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Hydrolysis of carbohydrate polymers such as cellulose, xylan and starch is used to produce fermentable sugars for bio-ethanol production.
Digestion of biomass material and its fermentation represents the best path forward for bio-ethanol production.
The use of lignocellulosic material to produce ethanol presents a number of challenges relative to the use of starch containing sources such as corn, which can be digested with amylases. In order to improve the ethanol yield from these polymers, scientists have turned to the use of specific enzymes from a number of different sources. Xylanase enzymes digest xylan polymers, which are a major constituent of the hemicellulose, into xylose.
Optimization of these enzymes in a laboratory or industrial setting requires experimentation with a number of different variables.
EnzCheck Ultra Amylase assay (cat # E33651), Amplex® Red Glucose assay (cat# A13261), and EnzCheck Ultra Xylanase assay (cat# E33650) kits as well as the fluorescent cellulase substrate (cat# E33953) were obtained from Life Technologies. Xylanase activity was measured using an EnzCheck Ultra Xylanase assay kit as described previously [2]. Cellulase activity was also measured directly using an EnzCheck Cellulase substrate as described previously [2]. In order to demonstrate the utility of microplates and microplate readers, three different commonly used enzymes responsible for the digestion of polysaccharides used as feed stocks for ethanol production were investigated for activity under a number of environmental conditions. The effect of pH activity was tested on amylase enzyme isolated from three different sources. Xylanase enzyme isolated from the fungi Thermomyces lanuginosus was found to be very temperature stable. Note that the effect of pH on the fluorescence of the reacted substrate must be accounted for when comparing results at different pH levels. Enzyme titrations performed at different pH levels corroborate the fixed concentration data. These data demonstrate the utility of microplate readers to perform classic enzymology experiments on critical enzymes necessary for the digestion of polysaccharide feed stock into fermentable monosaccharides for the production of ethanol. The use of cellulosic second-generation material to generate fermentable sugars has some marked advantages over first generation starch based grains. The hydrolysis of xylan results in the pentose sugars xylose and arabinose which are not fermented by wild strains of the yeast Saccharomyces cerevisiea.
The use of ethanol as a means to replace a portion of the transportation fuel used in the United States has had legislative support for a number of years. BioTek Instruments, Inc., headquartered in Winooski, VT, USA, is a worldwide leader in the design, manufacture, and sale of microplate instrumentation and software. The production of ethanol for use as a biofuel from grains or cellulosic material requires the hydrolysis of carbohydrate polymers such as cellulose, xylan and starch to monomeric sugars that are fermentable by cellular organisms such as bacteria and yeast. While there are a number of ways to produce ethanol, the most cost effective way is through alcoholic fermentation.
The different technologies used to produce the fermentation sugars necessary for ethanol production are often described as different generations depending on the technology employed. The ability to measure multiple samples simultaneously offers significant advantages in terms of time, expense and effort. The substrate used is a starch derivative labeled with BODIPY® FL to such a degree that the fluorescence is quenched.
In order to demonstrate the utility of microplates and microplate readers, three different commonly used enzymes responsible for the digestion of polysaccharides used as feed stocks for ethanol production were investigated for activity. Amylase enzyme from either the bacteria Bacillus subtilis or the fungus Aspergillus oryzae demonstrate significant amylase activity when measured using a fluorescent amylase substrate (Figure 5). Xylanase enzyme isolated from the thermophilic fungi Thermomyces lanuginosus, demonstrates significant concentration dependent activity towards the fluorescent xylanase substrate (Figure 6). Cellulase activity was determined using either a dedicated substrate or by the production of glucose. When carboxymethyl cellulose (CMC) is digested overnight (16 hr) by cellulase isolated from the soil fungus Aspergillus niger significant amounts of glucose are liberated.
These data demonstrate the utility of microplate readers to monitor the activity of critical enzymes necessary for the digestion of polysaccharide feed stock into fermentable monosaccharides for the production of ethanol.
