Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage

Hong, Jiong; Wang, Yiran; Ye, Xinhao; Zhang, Y.‐H. Percival

doi:10.1016/j.chroma.2008.04.048

Cited by 79 publications

(68 citation statements)

References 29 publications

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“…Mass concentration of the non-adsorbed TGC protein was measured based on fluorescence reading using a BioTek multi-detection microplate reader, as described elsewhere (Hong et al, 2007(Hong et al, , 2008a.…”

Section: Protein Mass Concentration Assaysmentioning

confidence: 99%

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

Zhu

Sathitsuksanoh

Vinzant

et al. 2009

Biotech & Bioengineering

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Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only $60% for the DA-pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a nonhydrolytic recombinant protein TGC were measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m 2 /g, nearly twice that of the DA-pretreated biomass (5.89 m 2 /g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA-and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility.

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Section: Protein Mass Concentration Assaysmentioning

confidence: 99%

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

Zhu

Sathitsuksanoh

Vinzant

et al. 2009

Biotech & Bioengineering

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200

164

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“…His-tagged proteins were purified by the Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA, USA). Fusion proteins containing a cellulose-binding-module and self-cleavage intein were purified through high-affinity adsorption on a large-surface-area regenerated amorphous cellulose 44,45 . Heat precipitation at 80°C for 20 min was used to purify ribose-5-phosphate isomerase, ribulose-5-phosphate epimerase, triosephosphate isomerase (TIM) and aldolase 46,47 .…”

Section: Methodsmentioning

confidence: 99%

A high-energy-density sugar biobattery based on a synthetic enzymatic pathway

Zhu

Tam²,

Sun³

et al. 2014

Nat Commun

247

194

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High-energy-density, green, safe batteries are highly desirable for meeting the rapidly growing needs of portable electronics. The incomplete oxidation of sugars mediated by one or a few enzymes in enzymatic fuel cells suffers from low energy densities and slow reaction rates. Here we show that nearly 24 electrons per glucose unit of maltodextrin can be produced through a synthetic catabolic pathway that comprises 13 enzymes in an air-breathing enzymatic fuel cell. This enzymatic fuel cell is based on non-immobilized enzymes that exhibit a maximum power output of 0.8 mW cm À 2 and a maximum current density of 6 mA cm À 2 , which are far higher than the values for systems based on immobilized enzymes. Enzymatic fuel cells containing a 15% (wt/v) maltodextrin solution have an energy-storage density of 596 Ah kg À 1 , which is one order of magnitude higher than that of lithium-ion batteries. Sugar-powered biobatteries could serve as next-generation green power sources, particularly for portable electronics.

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“…Cellulase activities of the purified recombinant proteins were determined on amorphous cellulose (acid-swollen cellulose [ASC]). ASC was prepared by the method of Hong et al (17). Each reaction mixture consisted of 100 l of 3% ASC, 300 l of 150 mM morpholineethanesulfonic acid (MES)-NaOH buffer (pH 6.8), and 100 l of enzyme solution.…”

Section: Methodsmentioning

confidence: 99%

CBM3d, a Novel Subfamily of Family 3 Carbohydrate-Binding Modules Identified in Cel48A Exoglucanase of Cellulosilyticum ruminicola

2011

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Previously, we found that exoglucanase Cel48A from Cellulosilyticum ruminicola H1 bound intensively to Avicel; however, no known carbohydrate-binding module (CBM) was observed in the protein. Bioinformatics suggested that a C-terminal fragment of 127 amino acids, named the Cellulosilyticum-specific paralogous module (CPM), could function in binding. CPM-appended proteins are all putative (hemi)cellulases from Cellulosilyticum spp. In the present work, we demonstrated that Cel48A without the CPM retained only exoglucanase activity and lost the Avicel-binding ability, while the isolated CPM exhibited a high affinity for Avicel. In addition, the CPM bound to chitin, but not to soluble polysaccharides, making it a type A CBM, which binds only insoluble polysaccharides. Phylogenetic analysis clustered the CPM and its homologs as a separate branch that was distantly related to CBM subfamilies 3a (28% identity), 3b (24% identity), and 3c (21% identity). Sequence alignment revealed distinct secondary structures of the new CBM 3 group, in particular, a conserved Pro66-Trp67 insert preceding strand ␤4, a deletion preceding strand ␤6, and incomplete strands ␤8 and ␤9. An alanine scan for six aromatic and three nonaromatic amino acid residues (D66, P66, and R111) by site-directed mutagenesis determined that Phe62, Pro66, Trp67, Tyr68, Arg111, and Trp117 were the functional residues for binding. Among them, Phe62, Pro66, and Trp67 were the newly determined key sites in the CPM for binding. Three-dimensional homolog modeling revealed two types of substrate-binding sites, planar and groove, in the CPM. Thus, a new subfamily, CBM family 3d, is proposed.

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Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage

Cited by 79 publications

References 29 publications

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

A high-energy-density sugar biobattery based on a synthetic enzymatic pathway

CBM3d, a Novel Subfamily of Family 3 Carbohydrate-Binding Modules Identified in Cel48A Exoglucanase of Cellulosilyticum ruminicola

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