scholarly journals UDP-glucose:solasodine glucosyltransferase from eggplant (Solanum melongena L.) leaves: partial purification and characterization.

1997 ◽  
Vol 44 (1) ◽  
pp. 43-53 ◽  
Author(s):  
C Paczkowski ◽  
M Kalinowska ◽  
Z A Wojciechowski
Keyword(s):  
Molecular Mass ◽  
Solanum Melongena ◽  
Divalent Metal ◽  
Partial Purification ◽  
Divalent Metal Ions ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Cytosol Fraction ◽  
Ammonium Sulphate Precipitation ◽  
Unripe Fruits

Uridine 5'-diphosphoglucose-dependent glucosyltransferase which catalyzes the glycosylation of solasodine i.e. UDP-glucose:solasodine glucosyltransferase, is present in leaves, roots, unripe fruits and unripe seeds of eggplant (Solanum melongena L.). The glucosylation product is chromatographically identical with authentic solasodine 3 beta-D-monoglucoside, a putative intermediate in the biosynthesis of solasodine-based glycoalkaloids characteristic of the eggplant. The enzyme was purified about 50-fold from crude cytosol fraction of eggplant leaves by ammonium sulphate precipitation and column chromatography on Q-Sepharose and Sephadex G-100. The native enzyme has a molecular mass of approx. 55 kDa and pH optimum of 8.5. Divalent metal ions are not required for its activity but the presence of free-SH groups is essential. Besides solasodine (Km = 0.04 microM), the enzyme effectively glucosylates tomatidine, another steroidal alkaloid of the spirosolane type, but it is virtually inactive towards the solanidane-type steroidal alkaloids such as solanidine or demissidine. The enzyme is specific for UDP-glucose (Km = 2.1 microM) since unlabelled ADP-, GDP-, CDP- or TDP-glucose could not effectively compete with UDP-[14C]glucose used as the sugar donor for solasodine glucosylation. Moreover, no synthesis of labelled solasodine galactoside was observed when UDP-[14C]glucose was replaced with UDP-[14C]galactose.

Partial Purification and Some Properties of a Hydroxycinnamoyl Glucosyltransferase from Tomato Fruits

1980 ◽  
Vol 35 (11-12) ◽  
pp. 967-972 ◽  
Author(s):  
A. Fleuriet ◽  
J. J. Macheix ◽  
R. Suen ◽  
R. K. Ibrahim
Keyword(s):  
Ferulic Acid ◽  
Metal Ions ◽  
Deae Cellulose ◽  
Divalent Metal ◽  
Partial Purification ◽  
Divalent Metal Ions ◽  
Tomato Fruits ◽  
Ammonium Sulphate Precipitation ◽  
Sh Group ◽  
Cherry Tomatoes

A glucosyltransferase was isolated from immature “cherry” tomatoes and was partially purified (200-fold) by ammonium sulphate precipitation and successive chromatography on Sephadex G-100 and DEAE-cellulose columns. The enzyme utilised the free hydroxycinnamic acids and UDP-glucose in the formation of their respective glucosides (pH 8.0) and glucose esters (pH 7.0); but did not accept the CoA thiolesters of HCAs in the presence of glucose-1-phosphate. The constant glucoside/glucose ester ratio observed during purification suggests that both reactions are catalysed by the same enzyme. The Km values for ρ-coumaric, caffeic, ferulic and sinapic acids were 0.8, 1.5, 1.4 and 2.5 μᴍ, respectively. With ferulic acid as substrate, the Km value for UDPG was 10 μᴍ. The enzyme required an -SH group for activity and the reaction was strongly inhibited by EDTA, divalent metal ions and UDP.

Purification and Characterization of Three Extracellular Proteinases Produced by Pseudomonas fluorescens INIA 745, an Isolate from Ewe's Milk

1999 ◽  
Vol 62 (5) ◽  
pp. 543-546 ◽  
Author(s):  
J. FERNÁNDEZ ◽  
A. F. MOHEDANO ◽  
P. GAYA ◽  
M. MEDINA ◽  
M. NUÑEZ
Keyword(s):  
Pseudomonas Fluorescens ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Divalent Metal ◽  
Divalent Metal Ions ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Extracellular Proteinases ◽  
Ewe's Milk

Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45°C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded β-casein, with preference to αs1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.

