scholarly journals Purification and characterization of an acetyl xylan esterase produced by Streptomyces lividans

1996 ◽  
Vol 319 (3) ◽  
pp. 881-886 ◽  
Author(s):  
Claude DUPONT ◽  
Nicole DAIGNEAULT ◽  
François SHARECK ◽  
Rolf MOROSOLI ◽  
Dieter KLUEPFEL
Keyword(s):  
Enzyme Activity ◽  
Culture Filtrate ◽  
Specific Activity ◽  
Streptomyces Lividans ◽  
Birchwood Xylan ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Acetyl Group ◽  
Assay Conditions

The acetyl xylan esterase cloned homologously from Streptomyces lividans [Shareck, Biely, Morosoli and Kluepfel (1995) Gene 153, 105–109] was purified from culture filtrate of the overproducing strain S. lividans IAF43. The secreted enzyme had a molecular mass of 34 kDa and a pI of 9.0. Under the assay conditions with chemically acetylated birchwood xylan the kinetic constants of the enzyme were: specific activity, 715 units/mg, Km 7.94 mg/ml and Vmax 1977 units/mg. Optimal enzyme activity was obtained at 70 °C and pH 7.5. Hydrolysis assays with different acetylated substrates showed that the enzyme is specific for deacetylating the O-acetyl group of polysaccharides and is devoid of N-deacetylation activity. Sequential hydrolysis shows that its action is essential for the complete degradation of acetylated xylan by the xylanases of S. lividans.

scholarly journals ʲ-mannanase of Streptomyces lividans 66: cloning and DNA sequence of the manA gene and characterization of the enzyme

1993 ◽  
Vol 290 (3) ◽  
pp. 857-863 ◽  
Author(s):  
N Arcand ◽  
D Kluepfel ◽  
F W Paradis ◽  
R Morosoli ◽  
F Shareck
Keyword(s):  
Dna Sequence ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Streptomyces Lividans ◽  
Gene Coding ◽  
Sds Page ◽  
Main Carbon Source ◽  
Assay Conditions ◽  
Main Carbon

The gene coding for a beta-mannanase was cloned homologously from Streptomyces lividans and its DNA sequence was determined. The fully secreted enzyme was isolated and purified from culture filtrates of the hyperproducing clone S. lividans IAF36 grown in mineral salt media containing galactomannan as the main carbon source. It had a molecular mass of 36 kDa and a specific activity of 876 i.u./mg of protein. Under the assay conditions used, the optimal enzyme activity was obtained at 58 degrees C and a pH of 6.8. The pI was 3.5. The kinetic constants of this mannanase determined with galactomannan as substrate were a Vmax. of 205 i.u./mg of enzyme and a Km of 0.77 mg/ml. Data from SDS/PAGE and Western blotting show that the cloned enzyme was identical to that of the wild-type strain.

scholarly journals (335) Purification and Characterization of ADP-glucose Pyrophosphorylase from Apple Leaves

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1083C-1083
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Purification And Characterization ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations ◽  
Dual Functions

Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.

scholarly journals Purification and characterization of an α-l-arabinofuranosidase from Streptomyces lividans 66 and DNA sequence of the gene (abfA)

1994 ◽  
Vol 302 (2) ◽  
pp. 443-449 ◽  
Author(s):  
C Manin ◽  
F Shareek ◽  
R Morosoli ◽  
D Kluepfel
Keyword(s):  
Dna Sequence ◽  
Avena Sativa ◽  
Specific Activity ◽  
Gel Filtration ◽  
Streptomyces Lividans ◽  
Native Protein ◽  
Purification And Characterization ◽  
Gene Encoding ◽  
Sds Page

The gene encoding an alpha-L-arabinofuranosidase (abfA) was homologously cloned in Streptomyces lividans and its DNA sequence was determined. The enzyme was purified from the cytoplasm of the hyperproducing clone S. lividans IAF116. Its M(r) was estimated by gel filtration and found to be approx. 380,000. Since SDS/PAGE indicated a native protein of M(r) 69,000, it can be concluded that the native protein consists of several subunits of that size. The pI value was 4.6. The kinetic constants determined with p-nitrophenyl alpha-L-arabinofuranoside as substrate were a Vmax of 180 units/mg of protein and a Km of 0.6 mM. The specific activity of the purified enzyme on this substrate was 153 units/mg of protein. Optimal enzyme activity was obtained at 60 degrees C and pH 6.0. The enzyme cleaved p-nitrophenyl alpha-L-arabinofuranoside, but had no activity on a variety of other p-nitrophenyl glycosides, except on p-nitrophenyl beta-D-xylopyranoside. The enzyme showed no activity on oat-spelts (Avena sativa) xylan or arabinogalactan, but acted on beet (Beta) arabinan or arabinoxylan. Hydrolysis occurred on arabino-oligoxylosides obtained from oat-splets xylan after digestion with xylanases. Since S. lividans normally does not secrete arabinofuranosidase, this enzyme may play a role in the assimilation of arabinose moieties from arabinose-containing xylo-oligosaccharides generated by beta-xylosidases or xylanases.

scholarly journals Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
Author(s):  
I Ishii-Karakasa ◽  
H Iwase ◽  
K Hotta ◽  
Y Tanaka ◽  
S Omura
Keyword(s):  
Enzyme Activity ◽  
Culture Medium ◽  
Gel Filtration ◽  
Partial Purification ◽  
Gastric Mucus ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Optimum Ph ◽  
New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

Purification and characterization of two isoforms of native α amylase from Ok-Rong mango (Mangifera indica Linn. cv. Ok-Rong)

2017 ◽  
Vol 42 (6) ◽  
Author(s):  
Raksmont Ubonbal ◽  
Saijai Porsoongnoen ◽  
Jureerut Daduang ◽  
Sompong Klaynongsruang ◽  
Sakda Daduang
Keyword(s):  
High Temperatures ◽  
Fruit Ripening ◽  
Specific Activity ◽  
Mangifera Indica ◽  
Ripening Stage ◽  
Purification And Characterization ◽  
Tropical Plant ◽  
Short Period ◽  
Structure Properties

AbstractIntroduction:The tropical plant amylases involved in the fruit ripening stage is outstanding for their high activities in converting starch to sugars within a short period at high temperatures over 40°C.Methods:The α amylase iso-enzymes from Ok-Rong mango (Results:The enzyme was purified 105-fold with a final specific activity of 59.27 U mgConclusion:Two α amylase iso-enzymes were classified as members of the low-pI group of amylases with identical structure, properties and functions. They are mesophilic with high possibilities for application for many purposes.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity

2013 ◽  
Vol 21 (6) ◽  
pp. 1522-1525 ◽  
Author(s):  
Ramazan Demirdağ ◽  
Veysel Çomaklı ◽  
Murat Şentürk ◽  
Deniz Ekinci ◽  
Ö. İrfan Küfrevioğlu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Carbonic Anhydrase ◽  
Purification And Characterization

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

1998 ◽  
Vol 64 (2) ◽  
pp. 789-792 ◽  
Author(s):  
Giuliano Degrassi ◽  
Benedict C. Okeke ◽  
Carlo V. Bruschi ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Naphthyl Acetate

ABSTRACT Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km ) andV max for α-naphthyl acetate were 1.54 mM and 360 μmol min−1 mg of protein−1, respectively.

Sign in / Sign up

Export Citation Format

Share Document