Purification and Characterization of a Thermostable  -Xylosidase from Thermomonospora fusca

Bachmann, Susan L.; McCarthy, Alan J.

doi:10.1099/00221287-135-2-293

Cited by 20 publications

(22 citation statements)

References 23 publications

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“…K m values of 0.48, 0.70, and 1.20 mM were obtained for peaks I, II, and III, respectively. These K m values are similar to those previously reported for b-D-xylosidases from barley (Lee et al, 2003) and numerous b-D-xylosidases from fungi and bacteria (Bachmann and McCarthy, 1989;Nanmori et al, 1990;Kumar and Ramon, 1996;Saha, 2003). The corresponding k cat /K m ratios of the three enzymes are not significantly different (Table II).…”

Section: Kinetic Properties and Enzymatic Characterizationsupporting

confidence: 88%

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

et al. 2004

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This work describes the purification and characterization of enzymes that exhibit b-D-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with b-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative a-L-arabinofuranosidase, and XYL4 and XYL1 as putative b-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-a-L-arabinofuranoside and XYL4 for p-nitrophenyl-b-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a b-D-xylosidase, while ARAf and XYL1 act as bifunctional a-L-arabinofuranosidase/b-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth. Plant cells are surrounded by an extracellular matrix known as the cell wall. The regulation of cell wall morphology and composition is important for the determination of cell size and shape, cellular interactions with the environment, mechanical resistance, and defense against pathogen attacks (Reiter, 2002). The cell wall is also involved in plant growth and development (Stolle-Smits et al., 1999;Obel et al., 2002). In addition, although cell walls consist mainly of polysaccharides broadly classified as celluloses, hemicelluloses, and pectins, their constitution varies, not only from plant to plant, but also in different tissues of the same plant (Heredia et al., 1995;Cosgrove, 1997;Popper and Fry, 2003). A coordinated series of biochemical processes occur during plant development resulting in the biosynthesis and degradation of cell wall components. Consequently, numerous enzymes must be implicated in these processes (Heredia et al., 1995;Cosgrove, 1997;Stolle-Smits et al., 1999;Obel et al., 2002;Reiter, 2002;Popper and Fry, 2003). However, to date, little information is available concerning such enzymes, their mode of action, and their physiological role. Increased research interest has focused on enzymes involved in the metabolism of hemicelluloses in ...

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Section: Kinetic Properties and Enzymatic Characterizationsupporting

confidence: 88%

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

et al. 2004

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“…To our knowledge, the P-xylosidase from T. ethanolicus described here exhibits the highest thermostability (the half-life at 82°C was longer than 3 h) and is stable over the broadest pH range (pH 5.5 to 8) of P-xylosidases described to date (1,3,6,9). Only a few thermostable ,-xylosidases have been purified.…”

Section: Discussionmentioning

confidence: 99%

“…High activity and thermostability of 3-xylosidase have attracted considerable attention as characteristics of an efficient enzyme. Some thermostable ,B-xylosidases have been purified and characterized (1,6,12). Furthermore, a few ,-xylosidases with an additional a-arabinosidase activity (3,9,14,16) or ,B-glucosidase activity (17) have been reported.…”

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confidence: 99%

“…These include the enzymes from the thermophilic aerobic bacterium Bacillus stearothermophilus (12), the anaerobic bacterium Clostridium acetobutylicum (9), and the "thermophilic" actinomycete Thermomonospora fusca (1). More recently, the P-xylosidase from the anaerobic thermophilic bacterium Caldocellum saccharolyticum, expressed in Escherichia coli, was described by Hudson et al (6).…”

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confidence: 99%

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Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus

Shao

Wiegel

1992

J Bacteriol

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A highly thermostable 13-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towardsp-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82°C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T0pt, s mInt the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93°C; however at 65 and 82°C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82°C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82°C in the absence of substrate reduced the activity to around 75%. At 86°C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.

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“…These data suggest that both activities may reside within a single bifunctional protein, which would be a rather novel enzyme. Xylosidases have been purified from Clostridium acetobutylicum (11), Bacillus pumilus (16), Bacillus coagulans (6), and Thermomonospora fusca (1), and all are multisubunit enzymes ranging in molecular weight from 130,000 to 224,000. None of these enzymes display arabinosidase activity.…”

Section: Pvuii Kpnl Ecorimentioning

confidence: 99%

The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region

Whitehead

Hespell

1990

J Bacteriol

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Genes coding for three xylan-degrading activities, xylanase, xylosidase, and arabinosidase, were simultaneously cloned from the colonic anaerobic organism Bacteriodes ovatus. The genes for the three enzymes were located on a 3.8-kilobase EcoRI genomic insert and were cloned by using plasmid pUC18. All three activities were expressed in Escherichia coli JM83, and all were cell associated. Expression of the xylanase gene was independent from expression of the xylosidase and arabinosidase genes, whereas expression of the latter two genes appeared to be coordinated. Restriction endonuclease analysis of the arabinosidase and xylosidase genes and partial purification of these enzyme activities from E. coli suggested that these activities were catalyzed by a bifunctional protein or two proteins of very similar molecular weight. All three enzyme activities were regulated in B. ovatus in response to the carbon source used for growth. This is the first report of the cloning and expression of B. ovatus genes.

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Purification and Characterization of a Thermostable -Xylosidase from Thermomonospora fusca

Cited by 20 publications

References 23 publications

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus

The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region

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