An investigation into the synergistic action of exo and endo-type cellulases on the hydrolysis of complex substrates

1. An investigation into the synergistic action of exo and endo-type cellulases on the hydrolysis of complex substrates MariskaThoresen Energy Postgraduate Conference 2013

2. Introduction DEPLETION OF FOSSIL FUELS • Need for alternative energy sources BIOMASS • Large scale availability • Low cost

3. Plant cell Cellulose Hydrogen bonding with and between cellulose chains • cd Microfibrils RECALCITRANT Macrofibrils Enzymes Cellulose Fiber β-1,4 glycosidic bonds

4. Cellulases 2. Cellobiohydrolases: release cellobiose from chain. • Endoglucanases: cut at random sites along the cellulose chain. 3. β-glucosidases: conversion of cellobiose to glucose. CBHI CBHII NR R Crystalline Amorphous Crystalline β-glucosidase

5. 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

6. Enzyme 2 Synergy Assays Enzyme 1 Enzyme 3 Enzyme 1 Designer enzyme cocktails Synergy >1 Synergy Synergy <1 Competition Synergy =1 No synergy

7. Synergistic relationships Exo Endo Synergy Endo Exo

8. 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)

9. CBHII:EGII CBHII:EGII CBHI:EGII CBHI:EGII Results CBHII:EGI CBHI:EGI CBHII:EGI CBHI:EGI Activity (mg/ml) supplemented with additional β-glucosidase (dotted lines) and the corresponding degrees of synergy (solid lines) on Avicelover 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

10. 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 cellulasescatalyze 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?

11. Prof. B.I. Pletschke (Rhodes University) • Dr. J.S. Van Dyk (Rhodes University) • Prof. W.H Van Zyl (Stellenbosch University) • Dr. Rden Haan (Stellenbosch University) • Lab 410 (Rhodes University) Acknowledgments

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