Purification and properties of endo-β-glucanase in the yeast Hanseniaspora valbyensis

1969 ◽  
Vol 15 (7) ◽  
pp. 697-701 ◽  
Author(s):  
Ahmed T. H. Abd-El-Al ◽  
H. J. Phaff
Keyword(s):  
Enzyme Activity ◽  
Cell Walls ◽  
Carbon Sources ◽  
Culture Fluid ◽  
Extracellular Enzyme ◽  
Enzyme Concentration ◽  
Random Pattern ◽  
Ph Optimum ◽  
Hanseniaspora Uvarum ◽  
Purification And Properties

Endo-β-glucanase was detected in Hanseniaspora valbyensis and Hanseniaspora uvarum. The extracellular enzyme activity of H. valbyensis was maximal with vigorous aeration at 30 C in a complete mineral medium with glucose and 0.2 M Na-succinate buffer. The enzyme concentration was lower when the buffer was 0.05 M Na-succinate. The use of a variety of carbon sources, including yeast cell walls, failed to induce higher enzyme activities. The enzyme was purified 34-fold from the culture fluid of H. valbyensis. The purified enzyme had a broad pH optimum between pH 3.5 and 4.5. Among the known β-glucan homopolymers, only laminarin (β-1 → 3 bonds) was hydrolyzed by the enzyme. The Km for laminarin was 0.11 mg/ml and the Vmax was 0.835 μmoles glucose equivalents/min mg protein. No action was detected with laminaribiose, laminaritriose, or oat glucan. Laminaritetraose, however, was hydrolyzed slowly in a random pattern. These results suggest the requirement for three consecutive β-1 → 3 bonds for activity.

THE HETEROGENEITY OF PANCREATIC DEOXYRIBONUCLEASE

1962 ◽  
Vol 40 (2) ◽  
pp. 165-175 ◽  
Author(s):  
G. C. Becking ◽  
R. O. Hurst
Keyword(s):  
Enzyme Activity ◽  
Ionic Strength ◽  
Enzyme Preparation ◽  
Deoxyribonucleic Acid ◽  
Paper Electrophoresis ◽  
Relative Activity ◽  
Enzyme Concentration ◽  
Uranyl Acetate ◽  
Ph Optimum

The action of crystalline pancreatic deoxyribonuclease on sodium oligonucleotides in the presence of manganous ions has been studied and a pH optimum of 6.6 observed. Inhibition of the enzyme activity by increased ionic strength of the digest occurred. The liberation of products soluble in uranyl acetate – trichloroacetate was found to vary with enzyme concentration and the relative activity of the enzyme on oligonucleotides was best determined by a logarithm-plot method. The activity of the enzyme towards deoxyribonucleic acid or sodium oligonucleotides as substrate was not affected by treatment with acetone. Evidence of heterogeneity in the crystalline enzyme preparation was obtained using paper electrophoresis and chromatography on carboxymethylcellulose. Two fractions were separated that showed different ratios of activity towards the two substrates employed.

Purification and Properties of the Soluble 17β-Hydroxysteroid Dehydrogenase of Rabbit Uterus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 726-737 ◽  
Author(s):  
Kunhard Pollow ◽  
Walter Eiger ◽  
Herrmann Heßlinger ◽  
Barbara Pollow
Keyword(s):  
Enzyme Activity ◽  
Dehydrogenase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Maximal Velocity ◽  
Ammonium Sulfate Precipitation ◽  
Ph Optimum ◽  
Mobility Data ◽  
Purification And Properties

Abstract 17 β-Hydroxysteroid dehydrogenase activity towards estradiol-17 β has been demonstrated in the 105,000 X g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromato­graphy yielded a single 17 β-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight or the enzyme, calculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 β-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 β reacts at a faster rate than testosterone. The Km for estradiol is 4.16X 10-5 mol/1 for the NAD-linked enzyme activity and 4.37 X 10-5 mol/1 when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.

