Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, six Streptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0–7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3–10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW < 3 kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04–0.62 mg·mL−1 and 0.08–1.25 mg·mL−1, respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975 μg·mL−1 (i.e., moderate toxicity) to Artemia salina. Identification of the peptide sequences of this fraction by liquid chromatography–tandem mass spectrometry yielded 130 proteins with retention times of 15.21–19.57 min. Eleven proteins with MWs of 1345.66–2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34–26.22 kcal·mol−1) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces, especially BLIS SCA-8, as antibacterial agents.

Download Full-text

THE PROTEOLYTIC ENZYMES OF MICROORGANISMS: IV. PARTIAL PURIFICATION AND SOME PROPERTIES OF EXTRACELLULAR PROTEASE FROM MORTIERELLA RENISPORA DIXON-STEWART

Canadian Journal of Botany ◽

10.1139/b52-047 ◽

1952 ◽

Vol 30 (6) ◽

pp. 685-692 ◽

Cited By ~ 2

Author(s):

L. R. Wetter

Keyword(s):

Culture Medium ◽

Considerable Increase ◽

Specific Activity ◽

Proteolytic Enzymes ◽

Partial Purification ◽

Ferrous Ions ◽

Ph Optimum ◽

Enzyme Preparations ◽

Wide Range ◽

Repeated Precipitation

A protease concentrate was obtained from the culture medium of Mortierella renispora Dixon-Stewart (PRL 26) by repeated precipitation with ammonium sulphate. The specific activity of the mold protease compared favorably with that of crystalline trypsin. The pH optimum was broad, with a maximum at a pH of 7.5 when hemoglobin was used as the substrate. A study of the pH stability characteristics showed that it was stable over a wide range (4.9 to 9.5) at 1 °C. and 25 °C. Ferrous ions caused a considerable increase in the activity of the enzyme preparations, other metals were ineffective as activators.

Download Full-text

EXTRACTION AND PURIFICATION OF VEGETABLE PEROXIDASE: A REVIEW

Periódico Tchê Química ◽

10.52571/ptq.v16.n31.2020.702_periodico31_pgs_692_703.pdf ◽

2019 ◽

Vol 16 (31) ◽

pp. 692-703

Author(s):

Aline HAAS ◽

Cleiton VAZ ◽

Aniela Pinto KEMPKA

Keyword(s):

Enzymatic Activity ◽

Specific Activity ◽

Extraction Methods ◽

Raw Material ◽

Partial Purification ◽

Buffer Solution ◽

Degradation Of Dyes ◽

Extraction And Purification ◽

Wide Range ◽

Purification Methods

Peroxidases are enzymes that catalyze the oxidation of various substrates, maintaining their enzymatic activity in wide ranges of pH and temperatures. These enzymes are used in processes for the degradation of dyes and phenolic compounds. Peroxidases are present in the tissues of several plants, and the search for new sources of this enzyme is necessary. This literature review aims to compile information about the extraction and/or purification of peroxidases contained in different plant tissues, presenting extraction methods, purification processes, enzymatic activities and their increments, according to the chemical and physical processes applied. Several plant sources can be raw material to obtain these enzymes, through different forms of extraction, where the processes of comminution predominate in the presence of buffer solution. For partial purification, are used precipitation with solvents (acetone and ethanol) and salts (ammonium sulfate) and centrifugation. For purification, chromatographic processes are used, in which molecular exclusion and affinity chromatography are prominent. It is concluded that there is a wide range of possibilities for obtaining the enzyme peroxidase from plants, with variability in the enzymatic activity when different extraction methods are applied. The purification methods used provide increases in the specific activity of the peroxidases.

Download Full-text

Antibacterial Activity, Cytotoxicity, and the Mechanism of Action of Bacteriocin from Bacillus subtilis GAS101

Medical Principles and Practice ◽

10.1159/000487306 ◽

2018 ◽

Vol 27 (2) ◽

pp. 186-192 ◽

Cited By ~ 11

Author(s):

Garima Sharma ◽

Shweta Dang ◽

Sanjay Gupta ◽

Reema Gabrani

Keyword(s):

