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Check out our new site currently under development, combining the Biotechnology and Science Learning Hubs with a new look and new functionality. The suffix ‘-ase’ is used with the root name of the substance being acted upon, for example,  when sucrose (sugar) is digested, it is acted upon by an enzyme called sucrase. The type of chemical reaction involved as the enzyme functions, for example, when sucrase acts on sucrose, it breaks it into a molecule of glucose and a molecule of fructose.
The following pathway summarises how starch present in a food like bread is broken down chemically into glucose, which can then be absorbed through the intestinal wall and into the bloodstream for transport to the liver and from there to other parts of the body.Mouth and duodenumStarch hydrolysed into maltose through the action of the enzyme amylase. 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.
Like all catalysts, enzymes work by lowering the activation energy for a reaction, thus dramatically increasing the rate of the reaction. Optical spectroscopy: Optical spectroscopy is a means of studying the properties of physical objects based on measuring how an object emits and interacts with light. Briefly mix this solution with a plastic micro spatula, close the lid to the spectrophotometer and begin the experimental run.
Record the time that the reaction began, observe the color and measure the absorbance after 3, 30, 60 and 90 minutes. Enzymes and, in particular, restriction enzymes have been invaluable assistants in the laboratory for decades, so it is not surprising that they have been instrumental in driving discoveries in epigenetics-related research as well. Enzymatic manipulation of methylated DNA has seen a tremendous amount of innovation over the last 30 years, making these DNA modifying enzymes great tools to have in your freezer for both today’s experiments and tomorrow’s innovation.
Long before bisulfite conversion, and pre-dating the advent of PCR, restriction enzymes were used to differentiate between DNA samples for their DNA methylation status. This pair of restriction enzymes has been particularly powerful as they recognize the same DNA sequence, but exhibit different sensitivities to DNA methylation modifications.
Today, MspI and HpaII are still going strong in protocols like the HELP assay (3), which made its debut in 2006 and, more recently, Methyl-Seq (4), which uses the restriction enzyme duo to pre-treat DNA samples before high-throughput sequencing.
Another simplified protocol that has been successful using smaller amounts of input genomic DNA samples, from both FFPE and other clinical tissue sources, is Reduced Representation Bisulfite Sequencing, or RRBS (5). This protocol uses MspI in an upstream digestion to decipher genomewide DNA methylation studies.
McrBC digestion was combined with microarray analysis back in 2005, by Martienssen and colleagues (7). In 2009, Irizarry and colleagues (8) incorporated McrBC into a new approach called Comprehensive High-throughput Arrays for Relative Methylation (CHARM). Scientists at New England Biolabs recently identified MspJI, one of the newest DNA methylation dependent restriction enzymes for DNA methylation research studies. In this guide, we highlight just a few of the key enzymes that have been instrumental in driving discoveries in DNA methylation studies. REBASE: a collection of information about restriction enzymes, methylases, the microorganisms from which they have been isolated, recognition sequences, cleavage sites, and methylation specificity. NEB Online Resources: a collection of online databases, interactive web tools, and extensive information about restriction enzymes, including their application in epigenetics research, can be found on our website. These enzymes have been used to drive innovation in protocols over the years, but they are great tools to consider for further developing protocols that address the future experimental challenges in DNA methylation analysis. Related PostsEnzymatic DNA Methylation Analysis Enzymatic analysis are tried and true method for cost effective, reliable characterization of DNA methylation. Digestive enzymes speed up the breakdown (hydrolysis) of food molecules into their ‘building block’ components. This reaction involves adding a water molecule to break a chemical bond and so the enzyme is a hydrolase. All digestive enzymes are hydrolases, whereas most of the enzymes involved in energy release for muscular contraction are oxidation-reduction enzymes such as oxidases, hydrogenases and dehydrogenases.Chemical structure of enzymesEnzymes are large protein molecules, all of which have their own specific 3D shape. 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.
As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity.
It can be used to measure attributes such as an object’s chemical composition, temperature, and velocity.
MspI and HpaII were an early duo that was used in some of the first DNA methylation analysis methods, which relied upon radioactive labeling for detection (1) and restriction landmark genome scanning, or RLGS. This enabled locus-specific discrimination between methylated and unmethylated DNA sequence, ushering in a new era of epigenetics. Both of these approaches provide broad coverage of DNA methylation marks with excellent levels of sensitivity. Using McrBC, this team removed the methylated fraction of the genome, and then co-hybridized it with an undigested sample on high-density microarrays, enabling them to identify regions of differential DNA methylation status.