Biofuel research has employed several different means to improve enzymatic digestion of polysaccharides. BioTek Instrument, Inc, est un leader mondial dans le domaine de la€™instrumentation et logiciel pour microplaque. Opinions expressed are those of the contributors and not necessarily those of the Foundation. Abstract Gelatinized wheat, potato and waxy maize starches were treated enzymatically in order to increase the degree of branching of the amylopectin fraction and thereby change the starch degradation profile towards a higher proportion of slowly digestible starch (SDS). This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0). The authors wish to change the description of preparation of samples at Experimental Section on their paper published in IJMS [1]. Moreover, throughout the paper text and in Tables and Figures the term “jet cooked” should be replaced with “gelatinized”. The exogenous digestion of these polymers on an industrial scale can be cost prohibitive without optimization of the enzyme reaction conditions. While starch based feedstocks have been used extensively, it is believed that lignocellulosic energy sources offer a better long-term prospect as a source for fermentable sugars. Using a combination of in vitro digestion, in conjunction with in vivo genetic manipulation of yeast and bacteria strains, the digestion efficiency to fermentable sugars cannot only be increased, but the ability to ferment monosaccharides other than glucose can be improved.
The ability to measure multiple samples rapidly in small volumes offers significant advantages in terms of time, expense and effort. Carboxymethyl cellulose (cat # 419273), xylanase from Thermomyces lanuginosus (cat# X2753), from Trichoderma longibrachiatum (cat # X2629), cellulase from Aspergillus niger (cat# C1184), α-amylase from Aspergillus oryzae (cat# 10065) from Bacillus licheniformis (cat# A4551) and Bacillus subtilis (Cat#10069) were purchased from Sigma-Aldrich.


In these experiments, equivalent concentrations of a reference standard were used to normalize data prior to plotting.
The direct assay has similar detection limits as the glucose determination, but does not require an overnight digestion.
Cellulosic materials do not compete directly with food stocks for resources such as tillable land, fertilizers and water.
Its linear chains of glucose moieties closely pack with one another forming hydrogen bonds between chains resulting in a crystalline material that is insoluble in water and many organic solvents. To address this limitation several different strains have been genetically modified in regards to xylanase and arabinose transport proteins and pentose fermentation pathways [4]. These technologies are used to aid life science research, facilitate drug discovery, provide rapid and cost-effective analysis, and enable sensitive, accurate quantification of molecules across diverse applications.
Here we describe fluorescence detection analysis in microplates of some of the enzymes that catalyze the digestive hydrolysis of polysaccharide polymers into monomeric constituents.
During this process, the hexose sugar glucose is split into two pyruvate molecules, generating two ATP and reducing two NAD+ molecules by a process known as glycolysis. First generation bio-ethanol production is from fermentable hexose sugars made available by the digestion of starches found in different feed stock sources such as corn or sugar cane.
Using a combination of in vitro digestion, in conjunction with in vivo genetic manipulation of yeast and bacteria strains not only can the digestion efficiency to fermentable sugars be increased, but the ability to ferment monosaccharides other than glucose has been improved.
The use of microplates is one way that large numbers of samples can be handled with minimal amounts of reagents and time. Carboxymethyl cellulose (cat # 419273), xylanase from Thermomyces lanuginosus (cat# X2753), cellulase from Aspergillus niger (cat# C1184), α-amylase from Aspergillus oryzae (cat# 10065) and Bacillus subtilis (Cat#10069) were purchased from Sigma- Aldrich. As the substrate is degraded by amylase activity the close proximity of fluorophores is removed and the quenching is relieved, yielding fluorescent fragments (Figure 2).
As demonstrated in Figure 7, there is a linear relationship between reacted Amplex red fluorescence and glucose concentration. As demonstrated in Figure 8, when aliquots of overnight digestions of CMC by cellulase are assayed for glucose an enzyme concentration dependent increase in glucose is observed. When the substrate is digested with cellulase an enzyme concentration dependent increase in fluorescence is observed (Figure 9).