Partial characterization of the extracellular carboxymethylcellulase activity produced by the rumen bacterium Bacteroides succinogenes

1983 ◽  
Vol 29 (5) ◽  
pp. 504-517 ◽  
Author(s):  
D. Groleau ◽  
C. W. Forsberg
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Culture Fluid ◽  
Extracellular Enzyme ◽  
Rumen Bacterium ◽  
Ph Optimum ◽  
Complex Protein ◽  
Culture Supernate ◽  
Triton X

In cultures of Bacteroides succinogenes, in which cellulose was the source of carbohydrate, from 70 to 80% of the carboxymethylcellulase (CMCase) activity was present in the culture fluid. The crude extracellular enzyme readily hydrolyzed acid-swollen cellulose with the production of glucose and cellobiose. Of this extracellular CMCase, 50–62% was associated with sedimentable membrane fragments, 9–13% with nonsedimentable material with a molecular weight greater than 4 × 106, and 28–38% with molecules having a molecular weight of approximately 45 000. Polyacrylamide gel electrophoresis (PAGE), in the presence of sodium dodecyl sulfate, revealed that both the nonsedimentable and the sedimentable fraction had complex protein compositions. The nonsedimentable and sedimentable CMCase fractions, after treatment with Triton X-100, were subjected to PAGE in the presence of 0.2% (w/v) Triton X-100. The results indicated the presence of fast- and slow-migrating CMCases in the former, and of a slow-migrating CMCase in the latter. An apparently uncharged CMCase, which probably corresponded to the slow-migrating component by PAGE, was partially purified from the concentrated culture supernate by solubilization in Triton X-100 and chromatography on DEAE–Sepharose, CM–Sepharose, and Phenyl–Sepharose. The partially purified CMCase had a pH optimum of 5.6–6.6 and a temperature optimum of 50 °C.

Cysteine proteinase of Phascolomyces articulosus: purification and properties

1986 ◽  
Vol 64 (11) ◽  
pp. 2441-2445 ◽  
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Enzyme Activity ◽  
Cysteine Proteinase ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Iodoacetic Acid ◽  
Phenylmethylsulfonyl Fluoride ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Salting Out ◽  
Purification And Properties

A proteinase from the mycelial extracts of Phascolomyces articulosus has been purified by salting out with ammonium sulphate, gel filtration, hydroxyapatite adsorption, and affinity chromatography. The proteinase rapidly hydrolysed haemoglobin but failed to hydrolyse any of the synthetic peptides tested. The enzyme is a glycoprotein with an apparent molecular weight of 12 800. The carbohydrate content was estimated to be 65%. It has a temperature optimum of 20 °C, pH optimum of 3.0, and has a Km value of 6.6 mg∙mL−1 for denatured haemoglobin. Iodoacetic acid, iodoacetamide, benzamidine, as well as all the heavy metals tested inhibited the enzyme activity. The enzyme activity was not enhanced by reducing agents such as cysteine, ethylenediaminetetra acetic acid, and dithiothreitol, the latter, however, reversed inhibition by phenylmethylsulfonyl fluoride. The inhibitor studies suggest that the enzyme belongs to the group of cysteine proteinases.

scholarly journals Isolation and Activity Determination of Enzyme Phosphatase Secreted by Aspergillus niger

2019 ◽  
Vol 9 (1) ◽  
pp. 41-45
Author(s):  
Tafadzwa Zharare ◽  
Rumbidzai Mangoyi
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Recombinant Dna ◽  
Extracellular Enzyme ◽  
Enzyme Concentration ◽  
Recombinant Dna Technology ◽  
Activity Determination ◽  
Pure Cultures ◽  
Industrial Use

The use of enzymes on industrial scale saves a lot of energy and avoids pollution, thus holding a promise for green and economically sustainable alternative strategies in industrial transformations.  Generally, the fungi Aspergillus niger secretes enzymes which can be used in different industries. Thus, coming up with these enzymes in large amounts will definitely result in reduced costs encountered in importing them for industrial use.  This study focussed on isolation and activity determination of an enzyme phosphatase secreted by Aspergillus niger.  This enzyme can be of great importance in molecular biology industries, particularly for recombinant DNA technology.  For this study, pure cultures of Aspergillus niger were used.  Aspergillus niger was resuscitated on potato dextrose agar and then subcultured in Adam’s medium, a medium specific for the production of phosphatase.  Cells were centrifuged and the filtrate was collected whilst the residue was discarded. The filtrate was expected to contain the crude enzyme phosphatase since Aspergillus niger secretes the extracellular enzyme into the medium. Disodium phenyl phosphate was used as a substrate for the determination of the phosphatase activity. The enzyme activity was determined spectrophotometrically by reading absorbance of phenol formed in the presence of enzyme and the substrate. The concentration of phenol liberated was then used to calculate the enzyme activity expressed in King Armstrong Units (KAU).  Further work on enzyme activity determination was done by varying enzyme and substrate concentrations.  Results showed that the isolated alkaline phosphatase had activity of 4.0 KAU and 4.5 KAU at 25 ºC and 37 ºC respectively. Acidic phosphatase had activity of 5 KAU and 7 KAU at 25 ºC and 37 ºC respectively. Rate of activity increased upon increasing enzyme concentration and substrate.  Thus, Aspergillus niger produces the enzyme phosphatase, however, there is need to induce the production of these enzymes for industrial use.

