Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

doi:10.1074/jbc.m115.673467

Cited by 69 publications

(65 citation statements)

References 37 publications

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“…Mutations were made by site directed mutagenesis (Zheng et al, ). Expression and purification was performed as previously described (Strobel et al, ). Briefly, SC‐Trp medium was inoculated with S. cerevisiae containing the Cel7A gene and grown for 3 days at 30°C, 220 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…The T. reesei Cel7A linker and CBM were produced with a homologous Cel7A CD from Talaromyces emersonii (Te‐Tr chimera) to enable efficient expression in Saccharomyces cerevisiae . Four residues in the CBM (Q2, H4, V18, and P30) were chosen for mutation based on favorable changes in cellulose/lignin adsorption selectivity when previously mutated to alanine (Strobel et al, ). Three additional residues (Y5, V27, and L28) were selected for mutation due to their hydrophobic nature.…”

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confidence: 99%

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Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition

Strobel

Pfeiffer

Blanch

et al. 2015

Biotech & Bioengineering

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Non-productive binding of cellulases to lignin inhibits enzymatic hydrolysis of biomass, increasing enzyme requirements and the cost of biofuels. This study used site-directed mutagenesis of the Trichoderma Cel7A carbohydrate binding module (CBM) and linker to investigate the mechanisms of adsorption to lignin and engineer a cellulase with increased binding specificity for cellulose. CBM mutations that added hydrophobic or positively charged residues decreased the specificity for cellulose, while mutations that added negatively charged residues increased the specificity. Linker mutations that altered predicted glycosylation patterns selectively impacted lignin affinity. Beneficial mutations were combined to generate a mutant with 2.5-fold less lignin affinity while fully retaining cellulose affinity. This mutant was uninhibited by added lignin during hydrolysis of Avicel and generated 40% more glucose than the wild-type enzyme from dilute acid-pretreated Miscanthus. Biotechnol. Bioeng. 2016;113: 1369-1374. © 2015 Wiley Periodicals, Inc.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition

Strobel

Pfeiffer

Blanch

et al. 2015

Biotech & Bioengineering

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“…Cellulases modified by succinylation to negatively charge the protein surface showed final higher conversion of Avicel in the presence of lignin compared with controls, suggesting that the electrostatic potential on a protein is also relevant for lignin‐mediated inactivation. Strobel et al investigated lignin adsorption behavior for a series of mutants of a family 1 CBM (Pfeiffer et al, ; Strobel et al, , ). Mutation of some residues lowered lignin adsorption without a corresponding loss of CBM1 binding to crystalline cellulose.…”

Section: Introductionmentioning

confidence: 99%

Insights into cellulase‐lignin non‐specific binding revealed by computational redesign of the surface of green fluorescent protein

Haarmeyer

Smith

Chundawat

et al. 2016

Biotech & Bioengineering

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Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28-0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases. Biotechnol. Bioeng. 2017;114: 740-750. © 2016 Wiley Periodicals, Inc.

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“…Strong bindings of Trichoderma reesei CBH-I and EG-I to both cellulose and lignin were observed using quartz crystal microgravimetry (Martín et al, 2013). Other studies reported that the cellulose binding domain of the effluent enzyme played a significant role in the unspecific binding of cellulases to lignin (Palonen et al, 2004;Rahikainen et al, 2013;Strobel et al, 2015Strobel et al, , 2016. T. longibrachiatum, which are taxonomically separable from T. reesei, could also act as a potential cellulase candidate with high activities (Kubicek et al, 1996).…”

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confidence: 98%

Characteristics of Lignin Fractions from Dilute Acid Pretreated Switchgrass and Their Effect on Cellobiohydrolase from Trichoderma longibrachiatum

Yao

Yoo

et al. 2018

Front. Energy Res.

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To investigate the interactions between acid pretreated switchgrass lignin and cellobiohydrolase (CBH), three different lignin fractions were isolated from dilute acid pretreated switchgrass by (i) ethanol extraction, followed by (ii) dioxane/H2O extraction, and (iii) cellulase treatment, respectively. Structural properties of each lignin fraction were elucidated by GPC, 13 C-NMR, and 2D-HSQC NMR analyses. The adsorptions of CBH to the isolated lignin fractions were also studied by Langmuir adsorption isotherms. Ethanolextractable lignin fraction, mainly composed of syringyl (S) and guaiacyl (G) units, had the lowest molecular weight, while dioxane/H2O-extracted lignin fraction had the lowest S/G ratio with higher content of p-coumaric acid (pCA) unit. The residual lignin fraction after enzymatic treatment had the highest S/G ratio without hydroxyphenyl (H) unit. Strong associations were found between lignin properties such as lignin composition and S/G ratio and its non-productive enzyme adsorption factors including the maximum adsorption capacity and binding strength.

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Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin

Cited by 69 publications

References 37 publications

Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition

Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition

Insights into cellulase‐lignin non‐specific binding revealed by computational redesign of the surface of green fluorescent protein

Characteristics of Lignin Fractions from Dilute Acid Pretreated Switchgrass and Their Effect on Cellobiohydrolase from Trichoderma longibrachiatum

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