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

1952 ◽  
Vol 30 (6) ◽  
pp. 685-692 ◽  
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.

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

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Muhammad A. Kurnianto ◽  
Harsi D. Kusumaningrum ◽  
Hanifah N. Lioe ◽  
Ekowati Chasanah
Keyword(s):  
Molecular Weight ◽  
Antibacterial Activity ◽  
Antimicrobial Agents ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
Proteinase K ◽  
Partial Purification ◽  
Chanos Chanos ◽  
Extracellular Metabolites ◽  
Wide Range

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.

Poly(β-L-malate) hydrolase from Plasmodia of Physarum polycephalum

1995 ◽  
Vol 41 (13) ◽  
pp. 192-199 ◽  
Author(s):  
Christian Korherr ◽  
Michael Roth ◽  
Eggehard Holler
Keyword(s):  
Hydrophobic Interaction ◽  
Malic Acid ◽  
Physarum Polycephalum ◽  
Culture Medium ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Reduced Form ◽  
Serine Esterase ◽  
Hydroxybutyric Acid ◽  
Ph Optimum

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.

scholarly journals EXTRACTION AND PURIFICATION OF VEGETABLE PEROXIDASE: A REVIEW

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.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals Preliminary characterization of the multiple forms of ram sperm hyaluronidase

1988 ◽  
Vol 252 (3) ◽  
pp. 875-882 ◽  
Author(s):  
R A Harrison
Keyword(s):  
Vinyl Alcohol ◽  
Specific Activity ◽  
Antigenic Determinants ◽  
Poly Vinyl Alcohol ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Low Concentrations ◽  
Poly Ethylene ◽  
Ram Sperm ◽  
Oligomeric Series

An investigation was made of the inter-relationships and characteristics of various hyaluronidase forms isolated from ram spermatozoa. They were shown to be members of an oligomeric series, apparently formed by intermolecular disulphide cross-linking. Two monomer species were detected, alpha (Mr 89,600) and beta (Mr 81,200). Although the alpha species predominated, the two were evenly distributed throughout the oligomer population, and they shared antigenic determinants; the beta species did not arise from the alpha species as a result of catabolism following cell disruption. The oligomeric series was of the form [Hyal]n, where n = 1, 2, 4, 5, 6, 7 etc.; no trimer was detectable. Though essentially cationic, part of the hyaluronidase population also had anionic characteristics, probably due to oxidation of free thiol groups. In the anionic subpopulation tetramers and higher oligomers predominated, whereas the non-anionic subpopulation was composed of monomers, dimers and tetramers. The pH optimum of the monomer was 4.3 in 0.2 M-NaCl/0.1 M-sodium citrate, whereas that of the anionic oligomers was 4.9. Both serum albumin and polylysine stimulated enzyme activity at pH 4.0 in the absence of NaCl; polylysine was particularly effective. NaCl diminished the stimulatory effects, and essentially suppressed them above the pH optimum. The specific activities of different oligomer populations were the same as that of the monomer, and conversion of oligomers into monomer by reduction had likewise no effect upon the specific activity. Low concentrations of poly(vinyl alcohol), poly(ethylene glycol) or polyvinylpyrrolidone stabilized soluble hyaluronidase activity by preventing the enzyme's binding to surfaces; solutions of anionic oligomers were further stabilized by NaCl. Enzyme preparations were stable for several months frozen in the presence of poly(vinyl alcohol) and salt.

scholarly journals Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

1977 ◽  
Vol 161 (2) ◽  
pp. 357-370 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Transport Chain ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.

scholarly journals Partial purification and properties of an AMP-specific soluble 5′-nucleotidase from pigeon heart

1990 ◽  
Vol 268 (1) ◽  
pp. 117-122 ◽  
Author(s):  
A C Skladanowski ◽  
A C Newby
Keyword(s):  
Ionic Strength ◽  
Molecular Mass ◽  
Formation Rate ◽  
Linear Increase ◽  
High Ionic Strength ◽  
Partial Purification ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Wide Range ◽  
Alpha Beta

