We briefly addressed what starch is in Lesson 5. Now, we’ll go into a little more depth. In plants, starch has two components: amylose and amylopectin. Amylose is a straight-chain sugar polymer. Normal corn has 25% amylose, high amylose corn has 50-70% amylose and waxy corn (maize) have less than 2%. The rest of the starch is composed of amylopectin. Its structure is branched and is most commonly the major part of starch. Animals contain something similar to amylopectin, called glycogen. The glycogen resides in the liver and muscles as granules.

You can visit howstuffworks.com to see a schematic of what amylopectin looks like in a granule (see 'How Play-Doh Works') and then strands of the compound. The figure below shows some micrographs of starch as it begins to interact with water. When cooking with starch, you can make a gel from the polysaccharide. (A) This part of the figure shows polysaccharides (lines) packed into larger structures called starch granules; upon adding water, the starch granules swell and polysaccharides begin to diffuse out of the granules. Heating these hydrated starch granules helps polysaccharide molecules diffuse out of the granules and form a tangled network. (B) This is an electron micrograph of intact potato starch granules. (C) This is an electron micrograph of a cooked flaxseed gum network.

Formation of polysaccharide gels. (A) Polysaccharides (lines) are packed into larger structures called starch granules; upon adding water, the starch granules swell and polysaccharides begin to diffuse out of the granules. Heating these hydrated starch granules helps polysaccharide molecules diffuse out of the granules and form a tangled network. (B) Electron micrograph of intact potato starch granules. (C) Electron micrograph of a cooked flaxseed gum network.

Credit: Science and Food Blog

Now, let’s look at the starch components on a chemical structure basis. Amylose is a linear molecule with the α-1,4-glucosidic bond linkage. Upon viewing the molecule on a little larger scale, one can see it is helical. It becomes a colloidal dispersion in hot water. The average molecular weight of the molecule is 10,000-50,000 amu, and it averages 60-300 glucose units per molecule. Figure 6.5 depicts the chemical structure of amylose.

Amylopectin is branched, not linear, and is shown in the figure below. It has α-1,4-glycosidic bonds and α-1,6-glycosidic bonds. The α-1,6-glycosidic branches occur for about 24-30 glucose units. It is insoluble compared to amylose. The average molecular weight is 300,000 amu, and it averages 1800 glucose units per molecule. Amylopectin is about 10 times the size of amylose.

Chemical structure of amylose. Each monomer is connected by the α-1,4-glycosidic bond. When looking at a larger scale, the polymer is helical.

Credit: Daily Kos and Amylose: from Wikipedia.org

Chemical structure of amylopectin. Each straight-chain monomer is connected by the α-1,4-glycosidic bond, while the branches are connected by an α-1,6-glycosidic bond.

Credit: The Science of Nutrition Blog and Amylose: from Wikipedia.org

Cellulose is the most abundant polysaccharide, and it is also the most abundant biomass on earth. The linkages are slightly different from starch, called β-1,4-glycosidic linkages, as the bond is in a slightly different configuration or shape. This bond causes the strands of cellulose to be straighter (not helical). The hydrogen on one polymer strand can interact with the OH on another strand; this interaction is known as a hydrogen bond (H-bond), although it isn't an actual bond, just a strong interaction. This is what contributes to the crystallinity of the molecule. [Definition: the H-bond is not a bond like the C-H or C-O bonds are, i.e., they are not covalent bonds. However, there can be a strong interaction between hydrogen and oxygen, nitrogen, or other electronegative atoms. It is one of the reasons that water has a higher boiling point than expected.] The strands of cellulose form long fibers that are part of the plant structure. The average molecular weight is between 50,000 and 500,000, and the average number of glucose units is 300-2500.

As seen in previous lessons, lignocellulosic biomass contains another component, hemicellulose. Rather than being a typical polymer where units repeat over and over again, hemicellulose is a heteropolymer. It has a random, amorphous structure with little strength. It has multiple sugar units rather than the one glucose unit we’ve seen for starch and cellulose, and the average number of sugar units is 500-3000 (glucose units with the starch and cellulose). The monomer units include xylose, mannose, galactose, rhamnose, and arabinose units. The various polymers of hemicellulose include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan.

Monomer sugars found in hemicellulose.

