scholarly journals Rapid partial purification of placental glucocerebroside β-glucosidase and its entrapment in liposomes

1977 ◽  
Vol 164 (2) ◽  
pp. 439-445 ◽  
Author(s):  
I P Braidman ◽  
G Gregoriadis
Keyword(s):  
Enzyme Activity ◽  
Organic Solvent ◽  
Affinity Chromatography ◽  
Rapid Method ◽  
Concanavalin A ◽  
Enzyme Preparation ◽  
Partial Purification ◽  
Glucosidase Activity ◽  
Beta Glucosidase

1. A glucocerebroside beta-glucosidase-rich detergent-free preparation was obtained from human placentas by a rapid method combining affinity chromatography on concanavalin A-Sepharose and organic-solvent precipitation. In a typical preparation about 11000 units of the enzyme purified 1500-fold were obtained from five placentas in 2 days. 2. The enzyme preparation also contained other hydrolases, but the extent of their purification was much smaller. 3. Studies on entrapment in liposomes showed that all glucocerebroside beta-glucosidase activity used could be incorporated in neutral egg phosphatidylcholine-cholesterol liposomes. Association with liposomes appeared to discriminate against other proteins, including some of the hydrolases, thus contributing to further purification of the enzyme. More than 95% of the liposome-associated enzyme activity was latent.

scholarly journals Partial purification and characterization of chick-embryo prolyl 3-hydroxylase

1979 ◽  
Vol 183 (2) ◽  
pp. 303-307 ◽  
Author(s):  
K Tryggvason ◽  
K Majamaa ◽  
J Risteli ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Chick Embryo ◽  
Ethylene Glycol ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Chick Embryo Extract

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.

scholarly journals Rapid method for partial purification of rice germ lipase by affinity chromatography.

1979 ◽  
Vol 43 (8) ◽  
pp. 1771-1772 ◽  
Author(s):  
Yoshiki AOYAGI ◽  
Hirotaka YAMASHITA ◽  
Shinji MATSUMOTO ◽  
Tetsujiro OBARA
Keyword(s):  
Affinity Chromatography ◽  
Rapid Method ◽  
Partial Purification

Aldosterone-induced proteins: characterization using lectin-affinity chromatography

1985 ◽  
Vol 249 (3) ◽  
pp. C215-C225 ◽  
Author(s):  
B. Blazer-Yost ◽  
M. Cox
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Specific Inhibitor ◽  
Partial Purification ◽  
Toad Urinary Bladder ◽  
Lectin Affinity Chromatography ◽  
Na Transport ◽  
Electrophoretic Polymorphism ◽  
Induced Proteins

Aldosterone-stimulated Na+ transport in toad urinary bladder is associated with the synthesis of a specific group of proteins whose induction appears to be related to the natriferic effect of the hormone. These aldosterone-induced proteins (AIPs) occur in two slightly different molecular weight classes (around 70 kDa), each class being composed of several proteins with discrete isoelectric points (range, 5.5-6.0). Because glycosylation is a common cause of such electrophoretic polymorphism and microheterogeneity, we examined whether these proteins are glycoproteins. Tunicamycin (a specific inhibitor of N-linked glycosylation) inhibited aldosterone-stimulated Na+ transport and AIP synthesis without affecting overall protein synthesis. The vast majority of epithelial cell proteins did not bind to the mannose-specific lectin, concanavalin A-sepharose. In contrast, both classes of AIPs bound to concanavalin A-sepharose, but the affinities of the higher and lower molecular weight proteins were markedly different: the former were readily eluted with 0.2 M alpha-methyl-D-mannoside alone, whereas the latter could only be eluted with 0.4 M alpha-methyl-D-mannoside in combination with high concentrations of NaCl (2.5-5.0 M). These studies indicate that 1) glycosylation is important in the natriferic response to aldosterone, 2) the AIPs are N-linked mannose-containing glycoproteins, and 3) the electrophoretic polymorphism of the AIPs is due, at least in part, to differences in glycosylation. Furthermore, concanavalin A-affinity chromatography provides a simple means for the partial purification of these putative "effectors" of the cellular action of aldosterone.

scholarly journals Optimization of a rapid method for studying the cellular location of beta-glucosidase activity in wine yeasts

2005 ◽  
Vol 99 (3) ◽  
pp. 558-564 ◽  
Author(s):  
M. Arevalo Villena ◽  
J.F. Ubeda Iranzo ◽  
R.R. Cordero Otero ◽  
A.I. Briones Perez
Keyword(s):  
Rapid Method ◽  
Wine Yeasts ◽  
Cellular Location ◽  
Glucosidase Activity ◽  
Beta Glucosidase

scholarly journals Effects of calcium on phosphatidylserine- and saposin C-stimulated glucosylceramide β-glucosidase activity

1995 ◽  
Vol 310 (2) ◽  
pp. 571-575 ◽  
Author(s):  
E M Prence
Keyword(s):  
Enzyme Activity ◽  
Fold Increase ◽  
Sphingolipid Metabolism ◽  
Lysosomal Hydrolase ◽  
Activator Protein ◽  
Glucosidase Activity ◽  
Saposin C ◽  
Membrane Bound ◽  
Beta Glucosidase

Glucosylceramide beta-glucosidase is a membrane-bound lysosomal hydrolase that is activated by acidic lipids, the most effective of which is phosphatidylserine (PtdSer), and an activator protein, saposin C. This report documents effects of Ca2+ ions on PtdSer- and saposin C-enhanced beta-glucosidase activity. Ca2+ either increased or decreased enzyme activity, depending on (1) the concentration of phospholipid, and (2) the presence or absence of saposin C. At PtdSer concentrations between 7.6 and 76 microM, in the absence of saposin C, Ca2+ caused an increase in beta-glucosidase activity up to 3 times that measured with PtdSer alone; this was due to both an increase in Vmax, and a decrease in Km. In contrast, at PtdSer concentrations greater than 100 microM, Ca2+ inhibited beta-glucosidase activity by 50%, due to a 2-fold increase in Km. Ca2+ was inhibitory at all PtdSer concentrations tested when both PtdSer and saposin C were present in the assay. Ca2+ ions were also shown to cause changes in the aggregation states of PtdSer. These results suggest that changes in Ca2+ concentration may play a role in regulating beta-glucosidase activity in vivo, thereby modulating sphingolipid metabolism. The implications of these findings are discussed.

Rapid Method for Partial Purification of Rice Germ Lipase by Affinity Chromatography

1979 ◽  
Vol 43 (8) ◽  
pp. 1771-1772
Author(s):  
Yoshiki Aoyagi ◽  
Hirotaka Yamashita ◽  
Shinji Matsumoto ◽  
Tetsujiro Obara
Keyword(s):  
Affinity Chromatography ◽  
Rapid Method ◽  
Partial Purification

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

scholarly journals Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Ahmad R. Attia ◽  
Reyed M. Reyed ◽  
Nadia A. Soliman
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Maximum Activity ◽  
Sodium Oxalate ◽  
Partial Purification ◽  
Bromophenol Blue ◽  
Bromocresol Purple ◽  
Laccase Enzyme

Abstract Background Due to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO were studied in the present investigation. Results Alcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C. Some metal ions Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents [ethylenediamine tetraacetic acid (EDTA), sodium citrate, and sodium oxalate] caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine. Conclusion The promising properties of the newly studied laccase enzyme from Alcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.

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