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.

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.

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 The purification and properties of pig spleen phosphofructokinase

1975 ◽  
Vol 151 (2) ◽  
pp. 327-336 ◽  
Author(s):  
P E Hickman ◽  
M J Weidemann
Keyword(s):  
Metal Ion ◽  
Protein Concentration ◽  
Deae Cellulose ◽  
Significant Inhibition ◽  
Free Form ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Phosphoenol Pyruvate ◽  
Sigmoidal Kinetics ◽  
Cellulose Column

Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors.

scholarly journals Ag-Functionalized Si Nanowire Arrays Aligned Vertically for SERS Detection of Captured Heavy Metal Ions by BSA

Coatings ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 685
Author(s):  
Ai-Huei Chiou ◽  
Jun-Luo Wei ◽  
Ssu-Han Chen
Keyword(s):  
Heavy Metal ◽  
Raman Scattering ◽  
Metal Ions ◽  
Metal Ion ◽  
Heavy Metal Ions ◽  
Great Promise ◽  
Ag Nps ◽  
Water Contact ◽  
Oh Groups ◽  
Si Nanowire

A novel surface-enhanced Raman scattering (SERS)-based probe to capture heavy metal ion (Zn2+) by bovine serum albumin (BSA) using Si-nanowire (SiNW) arrays with silver nanoparticles (AgNPs) was developed. A layer with AgNPs was deposited on the SiNW surface by RF magnetron sputtering for enhancement of SERS signals. Using a high-resolution transmission electron microscope (HRTEM), the observation reveals that the AgNP layer with depths of 30–75 nm was successfully deposited on SiNW arrays. The Ag peaks in EDS and XRD spectra of SiNW arrays confirmed the presence of Ag particles on SiNW arrays. The WCA observations showed a high affinity of the Ag–SiNW arrays immobilized with BSA (water contact angle (WCA) = 87.1°) and ZnSO4 (WCA = 8.8°). The results of FTIR analysis illustrate that the conjugate bonds exist between zinc sulfate (ZnSO4) and –OH groups/–NH groups of BSA. The resulting SiNWs/Ag NPs composite interfaces showed large Raman scattering enhancement for the capture of heavy metal ions by BSA with a detection of 0.1 μM. BSA and ZnSO4 conjugations, illustrating specific SERS spectra with high sensitivity, which suggests great promise in developing label-free biosensors.

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.

scholarly journals Purification and properties of two extracellular β-lactamases from Bacillus cereus 569/H

1970 ◽  
Vol 118 (3) ◽  
pp. 457-465 ◽  
Author(s):  
S. Kuwabara
Keyword(s):  
Molecular Weight ◽  
Bacillus Cereus ◽  
Zinc Sulphate ◽  
Deae Cellulose ◽  
Casein Hydrolysate ◽  
Crystalline Form ◽  
Casamino Acid ◽  
Fractional Precipitation ◽  
Purification And Properties ◽  
Rapid Production

1. When Bacillus cereus 569/H was grown in a casamino acid (casein-hydrolysate) medium containing zinc sulphate rapid production of extracellular β-lactamase II preceded that of β-lactamase I. 2. β-Lactamase I was separated from β-lactamase II by fractional precipitation with ammonium sulphate. 3. β-Lactamase I was purified by a process involving chromatography on Celite and DEAE-cellulose and β-lactamase II by chromatography on DEAE-cellulose after denaturation of β-lactamase I by heat. Both enzymes were obtained in crystalline form. 4. β-Lactamase II prepared in this way appeared to have a higher molecular weight than β-lactamase I and required Zn2+ as a cofactor for both cephalosporinase and penicillinase activities.

scholarly journals Purification and properties of α-d-mannosidase from the limpet, Patella vulgata

