A simple procedure for partial purification of an RNAase of Entamoeba invadens

1981 ◽  
Vol 27 (8) ◽  
pp. 856-859
Author(s):  
David L. Weller ◽  
Andrea Richman
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Sucrose Gradient ◽  
Specific Activity ◽  
Simple Procedure ◽  
Partial Purification ◽  
Step Procedure ◽  
Entamoeba Invadens

The behavior of an RNAase, present in centrifugally clarified homogenates of axenic trophozoites of Entamoeba invadens, on isoelectric focusing (IEF) and agarose–poly(G) chromatography is described. The results led to a simple two-step procedure for partial purification of the RNAase in which fractionation of the homogenate by IEF is followed by agarose–poly(G) chromatography. Recovery of enzyme activity has ranged from 50 to 80% of that present in homogenates, and increases of 80- to 160-fold in the specific activity have been obtained using the procedure. A single zone of activity was observed on analysis of the partially purified RNAase by sucrose gradient IEF and velocity zonal ultracentrifugation.

scholarly journals Isolation of β-N-acetylhexosaminidase from rabbit semen and its role in fertilization

1980 ◽  
Vol 191 (3) ◽  
pp. 827-834 ◽  
Author(s):  
A A Farooqui ◽  
P N Srivastava
Keyword(s):  
Enzyme Activity ◽  
Metal Ions ◽  
Seminal Plasma ◽  
Concanavalin A ◽  
Zona Pellucida ◽  
Specific Activity ◽  
Optimal Activity ◽  
Step Procedure ◽  
Km Value ◽  
Arylsulphatase A

Beta-N-Acetylhexosaminidase was purified from the rabbit seminal plasma by a three-step procedure involving hydroxyapatite, Sephadex G-200 and concanavalin A–Sepharose chromatography. The specific activity of the purified preparation was 56mu mol/min per mg of protein, which represented a 226-fold purification and a 54% yield of the enzyme activity. The purified enzyme was electrophoretically homogeneous. The homogeneous enzyme showed optimal activity at pH4.0. The apparent Km value and Vmax. were 1.4 mM and 56mu mol/min per mg of protein respectively. Metal ions such as Ag + and Hg2+ and p-chloromercuribenzoate strongly inhibited the enzyme activity. The treatment of rabbit ova with a mixture of Beta-N-acetylhexosaminidase and arylsulphatase A results in the swelling of the zona pellucida.

scholarly journals CHARACTERIZATION OF PARTIALLY PURIFIED TYROSINASE ISOLATED FROM BITTER KOLA (GARCINIA KOLA)

2022 ◽  
Author(s):  
B.O. Itakorode ◽  
O.E. Agboola ◽  
M.B. Adeboye ◽  
C.C. Benedict ◽  
K.N. Terkula ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Peer Review ◽  
Specific Activity ◽  
Ph Value ◽  
Inhibitory Effect ◽  
Enzymatic Browning ◽  
Partial Purification ◽  
Garcinia Kola ◽  
North East ◽  
Review Reports

Objective: Tyrosinase is a glycosylated, copper-containing oxidase that catalyzes the first two steps of mammalian melanogenesis as well as enzymatic browning events in damaged fruits during post-harvest handling and processing. Human skin hyperpigmentation and enzymatic browning in fruits are both undesirable. In this study, the properties and inhibitory effect of some compounds on bitter kola tyrosinase were investigated. Methods: Bitter kola tyrosinase was isolated and characterized using standard protocols. Partial purification was carried out on Sephadex G-100 loaded column chromatography.  Results: Bitter kola tyrosinase was purified with a specific activity of 3.5 U/mg protein, purification fold of 2.4 and a yield of 34%. The optimum pH value was found to be 6.0 while the optimum temperature value for maximum enzyme activity was observed at 60°C. The enzyme was stable at 40oC for 20 minutes. Metals such as NaCl, KCl, MgCl2 and CaCl2 had inhibitory effect on the activity; though MgCl2 and CaCl2 had minimal effect. Also, EDTA, β-marcaptoethanol and glutathione greatly inhibited the enzyme activity at all the tested concentration. Conclusion: The properties of bitter kola tyrosinase compare very well with the tyrosinase from other sources. Also, the study was able to establish the inhibitory effect of some compounds and this could be applied in food processing industries.                  Peer Review History: Received: 2 November 2021; Revised: 11 December; Accepted: 25 December, Available online: 15 January 2022 Academic Editor:  Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, [email protected] UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency.  Received file:                Reviewer's Comments: Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.5/10 Reviewers: Dr. Nazim Hussain, North East Frontier Technical University, Arunachal pradesh, India, [email protected] Ahmad Najib, Universitas Muslim Indonesia, Makassar, Indonesia, [email protected] Prof. Dr. Ali Gamal Ahmed Al-kaf, Sana'a university, Yemen, [email protected] Similar Articles: PHYTOCHEMICAL PURIFICATION OF ACTIVE CONSTITUENTS ISOLATED FROM ROOT OF THE MEDICINAL HERB, CARALLUMA QUADRANGULA

scholarly journals Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system

1981 ◽  
Vol 198 (2) ◽  
pp. 265-271 ◽  
Author(s):  
F Wuytack ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Transport System ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Partial Purification ◽  
Total Enzyme Activity ◽  
Dependent Atpase ◽  
Specific Activities ◽  
Total Enzyme

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.