Screening of newly isolated wild microorganisms can often identify strains with unique or superior characteristics. Enzymatic digestion of dietary carbohydrate (starch, maltose, and sucrose) in biological systems and the proposed inhibition of intestinal α-glucosidase by grape extracts (GE) and subsequent decrease or delay in blood sugar levels after eating a meal. The materials were characterized by single-pulse 1H HR-MAS NMR spectroscopy and in vitro digestion profile according to the Englyst procedure. Effect of Enzymatic Treatment of Different Starch Sources on the in Vitro Rate and Extent of Starch Digestion. We have previously shown the ability to quantitate these enzymes; here we describe the optimization of some of the reaction conditions for the enzymes that catalyze the digestive hydrolysis of polysaccharide polymers into monomeric constituents.
Lignocellulosic feedstocks, such as wood chips, corn stover or switchgrasses do not directly compete with food sources for land or consumption. The chain structure forms a stiff extended conformation that results in extensive hydrogen bonding with adjacent chains. Termites and ruminant herbivores digest cellulose through the action of symbiotic bacteria located in their intestines and ruminating chambers respectively.
Amylase enzyme isolated from different organisms were compared for their activity at different pH levels.
Enzyme concentration titration curves indicate that higher temperatures result in slightly more hydrolysis than ambient temperatures (Figure 7). Using the direct substrate assay, the effect of pH on Aspergillus niger cellulase enzyme activity was investigated. Grain based feed stock rich in starch requires amylase enzymatic activity, while plant and wood material needs cellulase and xylanase activity to break down the cellulose and xylan material contained in the plant fibers.
It can be chemically digested with a combination of high temperature and concentrated acids [3]. Ethanol is currently used as an additive to gasoline in the US to replace MBTE as a means to increase gasoline oxygen content. BioTek espouses a "Think Possible" approach that sets the tone for fresh ideas, unsurpassed customer service and original innovations. During alcoholic fermentation pyruvate is metabolized to ethanol and carbon dioxide with the regeneration of NAD+ (Figure 1). Second generation bioethanol production is the result of the fermentation of lignocellulosic biomass. The chain structure forms a stiff extended conformation that has extensive hydrogen bonding with adjacent chains. Here we describe the use of microplates to quantitate three of the enzymes that catalyze the hydrolysis of plant polysaccharides. Horseradish peroxidase then catalyzes the reaction between Amplex Red and H2O2 to form the fluorescent moiety resorufin (Figure 4). Using a glucose calibration curve, one can then determine glucose production as a function of cellulase enzyme concentration.
The direct assay has similar detection limits as the glucose determination, but does not require overnight digestion. Grain-based feed stock, rich in starch, requires amylase enzymatic activity, while plant and wood material needs cellulase and xylanase activity to break down the cellulose and xylan material contained in the plant fibers. The development and selection of genetically modified strains, where transport genes and metabolic pathways have been introduced can identify new strains that express greater amounts of enzyme activity or have superior fermentation abilities. Regardless of the source of feed stock, current production costs make them unpalatable as a fuel.
Primary and secondary walls contain cellulose, hemicellulose and pectin, albeit in different proportions.


The result is the formation of microfibrils with high tensile strength and poor water solubility.
Xylose is a five carbon monosaccharide that can form either furanose (5-member) or pyranose (6-member) ring structures. Here we describe the use of microplates to assess physical assay characteristics, such as pH, temperature and enzyme concentration, of some of the enzymes that catalyze the hydrolysis of plant polysaccharides. Correction for the influence of pH on fluorescent signal data was made by normalization each well of the initial unreacted read to a common value. Interestingly, temperatures very near ambient (25° C) had the greatest reduction in enzyme activity (Figure 4) when assessed using the fluorescent amylase substrate. The fluorescence was plotted after 30 minute incubation from assays run at different temperatures. As seen in Figure 10, Aspergillus niger cellulase exhibits significant activity in acidic pH levels. Regardless of the enzymatic requirements it is important that the conditions for maximal enzymatic activity be optimized. However, this technology is currently unfeasible from both an economic and hazardous waste standpoint. Working substrate solution was prepared from the stock solution immediately prior to use by diluting it 1:5 with 1X reaction buffer.
