Suggested improvements to the standard filter paper assay used to measure cellulase activity

Coward-Kelly, Guillermo; Aiello-Mazzari, Cateryna; Kim, Sehoon; Granda, Cesar B.; Holtzapple, Mark T.

doi:10.1002/bit.10620

Cited by 68 publications

(43 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…These assays often do not correlate well with each other, are often subject to exoenzyme interferences (30), and are often influenced by different stoichiometric reactivities based on the cello-oligomer length (31,32). Being spectrophotometric, they are also subject to optical limitations in that complex biomass samples cannot be assayed in this way, and being end-point assays, time resolution must be estimated by differentiating a series of stopped aliquots or separate parallel reactions.…”

Section: ) Endo-14-␤-d-glucan-4-glucanohydrolases (Eg)mentioning

confidence: 99%

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

Murphy

Cruys-Bagger

Damgaard

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s ؊1 at 30°C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

show abstract

Section: ) Endo-14-␤-d-glucan-4-glucanohydrolases (Eg)mentioning

confidence: 99%

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

Murphy

Cruys-Bagger

Damgaard

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The products of the various cellulases can be translated into activities measurements by using different substrates to elucidate modes of attack (Ghose, 1987). The standard filter paper activity assay is a good measure of total cellulase activity since filter paper is a nearly pure cellulose substrate and is readily available, with an established and accepted assay method (Coward-Kelly et al, 2003;Ghose, 1987;Xiao et al, 2004;Zhang et al, 2006). Microcrystalline cellulose, such as Avicel, can be used to measure exocellulase activity because it has a low degree of polymerization and relatively low accessibility (Wood and Bhat, 1988;Zhang et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

An optimized microplate assay system for quantitative evaluation of plant cell wall–degrading enzyme activity of fungal culture extracts

King

Donnelly

Bergstrom

et al. 2008

Biotech & Bioengineering

138

102

View full text Add to dashboard Cite

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose-or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.

show abstract

“…However, some of the major limitations of such colorimetric assays are poor stoichiometric correlations between the colorimetric oxidant (i.e., DNS, Cupric ions) and chain length of reducing oligosaccharidic sugar (Coward-Kelly et al, 2003;Sengupta et al, 2000). The colorimetric assays also suffer from non-specific interference due to protein and lignin present in the biomass hydrolysate (Rivers et al, 1984;Schwald et al, 1988).…”

Section: Bio-enzymatic Sugar Assaymentioning

confidence: 99%

“…However, the IUPAC FPA assay (Ghose, 1984) utilizing filter paper as the substrate is plagued by several disadvantages for high-throughput assays. Primary disadvantages are long assay periods, high labor inputs, high sensitivity, and poor reproducibility (Coward-Kelly et al, 2003). Poor reproducibility of the FPA assay is due to several factors such as filter paper properties (i.e., size, shape and folding) and accessory enzyme components (e.g., b-glucosidase).…”

Section: Introductionmentioning

confidence: 99%

High‐throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Chundawat

Balan

Dale

2008

Biotech & Bioengineering

118

113

View full text Add to dashboard Cite

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.

show abstract

Suggested improvements to the standard filter paper assay used to measure cellulase activity

Cited by 68 publications

References 7 publications

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

An optimized microplate assay system for quantitative evaluation of plant cell wall–degrading enzyme activity of fungal culture extracts

High‐throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Contact Info

Product

Resources

About