Preparation of crystalline, amorphous, and dyed cellulase substrates

Wood, Thomas M.

doi:10.1016/0076-6879(88)60103-0

Cited by 313 publications

(195 citation statements)

References 4 publications

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“…Activity and binding of Lic16A and its deletion derivatives were evaluated on lichenan, laminarin, Avicel CF1 and carboxymethylcellulose (Sigma-Aldrich), as well as on chitin, chitosan, xylan, bacterial crystalline cellulose from Sigma-Aldrich, pachyman from Megazym and pustulan from Roth. Phosphoric acid swollen cellulose was prepared from Avicel CF1 by the method of Wood (1988). Insoluble b-glucan from the yeast cell wall of Saccharomyces cerevisiae was prepared according to Bacon et al (1969).…”

Section: Methodsmentioning

confidence: 99%

Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

et al. 2009

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The multi-modular non-cellulosomal endo-1,3(4)-b-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca 2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca 2+ . The highest affinity to CBMX was shown for xylan (binding constant K53.1¾10 4 M "1 ), yeast cell wall glucan (K51.4¾10 5 M "1 ) and chitin (K53.3.10 5 M "1 in the presence of Ca 2+ ). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

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Section: Methodsmentioning

confidence: 99%

Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

et al. 2009

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“…Chemicals were obtained from Sigma-Aldrich/Fluka. Phosphoric acid swollen cellulose (PASC) was prepared from Avicel, as described previously (Wood, 1988).…”

Section: Methodsmentioning

confidence: 99%

Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases

2006

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Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65 % loss of dry mass within 98 days, and it produced endo-1,4-b-glucanase (EG), endo-1,4-b-xylanase, endo-1,4-b-mannanase, 1,4-b-glucosidase (BG), 1,4-b-xylosidase, 1,4-b-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and b-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2?6-2?8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed b-xylosidase and endoxylanase activities. The K m for carboxymethylcellulose was 3?5 g l "1 , with the highest activity at pH 3?5 and 70 6C. BG1 was a protein of 36 kDa with a pI around 2?6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K m for p-nitrophenyl-1,4-b-glucoside was 1?8 mM, with the highest activity at pH 4 and 60 6C, and the enzyme was competitively inhibited by glucose (K i =5?8 mM). The fungus produced mainly b-glucosidase and b-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and b-xylosidase were found in fungus-colonized wood.

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“…Plasmid pUC19 (Boehringer Mannheim) was used as cloning vector. Detection of activity on carboxy methyl cellulose (CMC, Sigma) or acid swollen cellulose (ASC) [26] was performed by incubation of grown cultures, cell suspensions, cell extracts or culture supernatants either on LB-agar plates supplemented with 1% CMC (w/v), or on thin agarose gels supplemented with 2% ASC (w/v), for 1-24 h at 37°C. Activity was detected by staining with Congo red (Sigma), as described [20].…”

Section: Strains Plasmids and Growth Conditionsmentioning

confidence: 99%

“…Concentrated cell extracts of recombinant E. coli/pUCel48C were mixed with an equal volume of 5% solutions of Avicel (Fluka), bacterial microcrystalline cellulose (BMCC, Monsanto) or ASC [26] in water, and incubated for 1 h at 4°C with gentle rotatory shaking. Samples were then centrifuged (16 000 g, Beckman), and the corresponding pellets washed for three times with the same buffer.…”

Section: Binding Assaysmentioning

confidence: 99%

Exo‐mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP‐23

Sánchez

Pastor

Dı́az

2003

European Journal of Biochemistry

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Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45°C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNPglycosides or 4-methylumbeliferyl a-D-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.

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Preparation of crystalline, amorphous, and dyed cellulase substrates

Cited by 313 publications

References 4 publications

Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases

Exo‐mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP‐23

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