Purification and characterization of an exoglucanase from <i>Streptomyces flavogriseus</i>

MacKenzie, Colin R.; Bilous, D.; Johnson, Kenneth G.

doi:10.1139/m84-183

Cited by 66 publications

(30 citation statements)

References 25 publications

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“…As has been reported for exoglucanases from both fungal and bacterial systems, Exo A showed little activity towards CMC (6,21,24,36). Carboxymethyl substitution of cellulose yielding three or more adjacent substituted glucosyl residues is very resistant to enzymatic attack (8,16).…”

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confidence: 52%

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1

1987

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An exo-0-1,4-glucanase (Exo A) from Ruminococcus flavefaciens FD-1 was purified to homogeneity and characterized. Enzyme activity was monitored during purification by using the substrate p-nitrophenyl-o-Dcellobioside (NPC). Over 85% of the NPC activity was found to be extracellular once the filter paper was degraded (7 days). Culture supernatant was harvested, and the protein was concentrated by ultrafiltration. The retentate (.300,000 Mr), containing most of the activity against NPC, was then fractionated with a TSK DEAE-5PW column. This yielded a sharp major peak of NPC enzyme activity, followed by a broader, less active area that appeared to contain at least six minor peaks of lower enzymatic activity. Further purification was achieved by chromatography with a hydroxylapatite column. Finally, gel ifitration chromatography yielded a homogeneous enzyme (Exo A) as determined by silver stains of both sodium dodecyl sulfate-and nondenaturing electrophoresis gels. Substrate specificity experiments and the products of cellulose digestion indicate that the enzyme was an exo-,3-1,4-glucanase. Exo Cellulolytic organisms produce several cellulase enzymes which have different specificities and modes of action. At least three types of cellulase enzymes are involved in the degradation of cellulose by fungi (25). Endo-P-1,4-glucanase (EC 3.2.1.4) is produced by all cellulolytic microorganisms and is more commonly referred to as carboxymethylcellulase (CMCase) or C, cellulase. Exo-P-1,4-glucanase (EC 3.2.1.91) is also referred to as C1 cellulase or cellobiohydrolase. ,-1,4-Glucosidase (EC 3.2.1.21) is widely distributed among organisms and is commonly known as cellobiase or P-glucosidase. The most efficient and complete hydrolysis of cellulose is thought to be the result of the combined synergistic action of both endoglucanase and exoglucanase.Two of the most important cellulolytic bacteria in the rumen are Ruminococcus flavefaciens and Ruminococcus albus (2, 11). Typically, these two species ferment cellulose, cellobiose, and xylan. The cellulase systems of both R. flavefaciens and R. albus appear to have many similarities. However, in general, R. flavefaciens degrades crystalline cellulose more efficiently than R. albus (2, 11). This observation suggests that R. flavefaciens possesses exoglucanase activity.Pettipher and Latham (29) investigated the cellulase complex from R. flavefaciens as a crude preparation and concluded that the most active enzymes present in the complex were of the exo-,-1,4-glucanase type. However, the authors did not purify any of the enzymes present or investigate the number and types of cellulolytic enzymes in the different molecular weight complexes. The authors concluded that the major enzyme type was exo-P-1,4-glucanase, based on the products of cellulolytic activity (cellobiose, cellotriose, and a small amount of glucose) and the changes in viscosity versus reducing sugar. However, valid interpretations of cellulase enzyme type cannot be made from data generated * Corresponding author. by a m...

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confidence: 52%

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1

1987

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“…In comparison, the cellodextrinase also functioned in an exo fashion, but it only had activity on cellooligosaccharides and did not attack acid-swollen cellulose or crystalline cellulose (23). The hydrolytic properties of the Cl--stimulated enzyme, however, appeared to be similar to those of some fungal exoglucanases and their bacterial counterparts in their ability to hydrolyze acid-swollen cellulose, the type of hydrolysis products formed, and their inability to degrade highly ordered cellulose extensively (10,12,17,22,31,34,43). It has been established that, in fungal cellulase systems, there is synergism between exoglucanses and endoglucanases that can affect the solubilization of crystalline cellulose (33,46).…”

Section: E E180mentioning

confidence: 92%

“…In the cases of several exo-type cellulases isolated from bacteria, the activity on carboxymethyl cellulose was found to be VOL. 170,1988 on present at a low level (12,31,34). The Cl--stimulated cellobiosidase had only negligible activity on crystalline cellulose, but it rapidly degraded cellotriose and longerchain-length cellooligosaccharides and acid-swollen cellulose, producing cellobiose and cellotriose.…”

Section: E E180mentioning

confidence: 99%

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85

1988

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A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gramnegative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39°C and pH 6.5, the activity of the enzyme with p-nitrophenyli--D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.o mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45°C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl -cellulose.Of the cellulolytic rumen bacteria, Bacteroides succinogenes has been described as a predominant cellulose degrader (8, 24). Its cellulase system has been the subject of extensive research in the past few years (16, 19-21, 23, 32, 37, 40). Both endoglucanase and cellobiase activities have been demonstrated in this bacterium for some time (19)(20)(21)37). Recently, two endoglucanases were purified from the extracellular-culture fluid of B. succinogenes (32). These two enzymes hydrolyzed cellooligosaccharides and amorphous cellulose but produced different end products. Examination of the exo-type cellulase activities in B. succinogenes has also been carried out. In a previous study, we identified an exo-type cellodextrinase in the periplasm of B. succinogenes cells grown on cellulose (23). This enzyme was capable of the successive removal of terminal cellobiose units from water-soluble cellodextrins, but not from longer cellulose chains, and therefore appeared to be different from the well-characterized fungal exoglucanases. We therefore decided to undertake further studies on exo-type cellulase activity.Here we report on the purification and characterization of a unique cellobiosidase from B. succinogenes, the activity of which was significantly enhanced by chloride and several other anions. This cellobiosidase may be involved in the initial steps of cellulose hydrolysis. MATERIALS AND METHODSOrganism and growth conditions. B. succinogenes S85 (previously obtained from M. P. Bryant, University of Illinois, Urbana) was grown continuously in a Multigen bench top fermentor (Multigen; New Brunswick Scientific Co., Inc., Edison, N.J.) equipped with a 2-liter chemstat vessel (C-30) and an automatic pH control, as described previously (23). The chemostat medium use...

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“…16) In the present study, we cloned the gene encoding CcEst1 from C. cinerea. The substrate specificities of the recombinant enzyme suggest that the enzyme released acetic and ferulic acids when mixed with native acetylated and ferulated xylan respectively.…”

Section: Coprinopsis Cinereamentioning

confidence: 99%

Extracellular Carbohydrate Esterase from the BasidiomyceteCoprinopsis cinereaReleased Ferulic and Acetic Acids from Xylan

Hashimoto

Kaneko

Yoshida

2010

Bioscience, Biotechnology, and Biochemistry

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Purification and characterization of an exoglucanase from Streptomyces flavogriseus

Cited by 66 publications

References 25 publications

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85

Extracellular Carbohydrate Esterase from the BasidiomyceteCoprinopsis cinereaReleased Ferulic and Acetic Acids from Xylan

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