1970 ◽  
Vol 117 (1) ◽  
pp. 129-137 ◽  
Author(s):  
Sybil M. Snaith ◽  
G. A. Levvy ◽  
A. J. Hay
Keyword(s):  
Metal Ion ◽  
Reversible Inactivation ◽  
Patella Vulgata ◽  
Full Activity ◽  
Acid Ph ◽  
Purification And Properties ◽  
Rate Of Reaction ◽  
Metal Ion Complex

1. α-Mannosidase from the limpet, Patella vulgata, was purified nearly 150-fold, with 40% recovery. β-N-Acetylglucosaminidase was removed from the preparation by treatment with ethanol. The final product was virtually free from β-galactosidase. 2. Limpet α-mannosidase was assayed at pH3.5 and at this pH it was necessary to add Zn2+ for full activity. At pH5, the enzyme had the same activity in the presence or absence of added Zn2+. 3. On incubation at acid pH, the enzyme underwent reversible inactivation, which was prevented by adding Zn2+. 4. EDTA accelerated inactivation and the addition of Zn2+ at once restored activity. No other cation was found to reactivate the enzyme. 5. Cl- had an unspecific effect on hydrolysis by limpet α-mannosidase. It increased the rate of reaction with substrate. The anion did not prevent or reverse inactivation by EDTA. 6. It is concluded that α-mannosidase is a metalloenzyme or enzyme–metal ion complex, dissociable at the pH of activity, and that it requires Zn2+ specifically.

scholarly journals Purification and properties of α-mannosidase II from Golgi-like membranes of baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells

1997 ◽  
Vol 324 (3) ◽  
pp. 951-956 ◽  
Author(s):  
Jianxin REN ◽  
Francis J. CASTELLINO ◽  
Roger K. BRETTHAUER
Keyword(s):  
Spodoptera Frugiperda ◽  
Reaction Pathway ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Mannose Residue ◽  
Lepidopteran Insect ◽  
Reducing Conditions ◽  
Apparent Homogeneity ◽  
Purification And Properties

An α-mannosidase II-like activity was identified in baculovirus-infected Spodoptera frugiperda (IPLB-SF21-AE) cells. The enzyme responsible was purified from Golgi-type membranes to apparent homogeneity by using a combination of steps including DEAE-cellulose, hydroxyapatite, concanavalin A–Sepharose and gel filtration chromatography. The molecular mass of this purified protein was approx. 120 kDa by SDS/PAGE under reducing conditions and approx. 240 kDa under non-reducing conditions, indicating that the enzyme is a disulphide-linked dimer. Substrates demonstrated to undergo hydrolysis with this enzyme were GlcNAc-Man5-GlcNAc-GlcNAc (non-reduced and reduced) and p-nitrophenyl α-d-mannopyranoside. The oligosaccharide substrate was converted into GlcNAc-Man3-GlcNAc-GlcNAc through an intermediate GlcNAc-Man4-GlcNAc-GlcNAc. Treatment of the isolated intermediate oligosaccharide with endoglycosidase H resulted in its conversion into GlcNAc-Man4-GlcNAc. This indicated that it contained the α-1,3-linked mannose residue on the α-1,6-linked mannose arm and showed that the α-1,6-linked mannose residue on the α-1,6-linked mannose arm had been preferentially hydrolysed by the mannosidase. The oligosaccharide lacking the β-1,2-linked GlcNAc residue on the α-1,3-linked mannose arm (Man5-GlcNAc-GlcNAc) was not hydrolysed in the presence of the enzyme. Metal ions were not required for enzymic activity on any of the substrates, but Cu2+ was strongly inhibitory. The activity of the enzyme was inhibited at low concentrations of swainsonine, but much higher concentrations of 1-deoxymannojirimycin were required to achieve inhibition. All of these properties are characteristic of mannosidase II enzymes from other eukaryotic tissues. The presence of mannosidase II in lepidopteran insect cells would allow entry of N-linked glycoproteins into the complex processing reaction pathway or into the terminal Man3-GlcNAc-GlcNAc pathway.

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