Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021

2019 ◽  
Author(s):  
Elham Noroozi ◽  
Naheed Mojgani ◽  
Elahe Motevaseli ◽  
Mohammad Hossein Modarressi ◽  
Majid Tebianian
Keyword(s):  
Molecular Weight ◽  
Lactobacillus Casei ◽  
Pathogenic Bacteria ◽  
Proteolytic Enzymes ◽  
Shigella Flexneri ◽  
Gel Filtration ◽  
Antimicrobial Protein ◽  
Purification Method ◽  
Ph Values ◽  
Subsequent Decrease

Background and Objectives: Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by au- tochthonous Lactobacillus casei TA0021 strain. Materials and Methods: The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed. Results: The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of ˃9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold. Conclusion: The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.

scholarly journals The release of collagenase as an inactive proenzyme by bone explants in culture

1972 ◽  
Vol 126 (2) ◽  
pp. 275-289 ◽  
Author(s):  
G. Vaes
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Culture Media ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Inhibitor Complex ◽  
Ph Values ◽  
Liver Lysosomes ◽  
Skin Explants ◽  
Extracellular Activity

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme–inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a `procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.

scholarly journals Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins

1989 ◽  
Vol 264 (2) ◽  
pp. 467-473 ◽  
Author(s):  
H Kirschke ◽  
B Wiederanders ◽  
D Brömme ◽  
A Rinne
Keyword(s):  
Cathepsin L ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Similar Rate ◽  
Cathepsin S ◽  
Purification Method ◽  
Cytoplasmic Granules ◽  
Bovine Kidney ◽  
Ph Values ◽  
C 50

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.

scholarly journals Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 87-94 ◽  
Author(s):  
MC Poon ◽  
OD Ratnoff
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Protease Inhibitors ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Native State ◽  
Noncovalent Bonds ◽  
Antihemophilic Factor

Abstract Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.

Renin Precursor and Its Activation Mechanism in Hog Kidney

1980 ◽  
Vol 59 (s6) ◽  
pp. 21s-24s ◽  
Author(s):  
Kazuo Murakami ◽  
Saori Takahashi ◽  
Shigehisa Hirose ◽  
Yukio Takii ◽  
Tadashi Inagami
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Activation Mechanism ◽  
Net Charge ◽  
Isoelectric Points ◽  
Binding Substance ◽  
Inactive Renin ◽  
Hog Kidney

1. A completely inactive renin was isolated from hog kidney extract by affinity chromatography on pepstatin-aminohexyl-Sepharose and on an Affi-Gel Blue column. 2. This inactive renin had a molecular weight of 43 000 ± 1500 as determined by gel filtration on Ultrogel AcA 44. Upon activation with trypsin, its molecular weight fell to 41 000 ± 1400. 3. The inactive renin lacked the ability to bind renin-binding substance whereas trypsin-activated renin was able to bind the renin-binding protein and to form high-molecular-weight renin. 4. Chymotrypsin as well as trypsin could activate the inactive renin although less effectively. 5. The active renins generated from the inactive renin by the action of different proteolytic enzymes differed in their net charge, reflecting the specificities of the proteases used; the isoelectric points of the native, the trypsin-activated and the chymotrypsin-activated forms of renin occurred at pH 5.3, 5.1 and 4.8 respectively.

Limited Proteolysis of the Purified Aα Chain of Human Fibrinosen by Plasmin

1975 ◽  
Author(s):  
L. Williams ◽  
G. Murano
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Mapping ◽  
Proteolytic Enzymes ◽  
Degradation Products ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Terminal Region ◽  
Low Molecular Weight ◽  
Human Fibrinogen

Based on evidence that a portion of circulating fibrinogen consists of a family of catabolic intermediates formed by proteolytic degradation of the COOH terminal region of Aα chains, we attempted to obtain early degradation products using the purified alkylated Aα chain derivative of human fibrinogen as the substrate and plasmin as the enzyme. Having established optimal conditions, a preparative quantity of material was digested in 0.1 M tris buffer pH = 9.5; time = 4 min; E/S ratio = 1/75 (mole/mole); temp = 37° C. Low molecular weight fragments were separated from the larger species, and further purified by gel filtration on Sephadex G-100. Selected early fragments were analyzed by polycrylamide gel electrophoresis, amino acid composition, peptide mapping and partial N-terminal amino acid sequence. Two of the earliest low molecular weight fragments released by plasmin were derived from the N-terminal region of the Aα chain. Their molecular size was estimated at about 10,000 daltons. One fragment contains fibrinopeptide A; both fragments extend beyond Met-51. Our data indicate that: a) the specificity of plasmin on the purified Aα chain differs from that on intact fibrinogen; or b) proteolytic enzymes other than or in addition to plasmin are responsible for the formation of early catabolic fibrinogen intermediates having a degraded Aα chain.(Supported by USPHS N. I. H. Grant HL 14142.)

scholarly journals Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum

1981 ◽  
Vol 199 (1) ◽  
pp. 9-15 ◽  
Author(s):  
M Janusz ◽  
K Starościk ◽  
M Zimecki ◽  
Z Wieczorek ◽  
J Lisowski
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Room Temperature ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Guanidinium Chloride ◽  
Terminal Amino Acid ◽  
Polyproline Ii ◽  
Terminal Amino

