Enzymatic Hydrolysis

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Biomass
Recalcitrance

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 Microorganisms:
LCB deconstruction
Biomass completely recycled in nature
Microorganisms possess enzymes
degrading complex LC network
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Trichoderma

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4
 Cellulases

I
II

Hydrolases, Glycoside hydrolases, O-Glycosides, Cellulose

Endo-b-1,4-glucanases (EC 3.2.1.4) (EG)
Randomly hydrolyze b-1,4-glucosidic
linkages
Amorphous regions
Cellobiohydrolase (CBH) (E.C. 3.2.1.91) b-Glucosidases
(EC 3.2.1.21) (bG)
Processively release G2 from ends
Cellobiose to D-glucose
Make up >70% of fungal cellulolytic secretome
Rate of CBH1 limits LCB hydrolysis
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 Cellulases Synergy

TrCel7A : CBH
TrCel5A : EG

Degree of Synergistic Effect:

Activity of synergistic mixture

=
sum of the activities of individual components

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Cellulases
Synergy

Cel7A : CBH
Cel7B : EG

SA: Specific Activity

DS: Degree of synergy
C7B : Mole fraction of EG

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 formation of productive ES
complex
CBH

CBM- recognition of cellulose

mediated chain end
binding to
cellulose
expulsion of
product hydrolysis of
next ES complex glycosidic bond

hydrolysis of CBH CBH EG

Cellulases
Synergy
cellulose synergy
CBH

EG accelerates
CBH action by degrading
amorphous regions.

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 CBH1: Processive Cycle

T. reesei Cel7A E binds with substrate

O-glycosylation Repeatedly cleaves glycosidic linkage

N-glycosylation
Release cellobiose
Eventually ES dissociates

polysaccharide
Rate not mechanically limited
Catalytic domain sufficient for
processive motion

High moving velocity on cellulose

weak interaction
Slow movement - high processivity
stronger interaction 9
 Hydrolysis yield
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑓𝑟𝑜𝑚 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑔 𝑥 0.9 𝑥 100
𝐺𝑙𝑢𝑐𝑎𝑛 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 % =
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑖𝑛 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 (𝑔)

𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑥𝑦𝑙𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑓𝑟𝑜𝑚 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑔 𝑥 0.88 𝑥 100

𝑋𝑦𝑙𝑎𝑛 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 % =
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑥𝑦𝑙𝑎𝑛 𝑖𝑛 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 (𝑔)

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 Hydrolysis yield
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑓𝑟𝑜𝑚 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑔 𝑥 0.9 𝑥 100
𝐺𝑙𝑢𝑐𝑎𝑛 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 % =
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑖𝑛 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 (𝑔)

𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑥𝑦𝑙𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑓𝑟𝑜𝑚 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑔 𝑥 0.88 𝑥 100

𝑋𝑦𝑙𝑎𝑛 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 % =
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑥𝑦𝑙𝑎𝑛 𝑖𝑛 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 (𝑔)

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CBH1 Mechanism 2

Retaining
Two carboxyl side chains
-COO- attacks C1 glycosidic bond and forms covalent 3
intermediate with inverted glycosidic bond
-COOH donates proton to glycosidic O
H2O attacks C1 carbon, inverting link
Retention of configuration

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 CBH2 Mechanism

Inversion
Two carboxyl groups in catalysis
COOH donates proton to glycosidic O
COO- removes a proton from H2O, attacks C1
Inverts linkage from b to a
Less processive

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Non-hydrolytic accessory proteins

GH45 domain
Expansins and expansin-like proteins
Loosen cell walls by disrupting H-bonds
Reduce cellulose crystallinity
Linker

Fungi: Swollenin
Expansin-like CBM
Displays endoglucanase activity
Major product - cellobiose

CBM

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Non-hydrolytic accessory proteins

Lytic polysaccharide monooxygenases (LPMOs)

AA (Auxiliary Activities)
Degrade surface cellulose nano-fibrils Enables
cellulases action on crystalline
Promotes faster degradation
Oxidation of C1 or C4 of b-1,4-glycosidic bonds
Produce oxidized chain-ends
Aldonolactone
4-Ketoaldose

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Cellulose
degradation

• SWO1: Swollenin
• AA9: lytic polysaccharide
monooxygenase (Auxiliary activity 9)
• CBD: cellobiose dehydrogenase heme
• CBH1 (reducing) and CBH2 (non-
reducing)

• SWO1 and AA9 : crystalline and less-

crystalline

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 Non-Hydrolytic Oxidative Cleavage

Cu, O2, e- donor

Cu (I) and Cu (II) cycling activate O2

Hydroxylation of C1 or C4

Lignin as e- donor

Balance between positive and

negative effect of lignin

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 LPMO Cu(II)-Superoxo Mechanism
Cycling of Cu between Cu(I) and Cu(II) Activates O2

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LPMO Cu(II)-oxyl Mechanism

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Carbohydrate-
Binding Modules
(CBM)
• Cellulases and hemicellulases
• Non-catalytic CBM
• Anchor catalytic site to
substrate
• Increases effective enzyme
concentration
• Enhances synergy

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