An extracellular proteolytic enzyme from Scopulariopsis brevicaulis. 1. Purification and properties

1971 ◽  
Vol 17 (8) ◽  
pp. 1029-1042 ◽  
Author(s):  
Kartar Singh ◽  
Claude Vézina
Keyword(s):  
Proteolytic Enzyme ◽  
Deae Cellulose ◽  
Alkaline Proteases ◽  
Casein Hydrolysis ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Sh Reagents ◽  
Extracellular Proteolytic Enzyme ◽  
Scopulariopsis Brevicaulis ◽  
Insulin Chains

A proteolytic enzyme present in culture filtrates of Scopulariopsis brevicaulis was purified about 200-fold by (NH4)2SO4 and ethanol fractionations followed by chromatography on DEAE-cellulose, DEAE-Sephadex, and hydroxylapatite. Ultracentrifugation of the purified enzymes showed only one sedimenting component and its molecular weight was estimated to be about 24 000. The protease hydrolyzed casein, urea-denatured hemoglobin, gelatin, fibrinogen, fibrin, insulin chains A and B, but not human serum albumin or ovalbumin. It also coagulated milk. The enzyme had no action on the various peptides tested and showed low esterase activity. Optimum pH for casein hydrolysis was 10.5 to 11; for hemoglobin hydrolysis 7.0–9.5, and for gelatin hydrolysis, 6.0–8.0. The enzyme activity was unaffected by most metal ions, SH-reagents, and some natural trypsin inhibitors. The protease was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride. Although similar in some respects to CA-7, the enzyme isolated from Aspergillus oryzae, and other alkaline proteases, the S. brevicaulis protease does not appear to be identical with any one of them.

Purification and properties of an extracellular proteolytic enzyme fromBacillus cereus

1973 ◽  
Vol 40 (3) ◽  
pp. 427-440 ◽  
Author(s):  
M. A. Islam ◽  
J. M. V. Blanshard
Keyword(s):  
Metal Ion ◽  
Absorption Maximum ◽  
Proteolytic Enzyme ◽  
Heavy Metal Ions ◽  
Freeze Drying ◽  
Deae Cellulose ◽  
Milk Clotting ◽  
Purification And Properties ◽  
Extracellular Proteolytic Enzyme ◽  
Metal Ion Activation

SummaryA milk-clotting proteolytic enzyme was isolated and purified from the culture filtrate ofBacillus cereusstrain x29 by fractionation with acetone or ammonium sulphate and subsequent column chromatography employing DEAE cellulose and DEAE Sephadex. The purified enzyme was found to be homogeneous by acrylamide gel electrophoresis from pH 3·5 to 8·6, with, a molecular weight of about 50000. The single absorption maximum of the native enzyme was at 277 nm and the value ofat 280 nm was 7·79. Purification resulted in a 9-fold enhancement of activity with 24 % yield. The optimum activity of the enzyme was at pH 8·0 at 40 °C with casein as the substrate. The enzyme was found to be most stable at pH 6·0 and was stable to freezing and freeze-drying. Heavy metal ions were found to inactivate the enzyme, but no metal ion activation was found. Enzyme activity was inhibited irreversibly by EDTA and reversibly by 1,10-phenanthroline. The enzyme has been identified as a Zn-containing neutral protease.

PURIFICATION AND PROPERTIES OF A PROTEASE FROM PENICILLIUM CYANEO-FULVUM

1960 ◽  
Vol 38 (1) ◽  
pp. 969-980 ◽  
Author(s):  
Kartar Singh ◽  
S. M. Martin
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Proteolytic Enzyme ◽  
Ethylenediaminetetraacetic Acid ◽  
Alkaline Proteases ◽  
Plasma Albumin ◽  
Metal Chelating ◽  
Bovine Plasma ◽  
Purification And Properties ◽  
Optimum Ph

A proteolytic enzyme present in culture filtrates of Penicillium cyaneo-fulvum was purified approximately 100-fold. In the ultracentrifuge the enzyme behaved as a homogeneous protein, but on electrophoresis some contamination was apparent. The molecular weight of the enzyme was estimated to be about 45,000.The protease hydrolyzed casein and denatured haemoglobin, gelatin, and native bovine plasma albumin but not native or denatured ovalbumin. It also coagulated milk. The optimum pH for caseolysis was 9.5 to 11.0. Metal chelating- and sulphydryl-reagents did not affect enzyme activity but zinc and mercurous ions inhibited the enzyme, the inhibition being reversed with ethylenediaminetetraacetic acid (EDTA). Soybean trypsin inhibitor was without effect on the enzyme whereas ovomucoid inhibited the enzyme. Although it is similar in some respects to other alkaline proteases, the P. cyaneo-fulvum enzyme does not appear to be identical with any one of them.

