Production of induced laccase by the fungus Rhizoctonia praticola

1986 ◽  
Vol 32 (11) ◽  
pp. 867-870 ◽  
Author(s):  
Kay L. Shuttleworth ◽  
Lori Postie ◽  
Jean-Marc Bollag
Keyword(s):  
Gel Electrophoresis ◽  
Inductive Effect ◽  
Laccase Production ◽  
Growth Media ◽  
Benzoic Acids ◽  
Substituted Phenols ◽  
Ph Optimum ◽  
Extracellular Laccase ◽  
Rhizoctonia Praticola

The ability of the fungus Rhizoctonia praticola to produce an induced extracellular laccase was examined. Potential inducers, including substituted phenols, anilines, and benzoic acids, were added at a concentration of 1 mM to the growth media of fungal cultures. Of the 11 compounds tested, 5 were found to have an inductive effect. The most effective inducer, p-anisidine (p-methoxyaniline), stimulated laccase production by a factor of 30. Other parameters which influenced the production of laccase were temperature, condition of the mycelia at the time of the induction, and the concentration of the inducer. A comparison of the p-anisidine induced laccase with the constitutive laccase showed noticeable similarities in Km, pH optimum, and mobility in gel electrophoresis, suggesting that the two enzymes may be similar.

Characterization of an enzyme from Rhizoctonia praticola which polymerizes phenolic compounds

1979 ◽  
Vol 25 (2) ◽  
pp. 229-233 ◽  
Author(s):  
Jean-Marc Bollag ◽  
Roy D. Sjoblad ◽  
Shu-Yen Liu
Keyword(s):  
Molecular Weight ◽  
Phenolic Compounds ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Phenol Oxidase ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Rhizoctonia Praticola

An extracellular phenol oxidase from the fungus Rhizoctonia praticola which polymerizes various xenobiotic phenols was isolated and characterized. The enzyme was purified by DEAE-cellulose and Sephadex G-200 chromatography followed by preparative polyacrylamide gel electrophoresis. Atomic absorption and EPR spectroscopy indicated the presence of copper, and SDS gel electrophoresis revealed a molecular weight of 78 000. With 2,6-dimethoxyphenol as substrate, the enzyme showed a pH optimum of 6.7–6.9, and a temperature optimum of 40 °C. According to these and additional characteristics it appears that the enzyme belongs to the class of laccases.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals Purification and properties of β-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller

1978 ◽  
Vol 175 (2) ◽  
pp. 743-750 ◽  
Author(s):  
P Calvo ◽  
A Reglero ◽  
J A Cabezas
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mixed Substrates ◽  
Ph Optimum ◽  
Terrestrial Mollusc ◽  
Buffer Solutions ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Ph Range

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8–4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.

Characterization and distribution of arginine kinase in the tissues of the scorpion, Palamneus phipsoni

1985 ◽  
Vol 63 (10) ◽  
pp. 2262-2266 ◽  
Author(s):  
A. V. Arjunwadkar ◽  
S. Raghupathi Rami Reddy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Alimentary Canal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Arginine Kinase ◽  
High Specificity ◽  
Ph Optimum ◽  
High Concentrations ◽  
Claw Muscle ◽  
Slight Inhibition

Arginine kinase in claw muscle extracts of the scorpion, Palamneus phipsoni, was characterized. The enzyme, with a pH optimum of 8.5 in the direction of phosphoarginine synthesis, showed activation by Mg2+, high specificity towards L-arginine as the guanidino substrate, slight inhibition by high concentrations of L-arginine and ATP, and a molecular weight of 33 500. On polyacrylamide gel electrophoresis at pH 8.3 the enzyme migrated to the anode as a single molecular species. In addition to the claw muscle, the enzyme activity was also found to be present in the heart, alimentary canal, hepatopancreas, and nervous system. In general, scorpion muscle arginine kinase appears to be similar in its properties to the enzyme from other arthropods.

Isozymes of α-galactosidase from Bacillus stearothermophilus

1980 ◽  
Vol 26 (8) ◽  
pp. 978-984 ◽  
Author(s):  
Dennis M. Pederson ◽  
Richard E. Goodman
Keyword(s):  
Gel Electrophoresis ◽  
Half Life ◽  
Protein Concentration ◽  
Mixed Type ◽  
Bacillus Stearothermophilus ◽  
Single Band ◽  
Molecular Forms ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Low Protein

Two molecular forms of α-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. α-Galactosidase I (with the substrate p-nitrophenyl α-D-galactopyranoside (PNPG)) has a pH optimum of 6 and a half-life at 65 °C of > 2 h at low protein concentration. α-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 °C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the β-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are α-galactosidase I, 280 000 ± 30 000 and α-galactosidase II, 325 000 ± 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 + 500 for α-galactosidase I and 84 000 ± 500 for α-galactosidase II, suggest that both enzymes consist of four subunits.

scholarly journals Purification of 2-oxoaldehyde dehydrogenase and its dependence on unusual amines

1975 ◽  
Vol 149 (3) ◽  
pp. 609-617 ◽  
Author(s):  
J Dunkerton ◽  
S P James
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
General Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Ph Values ◽  
Sheep Liver ◽  
Living Tissues

1. 2-Oxoaldehyde dehydrogenase was purified from sheep liver and gave one band on polyacrylamide-gel electrophoresis. 2. The enzyme was completely dependent for its activity on the presence of Tris or one of a number of related amines, all of general structure: (See article). When more than one R group was hydrogen no enzyme activity was observed. 3. Only one of these amines is known to exist in living tissues and large concentrations of all amines were required for maximum activity. L-2-Aminopropan-1-ol was the most effective amine on the basis of substrate Km and Vmax. values and the amine Km values. 4. The enzyme was activated by phosphate which lowered the Km values for methylglyoxal, amine and NAD+. 5. The pH optimum of the enzyme was 9.3 and there was no activity at pH values below 7.8. A search for activators that might produce activity at pH 7.4 proved unsuccessful. 6. The enzyme was inhibited by rather large concentrations of barbiturates (6-46 mM) and nitro-alcohol analogues of the activating amines (66-139 mM).

scholarly journals Purification and properties of β-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1079-1087 ◽  
Author(s):  
H Villarroya ◽  
J Williams ◽  
P Dey ◽  
S Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Electrophoretic Method ◽  
Germinated Seeds

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.

scholarly journals Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles

1987 ◽  
Vol 244 (1) ◽  
pp. 219-224 ◽  
Author(s):  
J M Jacobs ◽  
N J Jacobs
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chlorophyll Synthesis ◽  
Yeast Mitochondria ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Haem Biosynthesis ◽  
Triton X ◽  
Polypeptide Band

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.

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