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Title: Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

Abstract

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound highmore » molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1405916
Report Number(s):
NREL/JA-2700-70382
Journal ID: ISSN 2211-5501
DOE Contract Number:
AC36-08GO28308
Resource Type:
Journal Article
Resource Relation:
Journal Name: Current Biotechnology; Journal Volume: 6; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; lignin; protein binding; cellulase; xylanase

Citation Formats

Yarbrough, John M., Mittal, Ashutosh, Katahira, Rui, Mansfield, Elisabeth, Taylor, Larry E., Decker, Stephen R., Himmel, Michael E., and Vinzant, Todd. Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins. United States: N. p., 2017. Web. doi:10.2174/2211550105666160916170630.
Yarbrough, John M., Mittal, Ashutosh, Katahira, Rui, Mansfield, Elisabeth, Taylor, Larry E., Decker, Stephen R., Himmel, Michael E., & Vinzant, Todd. Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins. United States. doi:10.2174/2211550105666160916170630.
Yarbrough, John M., Mittal, Ashutosh, Katahira, Rui, Mansfield, Elisabeth, Taylor, Larry E., Decker, Stephen R., Himmel, Michael E., and Vinzant, Todd. Mon . "Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins". United States. doi:10.2174/2211550105666160916170630.
@article{osti_1405916,
title = {Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins},
author = {Yarbrough, John M. and Mittal, Ashutosh and Katahira, Rui and Mansfield, Elisabeth and Taylor, Larry E. and Decker, Stephen R. and Himmel, Michael E. and Vinzant, Todd},
abstractNote = {Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.},
doi = {10.2174/2211550105666160916170630},
journal = {Current Biotechnology},
number = 2,
volume = 6,
place = {United States},
year = {Mon Apr 24 00:00:00 EDT 2017},
month = {Mon Apr 24 00:00:00 EDT 2017}
}
  • Lignin, an abundant, naturally occurring biopolymer, is often considered 'waste' and used as a simple fuel source in the paper-making process. However, lignin has emerged as a promising renewable resource for engineering materials, such as carbon fibers. Unfortunately, the molecular architecture of lignin (in vivo and extracted) is still elusive, with numerous conflicting reports in the literature, and knowledge of this structure is extremely important, not only for materials technologies, but also for production of biofuels such as cellulosic ethanol due to biomass recalcitrance. As such, the molecular structures of solvent-extracted (sulfur-free) lignins, which have been modified using various acylmore » chlorides, have been probed using small-angle X-ray (SAXS) and neutron (SANS) scattering in tetrahydrofuran (THF) solution along with hydrodynamic characterization using dilute solution viscometry and gel permeation chromatography (GPC) in THF. Mass spectrometry shows an absolute molecular weight {approx}18-30 kDa ({approx}80-140 monomers), while GPC shows a relative molecular weight {approx}3 kDa. A linear styrene oligomer (2.5 kDa) was also analyzed in THF using SANS. Results clearly show that lignin molecular architectures are somewhat rigid and complex, ranging from nanogels to hyperbranched macromolecules, not linear oligomers or physical assemblies of oligomers, which is consistent with previously proposed delignification (extraction) mechanisms. Future characterization using the methods discussed here can be used to guide extraction processes as well as genetic engineering technologies to convert lignin into value added materials with the potential for high positive impact on global sustainability.« less
  • Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms ofmore » life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.« less