Molecular Weight ◽

Antibacterial Activity ◽

Bacillus Subtilis ◽

Vero Cell ◽

Cell Line ◽

Mode Of Action ◽

Proteolytic Enzymes ◽

Antibiofilm Activity ◽

Vero Cell Line ◽

Soil Isolate

Objective: The aim of this study was to purify and characterize bacteriocin from the soil isolate Bacillus subtilis GAS101, and to determine its antimicrobial as well as antibiofilm potential. The purified bacteriocin was further analyzed and evaluated for mammalian cell cytotoxicity and the possible mode of action. Material and Methods: Bacteriocin from B. subtilis GAS101 (an animal husbandry soil isolate) was partially purified and checked for antimicrobial and antibiofilm activity against gram-positive and gram-negative bacteria. The molecular weight of bacteriocin was determined using tricine SDS-PAGE gel. The stability of bacteriocin was investigated at various temperatures and pH levels, and its sensitivity towards 8 enzymes and 6 chemicals was determined. Cytotoxicity analysis was performed on a Vero cell line by a tetrazolium dye-based assay. Scanning electron microscopy (SEM) of bacteriocin-treated bacteria was carried out to determine the possible mode of action. Results: Bacteriocin from B. subtilis GAS101 was a potential inhibitor of both the indicator organisms (Staphylococcus epidermidis and Escherichia coli), and had a molecular weight of approximately 6.5 kDa. An in situ gel assay showed a zone of inhibition corresponding to the estimated protein band size. Bacteriocin was stable and showed antibacterial activity in broad ranges of temperature (30–121°C) and pH (2–12). It was sensitive to 4 proteolytic enzymes, which indicated its proteinaceous nature. Bacteriocin showed > 70% cell viability on the mammalian Vero cell line. SEM depicted that the bacteriocin was able to disrupt the bacterial cell membrane as its probable mode of action. Conclusion: Thermostable and pH-tolerant bacteriocin from B. subtilis GAS101, of about 6.5 kDa, showed broad-spectrum antimicrobial and antibiofilm activity.

Download Full-text

Immunoaffinity Chromatography of Canine High-Molecular-Weight Renin: Partial Purification and Characterization

Clinical Science ◽

10.1042/cs0650117 ◽

1983 ◽

Vol 65 (2) ◽

pp. 117-120 ◽

Cited By ~ 2

Author(s):

Fumihiko Ikemoto ◽

Victor J. Dzau ◽

Edgar Haber ◽

Kazuo Takaori ◽

Kenjiro Yamamoto

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Specific Activity ◽

Immunoaffinity Chromatography ◽

Partial Purification ◽

Low Molecular Weight ◽

Specific Method ◽

Disulphide Bonds ◽

Binding Substance ◽

Molecular Weight Form

1. Canine high-molecular-weight renin (mol. wt. 60 000) is believed to be a complex of renin (low-molecular-weight form, mol. wt. 40 000) and renin-binding substance. The immunocross-reactivity of high-molecular-weight renin and low-molecular-weight renin was demonstrated by using antibodies specific to low-molecular-weight renin. 2. Immunoaffinity chromatography with renin-specific antibodies coupled to Sepharose provided a simple and specific method for isolation of high-molecular-weight renin. High-molecular-weight renin with a specific activity of 137 600 ng of ANG I h−1 mg−1 of protein (19.6 Goldblatt units/mg of protein) was obtained. 3. This high-molecular-weight renin was stable in dithiothreitol (25 mmol/l), suggesting that disulphide bonds may not be involved in the binding mechanism between low-molecular-weight renin and renin-binding substance. 4. However, exposure to low pH (3.0) resulted in conversion of high-molecular-weight renin into the low-molecular-weight form.

Download Full-text

Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

Tikrit Journal of Pure Science ◽

10.25130/j.v23i10.756 ◽

2019 ◽

Vol 23 (10) ◽

pp. 46

Author(s):

Saif M. Hasan ◽

Firas T. Maher ◽

Nagham Q. Kadhim

Keyword(s):

Molecular Weight ◽

Specific Activity ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Maximum Velocity ◽

Partial Purification ◽

Diabetic Patients ◽

Kinetic Properties ◽

Topoisomerase Ib ◽

Electrophoresis Method

This study was done to partially purification of topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD). http://dx.doi.org/10.25130/tjps.23.2018.168

Download Full-text

Characterization of Low-Molecular-Weight Antiyeast Metabolites Produced by a Food-Protective Lactobacillus-Propionibacterium Coculture

Journal of Food Protection ◽

10.4315/0362-028x-71.12.2481 ◽

2008 ◽

Vol 71 (12) ◽

pp. 2481-2487 ◽

Cited By ~ 56

Author(s):

SUSANNE MIESCHER SCHWENNINGER ◽

CHRISTOPHE LACROIX ◽

STEFAN TRUTTMANN ◽

CHRISTOPH JANS ◽

CÄCILIA SPÖRNDLI ◽

...