A soluble 5′-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5′-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5′-deoxy-5′-isobutylthioinosine than by 5′-deoxy-5′-isobutylthioadenosine, but not by [alpha, beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5′-nucleotidase and from the previously purified IMP-specific 5′-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.

scholarly journals Characterization of Proteolytic and Collagenolytic Enzymes from the Larvae of Lucilia cuprina, the Sheep Blowfly

1988 ◽  
Vol 41 (2) ◽  
pp. 269 ◽  
Author(s):  
VM Bowles ◽  
PR Carnegie ◽  
RM Sandeman
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Proteolytic Enzymes ◽  
Chelating Agent ◽  
Collagenolytic Activity ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Highly Active ◽  
Wide Range ◽  
Secretory Enzyme

Isoelectric focusing was used to characterize proteolytic enzymes in homogenate and excretory-secretory preparations of the larvae of L. cuprina, the sheep blowfly. Zymogram overlays showed that the larvae produce a number of highly active proteases which have a wide range of isoelectric points and molecular weights. The alkaline and neutral pI proteases were inhibited by phenylmethyl-sulfonylfluoride, leupeptin and aprotinin; this indicated the presence of serine in the active site. Pepstatin and the metal chelating agent ethylenediaminetetraacetic acid had no effect Oil the activity of any of the proteases. Optimal pH for activity of the proteases was between 7 and 8. In addition, the proteases were found to be heat labile. Digestion of collagen fibrils confirmed the existence of collagenolytic activity in the excretory-secretory enzyme preparations. It is suggested that these enzymes may be involved in the nutrition of the larvae and in the pathogenesis of the lesion on the skin.

Purine catabolism in man: characterization of placental microsomal 5′-nucleotidase

1976 ◽  
Vol 54 (5) ◽  
pp. 462-469 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Specific Activity ◽  
Ph Optimum ◽  
Electrophoretic Variants ◽  
Molecular Weights ◽  
Wide Range ◽  
Stokes Radius ◽  
Michaelis Constants ◽  
Light Form ◽  
Molecular Weight Form

Human placental microsomal 5′-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence.There was a wide range of substrate specificity among nucleoside 5′-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68; IMP, 63; XMP, 28 and UDP–glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12–18 μM, from 33–67 μM and from 170–250 μM, respectively. Although 5′-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4–9.8.

scholarly journals Partial Purification and Characterization of Proteases in Tobacco Leaf and Callus

1984 ◽  
Vol 12 (4) ◽  
pp. 219-226
Author(s):  
D. Liu ◽  
S. J. Sheen
Keyword(s):  
Cation Exchange ◽  
Specific Activity ◽  
Partial Purification ◽  
Fraction I Protein ◽  
Ph Optimum ◽  
Tobacco Leaf ◽  
Exchange Column ◽  
Gel Permeation ◽  
Cation Exchange Column ◽  
Fraction I

AbstractAmmonium sulfate fra.ctionation, gel permeation and cation-exchange column chromatography were employed for panial purification of proteases from leaf laminae and callus tissues of Samsun NN tobacco (Nicotiana tabacum L). The predominant proteases in the leaf and callus are acidic sulfhydryl proteases which are activated by 2-mercaptoethanol and ethylenediamine tetraacetic acid, completely inhibited- by iodoacetic acid, and partially inhibited by phenylmethane sulfonyl fluoride and pepstatin A. With hemoglobin and tobaccl:l Fraction I protein as substrates, leaf and callus proteases showed a pH optimum of 5. However, specific activity was significandy higher in the callus than in the leaf. Tobacco proteases digested hemoglobin more effectively than Fraction I protein and showed the least activity with casein. Gel permeation resolved three -protease variants in leaf extracts but only two in callus samples. Rechromatography of the large molecular weight fraction in a cation-exchange column produced three and two variants for leaf and callus, respectively. The present results suggest that there are at least five variants of sulfhydryl protease in tobacco leaf and three in callus tissue and that tobacco Fraction I protein can be metabolized by both leaf and callus proteases.

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