Purification and properties of a nonspecific β-fructofuranosidase (inulinase) from the mushroom Panaeolus papillonaceus

1987 ◽  
Vol 33 (6) ◽  
pp. 520-524 ◽  
Author(s):  
Khana Mukherjee ◽  
S. Sengupta
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Activity Ratio ◽  
Optimum Temperature ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Inulinase Activity ◽  
Wide Ph Range ◽  
Total Molecular Weight ◽  
Ph Range

A nonspecific β-fructofuranosidase (inulinase) was purified to electrophoretic homogeneity from the culture filtrate of the mushroom Panaeolus papillonaceus. The enzyme is the first purified from a basidiomycete and consists of two subunits with a total molecular weight of 116 000. It is most active on sucrose, then on raffinose, stachyose, and inulin, in decreasing order. The sucrase/inulinase activity ratio (S/I) is 5.7. Fructose was detected as the liberated sugar from raffinose, stachyose, and inulin. The enzyme is highly thermostable with an optimum temperature range of 60–65 °C and a pH optimum of 6.0. The enzyme is stable over the pH range 4–10, and is also active over a wide pH range, exhibiting 50% activity even at pH 8.5. Iodoacetate, azide, and EDTA, at 20 mM concentration, and 1% (w/v) SDS have no effect on enzyme activity, whereas Ag+ and Hg2+ at 2 mM are highly inhibitory.

Cystathionine Synthase from Rat Liver: Partial Purification and Properties

1971 ◽  
Vol 49 (5) ◽  
pp. 484-491 ◽  
Author(s):  
F. Christine Brown ◽  
P. H. Gordon
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Pyridoxal Phosphate ◽  
Enzyme Concentration ◽  
Partial Purification ◽  
Sulfate Ions ◽  
Nitrobenzoic Acid ◽  
Assay Procedure ◽  
Purification And Properties ◽  
Rate Of Increase

Cystathionine synthase which has been purified about 1000-fold from rat liver has absorbance maxima at 280, 360, and 428 mμ. Treating the enzyme with cysteine apparently affects the removal of pyridoxal phosphate and destroys the enzyme activity. So does reduction with borohydride. However, in neither case is the spectrum affected. These observations suggest that pyridoxal phosphate may be bound to cystathionine synthase in an atypical fashion.Mercuric ions strongly inhibit the enzyme, but not in the presence of serine; hydroxylamine inhibits, but not in the presence of substrates. Other carbonyl reagents inhibit little if at all. Sulfate ions activate the enzyme.A new assay procedure for cystathionine synthase has been devised. In the presence of 5,5′-dithiobis-(2-nitrobenzoic acid), the enzyme catalyzes the degradation of cystathionine to serine and homocysteine. The rate of increase in absorbance at 412 mμ is a measure of enzyme concentration.

scholarly journals LIPASE IMMOBILIZATION ON FIBERS GRAFTED WITH POLYGLYCIDYL METHACHRYLATE

2019 ◽  
Vol 20 (1) ◽  
pp. 12-23
Author(s):  
Maan Alkhatib ◽  
Nik Adlin Bahrudin ◽  
HAMZAH M. SALLEH ◽  
Mohamed M. E. Nasef ◽  
Teo M. Ting
Keyword(s):  
Enzyme Activity ◽  
Storage Stability ◽  
Immobilized Enzyme ◽  
Immobilized Lipase ◽  
Enzyme Concentration ◽  
Free Enzyme ◽  
Effect Of Temperature ◽  
Ph Optimum ◽  
Lipase Enzyme ◽  
Central Composite

