Enzyme Immobilization Using the Cellulose-Binding Domain of a Cellulomonas Fimi Exoglucanase

Ong, Edgar; Gilkes, Neil R.; Warren, R. A. J.; Miller, Robert C.; Kilburn, Douglas G.

doi:10.1038/nbt0689-604

Cited by 73 publications

(49 citation statements)

References 102 publications

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“…Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al, 1989, Bio/Technology 76044071, each of the exposed tryptophans interacts reversibly with cellulose. …”

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confidence: 99%

Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

et al. 1996

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The cellulose-binding domain (CBD,,,) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the p-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 346993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDcex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDcex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDcex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDcex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 76044071, each of the exposed tryptophans interacts reversibly with cellulose.

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Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

et al. 1996

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“…For example, Ong et al (38) created a chimeric enzyme composed of a CBM from Cellulomonas fimi fused to a β-glucosidase from Agrobacterium sp. The specific activity of the resultant fused enzyme was 14% lower than the original enzyme, though its CBM sustained its binding activity.…”

Section: Discussionmentioning

confidence: 99%

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

Gefen

Anbar

Morag³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

114

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The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.

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“…The aforementioned example of glucose isomerase stability is only possible due to its immobilization permitting it to function at 60 °C for more than two years before it must be replaced [62]. Low-cost enzyme immobilization technologies are becoming widely-adopted, for example, cross-linking enzyme aggregate (CLEA) [78][79][80] and cellulose-binding module-tagged (CBM-tagged) protein immobilization [74,[81][82][83][84]. The CBM-tagged enzymes can be purified and immobilized in one step, which greatly decreases processing costs and operation complexity (Figure 7).…”

Section: Sypab Challenges and Opportunitiesmentioning

confidence: 99%

Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

Zhang

Mielenz

2011

Energies

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Abstract:The hydrogen economy presents an appealing energy future but its implementation must solve numerous problems ranging from low-cost sustainable production, high-density storage, costly infrastructure, to eliminating safety concern. The use of renewable carbohydrate as a high-density hydrogen carrier and energy source for hydrogen production is possible due to emerging cell-free synthetic biology technology-cell-free synthetic pathway biotransformation (SyPaB

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Enzyme Immobilization Using the Cellulose-Binding Domain of a Cellulomonas Fimi Exoglucanase

Cited by 73 publications

References 102 publications

Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

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