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.