Credit: Wikipedia page for each chemical, a) xylose wiki, b) mannose wiki, c) galactose wiki, d) rhamnose wiki, e) arabinose wiki

Example of polymers in hemicellulose (xylan).

Credit: Xylan: from Wikimedia Commons

Example of polymers in hemicellulose (arabinoxylan).

Credit: Otieno et al. (7)

So, we’ve identified the chemical structures of starch, cellulose, and hemicellulose. Now we’re going to look at what lignin is, chemically.

Vascular land plants make lignin to solve problems due to terrestrial lifestyles. Lignin helps to keep water from permeating the cell wall, which helps water conduction in the plant. Lignin adds support – it may help to “weld” cells together and provides stiffness for resistance against forces that cause bending, such as wind. Lignin also acts to prevent pathogens and is recalcitrant to degradation; it protects against fungal and bacterial pathogens (there is a discussion in Lesson 5 about recalcitrance). Lignin is comprised of crosslinked, branched aromatic monomers: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol; their structures are shown in the figures below and show how these building blocks fit into the lignin structure. p-Coumaryl alcohol is a minor component of grass and forage-type lignins. Coniferyl alcohol is the predominant lignin monomer found in softwoods (hence the name). Both coniferyl and sinapyl alcohols are the building blocks of hardwood lignin. The table below shows the differing amounts of lignin building blocks in the three types of lignocellulosic biomass sources.

Several different materials can be made from lignin, but most are not on a commercial scale. The table below shows the class of compounds that can be made from lignin and the types of products that come from that class of compounds. If an economic method can be developed for lignin depolymerization and chemical production, it would benefit the biorefining of lignocellulosic biomass.

There are also high molecular weight compounds. These include carbon fibers, thermoplastic polymers, fillers for polymers, polyelectrolytes, and resins, which can be made into wood adhesives and wood preservatives.

Cellulolysis is essentially the hydrolysis of cellulose. In low and high pH conditions, hydrolysis is a reaction that takes place with water, with the acid or base providing H+ or OH- to precipitate the reaction. Hydrolysis will break the β-1,4-glucosidic bonds, with water and enzymes to catalyze the reaction. Before discussing the reaction in more detail, let’s look at the types of intermediate units that are made from cellulose. The main monomer that composes cellulose is glucose. When two glucose molecules are connected, it is known as cellobiose – one example of a cellobiose is maltose. When three glucose units are connected, it is called cellotriose – one example is β -D pyranose form. And four glucose units connected together are called cellotetraose. Each of these is shown below.

Glucose structure with carbons numbered.

Credit: Palaeos.com

Cellobiose, or maltose (glucose + glucose) chemical structure.

Credit: Maltose Haworth: from Wikimedia Commons

Cellotriose in the β-D-pyranose form.

Credit: Answers.com

Cellotetraose.

Credit: Green Chem., 2012,14, 1284-1288

We’ve seen the types of intermediates, so now let’s see the reaction types that are catalyzed by cellulose enzymes. The steps are shown below.

1. Breaking of the noncovalent interactions present in the structure of the cellulose, breaking down the crystallinity in the cellulose to an amorphous strand. These types of enzymes are called endocellulases.
2. The next step is hydrolysis of the chain ends to break the polymer into smaller sugars. These types of enzymes are called exocellulases, and the products are typically cellobiose and cellotetraose.
3. Finally, the disaccharides and tetrasaccharides (cellobiose and cellotetraose) are hydrolyzed to form glucose, which are known as β-glucosidases.

Reaction sequence of cellulose with 1) endocellulase, 2) exocellulase, and 3) β-glucosidase.

Credit: Cellulase: from Wikipedia.org

Okay, now we have an idea of how the reaction proceeds. However, there are two types of cellulase systems: noncomplexed and complexed. A noncomplexed cellulase system is the aerobic degradation of cellulose (in oxygen). It is a mixture of extracellular cooperative enzymes. A complexed cellulase system is an anaerobic degradation (without oxygen) using a “cellulosome.” The enzyme is a multiprotein complex anchored on the surface of the bacterium by non-catalytic proteins that serve to function like individual noncomplexed cellulases but are in one unit. The figure below shows how the two different systems act. However, before going into more detail, we are now going to discuss what the enzymes themselves are composed of. The reading by Lynd provides some explanation of how the noncomplexed versus the complexed systems work.