Keyword(s):

Molecular Weight ◽

Carboxylic Acid ◽

Succinic Acid ◽

Solid Phase ◽

Immobilized Cells ◽

Proteinase K ◽

Partial Purification ◽

Low Molecular Weight ◽

Phenyllactic Acid ◽

High Mics

We developed a pH-controlled batch fermentation process with separately immobilized cells of the protective coculture of Lactobacillus paracasei subsp. paracasei SM20 and Propionibacterium jensenii SM11 in supplemented whey permeate medium yielding cell-free supernatants with high antiyeast activity against Candida pulcherrima and Rhodotorula mucilaginosa. The antiyeast compounds were resistant to proteinase K and pronase E treatments and showed high heat resistance (121°C for 15 min). Diafiltration (1,000-Da cutoff) revealed that the inhibitory metabolites have low molecular weights. Partial purification of active compounds was achieved by a microplate bioassay controlled procedure with solid-phase extraction (C18) followed by (i) gel filtration chromatography or (ii) semipreparative reverse-phase high-performance liquid chromatography (C18). In addition to propionic, acetic, and lactic acids, 2-pyrrolidone-5-carboxylic acid, 3-phenyllactic acid, hydroxyphenyllactic acid, and succinic acid were identified by chromatography and mass spectrometry. Accurate quantifications revealed only low concentrations (up to 7 mM) of 2-pyrrolidone-5-carboxylic acid, 3-phenyllactic acid, and hydroxyphenyllactic acid produced during fermentation in contrast to relatively high MICs (50 to more than 500 mM) determined at different pH values (4.0, 5.0, and 6.0). Succinic acid was present at higher concentrations (29 mM) in cell-free supernatants but with comparable high MICs (200 to more than 500 mM and pH 4.0, 5.0, and 6.0). Although none of these compounds was the main substance responsible per se for suppression of yeast growth, our study revealed a complex antiyeast mechanism with putative synergistic effects between several low-molecular-weight compounds.

Download Full-text

Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

Tikrit Journal of Pure Science ◽

10.25130/j.v24i7.913 ◽

2019 ◽

Vol 24 (7) ◽

pp. 59

Author(s):

Reemy M. Mohamed Saleh ◽

Firas T . Maher ◽

Nagham Q. Kadhim

Keyword(s):

Molecular Weight ◽

Polycystic Ovary Syndrome ◽

Specific Activity ◽

Polycystic Ovary ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Partial Purification ◽

Ovary Syndrome ◽

Electrophoresis Method ◽

High Level

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206). http://dx.doi.org/10.25130/tjps.24.2019.130

Download Full-text

Purification, Characterization and N-terminal Protein Sequencing of the Enzyme Dextransucrase Produced by Leuconostoc mesenteroides

Biosciences Biotechnology Research Asia ◽

10.13005/bbra/2915 ◽

2021 ◽

Vol 18 (2) ◽

pp. 287-295

Author(s):

Turki M. Dawoud ◽

Fatimah Alshehrei ◽

Khaizran Siddiqui ◽

Fuad Ameen ◽

Jameela Akhtar ◽

...

Keyword(s):

Molecular Weight ◽

Leuconostoc Mesenteroides ◽

Specific Activity ◽

Industrial Applications ◽

Protein Sequencing ◽

Partial Purification ◽

Terminal Protein ◽

Enzymatic Extract ◽

Branched Structure ◽

Wide Potential

Background: The wide use of dextran in many different applications, makes its industrial production a challenge and, hence, to obtain a control branched structure of this enzyme research is in progress. Objectives: In the present paper, the enzyme dextransucrase, produced by cultivation of the bacterium Leuconostoc mesenteroides CMG713, was purified and characterized. Methods: The produced dextransucrase was partially purified by PEG400 obtaining a purification factor of 29.4-fold and an overall yield of 18.3% from the initial crude enzymatic extract. Results: The partially purified dextransucrase had a specific activity of 24.0 U/mg and presented a molecular weight of about 200 kDa. In addition, the produced dextransucrase was stable at 30ºC and pH 5.5 for 3 days and led to a highly soluble dextran with wide potential industrial applications. The current study has successfully partial purification, characterization and conformation of dextransucrase produced by fermentation of the bacterium Leuconostoc mesenteroides CMG713.