This approach uses high-density arrays as well, but also uses a novel “smoothing algorithm” to more accurately measure methylation from raw microarray data. MspJI, covers a family of enzymes that are related to the known Mrr restriction enzymes in certain bacteria. They were discovered in Diplococcus pneumoniae in 1975, when they were considered unique because they cleaved DNA only at a methylated DNA sequence (11).
To learn more about these enzymes, or to discuss any technical specifications, NEB’s technical services group is always available for support. Zheng Y., et al (2010) A unique family of Mrr-like modification-dependent restriction endonucleases. Embedded within the shape is a region known as the ‘active site’, which can attract other suitably shaped molecules to bind to the site. 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. The use of microplates is one way that large numbers of samples can be handled with minimal amounts of reagents and time. 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. First, enzymes show high specificities toward their physiological substrates; compounds that differ only slightly from the natural substrate are often not acted upon. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. It involves visible, ultraviolet, or infrared light, alone or in combination, and is part of a larger group of spectroscopic techniques called electromagnetic spectroscopy. The MspJI family, first described in 2011, cleaves methylated cytosine when it is two nucleotides away from adenine or guanine, and leaves a four-base overhang on the 5’ side (9).
DpnI and DpnII recognize the same sequence, but different methylation patterns and can be useful in instances where researchers would like to discern two sample populations based on their DNA methylation status. Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. Maybe you haven’t kept up…Genome-wide and Gene-specific Analysis of DNA Methylation EpiGenie recently reviewed the text Epigenetics by Jorg Tost. The analogy that is often used to describe this mechanism is that of a key fitting into a lock. 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.
The direct oxidation of primary alcohols to carboxylic acids normally proceeds via the corresponding aldehyde, which is transformed via an aldehyde hydrate (R-CH(OH)2) by reaction with water before it can be further oxidized to the carboxylic acid. Second, activity of many enzymes can be finely tuned, allowing the metabolism proceed at a rate optimal for the well-being of the organism.
As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. This enzyme was recently used to sequence and map DNA methylation patterns in lung fibroblast genomic DNA, highlighting its potential in genome-wide applications (10).
DpnI cleaves methylated adenine to thymine in the 5’ to 3’ direction, while DpnII cleaves the same location, but only when adenine is unmethylated. The MspJI family of modification-dependent restriction endonucleases for epigenetic studies.
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.
Often it is possible to interrupt the oxidation of a primary alcohol at the aldehyde level by performing the reaction in absence of water, so that no aldehyde hydrate can be formed.
Third, and maybe the most facinating aspect of enzymes action is the fact that many enzymes bring about enormous rate accelerations. However, enzymes do differ from most other catalysts in that they are highly specific for their substrates.
However, in today’s modern world, there’s more than one way to…RRBS (Reduced representation bisulfite sequencing) Want the sensitivity of WGSBS without the cost?
If a solution of sugar is left in a sealed container, it breaks down into glucose and fructose extremely slowly.
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. For example, the enzyme rate constant kcat for decarboxylation of the amino acid arginine is 7*1019 times larger than the rate constant for the spontaneous decarboxylation of amino acids. RRBS brings down the scale and cost of WGSBS by only sequencing a reduced, representative sample of the whole genome. In the presence of a small amount of the enzyme sucrase, the rate of breakdown is millions of times faster.Sometimes, chemical substances other than substrates can bind with the active sites of enzymes, blocking their normal function. 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. We can use computational tools for gaining better understanding on how enzymes bring about such enormous rate accelerations. A few RNA molecules called ribozymes also catalyze reactions, with an important example being some parts of the ribosome. First the…Stay in the know with our twice monthly news update delivered to your inbox. For example, water-soluble compounds of arsenic and mercury are extremely poisonous because they can permanently bind to some enzyme systems, markedly reducing their efficiency. 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. Depending on the dose, the end result could be death.Digestive enzymesDigestive enzymes all belong to the hydrolase class, and their action is one of splitting up large food molecules into their ‘building block’ components.
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.
Another unique property is that they are extracellular enzymes that mix with food as it passes through the gut.
The majority of other enzymes function within the cytoplasm of the cell.The chemical digestion of food is dependent on a whole range of hydrolase enzymes produced by the cells lining the gut as well as associated organs such as the pancreas. The end goal is to break large food molecules into very much smaller ‘building block’ units.



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