Microbial Degradation of Plant Biomass and Cellulases

Flashcards covering the microbial degradation of plant biomass, including cellulose structure, enzyme mechanisms (free cellulases vs. cellulosomes), and biotechnological applications.

What is the primary structural component of plant cell walls, accounting for approximately $50\%$ of global biomass?

Cellulose

What specific chemical bonds link glucose molecules together to form the polymer cellulose?

$\beta-1,4$-glycosidic bonds

Which region of a cellulose microfibril is characterized by being highly ordered?

The crystalline central core

What creates the extensive network that makes cellulose chains tightly bound and strong?

Hydrogen-bonding

What are the two types of termini found on each cellulose chain?

One reducing end and one non-reducing end

How does the hydrophobicity of the environment between glucose planes affect cellulose degradation?

It makes it very difficult to solubilise or break down with acid

What is the primary line of defense for a plant against pathogens and harmful substances?

The cellulose-rich cell wall

Why must the complex plant cell wall remain porous?

To enable vital communication and transport between adjoining plant cells

Name the three main components that create the intricate, tough network of the plant cell wall.

Cellulose microfibrils, hemicellulose, and pectin

Which biological entities rely on tens of thousands of microbial cellulases in their gut for nutrition?

Herbivores, such as cows

What are three biological roles of microorganisms secreting cellulases outside of nutrition?

Remodelling biofilms, invading plant cells, and invading amoeba cells (pathogenicity)

How is bioethanol produced from agricultural waste like stalks and sawdust?

Cellulases break down cellulose into fermentable sugars, which are then fermented into alcohol

What is the primary benefit of using cellulose for bioethanol compared to corn-based ethanol?

It solves the Food vs. Fuel conflict by utilizing waste products rather than food crops

How does the abundance of cellulosic waste compare to known oil reserves?

There is a significantly larger quantity of cellulosic waste than reserves of oil

Why does fuel produced from cellulose have a low net greenhouse effect?

The carbon dioxide released during burning is offset by the carbon dioxide the plants absorbed while growing

What biotechnology application uses cellulases for stone-washing denim?

Textiles and laundry

In the food and drink industry, what is one function of cellulases regarding fruit juices?

Clarifying fruit juices

How does cellulose in the diet affect gut flora?

It acts as dietary fiber, and gut flora composition adjusts based on the amount of fiber ingested

What beneficial byproduct is produced by gut microorganisms when they degrade high-fiber plant material?

Short-chain fatty acids

What is the health benefit of short-chain fatty acids produced by gut flora?

They help reduce the incidence of bowel and inflammatory diseases

In enzyme synergy, what is meant by the final activity level?

The combined activity is greater than the sum of their individual activities

What is the primary function of endoglucanases in cellulose degradation?

They attach to the middle of the chain (often in amorphous regions) and cut it to create new ends

What is the physical shape of an endoglucanase active site?

Cleft-like

Which class of cellulases is characterized by a tunnel-like structure?

Cellobiohydrolases (CBHs)

From which specific end does Cellobiohydrolase I ($CBH1$) attack the cellulose chain?

The reducing end

From which specific end does Cellobiohydrolase II ($CBH2$) attack the cellulose chain?

The non-reducing end

What is the primary product released by cellobiohydrolases?

Cellobiose (two linked glucose molecules)

What is the role of $\beta$-glucosidases in the enzyme cocktail?

They cleave cellobiose into individual glucose molecules

Why are $\beta$-glucosidases typically kept inside the microorganism?

To prevent free glucose from being stolen by competitors in the surrounding environment

Which auxiliary enzymes use oxidation to help create more chain ends?

Lytic Polysaccharide Monooxygenases ($LPMOs$)

What is the "obligatory" unit of a cellulase that performs chemical cleavage?

Catalytic Module ($CM$)

What is the simplest modular form of a cellulase?

An isolated Catalytic Module ($CM$)

Why might an enzyme feature duplicated Catalytic Modules?

