Purification and properties of polygalacturonic acid trans-eliminase from Bacillus stearothermophilus

1980 ◽  
Vol 26 (3) ◽  
pp. 377-384 ◽  
Author(s):  
A. Karbassi ◽  
R. H. Vaughn
Keyword(s):  
Bacillus Stearothermophilus ◽  
Ethylenediaminetetraacetic Acid ◽  
Thermophilic Bacteria ◽  
Maximum Activity ◽  
Polygalacturonic Acid ◽  
Pectic Acid ◽  
Heat Stable ◽  
Pectolytic Activity ◽  
Purification And Properties ◽  
Optimal Ph

A strain of thermophilic bacteria, Bacillus stearothermophilus, with pectolytic activity has been isolated. It produced an endo-polygalacturonic acid trans-eliminase (endo-PATE, EC 4.2.2.1) extracellularly when grown at 65 °C on a pectic acid medium. The PATE was purified 62-fold by the rapid affinity chromatographic method on a Sepharose–polygalacturonamide linked matrix. The absorbed PATE was eluted from the column with a continuous gradient of 0–10−3 M ethylenediaminetetraacetic acid (EDTA) in phosphate buffer at pH 7.6.The endo-PATE of this organism was much more heat stable than similar enzymes from the mesophilic Bacillus polymyxa and the thermotolerant Bacillus pumilus. The maximum activity of the enzyme occurred at 70 °C. With pectic acid as the substrate, the endo-PATE had an optimal pH of 9.0, the highest optimal pH compared with those of similar enzymes from other species of the genus.The molecular weight of the endo-PATE, as determined by chromatography on a Sephadex G-100 gel column, was 24 000.

scholarly journals Enzymatic Degradation of Polygalacturonic Acid by Yersinia and Klebsiella Species in Relation to Clinical Laboratory Procedures

1977 ◽  
Vol 6 (4) ◽  
pp. 379-386
Author(s):  
Mortimer P. Starr ◽  
Arun K. Chatterjee ◽  
Phoebe B. Starr ◽  
Gordon E. Buchanan
Keyword(s):  
Yersinia Pseudotuberculosis ◽  
Clinical Laboratory ◽  
Soft Rot ◽  
Polygalacturonic Acid ◽  
Pectic Acid ◽  
Klebsiella Species ◽  
Pectolytic Activity ◽  
Pectolytic Enzymes ◽  
Laboratory Procedures ◽  
Culture Supernatants

As scored by several specified plating procedures, clinical and environmental strains of Yersinia enterocolitica, Yersinia pseudotuberculosis , and Klebsiella pneumoniae “Oxytocum” showed detectable, albeit generally weak, ability to digest polygalacturonic (pectic) acid. None of these bacterial strains had the vigorous and rapid pectolytic activity on these polygalacturonic acid-containing media that is typical of soft-rot Erwinia species, although some of the Oxytocum strains came fairly close. Analyses of the pectolytic enzyme contents of the cells and culture supernatants of the Yersinia and Klebsiella species revealed that readily detectable quantities of cell-bound polygalacturonic acid trans -eliminase and hydrolytic polygalacturonase were formed by the Yersinia and Klebsiella species; however, the total units of enzyme activity produced by these bacteria were, in general, lower than were produced by soft-rot Erwinia species. Furthermore, unlike the situation in soft-rot Erwinia cultures, these pectolytic enzymes of Yersinia and Klebsiella species were not excreted rapidly and massively into the growth medium. Cultures of other enterobacteria ( Citrobacter species, Enterobacter species, Erwinia amylovora, Erwinia herbicola, Escherichia coli, Proteus species, Salmonella typhimurium , and Serratia marcescens ) showed no pectolytic ability whatsoever by any of the plating procedures used and (to the extent they were so examined) produced no pectolytic enzymes detectable either in their cells or culture supernatants. This slow or weak release of pectolytic enzymes by Yersinia and Klebsiella species has a bearing on clinical laboratory procedures suitable for detecting their pectolytic activity; methods adequate for this purpose are detailed.

