Assessing Substrate Accessibility to Enzymatic Hydrolysis by Cellulases

Chandra, Richard P.; Esteghlalian, Ali R.; Saddler, John N.

doi:10.1002/9781444305425.ch4

Cited by 20 publications

(31 citation statements)

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“…Simon's staining of lignocellulose for determining porosity was performed as described by Chandra et al, , and others who have shown that extent of hydrolysis correlates with surface area (Arantes and Saddler, , 2009; Burns et al, ; Chandra et al, ; Del Rio et al, ; Grethlein, ; Ishizawa et al, ; Wong et al, ; Yu and Atalla, ). (Refer to Supporting Information).…”

Section: Methodsmentioning

confidence: 99%

Hydrolysis‐determining substrate characteristics in liquid hot water pretreated hardwood

Kim

Kreke

et al. 2015

Biotech & Bioengineering

118

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Fundamental characterization of pretreated hardwood and its interactions with cellulolytic enzymes has confirmed that a pathway exists for dramatically reducing the loading of cellulase required for hydrolysis of pretreated biomass. We demonstrate that addition of protein effecting a seven-fold decrease in the specific activity of cellulases enables a ten-fold reduction in enzyme loading while maintaining a high level of cellulose hydrolysis in pretreated hardwood. While use of protein and other additives that adsorb on lignin have been reported previously, the current work demonstrates the effect in a dramatic manner and brings the rationale for this change into clear focus. The key to this result is recognizing and mitigating the pretreatment conundrum where increasingly severe pretreatment conditions enhance accessibility of the enzymes not only to cellulose, but also to lignin. The lignin adsorbs enzyme protein causing loss of cellulase activity. More enzyme, added to compensate for this lost activity, results in a higher cellulase loading. The addition of a different protein, such as BSA, prevents cellulase adsorption on lignin and enables the enzyme itself to better target its glucan substrate. This effect dramatically reduces the amount of cellulase for a given level of conversion with enzyme loadings of 15 FPU and 1.3 FPU/g solids both achieving 80% conversion. The understanding of this phenomenon reinvigorates motivation for the search for other approaches that prevent cellulase adsorption on lignin in order to achieve high glucose yields at low enzyme loadings for pretreated lignocellulose.

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Section: Methodsmentioning

confidence: 99%

Hydrolysis‐determining substrate characteristics in liquid hot water pretreated hardwood

Kim

Kreke

et al. 2015

Biotech & Bioengineering

118

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“…However, the largest challenge of the biological-based platform is how to achieve a high amount of fermentable sugars via enzymatic saccharification. Technically, successful saccharification can be achieved by "opening up" the ultrastructure of the lignocellulosics through pretreatment or some other manner of pre-enzymatic treatment (Mansfield et al 1999;Chandra et al 2008). However, hurdles remain that must be overcome to ensure a successful rate and extent of substrate enzymatic hydrolysis, inter alia, lignin structure and distribution, cellulose crystallinity, the degree of cellulose polymerization, particle size, and pore volume (Mooney et al 1998;Hall et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Molecular Changes in Corn Stover Lignin Resulting from Pretreatment Chemistry

et al. 2017

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Lignin is an amorphous polymer that limits the enzymatic conversion of polysaccharides to fermentable sugars. Thus, a pretreatment that can enhance the accessibility of carbohydrates is a key step of successful biofuel conversion schemes. In this study, corn stover was fractioned into stem, cob, and leaf because their lignin is different. To elucidate the lignin changes, autohydrolysis, diluted acid, and alkali pretreatments were applied on the samples, followed by the isolation of cellulolytic enzyme lignin preparations. Alkaline nitrobenzene oxidation, 13 C-Nuclear Magnetic Resonance (NMR), and 1 H-13 C heteronuclear single quantum coherence NMR were used to profile the lignin changes. The results indicated that corn stover lignin is a p-hydroxyphenyl-guaiacyl-syringyltype lignin that incorporates p-coumarate and ferulate esters. The β-arylether was the most abundant inter-unit linkage, followed by condensed linkages, e.g. pino-/syringaresinol, phenylcoumaran, and spirodienone. As for the non-pretreated samples, leaf lignin was more condensed than stem lignin and cob lignin. More lignin was removed by the alkali pretreatment due to more cleavage of β-aryl-ether linkages. As a comparison, more condensed linkages were generated by the acidic pretreatments. The decrease of the syringyl/guaiacyl ratio indicated that the residual lignin became more condensed and confirmed that guaiacyl and p-hydroxyphenyl units were more stable than syringyl units during the pretreatment.

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“…Several groups have tried to develop methods of accurately quantifying changes in the accessibility of lignocellulosic substrates, with many of these techniques modified from traditional pulp and paper procedures [54]. Although these techniques have previously only been used to assess the overall accessibility of the substrate, including accessibility to the lignin and hemicelluloses as well as the cellulose, some of these techniques also have potential for quantifying the amorphogenesis step.…”

Section: Introductionmentioning

confidence: 99%

“…Or, performing techniques such as solute exclusion, differential scanning calorimetry, time-domain nuclear magnetic resonance, Simons’ Staining and protein adsorption (Reviewed in [54]). One of the benefits of these modified pulp and paper techniques is that they can monitor changes in the cellulose accessibility at the macroscopic, microscopic and nanoscopic levels.…”

Section: Introductionmentioning

confidence: 99%

Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis

Gourlay

Arantes

Saddler

2012

Biotechnol Biofuels

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BackgroundCellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process.ResultsIn this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique.ConclusionsThe adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix.

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Assessing Substrate Accessibility to Enzymatic Hydrolysis by Cellulases

Cited by 20 publications

References 0 publications

Hydrolysis‐determining substrate characteristics in liquid hot water pretreated hardwood

Hydrolysis‐determining substrate characteristics in liquid hot water pretreated hardwood

Molecular Changes in Corn Stover Lignin Resulting from Pretreatment Chemistry

Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis

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