Purification of lactate dehydrogenase isoenzyme-5 from human liver.

1981 ◽  
Vol 27 (1) ◽  
pp. 88-93 ◽  
Author(s):  
S M Pettit ◽  
D A Nealon ◽  
A R Henderson
Keyword(s):  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Major Protein ◽  
Dehydrogenase Isoenzyme ◽  
Major Protein Band

Abstract We present a method for preparing human liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase; EC 1.1.1.27) isoenzyme-5 by sequential ion-exchange chromatography, general-ligand (AMP analog) affinity chromatography, and preparative isoelectric focusing. The yield ws 40%, with a 493-fold purification. The final specific activity was 458 kU per gram of protein. The preparation contained less than 0.2% of lactate dehydrogenase isoenzyme-4, was homogeneous by agarose gel electrophoresis and also by polyacrylamide gel electrophoresis at pH 8.9 and 6.9, and showed one major protein band (containing all the enzyme activity) and one minor anodic contaminant (containing no enzyme activity) by analytical isoelectric focusing. The enzyme had a mean pI value of 9.59 (SD 0.04) (n = 5) at 5 degrees C. By comparison, the pI value of a preparation of rabbit lactate dehydrogenase-5 was 9.16 (5 degrees C).

Rat prostatic acid phosphatase: androgenic control of isoelectric focusing patterns

1983 ◽  
Vol 61 (7) ◽  
pp. 744-749 ◽  
Author(s):  
J. Downey ◽  
D. Mahan ◽  
T. G. Flynn ◽  
C. E. Bird ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Isoelectric Focusing ◽  
Specific Activity ◽  
Electrophoretic Pattern ◽  
Prostatic Acid Phosphatase ◽  
Staining Intensity ◽  
Adult Rats ◽  
Enzyme Specific Activity ◽  
Ph Range

To further characterize the androgen dependence of prostatic acid phosphatase (AP), the isoelectric focusing patterns of enzyme activity have been examined for normal and castrated adult rats and for rats receiving androgen injections. Isoelectric focusing was performed in polyacrylamide gels over the pH range 4–8. Naphthyl phosphate was used as substrate for staining. For normal rats there was a single lysosomal band (isoelectric point (pI) = 7.35 ± 0.04), four closely migrating secretory bands (pI = 5.96–5.63), and an androgen-dependent band (pI = 6.37 ± 0.05) which as yet has not been identified as either lysosomal or secretory. Following castration the secretory bands decreased significantly in staining intensity, the androgen-dependent band disappeared, and two new lysosomal bands (pI's = 7.13 ± 0.03 and 7.00 ± 0.03) appeared. With androgen replacement the latter two bands disappeared, the androgen-dependent band reappeared, and the secretory bands increased in staining intensity but with the most anodic of the four appearing before the others. This suggests that it could be a precursor to the others. The isoelectric focusing patterns of AP activity appear to be a better method of assessing the androgen status of the prostate than are the previously used parameters, namely, enzyme specific activity, degree of inhibition by tartrate, and polyacrylamide gel electrophoretic pattern.

scholarly journals Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

2018 ◽  
Vol 22 (2) ◽  
pp. 47
Author(s):  
Akhmad Solikhin ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Wendry Setiyadi Putranto
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Iodoacetic Acid ◽  
Aspartic Protease ◽  
Partial Purification ◽  
Antioxidant Agents

   Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

scholarly journals Partial purification of goat kidney β-mannosidase

1988 ◽  
Vol 249 (3) ◽  
pp. 871-875 ◽  
Author(s):  
J I Frei ◽  
K T Cavanagh ◽  
R A Fisher ◽  
R P Hausinger ◽  
M Dupuis ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Cation Exchange ◽  
Anion Exchange ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Fast Protein Liquid Chromatography ◽  
Partial Purification

1. Goat kidney beta-mannosidase was purified 8500-fold to a specific activity of 65,000 nmol/h per mg of protein with a 6% yield by using multiple steps including cation-exchange and anion-exchange fast protein liquid chromatography. This is the first description of a highly purified preparation from goat tissue; however, it was not homogeneous, as judged by silver-stained SDS/polyacrylamide-gel electrophoresis. 2. The enzyme exhibited microheterogeneity when analysed by isoelectric focusing (pI 5.5-6.5). 3. Purified beta-mannosidase hydrolysed the terminal beta-(1→4)-linkage of oligosaccharides that accumulate in beta-mannosidosis.

