Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus

1983 ◽  
Vol 29 (11) ◽  
pp. 1526-1531 ◽  
Author(s):  
Susan E. Jensen ◽  
Donald W. S. Westlake ◽  
Saul Wolfe
Keyword(s):  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Isopenicillin N

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate – polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.

Purification of isopenicillin N synthetase from Streptomyces clavuligerus

1986 ◽  
Vol 32 (12) ◽  
pp. 953-958 ◽  
Author(s):  
Susan E. Jensen ◽  
Brenda K. Leskiw ◽  
Leo. C. Vining ◽  
Yair Aharonowitz ◽  
Donald W. S. Westlake ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
High Performance Liquid Chromatographic ◽  
Inhibitory Effect ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 × 10−3 units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.

Partial purification and characterization of prolyl hydroxylase from the free-living nematode Panagrellus silusiae

1978 ◽  
Vol 56 (2) ◽  
pp. 159-165 ◽  
Author(s):  
J. R. A. Leushner ◽  
J. Pasternak
Keyword(s):  
Enzyme Preparation ◽  
Atmospheric Oxygen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Prolyl Hydroxylase ◽  
Partial Purification ◽  
Free Living ◽  
Free Living Nematode

Prolyl hydroxylase was isolated from the free-living nematode Panagrellus silusiae by ammonium sulfate precipitation and calcium phosphate gel ion exchange from Triton X-100 treated homogenates. The enzyme preparation was highly purified but not to homogeneity as judged by agarose gel filtration and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. Gel filtration indicated that the molecular weight of the enzyme approximated 285 000. Analysis by SDS–acrylamide electrophoresis revealed subunits of ~67 000. Complete enzymic activity required α-ketoglutarate, Fe2+, ascorbate, catalase, atmospheric oxygen, and dithiothreitol. This activity was dramatically inhibited by α, α-dipyridyl, phenanthroline, and polyproline II. The purified enzyme preparation synthesized 50 to 100 μg hydroxyproline per milligram chick protocollagen per hour at 30 °C with an estimated Km for the substrate of 80 μg. Thus, a purified prolyl hydroxylase from a simple invertebrate possesses many of the properties of the prolyl hydroxylases of various vertebrates.

Partial purification and properties of a β-glucosidase from Erwinia herbicola Y46

1975 ◽  
Vol 21 (4) ◽  
pp. 513-520 ◽  
Author(s):  
A. Garibaldi ◽  
L. N. Gibbins
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Basal Medium ◽  
Plant Diseases ◽  
Partial Purification ◽  
Major Protein ◽  
Erwinia Herbicola ◽  
Major Protein Band

A constitutive β-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant β-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight." The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-β-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against β-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of β-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells. The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0–7.5 (100% activity) and 4.5–>8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 °C, but only 25% after 30 min at 60 °C. The enzyme had a mean Km value (phloridzin) of 1.35 × 10−4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5–7.0.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria

1984 ◽  
Vol 224 (1) ◽  
pp. 171-179 ◽  
Author(s):  
I R Cottingham ◽  
A L Moore
Keyword(s):  
Nadh Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Properties ◽  
Nadh Dehydrogenases ◽  
Arum Maculatum ◽  
A Minor ◽  
Considerable Loss

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.

scholarly journals Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

1982 ◽  
Vol 207 (1) ◽  
pp. 133-138 ◽  
Author(s):  
M G Battelli ◽  
E Lorenzoni
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Xanthine Dehydrogenase ◽  
Enzymic Activity ◽  
Oxidized Glutathione ◽  
Adult Rats ◽  
Purification And Properties ◽  
Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

scholarly journals Properties of Peanut (KAC431) Protein Hydrolysates and Their Impact on the Quality of Gluten-Free Rice Bread

Foods ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 942 ◽  
Author(s):  
Suphat Phongthai ◽  
Nuttapon Singsaeng ◽  
Rossarin Nhoo-ied ◽  
Thipubol Suwannatrai ◽  
Regine Schönlechner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Functional Properties ◽  
Positive Impact ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Hydrolysates ◽  
Degree Of Hydrolysis ◽  
Major Protein ◽  
Gluten Free ◽  
Major Protein Band

Protein hydrolysates (PH) with a degree of hydrolysis (DH) of 5%, 10%, and 13% from two varieties of peanut were prepared using two commercial enzymes, Alcalase and Flavourzyme. The content of essential amino acids (30,290 mg/100 g) and hydrophobic amino acids (34,067 mg/100 g) of the peanut variety Kalasin 2 (KAC431) protein was higher than that of a common variety, Kalasin 1 (KAC1) (p < 0.05). The protein molecular weight distributions of the two varieties of peanut detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were similar, ranging from 15 to 75 kDa, with a major protein band at 50–75 kDa. The antioxidant and functional properties of derived PHs were influenced by DH. Although the foaming ability of protein was improved by DH5%, it was obviously decreased upon increasing DH further. The best emulsifying properties were observed in PH with DH5% (p < 0.05). The incorporation of PH with a small DH, especially when produced using Flavourzyme, had a highly positive impact on the specific volume and relative elasticity of gluten-free bread. The effect of PHs on bread quality was highly correlated with their functional properties. This study suggests that partially enzymatically modified proteins are suitable for incorporation in food products such as bread and other gluten-free products.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

Further purification and properties of rat uterine peroxidase

1981 ◽  
Vol 59 (11-12) ◽  
pp. 916-920 ◽  
Author(s):  
Hugh S. Keeping ◽  
Shioko Kimura ◽  
Jane Lovsted ◽  
Peter H. Jellinck
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Prolonged Storage ◽  
High Recovery ◽  
Partial Protection ◽  
Purification And Properties ◽  
Single Protein ◽  
Protein Molecular Weight

Peroxidase was purified 3700-fold from homogenates of estradiol-treated rat uteri by affinity chromatography on concanavalin A (ConA) – Sepharose followed by gel filtration on Bio-Gel P-150 with high recovery of enzyme. A single protein (molecular weight (MW) 45 000) staining for heme was shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis to be present in the peak fractions of enzymic activity eluted from the ConA–Sepharose column. This protein had the same mobility as bovine lactoperoxidase (MW 78 000) in a cationic gel electrophoretic system under nondenaturing conditions. Peroxidase activity in a NaCl extract of the uterus was lower than that in a CaCl2 extract but was unaffected by prolonged storage at −20 °C. In contrast, the CaCl2-extracted enzyme lost much, of its activity under these conditions by a process which could be prevented by the addition of glycerol. The sulfhydryl reagent, N-ethylmaleimide, which caused a marked increase in the activity of uterine peroxidase, provided only partial protection against inactivation during storage of CaCl2 extracts of this enzyme at low temperature.

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