Cellulases

Why do microorganisms degrade cellulose

nutrition, biofilm remodelling and pathogenecity

What bonds make up cellulose

glucose beta 1,4-glycosidic bonds

What are the uses of gut flora degrading cellulose

adjusting to fibre content and bowel and inflammatory disease

Why is degradation of cellulose important in biotech?

for use in textiles, paper, detergents, feed, soil fertility, food industries and bioethanol

Why is conflict over cellulose minimal?

as it is a waste product in agriculture, forestry, municipal (household), and food processing

Benefits of cellulose bioethanol?

less conflict, waste product, larger quantity, lower net greenhouse effect, and positive net energy balance

what are the names of the enzymes used in microbial cellulose degradation

Cellobiohydrolase, endoglycanase, and beta-1,4-glucosidases

What is the role of cellobiohydrolase

Work progressively from ends of cellulose chians, typically releasing cellobiose (disaccharide of glucose) as main produc

What end is CBH I on?

non-reducing end

what is function of endo-beta-1,4-glucanase

Enzymes initiate attack by randomly cleaving internal beta-1,4-glycosidic bonds wihtin amorphous (less ordered) regions of cellulose polymer

what is function of beta-1,4-glucosidases

Enzymes hydrolyze the cellobiose and other short soluble cellooligosaccharides produced by Egs and CBHs into individual glucose monomers

what does LPMO do?

Introduce oxidative breaks into highly crystalline regions of cellulose fibres, making recalcitrant structures more amenable to attack by hydrolytic enzymes such as endoglucanases

what does cellobiose dehydrogenase do?

Also contributes to oxidative cellulose cleavage enhancing cellulase efficiency by potentially providing electrons for LPMO activity

How many glyocsidic bonds do cellulases cleave per second

1000

what are the two GH enzyme mechanisms

retention of configuration and inversion of configuration

What does CM stand for

catalytic module aka glycoside hydrolase

what happens in retention of configuration mechanism of CM

nucleophilic attack on C1, water attacks glycosyl-enzyme, oxocarbenium ion-like TS.

what happens in inversion of configuration mechanism of CM

Oxocarbenium ion TS, acidic groups are further apart, and no glycosyl-enzyme intermediate

what is role of CBM

target and disrupt glycosidic bonds

where are CBMs distributed

N or C terminus, between CM, or flanking

why do CBMs weakly interact

to allow detachment

How can you enhance CBM binding?

glycosylation, multiple copies of multiple binding sites

What are CBMs utilised for

affinity purification/immobilisation, targeting CM, fine-tune CM

Where in CBH I does hydrolysis occur

between -1 and +1

What is special about -7 to -4 in CBHI cellulose binding domain

it is undistorted. Water mediated hydrogen bonds and aromatic stacking. All non-specific interactions so it slides

What is special about -3 to -2 in CBHI cellulose binding domain

They twist with strong protein hydrogen bonds

What is special about -1 in CBHI cellulose binding domain

it distorts to 1,4B six protein hydrogen bonds

What is special about +1 in CBHI cellulose binding domain

interacts with Tryptophan and +2 specific hydrogen bonds

What are the steps of CBH1 catalytic cycle?

adsorption, decrystallisation and threading, catalysis, and desorption post catalysis

What happens in catalysis step?

processive motion in slide mode, then catalytic activation to michaelis complex, then glycosylation to unprimed GEI, then product movement to primed GEI, then deglycosylation via hydrolysis then product is expelled and back to pre-slide mode

What mechanisem does CBHII hydrolysis use?

inverting mechanism

where do three glucose bind favourably in CBHII

-2, +1, +2

What happens at -2/-1 in CBHII hydrolysis

rotations. -1 is perpendicular and twisted

What is CBHII hydrloysis catalytic cycle

pre-slide mode is processed into slide mode, loop closure/activation to michaelis complex, then hydrolysis to substrate-product complex then product expulsion to pre-slide mode

What are cellulosomes

Anaerobic microbes that produce enzymes for degradation of plant cell wall.

What are the sizes of cellulosomes after scaffoldin help?

1.5 to 6MDa

what is the dissociation constant for cohesin-dockerin?

10^-10M^-1

What is the function of X-modules

provide stability, extend from surface, linkers (flexible, pleated in complex)

Why are cellulosomes used?

tethering enzymes to substrate, proximity, spectrum of activities