A proline-rich polypeptide isolated from sheep colostrum is described. The molecular weight of the polypeptide determined by gel filtration is 17 200. However, in the presence of guanidinium chloride the molecular weight found is about 6000. The polypeptide contains about 22% of proline, a high proportion of non-polar amino acids, a low percentage of glycine, and no alanine, arginine and cysteine residues. The only N-terminal amino acid found is leucine. C.d. spectra in water and in 50% (v/v) trifluoroethanol suggest the presence of block sequences of proline residues forming helices of polyproline II type. The proline-rich polypeptide is soluble at 4 degrees C but is reversibly precipitated on warming to room temperature. Maximal precipitation is observed at pH 4.6 and at ionic strength above 0.6. The precipitation depends on the concentration of the polypeptide. No effect of other proteins, Ca2+ and Zn2+ ions on the precipitation of the polypeptide was found. The proline-rich polypeptide is not an amphipathic protein. The lack of effect of the polypeptide on proteolytic enzymes ruled out the possibility that it is an inhibitor of proteinases.

scholarly journals Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 87-94
Author(s):  
MC Poon ◽  
OD Ratnoff
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Protease Inhibitors ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Native State ◽  
Noncovalent Bonds ◽  
Antihemophilic Factor

Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.

scholarly journals Antimicrobial properties of the peptide substance Bacillus Subtilis PSF-19

2019 ◽  
pp. 22-26
Author(s):  
Viktor Danilovich Pokhilenko ◽  
Vladimir Vladimirovich Perelygin ◽  
Timur Akhmerovich Kalmantaev ◽  
Konstantin Vladimirovich Detushev ◽  
Irina Anatolevna Chukina
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Pathogenic Bacteria ◽  
Proteolytic Enzymes ◽  
Raw Materials ◽  
Antimicrobial Properties ◽  
Culture Fluid ◽  
Fluid Fraction ◽  
Intestinal Pathogens ◽  
Vegetable Raw Materials

The subject of the study is a strain of Bacillus subtilis PSF-19 isolated by us from vegetable raw materials (Passiflora preparation), which is capable of producing antimicrobial substance (AMV) suppressing pathogenic bacteria. The article discusses the method and conditions of extraction from the culture fluid fraction of AMV, active against Listeria monocytogenes – one of the dangerous intestinal pathogens that infect food. Using biochemical methods and mass spectroscopy, the molecular weight and the peptide nature of the active fraction of AMV were determined. A fraction of AMV with a molecular weight of 3,4–3,6 kDa has bactericidal activity, which is destroyed by treatment with proteolytic enzymes, which allowed it to be attributed to the group of low-molecular antimicrobial peptides – bacteriocins. The studies allow to consider the strain Bacillus subtilis PSF-19 as a producer of bacteriocin, especially effective against pathogens of intestinal listeriosis. The establishment of the fact of destructibility of proteolytic enzymes, finding the conditions of microbiological synthesis of AMV, its isolation and accumulation for research, provide opportunities for practical use as a means for decontamination of the environment instead of traditional antibiotics.

scholarly journals Potential Screening of Bacteriocinogenic-Lactic Acid Bacteria from Mangrove Sediment of Logending Beach for Fisheries Product Preservation

2021 ◽  
Vol 6 (1) ◽  
pp. 61927
Author(s):  
Dyah Fitri Kusharyati ◽  
Taruna Dwi Satwika ◽  
Afifah Mariana ◽  
Anwar Rovik
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Pathogenic Bacteria ◽  
Proteolytic Enzymes ◽  
Shigella Flexneri ◽  
Salmonella Typhi ◽  
Mangrove Sediment ◽  
In Vitro Test ◽  
Cell Free Supernatant

The meat and fisheries products have high nutritional content which is highly ideal for bacterial growth. Lactic Acid Bacteria (LAB) have several potential advantages as a bio-preservative agent in the food industry because they produce antimicrobial substances against pathogenic bacteria e.g. bacteriocin. Our previous study has succeeded in isolating and characterizing LAB from the mangrove sediments of Logending Beach, Kebumen. This present study aimed to determine the activity of bacteriocinogenic-LAB against food-borne pathogens and their potential for fisheries product preservation. The study consisted of five serial stages, as follows: screening of LAB isolates, cell-free supernatant production and its inhibition activity, extraction of partially purified bacteriocin, bacteriocin confirmation against proteolytic enzymes, and in-vitro test of partially-purified bacteriocin against Listeria monocytogenes, Shigella flexneri, and Salmonella typhi. A total of 25 out of 99 isolates were able to grow on MRSA+1% CaCO3 medium. Initial screening showed that the cell-free supernatant of 14 LAB isolates was able to inhibit the growth of S. thypi, S. flexneri, and L. monocytogenes. There was an increased inhibitory activity of partially purified bacteriocin when compared with the cell-free supernatant which was statistically different (p<0.01). It indicated that the purification was successfully performed. Bacteriocin expressed a lower inhibition against S. typhi than L. monocytogenes and S. flexneri. The ANOVA test showed that each indicator pathogenic-bacterium expresses a very significant sensitivity to the partially purified bacteriocin.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

Sign in / Sign up

Export Citation Format

Share Document