To provide slightly different cleavage specificities, allowing the protein to attack in multiple ways

What is the function of the Carbohydrate Binding Module ($CBM$)?

It anchors the enzyme to the insoluble cellulose substrate, increasing turnover efficiency

What component connects the Catalytic Module ($CM$) and the Carbohydrate Binding Module ($CBM$)?

Linkers

What are three characteristics of the linkers in cellulases?

Extended, flexible, and heavily glycosylated

What is the purpose of glycosylation on cellulase linkers?

It keeps the linker extended, prevents sticking to the binding module, and protects it from proteases

How does the presence of a $CBM$ affect the local concentration of the catalytic module?

It drastically increases the local concentration near the cellulose surface

What classification is given to Catalytic Modules that use water to break glycosidic bonds?

Glycoside Hydrolases ($GH$)

How many acidic amino acids are typically used in the active site of a $GH$ enzyme for catalysis?

$$2$$

What is the transition state in the $GH$ catalytic mechanism?

Oxocarbenium ion-like transition state

At what distance between acidic residues does the enzyme typically use a retaining mechanism?

$$\sim 5.5\,\text{\AA}$$

At what distance between acidic residues does the enzyme typically use an inverting mechanism?

$$\sim 10\,\text{\AA}$$

How many different $GH$ families contain $\beta-1,4$ cleaving modules?

$$20$$

What does the "$3$" represent in the enzyme category $EC\,3.2.1.4$?

It is a hydrolase

What does the "$2$" represent in the enzyme category $EC\,3.2.1.4$?

It acts on glycosyl bonds

Which database groups enzymes into families based on amino acid sequences?

CAZy (Carbohydrate-Active enZYmes)

What is a "GH Clan"?

A group of GH families that share a common $3D$ structural fold

What is the approximate half-life of $\beta-1,4$-glycosidic bonds in plain water?

$5 \times 10^6$ years

At what rate can $GH$ catalytic modules cleave glycosidic bonds?

Roughly $1,000$ times per second

What is the most important preliminary step in the $GH$ catalytic mechanism?

Physically distorting the sugar substrate from its classic conformation

What is the classic, stable conformation of a glucose molecule?

$4C1$ conformation

In a retaining mechanism, what intermediate is formed between the enzyme and the sugar?

A covalent glycosyl-enzyme intermediate

Why does a $10\,\text{\AA}$ distance between residues allow for an inverting mechanism?

It allows room for a water molecule and sugar to fit into the active site simultaneously

What common structural fold do many $CBMs$ form?

A $\beta$-sandwich structure

Which aromatic amino acids are frequently exposed on the surface of $CBMs$?

Tryptophan or tyrosine

What is the spacing of aromatic amino acids in a $CBM$ to match cellulose?

Exactly $\sim 10.3\,\text{\AA}$

What is the typical binding affinity ($K_d$) range for $CBM$ binding to cellulose?

The micromolar range

What is the purpose of weak and reversible $CBM$ binding?

To allow the enzyme to detach and slide down the cellulose microfibril

What do Type A $CBMs$ specifically bind to?

Flat, crystalline cellulose

What is the binding target of Type C $CBMs$?

The termini (ends) of the cellulose chains

Which fungus is known for being a hyper-producer that secretes "free" cellulase enzymes?

Trichoderma reesei

Approximately what percentage of the cellulases produced by T. reesei is composed of $CBH1$?

$$50\%$$

What is the characteristic "slide and peel" mechanism of T. reesei enzymes?

A processive movement across the cellulose surface

How many exoglucanases and endoglucanases are typically found in the T. reesei enzyme cocktail?

$2$ exoglucanases ($CBH1$ and $CBH2$) and $5$ endoglucanases

Why does T. reesei need five different endoglucanases?

To recognize and cleave different structural variations within irregular amorphous cellulose regions

In T. reesei $CBH1$, what is the relationship between the $CBM$ exchange rate and the $CM$ hydrolysis rate?