An extracellular proteolytic enzyme from Scopalariopsis brevicaulis. II. Hydrolysis of poly amino acids

1972 ◽  
Vol 18 (7) ◽  
pp. 1165-1167 ◽  
Author(s):  
Kartar Singh ◽  
Claude Vézina
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Proteolytic Enzyme ◽  
Ph Values ◽  
Optimum Ph ◽  
Extracellular Proteolytic Enzyme ◽  
Scopulariopsis Brevicaulis ◽  
Hydrolysis Of

Scopulariopsis brevicaulis protease hydrolyzed poly-L-lysine and poly-L-glutamic acid; optimum pH values for hydrolysis were 10.6 and 4.7 respectively. Final products of poly-L-lysine digestion by the protease were intermediate peptides from tetramer upwards. Pentalysine was not hydrolyzed by the enzyme. The protease had no action on poly-L-aspartic acid, poly-L-alanine, poly-L-glycine, poly-L-valine, or poly-L-leucine.

PURIFICATION AND PROPERTIES OF A PROTEASE FROM PENICILLIUM CYANEO-FULVUM

1960 ◽  
Vol 38 (9) ◽  
pp. 969-980 ◽  
Author(s):  
Kartar Singh ◽  
S. M. Martin
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Proteolytic Enzyme ◽  
Ethylenediaminetetraacetic Acid ◽  
Alkaline Proteases ◽  
Plasma Albumin ◽  
Metal Chelating ◽  
Bovine Plasma ◽  
Purification And Properties ◽  
Optimum Ph

A proteolytic enzyme present in culture filtrates of Penicillium cyaneo-fulvum was purified approximately 100-fold. In the ultracentrifuge the enzyme behaved as a homogeneous protein, but on electrophoresis some contamination was apparent. The molecular weight of the enzyme was estimated to be about 45,000.The protease hydrolyzed casein and denatured haemoglobin, gelatin, and native bovine plasma albumin but not native or denatured ovalbumin. It also coagulated milk. The optimum pH for caseolysis was 9.5 to 11.0. Metal chelating- and sulphydryl-reagents did not affect enzyme activity but zinc and mercurous ions inhibited the enzyme, the inhibition being reversed with ethylenediaminetetraacetic acid (EDTA). Soybean trypsin inhibitor was without effect on the enzyme whereas ovomucoid inhibited the enzyme. Although it is similar in some respects to other alkaline proteases, the P. cyaneo-fulvum enzyme does not appear to be identical with any one of them.

scholarly journals Purification and properties of a proteolytic enzyme from French beans

1965 ◽  
Vol 97 (1) ◽  
pp. 228-235 ◽  
Author(s):  
JRE Wells
Keyword(s):  
Proteolytic Enzyme ◽  
Deae Cellulose ◽  
Basic Amino Acid ◽  
Animal Origin ◽  
Prolonged Storage ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
French Beans ◽  
Purification And Properties ◽  
Endopeptidase Activity

1. A proteolytic enzyme with some features of a carboxypeptidase has been purified some 1180-fold from the sap of French beans (Phaseolus vulgaris var. Prince). A bright blue protein, plastocyanin, was separated from the enzyme by DEAE-cellulose chromatography. 2. Unlike carboxypeptidase A or B of animal origin, there is no evidence that the enzyme is a metalloprotein. There was no stimulation of activity by a number of metal ions, reducing agents or 2-mercapto-ethanol. Neither EDTA nor 1,10-o-phenanthroline inhibited the enzyme. 3. The proteolytic enzyme from beans, readily soluble at neutral or slightly acidic pH values, has a pH optimum of pH5.6 for the hydrolysis of leucine from benzyloxy-carbonylglycyl-l-leucine. Solutions of the enzyme in 0.1m-sodium acetate, pH5.5, lose about 2% of their activity/week at 4 degrees . Virtually no loss of activity results after prolonged storage at -15 degrees . 4. Incubation of the bean enzyme with peptides indicates that the enzyme will release acidic, neutral and basic amino acid residues as well as proline, although adjacent acidic residues in a peptide appear to inhibit the enzyme. The possibility of endopeptidase activity in the purified preparation requires further examination.

scholarly journals A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

2021 ◽  
Author(s):  
◽  
Graeme Lyall Dick
Keyword(s):  
Deae Cellulose ◽  
Effect Of Temperature ◽  
Endogenous Inhibitor ◽  
Catalysed Reaction ◽  
Purification And Properties ◽  
Isoelectric Points ◽  
Costelytra Zealandica ◽  
Optimum Ph ◽  
Effects Of Ph ◽  
The Stability

