. These findings strongly suggest that the association of cellular and secretory proteinase pattern could represent a useful marker to help trypanosomatid identification.

scholarly journals Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

2007 ◽  
Vol 73 (7) ◽  
pp. 2247-2250 ◽  
Author(s):  
Sirinat Srionnual ◽  
Fujitoshi Yanagida ◽  
Li-Hsiu Lin ◽  
Kuang-Nan Hsiao ◽  
Yi-sheng Chen
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Proteinase K ◽  
Mass Spectrometry Analysis ◽  
Fermented Fish ◽  
Spectrometry Analysis ◽  
Weissella Cibaria ◽  
Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

Isolation and Preliminary Characterization of a Bacteriocin Produced by Lactobacillus plantarum N014 Isolated from Nham, a Traditional Thai Fermented Pork

2006 ◽  
Vol 69 (8) ◽  
pp. 1937-1943 ◽  
Author(s):  
PONGSAK RATTANACHAIKUNSOPON ◽  
PARICHAT PHUMKHACHORN
Keyword(s):  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Exponential Growth Phase ◽  
Plasmid Isolation ◽  
Inhibitory Spectrum ◽  
Lipase A

Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gram-negative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as α-amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and pH values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was pH-dependent. No bacteriocin adsorption was detected at pH 1 to 3, whereas 100% bacteriocin adsorption was found at pH 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kDa.

Purification and characterization of enterocin MC13 produced by a potential aquaculture probiontEnterococcus faeciumMC13 isolated from the gut ofMugil cephalus

2011 ◽  
Vol 57 (12) ◽  
pp. 993-1001 ◽  
Author(s):  
R. Satish Kumar ◽  
P. Kanmani ◽  
N. Yuvaraj ◽  
K.A. Paari ◽  
V. Pattukumar ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Enterococcus Faecium ◽  
Vibrio Vulnificus ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Probiotic Bacterium ◽  
Mass Spectrometry Analysis ◽  
Native Page ◽  
Spectrometry Analysis

A bacteriocin producer strain MC13 was isolated from the gut of Mugil cephalus (grey mullet) and identified as Enterococcus faecium . The bacteriocin of E. faecium MC13 was purified to homogeneity, as confirmed by Tricine sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE). Reverse-phase high-performance liquid chromatography (HPLC) analysis showed a single active fraction eluted at 26 min, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analysis showed the molecular mass to be 2.148 kDa. The clear zone in native PAGE corresponding to enterocin MC13 band further substantiated its molecular mass. A dialyzed sample (semicrude preparation) of enterocin MC13 was broad spectrum in its action and inhibited important seafood-borne pathogens: Listeria monocytogenes , Vibrio parahaemolyticus , and Vibrio vulnificus . This antibacterial substance was sensitive to proteolytic enzymes: trypsin, protease, and chymotrypsin but insensitive to catalase and lipase, confirming that inhibition was due to the proteinaceous molecule, i.e., bacteriocin, and not due to hydrogen peroxide. Enterocin MC13 tolerated heat treatment (up to 90 °C for 20 min). Enterococcus faecium MC13 was effective in bile salt tolerance, acid tolerance, and adhesion to the HT-29 cell line. These properties reveal the potential of E. faecium MC13 to be a probiotic bacterium. Enterococcus faecium MC13 could be used as potential fish probiotic against pathogens such as V. parahaemolyticus, Vibrio harveyi , and Aeromonas hydrophila in fisheries. Also, this could be a valuable seafood biopreservative against L. monocytogenes.

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.

scholarly journals Production optimization using Plackett-Burman and Box-Behnken designs with partial characterization of amylase from marine actinomycetes

2021 ◽  
Author(s):  
Sarah I Bukhari ◽  
Mohamed H Al-Agamy ◽  
Mahmoud S Kelany ◽  
Mohammad R Al Hazani ◽  
Moaz M Hamed
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Fermentation Medium ◽  
Culture Conditions ◽  
Production Optimization ◽  
Activity Measurement

Abstract Amylase is an industrial enzyme that is used in the food and biofuel industries. We screened four actinomycetes strains for amylase biosynthesis. The Streptomyces rochei strain had a larger hydrolytic zone (24 mm) on starch agar plates, than the other isolates. Plackett-Burman’s experimental design was implemented to optimize the conditions for amylase production by the selected strains. Growth under optimized culture conditions led to 1.7, 9.8, 7.7, and 3.12 -fold increases for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specific activity measurement in comparison with growth under primary conditions. When applying the Box-Behnken design on S. rochei using the most significant parameters starch, K2HPO4, pH, and temperature, there was a 12.22-fold increase in the specific activity measurement: 7.37 U/mg. The optimal fermentation medium formula was kept at 30.6°C for seven days. The amylase from S. rochei was partially purified, and its molecular weight was determined using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was found to be 45, 43, and 53 kDa. Amylase was particularly active at pH 6 and 65°C. The purified enzyme was most active at 65°C and a pH of 6, thermal stability of 70°C for 40 min and salt concentration of 1 M with a Km and Vmax of 6.58 mg/ml and 21.93 mg/ml/min, respectively. The amylase improved by adding Cu + 2, Zn + 2, and Fe + 2 (152.21%, 207.24%, and 111.89%). Increased production of amylase enzyme by Streptomyces rochei KR108310 attracts the production of industrially significant products.