Polygalacturonase isolated from the culture of the psychrophilic fungus Sclerotinia borealis

1997 ◽  
Vol 43 (5) ◽  
pp. 417-424 ◽  
Author(s):  
Toshihide Takasawa ◽  
Keiko Sagisaka ◽  
Koichi Yagi ◽  
Kyoko Uchiyama ◽  
Atsushi Aoki ◽  
...  
Keyword(s):  
Culture Medium ◽  
Enzyme Reaction ◽  
Wheat Seedling ◽  
Maximum Activity ◽  
Reducing Sugar ◽  
Pectic Acid ◽  
Snow Mold ◽  
Viscosity Change ◽  
Optimum Ph ◽  
Psychrophilic Fungus

A polygalacturonase was isolated from the culture medium of Sclerotinia borealis, a psychrophilic fungus that grows on lawn and wheat seedling under the snow in winter and induces the snow mold disease. Pectic acid was a better substrate of this enzyme than pectin when the activity was determined by measuring the reducing sugar produced. However, when the activity was measured by viscosity change, the viscosity of pectin decreased more rapidly than that of pectic acid. The results of viscosity change apparently indicate that the polygalacturonase catalyzes pectin hydrolysis as an endo-type enzyme. Highly methyl-esterified pectin was a poor substrate, as determined by measurements of reducing sugar production and viscosity change. It is suggested from the results that the methoxy group of pectin affects the polygalacturonase reaction. A reaction mechanism was proposed for the polygalacturonase reaction. Molecular mass of this enzyme was 40 kDa and its isoelectric point was pH 7.5. Optimum pH of the enzyme reaction was 4.5 and its optimum temperature was 40–50 °C. Thirty percent of the maximum activity was observed at 5 °C, but it was only slightly active above 60 °C. The activity was preserved for more than 2 years at 5 °C and pH 4.5, but it was lost when kept at room temperature overnight or heated at 50 °C for 30 min. The amino acid sequence of the N-terminal region of the psychrophilic polygalacturonase of Sclerotinia borealis is compared with those of polygalacturonases of mesophilic fungi. The function of this enzyme against the target plants is discussed with reference to the reaction of polygalacturonases of mesophilic fungi.Key words: polygalacturonase, pectin-hydrolyzing enzyme, psychrophilic fungi, snow mold disease, Sclerotinia borealis.

Purification and properties of glutamate-phenylpyruvate aminotransferase from the ruminal protozoan Entodinium caudatum

2004 ◽  
Vol 55 (9) ◽  
pp. 991
Author(s):  
Md. Ruhul Amin ◽  
Ryoji Onodera ◽  
R. Islam Khan ◽  
R. John Wallace ◽  
C. Jamie Newbold
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Maximum Activity ◽  
Purification And Properties ◽  
Sds Page ◽  
Wide Range ◽  
S 100 ◽  
A Cell

Entodinium species are important in catabolic protein metabolism by the mixed ruminal microbial population. This study was conducted to purify, and investigate properties of one of the enzymes involved in amino acid metabolism by Entodinium caudatum, glutamate-phenylpyruvate aminotransferase (GPA; EC 2.6.1.64). GPA was purified 74-fold from a cell-free extract by ammonium sulfate precipitation and column chromatography with phenyl-superose, DEAE-Toyopearl 650M, Sephacryl S-100 HR, and Sephadex G-100. The molecular mass of GPA was estimated by SDS–PAGE to be 65.0 kDa. The optimum pH was 6.0 and it was found to be reactive over a wide range of pH from 5.0 to 10.5. Maximum activity of GPA occurred at 45°C and the activity declined at temperatures over 55°C. GPA was stable below 60°C. Aminooxyacetate and phenylhydrazine were highly inhibitory, and SDS, EDTA, and some heavy metal ions also inhibited activity. The purification and characterisation of the enzyme will help to isolate the gene and ultimately to understand the role of GPA in both anabolic and catabolic amino acid metabolism by Entodinium caudatum.

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.

The purification and properties of an amino acid arylamidase from bovine milk

1969 ◽  
Vol 47 (2) ◽  
pp. 173-178 ◽  
Author(s):  
A. Mellors
Keyword(s):  
Amino Acid ◽  
Milk Fat ◽  
Specific Activity ◽  
Bovine Milk ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Divalent Metal Ions ◽  
Purification And Properties ◽  
Milk Fat Globule ◽  
High Concentrations

An amino acid arylamidase is present in bovine milk and is associated with the "microsomes" of the milk-fat globule membrane. It has been purified by DEAE-cellulose chromatography of a 0.1 M NaCl extrast of milk microsomes. The specific activity of the purified arylamidase was increased 12 700-fold over that of the milk. Three peaks of arylamidase activity could be recognized after the chromatography. One form was apparently bound to casein. The major peak of arylamidase activity hydrolyzes lysyl-, alanyl-, valyl-, and arginyl-β-naphthylamides at similar rates, with little activity against glycyl- and histidyl-β-naphthylamides. The arylamidase requires the restoration of sulfhydryl groups by dithiothreitol for maximum activity. It is inhibited by EDTA and some divalent metal ions, and only calcium ions restore the EDTA-inactivated enzyme. The optimum pH for the hydrolysis of lysyl-β-naphthylamide is pH 7.7, and high concentrations of this substrate are inhibitory.

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