Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition

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

doi:10.1002/bit.25889

Cited by 48 publications

(47 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Protein surface‐binding relationships evaluated in the present contribution suggest a controllable relationship between designed protein net charge and protein lignin binding capacity, consistent with recent efforts (Nordwald et al, ; Strobel et al, ). However, some low lignin‐binding designs had a positive net charge, showing that net charge alone is not sufficient to explain the entire dataset.…”

Section: Discussionsupporting

confidence: 90%

“…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%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 90%

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

View full text Add to dashboard Cite

show abstract

“…1 and 2 lignin. In the aliphatic region, the signals from -OCH3 at 55.8 ppm, β-O-4′ at around 72.3 ppm (Cα in β-O-4′), 85.2 ppm (Cβ in S type β-O-4′) and 60.1 ppm (Cγ in β-O-4′), β-5 at about 86.8 ppm (Cα) and Cγ in β-β′ at ~71.7 ppm (Samuel et al, 2010;Strobel et al, 2016) were readily assigned. structural information of lignin Fractions using 2D-hsQc nMr analysis HSQC is the most applied 2D NMR techniques for the structure determination of lignin samples with enhanced spectral resolution.…”

Section: Nmr Analysismentioning

confidence: 99%

“…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).…”

mentioning

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.

View full text Add to dashboard Cite

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.

show abstract

“…Next, the CD cleaves the cellulose chains on the regions of low crystallinity to yield oligosaccharides and/or cellobiose [5]. The linker performs various functions, such as providing cold adaptation to cellulase of an Antarctic bacterium [4], separating the CD and CBM by a necessary distance, facilitating the dynamic adsorption of CBM on the substrate [6], acting as a molecular spring between two functional modules [6], and affecting the affinity of cellulase for lignin and cellulose [7]. Kont et al recently reported that the synergism between the CBM-linker and CD plays an important role in movement of the cellulose chain into the active site of the processive endoglucanase Cel7A from Trichoderma reesei [8].…”

Section: Introductionmentioning

confidence: 99%

The PT/S-Box of Modular Cellulase AcCel12B Plays a Key Role in the Hydrolysis of Insoluble Cellulose

Wang

et al. 2018

Catalysts

View full text Add to dashboard Cite

Abstract:Cellulases play key roles in the degradation of lignocellulosic materials. The function and mechanism of the catalytic domain (CD) and carbohydrate-binding module (CBM) of cellulases were earlier revealed by analysis and characterization of protein structure. However, understanding of the catalytic mechanism of the entire enzyme, and the analysis of the catalytic model, were inadequate. Therefore, the linker chain between CD and CBM has been extensively studied to bridge this gap. Cellulase AcCel12B and three mutants with different linker lengths (with no or 1-3 PT/S-box units) were successfully constructed and purified. Results showed that the activity of cellulases on Avicel and regenerated amorphous cellulose (RAC) increased with the number of PT/S-box units. Furthermore, the desorption of AcCel12B and its mutants from RAC and Avicel were significantly different. The energy of desorption of wild-type and mutant AcCel12B from cellulose decreased with the number of PT/S-box units. Thus, AcCel12B containing more PT/S-box units was more easily desorbed and had more opportunity to hydrolyze cellulose than other samples. The number of PT/S-box units in endocellulase affected the desorption of the enzyme, which is possibly responsible for the differences in the activity of wild-type and mutant AcCel12B on Avicel and RAC.

show abstract

Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition

Cited by 48 publications

References 21 publications

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

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

The PT/S-Box of Modular Cellulase AcCel12B Plays a Key Role in the Hydrolysis of Insoluble Cellulose

Contact Info

Product

Resources

About