scholarly journals Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 132-140 ◽  
Author(s):  
DP O'Brien ◽  
KM Gale ◽  
JS Anderson ◽  
JH McVey ◽  
GJ Miller ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Vii ◽  
Rabbit Brain ◽  
Bleeding Tendency ◽  
Wild Type ◽  
Undetectable Plasma ◽  
Reduced Activity

Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one- stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.

Isolation and Purification of Colicin ECL 12, a Bacteriocin That Inhibits Strains of Escherichia coli O157:H7†

1997 ◽  
Vol 60 (12) ◽  
pp. 1520-1528 ◽  
Author(s):  
WANDA J. LYON ◽  
DENNIS G. OLSON
Keyword(s):  
Escherichia Coli ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Viable Cell ◽  
Exchange Chromatography ◽  
Isolation And Purification ◽  
E Coli ◽  
Log Reduction ◽  
Cell Counts

A swine fecal isolate, identified as Escherichia coli ECL12, was found to produce an antimicrobial substance designated as colicin ECL12. Colicin ECL12 was inhibitory against 20 strains of E. coli O157:H7 previously isolated from both human and bovine feces. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Colicin ECL12 was sensitive to several proteolytic enzymes. Adsorption of colicin ECL12 to sensitive cells of E. coli O157:H7 was bactericidal, resulting in a 2 log reduction in viable cell counts. Colicin ECL12 was purified from strain ECL12 by cell extraction and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of colicin ECL12 resolved a single protein with a molecular weight of approximately 65,000.

scholarly journals Immunologic studies of native and modified human factor VIII/von Willebrand factor

Blood ◽  
1979 ◽  
Vol 54 (2) ◽  
pp. 310-321 ◽  
Author(s):  
ME Switzer ◽  
PA McKee
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Procoagulant Activity ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII/von Willebrand factor (FVIII/vWF) is a glycoprotein with a molecular weight greater than one-million daltons. Two activities are associated with this large molecule: FVIII procoagulant activity and vWF activity. Incubation of FVIII/vWF with proteolytic enzymes causes rapid inactivation of the FVIII procoagulant activity but has little effect on the vWF activity or antigenicity. In an attempt to gain insight into the structural features required for these two activities, antisera were raised in rabbits to normal, thrombin-inactivated, and plasmin-inactivated FVIII/vWF. All of these proteolytically modified forms of FVIII/vWF cross-reacted with each of the rabbit antisera; each blocked the ability of a human inhibitor to inactivate native active FVIII/vWF. Each of the antisera was a potent inhibitor of vWF activity and inactivated vWF activity at the same titer. The antisera were much less potent inhibitors of FVIII activity than of vWF activity. Antibodies to thrombin-inactivated FVIII/vWF or normal FVIII/vWF had about the same ability to inactivate FVIII procoagulant activity. Surprisingly, those to plasmin-inactivated FVIII/vWF still retained about 50% of this inhibitory capacity. A comparison of the three types of antigens by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-6 M urea demonstrated that the structure of thrombin- inactivated FVIII/vWF was indistinguishable from that of normal FVIII/vWF, while plasmin-inactivated FVII/vWF was completely cleaved to lower molecular weight fragments. Some of the reported variations in the ability of rabbit antibodies to inhibit procoagulant activity may be due to partial degradation of the starting antigen. The retention by FVIII/vWF protein of its immunologic properties even after extensive proteolytic degradation suggests that under nondenaturing conditions, the conformation of the native and degraded molecules are very similar.

Proteolytic expression inBlastocrithidia culicis: influence of the endosymbiont and similarities with virulence factors of pathogenic trypanosomatids

Parasitology ◽  
2004 ◽  
Vol 130 (4) ◽  
pp. 413-420 ◽  
Author(s):  
C. M. D'AVILA-LEVY ◽  
F. M. ARAUJO ◽  
A. B. VERMELHO ◽  
R. M. A. SOARES ◽  
A. L. S. SANTOS ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Life Cycles ◽  
Western Blots ◽  
Sds Page ◽  
Bacterial Endosymbionts ◽  
Proteolytic Activities ◽  
Qualitative Changes

Blastocrithidia culicisis an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of theB. culicisproteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids,Leishmaniaspp. andTrypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.

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