They are perfectly matched/synchronized, allowing dynamic attachment

What structural fold does the $CM$ of $CBH1$ form?

A $\beta$-sandwich (GH7 family)

How long is the enclosed tunnel in the $CBH1$ Catalytic Module?

$$50\,\text{\AA}$$

How many glucose molecules can bind simultaneously in the $CBH1$ tunnel?

$$10$$

In $CBH1$, at what positions (-7 through +1) does the sugar remain undistorted?

Positions $-7$ to $-4$

Into what high-energy conformation is the $-1$ sugar distorted in the $CBH1$ active site?

$1,4$ boat conformation

How many specific hydrogen bonds lock the $-1$ sugar into its distorted state in $CBH1$?

$$6$$

What experimental technique was used to visualize $CBH1$ sliding along cellulose?

Atomic Force Microscopy ($AFM$) with gold particles

What speed was observed for $CBH1$ sliding along a cellulose fibril?

$50\,\text{nm}$ in $3\,\text{s}$

Where is the $CBM$ located in $CBH2$ compared to $CBH1$?

At the N-terminus (whereas in $CBH1$ it is at the C-terminus)

What is the structural fold of the $CBH2$ Catalytic Module?

A TIM barrel fold ($7$-stranded $\beta/\alpha$)

Where is the active site located in the TIM barrel of $CBH2$?

At the C-terminus of the barrel

How long is the tunnel formed by flexible loops in $CBH2$?

$$20\,\text{\AA}$$

What is the function of the tunnel structure in $CBH1$ and $CBH2$?

It allows the enzymes to act processively by threading the chain and sliding without detaching

What is the distance between the two catalytic acidic residues in $CBH2$?

$$10\,\text{\AA}$$

Does $CBH2$ use a retaining or inverting mechanism?

Inverting mechanism

In $CBH2$, in what plane do the three glucoses at positions $-2$, $+1$, and $+2$ bind?

The horizontal plane

Into what conformation is the $-1$ sugar distorted in $CBH2$?

Skew boat conformation

What often acts as the rate-limiting step in cellulase catalysis?

Forcing the stable sugar substrate into a distorted, high-energy conformation

Which endoglucanase makes up $15\%$ of T. reesei's total cellulases?

Endoglucanase I

How does the active site of Endoglucanase I differ from $CBH1$?

It has much shorter loops, resulting in a much more open active site cleft

What is the evolutionary theory regarding the origin of endoglucanases from CBHs?

An ancestral cellobiohydrolase lost its loops to gain the function of cutting in the middle of chains

Which T. reesei endoglucanase lacks a $CBM$ and makes up only $1\%$ of the cocktail?

Endoglucanase III

Which microbes produce the cellulosome, a massive extracellular multi-enzyme complex?

Anaerobic microbes (e.g., Clostridia)

What is the protein backbone of the cellulosome called?

Scaffoldin

What are the 'sockets' on the scaffoldin that bind catalytic modules?

Cohesins ($Coh$)

What modules on the catalytic enzymes act as 'plugs' for the cohesins?

Dockerins ($Doc$)

How many cohesin units are typically found in a single scaffoldin?

At least $4$, sometimes up to $9$

How do cohesin-dockerin units bind to the scaffoldin?

Cooperatively but randomly

What is the role of the single $CBM$ found in the primary scaffoldin?

It targets the entire massive complex to the cellulose substrate

What module is linked to the Type II dockerin at the end of the scaffoldin to provide stability?

X-module

What do Type II dockerins plug into on the bacterial cell surface?

An anchoring subunit containing a Type II cohesin

What is the maximum molecular weight a cellulosome complex can reach?

$$6\,MDa$$

How is a cellulosome substrate-specific, or 'LEGO-like'?

The bacteria produces different CMs (like xylanases or cellulases) based on the substrate, which plug into standard connectors