Pectic enzymes produced by Pseudomonas fluorescens, an organism associated with “pink eye” disease of potato tubers

1972 ◽  
Vol 50 (12) ◽  
pp. 2479-2488 ◽  
Author(s):  
S. S. Hagar ◽  
G. A. McIntyre
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pectin Methylesterase ◽  
Viscosity Reduction ◽  
Cellulose Acetate Electrophoresis ◽  
Pectic Enzymes ◽  
Ph Range ◽  
Two Peaks ◽  
Layer Chromatography

No pectin methylesterase (PME) activity was observed in crude or dialyzed extracts from macerated potato tuber tissue inoculated with Pseudomonas fluorescens; however, pectic lyase (syn. polygalacturonic acid transeliminase, PATE) activity was observed. Two PATE enzymes (peaks 1 and 2) were eluted from a pH 9.4 DEAE-cellulose column using a gradient of pH 7.6 Tris-HCl buffer (0.01–0.1 M). Enzyme in peak 1 was about 6 times more active than enzyme in peak 2 based on reducing group assays, and 10 times more active in viscosity reduction of 1% Na-polypectate (NaPP) at pH 8.5. No increase in absorbancy was observed at 515 nm of clarified reaction mixtures, indicating that saturated oligouronides did not accumulate. Other properties of the two peaks: optimum pH range was 8.5–9.5, substrate preference was NaPP vs. pectin, addition of Ca2+ (0.001 M) enhanced activity while EDTA (0.001 M) decreased activity to [Formula: see text], cellulose acetate electrophoresis revealed one band of protein per peak, and heat of inactivation was 51–60C. Thin-layer chromatography of hydrolytic products from NaPP revealed unsaturated uronides and pectic fragments after 2 h hydrolysis; after 96 h hydrolysis only unsaturated uronides were observed. Molecular weight estimations by Sephadex G-200 gel filtration were about 18 000 (peak 1) and 22 500 (peak 2). Enzyme in either peak macerated 400-μ sections of potato tissue.

Purification and characteristics of the chloride transport stimulating factor from locust corpora cardiaca: a new peptide

1980 ◽  
Vol 58 (10) ◽  
pp. 1851-1860 ◽  
Author(s):  
J. E. Phillips ◽  
W. Mordue ◽  
J. Meredith ◽  
J. Spring
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Gel Filtration ◽  
Short Circuit Current ◽  
Water Soluble ◽  
Cellulose Acetate Electrophoresis ◽  
Corpora Cardiaca ◽  
Ph Range ◽  
And Storage ◽  
Layer Chromatography

Corpora cardiaca (CC), cAMP, and hemolymph all increase short-circuit current (Isc) and electropotential difference (PD) across locust rectum by stimulating electrogenic transport of Cl− from the lumen. Using ΔIsc as a bioassay, we have purified the water-soluble stimulant (CTSH) from CC using gel filtration chromatography, DEAE-Sephadex anion exchange, cellulose acetate electrophoresis, and thin-layer chromatography. A single peak of CTSH activity was observed after all these procedures, although small amounts of CTSH activity occasionally remained in the high molecular weight (MW) protein precipitate. CTSH was purified more than 100-fold on Bio-Gel P-30 columns. It has a MW of 8 000 – 12 000, is destroyed by trypsin digestion, and has a net negative charge over the pH range (5–10) at which it is most stable. Various properties (i.e., stability at 20 °C, localization in CC, MW, Rf values) and reciprocal bioassay s indicate that CTSH is different from diuretic, antidiuretic, and adipokinetic hormones from locust CC. No difference in the properties of CTSH from glandular (GL) and storage lobes (SL) of CC were noted, although 80% of activity was in the SL. The concentration of purified CTSH required to cause maximal stimulation of rectal Isc is less than 7 nM.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

Isolation and some properties of extracellular heat-stable lipases fromPseudomonas fluorescensstrain AFT 36

1983 ◽  
Vol 50 (1) ◽  
pp. 77-89 ◽  
Author(s):  
Patrick F. Fox ◽  
Leszek Stepaniak
Keyword(s):  
Phosphate Buffer ◽  
Lipolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Lipase Production ◽  
Maximum Activity ◽  
Temperature Range ◽  
Milk Serum ◽  
Heat Stable ◽  
Ph Range

SummaryAeration increased the growth and lipase production in milk byPseudomonas fluorescensstrain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented ∼ 71 % of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8·0 and 35 °C; it had aKmon tributyrin of 3·65 mM. and was inhibited by concentrations of substrate > ∼ 17 mM. The enzyme was very stable over the pH range 6–9; it was relatively heat-labile in phosphate buffer in the temperature range 60–80 °C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100–150 °C: theDvalues at 150 °C were ∼ 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the correspondingZvalues in the temperature range 100–150 °C were ∼ 40 and ∼ 42 °C and theEafor inactivation were 7·65 × 104J mol-1and 6·97 × 104J mol-1respectively.

Bovine hepatic carboxylesterases chromatographic fractionation, gel filtration, and molecular weight estimation

1969 ◽  
Vol 47 (8) ◽  
pp. 799-805 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bovine Liver ◽  
Weight Estimation ◽  
Molecular Weights ◽  
Starch Gel ◽  
Two Peaks ◽  
Second Peak

The carboxylesterase activity of bovine liver was fractionated by DEAE-cellulose chromatography into two peaks of activity. Electrophoresis in starch gel showed that the peak eluted first was composed of a group of five electrophoretically slow bands while the second peak was a single, rapidly migrating band. Gel filtration of the crude extract on Sephadex G-100 and G-200 yielded a single peak of esterase activity containing both the electrophoretically slow and fast bands. The determination of molecular weights by gel filtration on Sephadex G-200 and G-100 yielded estimates of 52 000 and 55 000, respectively. The molecular weight estimates of the DEAE-cellulose fractionated electrophoretically slow and fast bands on Sephadex G-100 were identical, namely 55 000.

