An investigation into
the synergistic action
of exo and endo-type
cellulases on the
hydrolysis of
complex substrates
Mariska Thoresen
Energy Postgraduate Conference 2013
Introduction
DEPLETION OF FOSSIL FUELS
• Need for alternative energy sources
BIOMASS
• Large scale availability
• Low cost
Cellulose
• cd
Plant cell
Hydrogen bonding with and
between cellulose chains
Microfibrils
RECALCITRANT
Macrofibrils
Cellulose
Fiber
Enzymes
β-1,4 glycosidic bonds
Cellulases
1.
Endoglucanases: cut at random sites along the cellulose
chain.
2. Cellobiohydrolases: release cellobiose from chain.
3. β-glucosidases: conversion of cellobiose to glucose.
CBHI
CBHII
NR
R
Crystalline
β-glucosidase
Amorphous
Crystalline
What is Synergy?
• Understanding how enzymes cooperate to degrade complex
substrates
• Synergy = Enzymes working together display a higher activity
than when used individually
• Degree of synergy or synergism =
(Actual observed activity of enzymes in combination )
(Theoretical sum of activities of individual enzymes)
• Basis of synergy
– Enzymes target different parts of the substrate, removing
obstacles and creating new sites for other enzymes
Synergy Assays
Enzyme 11
Enzyme
Enzyme 2
Enzyme 3
Designer
enzyme
cocktails
Synergy <1
Competition
Synergy =1
No synergy
Synergy >1
Synergy
Synergistic relationships
Exo
Endo
Synergy
Endo
Exo
Methodology
• Enzymes Obtained from Stellenbosch University
• Assessment of protein purity (SDS-PAGE)
• Protein determination (Bradford Assay)
• Enzyme activity assays (DNS method)
• Synergy Studies (Bi-synergy)
– Enzyme ratios ranging from 0-100% were tested. Total
protein concentration in all assays were kept constant
(40 ug)
– Avicel
– Time studies (24h, 48h, 72h)
Results
CBHI:EGII
CBHI:EGI
CBHII:EGII
CBHII:EGI
CBHII:EGI
CBHI:EGI
Activity (mg/ml) supplemented with additional β-glucosidase (dotted lines) and the corresponding degrees of synergy (solid lines) on Avicel
over 72 hours. The enzyme assays were set up using different combinations of CBHI, CBHII, EGI, EGII and BGL with a total protein
concentration of 20 ug/ml Values are shown as the average SD. (A): CBHI/EGII; (B); CBHII/EGII; (C): CBHI/EGI; (D): CBHII/EGI
Conclusions
• CBHI and CBHII are key enzymes in cellulose hydrolysis
• Degree of Synergy increases over time
• Major gaps in our understanding of the mechanism by which
cellulases catalyze the hydrolysis of crystalline cellulose
• By gaining a comprehensive understanding of the enzyme
synergy that exists between different cellulases, we could optimise
enzyme cocktail design for cellulose hydrolysis
• Defined vs complex substrates?
Acknowledgments
•
•
•
•
•
Prof. B.I. Pletschke (Rhodes University)
Dr. J.S. Van Dyk (Rhodes University)
Prof. W.H Van Zyl (Stellenbosch University)
Dr. R den Haan (Stellenbosch University)
Lab 410 (Rhodes University)
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