Schematic representation of the hydrolysis of amorphous and microcrystalline cellulose in A) noncomplexed and B) complexed cellulase systems.

Click for a text description of the schematic above.

This image is a comparative illustration of two distinct mechanisms for cellulose degradation, presented in two labeled panels: A and B. Panel A focuses on the free enzyme system, where individual enzymes act independently to break down cellulose. At the top, the structure of cellulose is shown, highlighting both crystalline and amorphous regions. Below, various enzymes—including endoglucanase, exoglucanase (CBHI and CBHII), and β-glucosidase—are illustrated interacting with the cellulose fibers. These enzymes work in concert to hydrolyze the cellulose into simpler sugars such as glucose, cellobiose, and cello-oligosaccharides.

Panel B illustrates the cellulosome-mediated degradation pathway, a more structured and synergistic approach. At the top, bacterial cell walls are shown with scaffoldin proteins anchored to them. These scaffoldins contain cohesin moieties that specifically bind to dockerin-containing enzymes like endoglucanase (CelF/CelS) and exoglucanase (CelE). The bottom part of this panel shows these enzymes forming a multi-enzyme complex, working together to efficiently degrade both crystalline and amorphous cellulose into simpler sugars.

A legend at the bottom of the image provides symbolic representations for the various components involved, including different enzymes, sugar products, and structural modules like carbohydrate-binding modules (CBMs) and phosphorylases. Overall, the image serves as a detailed visual comparison of the free enzyme system versus the cellulosome system in the biochemical breakdown of cellulose.

Credit: Lynd et al., 2002

The first place to start is to describe the structure of a cellulase using typical terms in biochemistry. A modular cellobiohydrolase (CBH) has a few aspects in common; the common features include 1) a binder region of the protein, 2) a catalytic region of the protein, and 3) a linker region that connects the binder and catalytic regions. the first figure below shows a general diagram of the common features of a cellulase. The CBH acts on the terminal end of a crystalline cellulosic substrate, where the cellulose binding domain (CBD) is embedded in the cellulose chain, and the strand of cellulose is digested by the enzyme catalyst domain to produce cellobiose. This type of enzyme is typical of exocellulases. The second figure shows a more realistic model, where the linker is attached to the surface of the cellulose.

One of the main differences between glycosyl hydrolases (a type of cellulase) and the other enzymes is how the catalytic domain functions. There are three types: 1) pocket, 2) cleft, and 3) tunnel. Pocket or crater topology is optimal for the recognition of a saccharide non-reducing extremity and is encountered in monosaccharidases. Exopolysaccharidases are adapted to substrates having a large number of available chain ends, such as starch. On the other hand, these enzymes are not very efficient for fibrous substrates such as cellulose, which has almost no free chain ends. Cleft or groove cellulase catalytic domains are “open” structures, which allow a random binding of several sugar units in polymeric substrates and are commonly found in endo-acting polysaccharidases such as endocellulases. Tunnel topology arises from the previous one when the protein evolves long loops that cover part of the cleft. Found so far only in CBH, the resulting tunnel enables a polysaccharide chain to be threaded through it. The red portions on each catalytic domain are supposed to be the carbohydrates being processed, although it is difficult to see in this picture.

The other main feature of these enzymes is the cellulose binding domain or module (CBD or CBM). Different CBDs target different sites on the surface of the cellulose; this part of the enzyme will recognize specific sites, help to bring the catalytic domain close to the cellulose and pull the strand of cellulose molecule out of the sheet so the glycosidic bond is accessible.

So now, let’s go back to noncomplexed versus complexed cellulase systems. The first figure below is another comparison of noncomplexed versus complexed cellulase systems, but this time, it focuses on the enzymes. Notice in Figure A below, the little PacMan look-alike figures for enzymes. The enzymes are separate but work in concert to break down the cellulose strands into cellobiose and glucose. Recall that this process is aerobic (in oxygen).

Now look at Figure B and the complexed system. The enzymes are attached to subunits that are attached to the bacterium cell wall. The products are the same, but recall that this system is anaerobic (without oxygen), and these enzymes all work together to produce cellobiose and glucose.