A proteolytic pseudomonad from skin lesions of rainbow trout (Salmo gairdnerii). II. Some properties of the proteinase

1968 ◽  
Vol 14 (8) ◽  
pp. 875-880 ◽  
Author(s):  
M. F. Li ◽  
Carol Jordan
Keyword(s):  
Rainbow Trout ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Skin Lesions ◽  
Complexing Agents ◽  
Bivalent Cations ◽  
Wide Ph Range ◽  
Ph Range ◽  
Trout Muscle ◽  
Cellulose Column

An extracellular proteinase from a pseudomonad pathogenic to rainbow trout was purified 33-fold by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G 75 gel filtration. The purified enzyme was active over a wide pH range, from pH 5.0 to 10.0. Heating at 98 °C for 1 h did not completely inactivate the enzyme. Its observed temperature optimum was 45 °C. Michaelis–Menten constants were found to be 0.625% for casein and 0.263% for rainbow trout muscle albumin. Activation energies calculated for these substrates were 8.1 kcal and 11.4 kcal per mole, respectively. The involvement of bivalent cations and free sulfhydryl groups in the enzymatic activity was demonstrated by the inhibition caused by metal-complexing agents and p-chloromercuribenzoate, respectively.

scholarly journals Biochemical properties and positional specificity of Lipase from hepatopancreas of Tra catfish (Pangasius)

2013 ◽  
Vol 16 (4) ◽  
pp. 85-91
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Ion Exchange ◽  
Lipase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Biochemical Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Positional Specificity ◽  
Tra Catfish ◽  
Ph Range

Lipase from the hepatopancreas of Tra catfish (Pangasius) was precipitated by ammonium sulfate fractionation, purified by ion-exchange chromatography on DEAE cellulose and gel filtration on Sephadex G- 75. On substrate triolein, purified lipase has Km= 1.381 mM and Vmax= 0.063 mM/min. The lipase was stable at a pH range of 7.0- 9.0 and in temperatures of 35-50°C. At 500C the enzyme loosed 44,7% activity after 120 min. The enzyme was specific for the α- positions (1, 3) of triglyceride. In bile salt solution of 0.015M NaTC, lipase activity of the enzyme increased in 3.08 folds in comparison of sample without NaTC.

scholarly journals A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake MushroomLentinus edodes

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Guo-Qing Zhang ◽  
Ying-Ying Wu ◽  
Tzi-Bun Ng ◽  
Qing-Jun Chen ◽  
He-Xiang Wang
Keyword(s):  
Sequence Similarity ◽  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Phytase Activity ◽  
Ph Tolerance ◽  
Phosphorylated Compounds ◽  
Terminal Amino ◽  
Ph Range

A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroomLentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate >β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+at a tested concentration of 5 mM.

scholarly journals Purification and characterization of a novel laccase from the mushroom Pleurotus nebrodensis.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Guo-Ting Tian ◽  
Guo-Qing Zhang ◽  
He-Xiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Slight Reduction ◽  
Pleurotus Nebrodensis ◽  
Precipitous Decline ◽  
Ph Range ◽  
Purification Protocol

A novel laccase with a molecular mass of 64 kDa and the N-terminal sequence AIGPDDTINF was isolated from fresh fruiting bodies of the mushroom Pleurotus nebrodensis. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but not on CM-cellulose. It demonstrated an optimal temperature of 70°C. The enzyme activity increased steadily over the temperature range 20°C-70°C. There was only a slight reduction in activity at 80°C. However, all activity disappeared following exposure to 100°C for 10 minutes. The enzyme activity changed only slightly over the pH range 3-5, with the optimum at pH 5, but underwent a precipitous decline when the pH was elevated to 6, and was undetectable at pH 8 and pH 9.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

scholarly journals Isolation, Purification and Analysis of Pancreatic Lipase from ‘Gallus gallus domesticus’

2020 ◽  
Vol 5 (7) ◽  
pp. 590-594
Author(s):  
Sandhya. B ◽  
Nagamani. T.S
Keyword(s):  
Pancreatic Lipase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gallus Gallus ◽  
Gallus Domesticus ◽  
Biodiesel Production ◽  
Gallus Gallus Domesticus ◽  
Pharmaceutical Industries ◽  
Ph Range ◽  
Hydrolysis Of

This article discusses the isolation of pancreatic lipase enzyme from the pancreas of Gallus gallus domesticus. Whereas lipase catalyses the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, it involves applications like organic syntheses, hydrolysis of fats, oils, modification of fats, flavor enhancement in food processing, detergent industries, pharmaceutical industries, chemical analyses, and biodiesel production. Pancreatic lipase was purified to the homogeneity by 70% saturated Ammonium sulphate further, it was dialysate using the dialysis membrane and then gel filtration chromatography was carried out by Sephadex G-75 and DEAE cellulose. The molecular weight of purified lipase sample was determined by SDSPAGE, it was found to be 98KDa. The lipase was active in the pH range of 5-10 with an optimum pH of 6.0. The optimum temperature for the hydrolysis of olive oil was 37ºC in the range of 25ºC - 50ºC.

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