Because of the 1:1 stoichiometry of these reactions glucose concentration can be measured by the proportional increase in resorufin fluorescence. Because microplates provide the ability to test large numbers of samples in a single experiment they are an ideal tool to test multiple experimental conditions. Towards that end, researchers investigating digestive enzymes necessary for production of fermentable sugars used in the production of biofuels (e.g. Johnson, I (2007) An Ultrasensitive, Continous Assay for Xylanase using the Fluorogenic substrate 6,8-difluoro- 4-methylumbelliferyl beta-D-xylobioside. The proportion of SDS was significantly increased when using hydrolytic enzymes after treatment with branching enzyme but it was only for waxy maize that the proportion of ?-(1-6) bonds and the in vitro digestion profile was significantly correlated.
The secondary walls of woody tissue and grasses are composed predominantly of cellulose, lignin, and hemicellulose. In order to correct for the influence of pH on the fluorescent signal data was normalized using a fluorescence reference supplied by the assay kit at each pH. The effect of temperature was measured by running separate assays at defined temperatures using the Synergy H4 Hybrid Multi- Mode Microplate Reader to maintain temperature.
Subsequent data was normalized using the same factor and expressed as the change in signal of the initial unreacted fluorescence.
Because microplates provide the ability to test large numbers of samples they are an ideal tool to test multiple experimental conditions. Towards that end, efforts have focused on ways to reduce the amount of acid used as a treatment by using a combination of dilute acids in conjunction with enzymatic hydrolysis. Because it is currently being used in large quantities the infrastructure to deliver the fuel already exists. Depending on the feed stock source, different enzymatic means are used to extract the fermentable saccharides from the polysaccharide polymer. Fluorescence for all experiments was measured kinetically for 30 minutes with a Synergy™ H4 Multi-Mode Microplate Reader. The effect of temperature was measured by running separate assays at defined temperature settings using the Synergy™ H4 Hybrid Multi-Mode Microplate Reader to control temperature. Fluorescence for all experiments was measured kinetically with a Synergy H4 Hybrid Multi-Mode Microplate Reader. Enzymes that can be shown to work well in high temperatures in an acidic environment would provide a distinct advantage over those that are labile under the same conditions.
Fluorescence for all experiments was measured kinetically for 30 minutes with a Synergy H4 Multi-Mode Microplate Reader. The fluorescent assays described provide the ability to assess lysates from microorganisms for enzyme activity. Cross-linking of this network is believed to result in the elimination of water from the wall and the formation of a hydrophobic composite that limits accessibility of hydrolytic enzymes and is a major contributor to the structural characteristics of secondary walls. As demonstrated in Figure 6, enzymes from these different sources have markedly different responses to differing pH levels.
Likewise the xylanase enzymes that work effectively under similar conditions are desirable as xylan is a significant component of most cellulosic feed stocks. Fluorescence for all experiments was measured kinetically with a Synergy H4 Multi-Mode Microplate Reader. Xylan, which accounts for up to 30% of the mass of the secondary walls in wood and grasses, contributes to the recalcitrance of these walls to enzymatic degradation.
The xylanase assay kit uses the substrate 6,8-difluoro- 4-methylumbelliferyl β-D-xylobioside. Reaction fluorescence was measured after a 10 minute incubation with a Synergy™ H4 Multi-Mode Microplate Reader using an excitation of 528 nm and an emission of 590 nm. The background fluorescence from an unreacted sample (no enzyme) at that pH raw was first subtracted and then the blanked data normalized by a known fluorescence reference at the same pH. This substrate has an advantage of having a greater pH range than the chromogenic substrate o-nitrophenyl-β-Dxylobioside, which is particularly important with acidic xylanase enzymes [1].
This non-fluorescent molecule mimics xylan and is hydrolyzed by xylanase enzyme, liberating the fluorescent coumarin moiety (Figure 3).



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