So, what are those subunits that are essentially the connectors in the enzyme? The figure below shows a schematic of the types. The cellulosome is designed for the efficient degradation of cellulose. A scaffoldin subunit has at least one cohesin module that is connected to other types of functional modules. The CBM shown is a cellulose-binding module that helps the unit anchor to the cellulose. The cohesin modules are major building blocks within the scaffoldin; cohesins are responsible for organizing the cellulolytic subunits into the multi-enzyme complex. Dockerin modules anchor catalytic enzymes to the scaffoldin. The catalytic subunits contain dockerin modules; these serve to incorporate catalytic modules into the cellulosome complex. This is the architecture of the C. thermocellum cellulosome system. (Alber et al., CAZpedia, 2010). Within each cellulosome, there can be many different types of these building blocks. The last figure shows a block diagram of two different structures of T. neapolitana LamA and Caldicellulosiruptor strain Rt8B.4 ManA in a block diagram form. Due to the level of this class, we will not be going into any greater depth about these enzymes.

Hemicellulases work on the hemicellulose polymer backbone and are similar to endoglucanases. Because of the side chain, “accessory enzymes” are included for side-chain activities. An example of hemicellulase activity on arabinoxylan and the places where bonds are broken by enzymes are shown (blue) in the first figure below. The second figure shows another example of how hemicellulose breaks down hemicellulose, a complex mixture of enzymes, to degrade hemicellulose. The example depicted is cross-linked glucurono arabinoxylan.

The complex composition and structure of hemicellulose require multiple enzymes to break down the polymer into sugar monomers—primarily xylose, but other pentose and hexose sugars also are present in hemicelluloses. A variety of debranching enzymes (red) act on diverse side chains hanging off the xylan backbone (blue). These debranching enzymes include arabinofuranosidase, feruloyl esterase, acetylxylan esterase, and alpha-glucuronidase [The table below shows enzyme families for degrading the hemicellulose]...As the side chains are released, the xylan backbone is exposed and made more accessible to cleavage by xylanase. Beta-xylosidase cleaves xylobiose into two xylose monomers; this enzyme also can release xylose from the end of the xylan backbone or a xylo-oligosaccharide. (U.S. DOE, 2006)

Example of hemicellulase activity on arabinoxylan, showing bonds that are broken. The hemicellulases are shown in blue.

Credit: U.S. DOE. 2006. Breaking the Biologic Barriers to Cellulosic Ethanol

A complex mixture of enzymes for degrading hemicelluloses. Instead of using chemical structures, this example uses abbreviations for different parts of the glucurono arabinoxylan so the connections can be observed more easily. The backbone is shown in blue, while the hemicellulases are shown in red.

Credit: Ji, Xiao-Jun & Huang, He & Nie, Zhi-Kui & Qu, Liang & Xu, Qing & Tsao, George. (2012). Fuels and Chemicals from Hemicellulose Sugars. Advances in biochemical engineering/biotechnology. 128. 199-224.

Lignin-degrading enzymes are different from hemicellulases and cellulases. They are known, as a group, as oxidoreductases. Lignin degradation is an enzyme-mediated oxidation, involving the initial transfer of single electrons to the intact lignin (this would be a type of redox reaction or reduction-oxidation reaction). Electrons are transferred to other parts of the molecule in uncontrolled chain reactions, leading to the breakdown of the polymer. It is different from carbohydrate hydrolysis because it is an oxidation reaction, and it requires oxidizing power (e.g., hydrogen peroxide, H₂O₂) to break the lignin down. In general, it is a significantly slower reaction than the hydrolysis of carbohydrates.

Examples of lignin-degrading enzymes include lignin peroxidase (aka ligninase), manganese peroxidase, and laccase, which contain metal ions involved in electron transfer. Lignin peroxidase (previously known as ligninase) is an iron-containing enzyme, that accepts two electrons from hydrogen peroxide (H₂O₂), and then passes them as single electrons to the lignin molecule. Manganese peroxidase acts similarly to lignin peroxidase but oxidizes manganese (from H₂O₂) as an intermediate in the transfer of electrons to lignin. Laccase is a phenol oxidase, which directly oxidizes the lignin molecule (contains copper). There are also several hydrogen-peroxide-generating enzymes (e.g., glucose oxidase), which generate H₂O₂ from glucose. (Microbial World, The University of Edinburgh).

Lesson 6 will discuss the process of ethanol production after the use of cellulases on cellulose.