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A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial beta-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.

This study reports differential expression of endoglucanase (EG) and beta-glucosidase (betaG) isoforms of Aspergillus terreus. Expression of multiple isoforms was observed, in presence of different carbon sources and culture conditions, by activity staining of poly acrylamide gel electrophoresis gels. Maximal expression of four EG isoforms was observed in presence of rice straw (28 U/g DW substrate) and corn cobs (1.147 U/ml) under solid substrate and shake flask culture, respectively. Furthermore, the sequential induction of EG isoforms was found to be associated with the presence of distinct metabolites (monosaccharides/oligosaccharides) i.e., xylose (X), G(1), G(3) and G(4) as well as putative positional isomers (G(1)/G(2), G(2)/G(3)) in the culture extracts sampled at different time intervals, indicating specific role of these metabolites in the sequential expression of multiple EGs. Addition of fructose and cellobiose to corn cobs containing medium during shake flask culture resulted in up-regulation of EG activity, whereas addition of mannitol, ethanol and glycerol selectively repressed the expression of three EG isoforms (Ia, Ic and Id). The observed regulation profile of betaG isoforms was distinct when compared to EG isoforms, and addition of glucose, fructose, sucrose, cellobiose, mannitol and glycerol resulted in down-regulation of one or more of the four betaG isoforms.

Spectroscopic characterization of both untreated and treated material is being performed in order to determine changes in the biomass and the effects of pretreatment on crystallinity, lignin content, selected chemical bonds, and depolymerization of hemicellulose and lignin. The methods used are X-ray diffraction for determination of cellulose crystallinity (CrI); diffusive reflectance infrared (DRIFT) for changes in C-C and C-O bonds; and fluorescence to determine lignin content. Changes in spectral characteristics and crystallinity are statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that govern its enzymatic conversion to monomeric sugars. Models of the hydrolysis initial rate and 72 h extent of conversion were developed and evaluated. Results show that the hydrolysis initial rate is most influenced by the cellulose crystallinity, while lignin content most influences the extent of hydrolysis at 72 h. However, it should be noted that in this study only crystallinity, lignin, and selected chemical bonds were used as inputs to the models. The incorporation of additional parameters that affect the hydrolysis, like pore volume and size and surface area accessibility, would improve the predictive capability of the models.

Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in beta-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and beta-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.

The effects of carbon source on xylanase and cellulase production were studied. The extract of steam-exploded corn stover was found to be the best raw material for producing cellulase-free xylanase. The xylanolytic enzymes of Aspergillus niger An-76 were purified by chromatography, and the properties of the four purified components were analyzed. When the enzyme was used to treat the birch Kraft pulp, and followed by a subsequent CEH bleaching, a brightness of 6.8% SBD more than that of the untreated one using the same chlorine dosage, or a saving of nearly 50% of chlorine consumption with the same brightness, was realized.