Partial Purification and Characterization of Membrane-Associated 3-Hydroxy-3-methylglutaryl-Coenzyme A Lyase from Radish Seedlings

1993 ◽  
Vol 48 (5-6) ◽  
pp. 444-450 ◽  
Author(s):  
Thomas Weber ◽  
Thomas J. Bach
Keyword(s):  
Activation Energy ◽  
Molecular Mass ◽  
Coenzyme A ◽  
Arrhenius Plot ◽  
Linear Part ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Radish Seedlings

Abstract We solubilized from radish membranes and purified by 154-fold 3-hydroxy-3-methyl-glutaryl-CoA lyase (HMGL, EC 4.1.3.4) catalyzing the conversion of 3-hydroxy-3-methyl-glutaryl-(HMG -)CoA into acetyl-CoA and acetoacetate. The apparent molecular mass under non-denaturating conditions is 70 kDa. The enzyme has a broad pH optimum around 8.0 and its activation energy as determined from the linear part of an Arrhenius plot is 137.1 kJ/mol. The Km with respect to (5)-H MG-CoA is 40 μM . The enzyme is extremely unstable and rapidly loses activity even when kept on ice, but retains some activity over several weeks when stored at -80 °C.

Purification and characterization of a lectin from Xanthomonas campestris NCIM 5028

1996 ◽  
Vol 42 (6) ◽  
pp. 609-612 ◽  
Author(s):  
Bhagyashree Joshi ◽  
Jayant M. Khire ◽  
Hephzibah SivaRaman ◽  
M. Islam Khan
Keyword(s):  
Molecular Mass ◽  
Host Plant ◽  
Xanthomonas Campestris ◽  
Simple Procedure ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Molecular Masses ◽  
Lectin Purification

A lectin was isolated from culture filtrates of Xanthomonas campestris NCIM 5028, by a simple procedure of hydrophobic chromatography on phenyl-Sepharose after ammonium sulphate precipitation. The lectin was a heterodimer, with subunit molecular masses of 30 000 and 28 000. Gel filtration on S-300 column, calibrated with markers, showed its molecular mass to be approximately 70 000. Its isoelectric point was 7.2. The agglutination of the rabbit erythrocytes by the lectin was inhibited by fetuin glycopeptides and host plant (Brassica oleracea) extracts.Key words: Xanthomonas campestris, lectin, purification.

Identification, Properties and Genetic Control of UDP-ʟ-Rhamnose: Anthocyanidin 3-O-Glucoside, 6″-O-Rhamnosyltransferase Isolated from Retals of the Red Campion (Silene dioica)

1980 ◽  
Vol 35 (3-4) ◽  
pp. 249-257 ◽  
Author(s):  
John Kamsteeg ◽  
Jan van Brederode ◽  
Gerrit van Nigtevecht
Keyword(s):  
Biosynthetic Pathway ◽  
Dominant Gene ◽  
Divalent Metal ◽  
Hydroxyl Group ◽  
Silene Dioica ◽  
Divalent Metal Ions ◽  
Ph Optimum ◽  
Red Campion ◽  
Km Value ◽  
Reduced Rate

Abstract An enzyme catalyzing the transfer of the rhamnosyl moiety of UDP-ʟ-rhamnose to the 6 -hydroxyl group of the 3-O-bound glucose of anthocyanidin 3-O-glucosides has been demonstrated in petal extracts of Silene dioica plants. The enzyme activity is controlled by a single dominant gene N; no rhamnosyltransferase activity is found in petals of n/n plants. The 60-fold purified rhamno-syltransferase exhibits a pH optimum of 8.1, has a molecular weight of about 45000 daltons, is stimulated by the divalent metal ions Mg2+, Mn2+ and Co2+, and has a “true Km” value of 0.09 mᴍ for UDP-ʟ-rhamnose and 2.2 mᴍ for cyanidin 3-O-glucoside. Pelargonidin 3-O-glucoside and delphinidin 3-O-glucoside can also serve as acceptor. The enzyme can also catalyze the rhamnosylation of anthocyanidin 3,5-diglucosides although at reduced rate. The biosynthetic pathway for the synthesis of cyanidin 3-rhamnosylglucoside-5-glucoside in petals of S. dioica is discussed.

Partial Purification and Characterization of S-Adenosyl-ʟ-Methionine: Norreticuline N-Methyltransferases from Berberis Cell Suspension Cultures

1986 ◽  
Vol 41 (1-2) ◽  
pp. 126-134 ◽  
Author(s):  
Chi-Kit Wat ◽  
Paul Steffens ◽  
Meinhart H. Zenk
Keyword(s):  
Cell Suspension ◽  
Cell Suspension Cultures ◽  
Apparent Molecular Weight ◽  
Suspension Cultures ◽  
Partial Purification ◽  
Enzymatic System ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Ph Optimum

Abstract Two new N-methyltransferases (NMT-I and NMT-II) were found to occur in Berberis vulgaris cell suspension cultures. One of these enzymes (NMT-I) was partially purified (100-fold) and characterized. This enzyme is specific for tetrahydrobenzylisoquinoline alkaloids and S-adenosyl-ʟ-methionine serves as the methyl donor. The apparent molecular weight of the enzyme is 68,000. The pH optimum of the enzyme is 7.6, the temperature optimum 35 °C. Apparent KM values for (R)-tetrahydropapaverin as substrate were 0.2 mᴍ and for SAM 0.04 mᴍ. The preparation of the same type of enzyme from B. wilsoniae var. subcaulialata was utilized as an efficient enzymatic system for the synthesis of stereochemically pure (R)-as well as (S)-reticuline labelled with tritium or 14C at the N-CH3 group. Enzymes catalyzing this type of reactions are named S-adenosyl-ʟ-methionine: norreticuline N-methyltransferases.