Download Full-text

Modifications of Isoalantolactone Leading to Effective Antibacterial and Antiviral Compounds

Letters in Drug Design & Discovery ◽

10.2174/1570180817999201211193151 ◽

2020 ◽

Vol 17 ◽

Author(s):

Sergey S. Patrushev ◽

Lyubov G. Burova ◽

Anna A. Shtro ◽

Tatyana V. Rybalova ◽

Dmitry S. Baev ◽

...

Keyword(s):

Antibacterial Activity ◽

Antiviral Activity ◽

Influenza A ◽

Antimicrobial Agents ◽

Sesquiterpene Lactones ◽

Biological Properties ◽

Bacterial Strains ◽

E Coli ◽

Wide Range

Background: Natural sesquiterpene lactones are an important class of heterocyclic compounds in drug discovery since they are possessed a wide range of biological properties including antibacterial activity. Objective: The objective of this study was to synthesize of isoalantolactone derivatives with a furo[2,3-d]pyrimidin-2-оne moiety and to evaluate their antibacterial and antiviral activity. Methods: The Sonogashira cross-coupling and subsequent Ag-catalyzed cyclization reactions were the main routes of synthesis. The antibacterial activity and the ability to inhibit biofilms formation on E. coli, S. aureus, A. viscosus, P. aeruginosa and E. faecalis bacterial strains were evaluated. A study of the molecular interactions of new compounds with the multiple virulence factor regulators was performed using docking simulations. The antiviral activity against influenza A virus and human orthopneumovirus H-2А was also studied. Results: The in vitro antibacterial activity for 4 (MIC = 58.33±4.41 μg/mL) concerning E. coli and 5 (MIC = 96.5±3.25 μg/mL) against A. viscosus and the inhibition of biofilm formation for compounds 2, 4, and 5 on E. coli, S. aureus, P. aeruginosa and E. faecalis bacterial strains has been of interest for the search of improved antimicrobial agents. Compound 3 was endowed with antiviral activity to human orthopneumovirus H-2А with SI >33. The activity of the new type of hybrid compounds is depended on the substituent in the 6th position of furo[2,3-d]pyrimidin-2-one fragment. Conclusion: The decoration of isoalantolactone with a furo[2,3-d]pyrimidin-2-one fragment led to perspective antiviral and antimicrobial agents. Due to antimicrobial activity, pyridine-4-yl substituted isoalantolactone-furopyrimidinone hybrid is considered as a candidate compound to participate in further research.

Download Full-text

Artichoke leaf extracts: Proteolytic activity, coagulant and HPLC analysis

Ciência e Agrotecnologia ◽

10.1590/1413-7054202145001721 ◽

2021 ◽

Vol 45 ◽

Author(s):

Gabriela Muricy de Souza Silva ◽

Jessyka Silva da Costa ◽

Janaina Oliveira Freire ◽

Leandro Soares Santos ◽

Renata Cristina Ferreira Bonomo

Keyword(s):

Proteolytic Activity ◽

Hplc Analysis ◽

Specific Activity ◽

Proteolytic Enzymes ◽

Protein Profile ◽

Leaf Extracts ◽

Citrate Buffer ◽

Cynara Scolymus ◽

Milk Clotting ◽

Wide Range

ABSTRACT The search for origin plant-based proteases increases gradually due to their diversity and stability over a wide range of pH and temperature. Artichoke (Cynara scolymus) flowers are a proteolytic vegetable source already studied, but their leaves are scarce in this respect. Thus, the objective of this research was to obtain extracts of artichoke leaves with different buffers and extraction methods as an alternative proteolytic source and plant coagulant, as well as the separation and comparison of the protein profile of these extracts. The methodology used was based on extraction with sodium citrate buffer (pH 3), sodium acetate (pH 5) and Tris-HCl (pH 7) by mechanical stirrer (MS) and ultrasound (US); protein determination; proteolytic activity (PA) and specific activity (SA); milk clotting activity (MCA) and rennet substitution potential (RSP); high- performance liquid chromatography analysis (HPLC) with UV-Vis detector and principal component analysis (PCA). Extracts of Cynara scolymus leaves showed high results with Citrate-US for the parameters PA (14.38), SA (19.71), MCA (440) and RSP (30.60) compared to other treatments. The extracts with citrate and acetate presented a quick coagulation time (max 3 min). The HPLC analysis enabled the separation of the different protein compounds present in the extracts and most expressive peaks in the samples with Citrate-MS and Acetate-MS; and isolated peaks for Citrate-US. It was concluded that extracts of artichoke leaves with citrate and acetate buffer attributed satisfactory results to act as plant coagulant, as well as to carry out further studies for the purification of proteolytic enzymes and application in cheeses.

Download Full-text