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

Purification and Properties of Ribonuclease From Buffalo Milk Whey

1977 ◽  
Vol 40 (6) ◽  
pp. 375-377 ◽  
Author(s):  
AZZA A. ISMAIL ◽  
N. S. AHMED ◽  
M. A. KHORSHID
Keyword(s):  
Deae Cellulose ◽  
Ribonuclease A ◽  
Buffalo Milk ◽  
Optimum Temperature ◽  
Isolation And Identification ◽  
Milk Whey ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Ribonuclease B

A procedure was developed for isolation and identification of ribonuclease from buffalo milk whey. Ribonuclease was precipitated with (NH4)2SO4 between 65 and 90% saturation. The precipitate was dissolved, dialyzed, and fractionated on DEAE-cellulose. Two ribonuclease-rich fractions were collected, i.e. ribonuclease A and B. Ribonuclease A had an optimum pH of 7 .0, and ribonuclease B had an optimum pH of 8.6. Both had an optimum temperature at 38 C. The ribonucleases in the purified state were unstable to heat and their activity decreased as the time of exposure increased. Both enzyme fractions were sensitive to inhibitors. NaCl and NaN3 were stimulatory for ribonuclease A, while ribonuclease B was stimulated only by NaCl.

scholarly journals A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

2021 ◽  
Author(s):  
◽  
Graeme Lyall Dick
Keyword(s):  
Deae Cellulose ◽  
Effect Of Temperature ◽  
Endogenous Inhibitor ◽  
Catalysed Reaction ◽  
Purification And Properties ◽  
Isoelectric Points ◽  
Costelytra Zealandica ◽  
Optimum Ph ◽  
Effects Of Ph ◽  
The Stability

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

AN EXTRACELLULAR PROTEASE FROM ASPERGILLUS FUMIGATUS

1965 ◽  
Vol 43 (10) ◽  
pp. 1745-1753 ◽  
Author(s):  
S. M. Martin ◽  
A. G. Jönsson
Keyword(s):  
Aspergillus Fumigatus ◽  
Column Chromatography ◽  
Proteolytic Enzyme ◽  
Heat Inactivation ◽  
Alkaline Proteases ◽  
Polyglutamic Acid ◽  
Extracellular Proteolytic Enzyme ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Single Enzyme

An extracellular proteolytic enzyme produced by Aspergillus fumigatus has been isolated and purified. Preparations showed two distinct peaks of activity when allowed to act on proteins buffered at different pH's (pH 7 and 10 with casein and pH 4 and 9 with hemoglobin). Although this might suggest the presence of distinct neutral and alkaline proteases, all other observations (column chromatography, electrophoresis, heat inactivation, etc.) indicated that a single enzyme was involved. The enzyme hydrolyzed polyglutamic acid and polylysine optimally at about pH 4 and 10 respectively, with the rate of hydrolysis of pofyglutamic acid being about 10 times that of polylysine. The products of digestion of polyamino acids were the corresponding peptides, no trace of monomer being found.

scholarly journals Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney

1989 ◽  
Vol 261 (3) ◽  
pp. 761-768 ◽  
Author(s):  
D R Deshmukh ◽  
S M Mungre
Keyword(s):  
Rat Kidney ◽  
Deae Cellulose ◽  
Dicarboxylic Acids ◽  
Structural Similarity ◽  
Appreciable Amount ◽  
Kinetic Properties ◽  
Bovine Kidney ◽  
Purification And Properties ◽  
Kidney Enzyme ◽  
Structural Differences

Previous studies with rat kidney preparations indicated that 2-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are properties of a single protein. We found that bovine kidney contains an appreciable amount of AadAT activity, but lacks KAT activity. AadAT from bovine and rat kidney extracts were purified to electrophoretic homogeneity. The purification procedure included fractionation with (NH1)2SO1, heat treatment, DEAE-cellulose chromatography and hydroxyapatite chromatography. Physical and kinetic properties, such as pH optima, Km for substrates, Mr, electrophoretic mobility and inhibition by dicarboxylic acids of bovine kidney AadAT, were similar to those of the rat kidney enzyme. However, bovine kidney AadAT differed from rat kidney AadAT in substrate specificity, amino acid composition and stability when stored. The titration curve of bovine kidney AadAT was also different from that of the rat kidney enzyme. The results suggest that bovine kidney AadAT may have some structural similarity to rat kidney AadAT and that the structural differences observed between the two enzymes may explain the absence of KAT activity in bovine kidney.

Sign in / Sign up

Export Citation Format

Share Document