The purification and characterization of a β-glucosidase from Alcaligenes faecalis

1986 ◽  
Vol 64 (9) ◽  
pp. 914-922 ◽  
Author(s):  
Anthony G. Day ◽  
Stephen G. Withers
Keyword(s):  
Glucose Production ◽  
High Specificity ◽  
Alcaligenes Faecalis ◽  
Divalent Metal ◽  
Exchange Chromatography ◽  
Good Choice ◽  
Divalent Metal Ions ◽  
Purification And Characterization ◽  
Glucanase Activity

The β-glucosidase from Alcaligenes faecalis has been purified to homogeneity (880-fold purification, 11% yield) using a combination of classical techniques and medium pressure ion-exchange chromatography. It is a dimeric enzyme of monomer molecular weight 50 000 and has no specific requirement for divalent metal ions. It has a high specificity for β-glucosides and hydrolyses a wide variety of different chemical types with retention of configuration at the anomeric centre. It has no exo-β-1,4-glucanase activity. It is reversibly inhibited by a variety of sugars which have been shown previously to be very active against glucosidases, suggesting a normal mechanism of action. Measured Km values for cellobiose and p-nitrophenyl β-D-glucopyranoside are quite low (0.70 and 0.08 mM, respectively), making this a good choice for cocloning into a cellulase system optimized for glucose production.

Separation, Partial Purification and Characterization of a Fatty Acid Hydroperoxide Cleaving Enzyme from Apple and Tomato Fruits

1982 ◽  
Vol 37 (3-4) ◽  
pp. 165-173 ◽  
Author(s):  
P. Schreier ◽  
G. Lorenz
Keyword(s):  
Fatty Acid ◽  
Inhibitory Effect ◽  
Fatty Acid Hydroperoxide ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isoelectric Points ◽  
Acid Hydroperoxide ◽  
Membrane Bound

Abstract A membrane-bound enzyme catalysing the cleavage of 13-hydroperoxy-(Z)-9,(E)-11-oc-tadecadienoic acid (13-LHPO) and 13-hydroperoxy-(Z)-9,(E)-11,(Z)-15-octadecadienoic acid (13-LnHPO) to C6-aldehydes was isolated and partially purified from apples and tomatoes. Attempts to employ Ultrogel AcA 34 and AcA 22 in a gel chromatographic purification step were partially frustrated by reaggregation phenomena. However, by using Sepharose CL-4 B an enzyme fraction (MW 200 000 Da) with lipoxygenase and fatty acid hydroperoxide cleaving activity could be separated from a high molecular-weight active eluate. By applying preparative isoelec­ tric focussing to the tomato protein we succeeded in separating the fatty acid cleaving activity from the lipoxygenase, because o f their different isoelectric points of pH 5.8 -6 .1 and pH 5.0, respectively, An 8.4-fold purification of the fatty acid cleaving activity was achieved. A pH-optimum of 5.5 and a Km-value of 2.6 × 10-5 м/1 for the 13-hydroperoxide of linoleic acid were measured. p-Chloromercuribenzoic acid (1 mм) showed significant inhibitory effect on the fatty acid hydroperoxide cleaving enzyme, but no evidence o f inhibition was found with 1 mм H2O2, KCN, DABCO and EDTA or superoxide dismutase (270 U). The maximum amount of fatty acid hydroperoxide decomposition (C6-aldehyde formation) was determined to be 59%.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Partial purification and properties of an AMP-specific soluble 5′-nucleotidase from pigeon heart

1990 ◽  
Vol 268 (1) ◽  
pp. 117-122 ◽  
Author(s):  
A C Skladanowski ◽  
A C Newby
Keyword(s):  
Ionic Strength ◽  
Molecular Mass ◽  
Formation Rate ◽  
Linear Increase ◽  
High Ionic Strength ◽  
Partial Purification ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Wide Range ◽  
Alpha Beta

A soluble 5′-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5′-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5′-deoxy-5′-isobutylthioinosine than by 5′-deoxy-5′-isobutylthioadenosine, but not by [alpha, beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5′-nucleotidase and from the previously purified IMP-specific 5′-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.

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