ABSTRACT: Lipase enzyme originated from wheat germ was immobilized on nylon -6- grafted with polyglycidyl methachrylate (PGMA). The immobilization of enzyme experiments were designed and studied using face centred central composite design (FCCCD) under response surface methodology (RSM). Prior to immobilization, the polymer was activated with diethyl amine/ethanol to introduce an amine functional group to facilitate covalent bonding with the enzyme. The immobilized and free enzymes were characterized for effect of temperature and pH on enzyme activity, stability, storage and reusability as well as kinetics studies. ANOVA revealed that optimum lipase activity of 0.287 U/ml was achieved at immobilization time of 5 h, pH of 6 and 1.0 mg/ml for enzyme concentration. The optimum temperatures and pH for immobilized and free enzymes were 45 °C and 35 °C, and 8 and 7, respectively. The immobilized enzyme showed higher stability compared to free enzyme. The immobilized enzyme retained 18% of its activity after being recycled 8 times. In a storage stability test, immobilized lipase was able to retain 70% of its activity after being stored for 30 days, while free enzyme activity dropped to 15 % after 20 days of storage. ABSTRAK:Enzim Lipase telah dihasilkan daripada mikroorganisma pegun gandum di atas nilon -6- dan digraf bersama poliglisidel methakrilet (PGMA). Enzim pegun ini direka dan dikaji secara eksperimen menggunakan reka bentuk campuran pusat pada permukaan (FCCCD) di bawah kaedah tindak balas permukaan (RSM). Sebelum menjadi pegun, polimer ini telah diaktifkan dengan dietil amine/ethanol bagi menghasilkan kumpulan fungsi amine bagi membantu ikatan kovalen atom pada enzim. Enzim pegun dan bebas ini telah dikategorikan mengikut kesan enzim ke atas suhu, aktiviti enzim ke atas kesan pH, kestabilan, keboleh-simpanan dan keboleh-gunaan balik, serta ujian tindak balas kinetik. ANOVA membuktikan bahawa aktiviti optimum enzim lipase ini adalah sebanyak 0.287 U/ml telah terhasil selama 5 jam pegun, pada pH 6 dan kepekatan enzim sebanyak 1.0 mg/ml. Suhu dan pH optimum, pada enzim pegun dan enzim bebas ini adalah pada 45 °C dan 35 °C, dan pH 8 dan 7, masing-masing. Enzim pegun ini menunjukkan lebih stabil daripada enzim bebas. Enzim pegun dilihat kekal 18% daripada aktivitinya selepas 8 kali ulangan. Melalui ujian kestabilan simpanan, enzim lipase pegun dapat mengekalkan 70% daripada aktivinya selepas disimpan selama 30 hari, manakala aktiviti enzim bebas telah menurun kepada 15% selepas 20 hari dalam simpanan.

THE HETEROGENEITY OF PANCREATIC DEOXYRIBONUCLEASE

1962 ◽  
Vol 40 (1) ◽  
pp. 165-175 ◽  
Author(s):  
G. C. Becking ◽  
R. O. Hurst
Keyword(s):  
Enzyme Activity ◽  
Ionic Strength ◽  
Enzyme Preparation ◽  
Deoxyribonucleic Acid ◽  
Paper Electrophoresis ◽  
Relative Activity ◽  
Enzyme Concentration ◽  
Uranyl Acetate ◽  
Ph Optimum

The action of crystalline pancreatic deoxyribonuclease on sodium oligonucleotides in the presence of manganous ions has been studied and a pH optimum of 6.6 observed. Inhibition of the enzyme activity by increased ionic strength of the digest occurred. The liberation of products soluble in uranyl acetate – trichloroacetate was found to vary with enzyme concentration and the relative activity of the enzyme on oligonucleotides was best determined by a logarithm-plot method. The activity of the enzyme towards deoxyribonucleic acid or sodium oligonucleotides as substrate was not affected by treatment with acetone. Evidence of heterogeneity in the crystalline enzyme preparation was obtained using paper electrophoresis and chromatography on carboxymethylcellulose. Two fractions were separated that showed different ratios of activity towards the two substrates employed.

scholarly journals In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.

1983 ◽  
Vol 258 (19) ◽  
pp. 11430-11433 ◽  
Author(s):  
C Edelstein ◽  
J I Gordon ◽  
K Toscas ◽  
H F Sims ◽  
A W Strauss ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Extracellular Enzyme ◽  
Extracellular Enzyme Activity ◽  
Partial Characterization
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