Deoxyribonucleic Acid Polymerase from Immature Testes of Salmon

1971 ◽  
Vol 49 (1) ◽  
pp. 19-27 ◽  
Author(s):  
H. L. A. Tarr ◽  
Linda Gardner
Keyword(s):  
Dna Polymerase ◽  
Nearest Neighbor ◽  
Specific Activity ◽  
Simple Procedure ◽  
Buoyant Density ◽  
Partial Purification ◽  
Alternating Copolymer ◽  
Copolymer Poly ◽  
Deoxyribonucleoside Triphosphate ◽  
Limited Digestion

A simple procedure for partial purification of a DNA polymerase from immature testes of salmon (Oncorhynchus nerka) in which 20% glycerol and 2 mM dithiothreitol are used to protect the enzyme is described. The enzyme has a specific activity of 17–45 nmoles of deoxyribonucleoside triphosphate (dTTP) incorporated into DNA per milligram protein per hour at 25 °C. It has a specific requirement for Mg2+ or Mn2+, for four deoxyribonucleoside triphosphates, and for a DNA primer. The latter is best supplied by a DNA duplex that has undergone limited digestion with pancreatic DNase or by polydeoxyadenylate polydeoxythymidylate alternating copolymer (poly d(A–T)) that has been similarly treated. Native and heat-denatured DNA are poor primers. The enzyme is strongly inhibited by pancreatic DNase and pyrophosphate, less strongly by actinomycin D, and is markedly stimulated by EDTA. Addition of KCl and NaCl causes inhibition that at 100 mM concentration is 70% with KCl and 80% with NaCl. The enzyme loses 50% of its activity in 15 min at 35 °C and in 3 h at 30 °C. At 30 °C glycerol (47%), DNA, and bovine serum albumin (2 mg/ml) retard loss in activity. With poly d(A–T) primer, incorporation of α-32P-dATP into DNA proceeds for only a short time and then ceases unless dTTP is present, when incorporation continues. When the products formed in such reactions are subjected to nearest-neighbor frequency analysis only 3′-dTMP possesses radioactivity. Polydeoxyguanylate polydeoxycytidylate linear polymer has no priming activity. The product of the DNA polymerase formed a band in a buoyant density gradient of CsCl at a density similar to that of the DNA primer.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Pengaruh Penambahan Molases Pada Kulit Pisang Kapendis Untuk Media Produksi Xilanase B.Stearothermophillus DSM 22

2019 ◽  
Vol 15 (3) ◽  
Author(s):  
Trismillah
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Banana Peel ◽  
Partial Purification ◽  
Temperature Conditions ◽  
Cavendish Banana ◽  
Working Volume ◽  
Initial Ph ◽  
Xylanase Enzyme

Cavendish banana peel can be used as a substitute for the expensive xylan, while molasses than as a source of carbon as well as nitrogen, minerals and nutrients needed for the growth of microbes that can produce the enzyme. Xylanase produced from Bacillus stearothermopillus DSM 22, using media cavendish banana peels with the addition of molasses 1%, 2%, and 3%. Fermentation is done in a shaker incubator at 550C temperature conditions, initial pH 8, and 250 rpm agitation. The result showed the highest enzyme activity of 4,14 ± 0,16 U/mL min., on the addition 2% molasses after 24 hours. Further fermentation carried out in the fermenter working volume of 3.5 liters, with the condition of temperature 550C, pH 8, aeration 1 vvm, agitation 250 rpm, the highest spesific enzyme of activity of 51,62 ± 0,16 U/mg after 24 hours. Partial purification of xylanase enzyme fermentation is done with the results of microfiltration, ultrafiltration, ammonium sulfate (0-80%) and dialysis. There is an increase in the purity of the enzyme at each stage of purification, the highest purity on dialysis 3.23 times of crude enzymes.Kulit buah pisang kapendis dapat digunakan sebagai pengganti xilan yang harganya mahal, sementara molases selain sebagai sumber karbon serta nitrogen, mineral dan nutrisi dibutuhkan untuk pertumbuhan mikroba yang dapat menghasilkan enzim. Xilanase yang dihasilkan dari Bacillus stearothermopillus DSM 22, menggunakan media kulit pisang kapendis dengan penambahan molase 1%, 2%, dan 3%. Fermentasi dilakukan dalam shaker inkubator pada temperatur 550C, pH awal 8, dan agitasi 250 rpm. Hasilnya menunjukkan aktivitas enzim tertinggi 4,14 ± 0,16 U/mL min., pada penambahan 2% molases setelah 24 jam. Selanjutnya fermentasi dilakukan di dalam fermentor, volume kerja dari 3,5 liter, dengan kondisi temperatur 550C, pH 8, aeration 1 vvm, agitasi 250 rpm, aktivitas spesifik tertinggi 51,62 ± 0,16 U/mg setelah 24 jam. Pemurnian parsial fermentasi enzim xilanase dilakukan dengan hasil mikrofiltrasi, ultrafiltrasi, amonium sulfat (0-80%) dan dialisis. Ada peningkatan kemurnian enzim pada setiap tahap pemurnian, kemurnian tertinggi pada dialisis 3,23 kali dari enzim kasar.Keywords: Xylanase, B. stearothermophillus DSM 22, Cavendish banana peel, molasses, enzyme activity

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