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The Development of Recombinant Fungal Enzyme Cocktails for the
Hydrolysis of Cellulosic Waste Products
Rosemary DobsonUniversity of Stellenbosch
Energy Postgraduate Conference 2013
Introduction
• Alternative fuels• Renewable resources =
sustainable• Bioconversion of
lignocellulosic biomass• Abundant resource• Hydrolysis = major
bottleneck• Enzymatic hydrolysis
• Current enzyme mixtures not sufficient
• Cellulases are expensive
http://www.ijbs.com/v05p0578.htm
β-glucosidase (Bgl)
Cellobiohydrolase (Cbh)
Endoglucanase (Eg)Glucose
Cellobiose
• Hydrolysis of cellulosic biomass
• Mixtures of hydrolytic enzymes
• Collectively known as cellulases
• T. reesei & A. niger produce large amounts of extracellular cellulolytic enzymes
• Synergistic manner
Cellulases
• Develop a recombinant fungal enzyme cocktail for
effective hydrolysis of paper sludge • Select core enzymes (cellulases)
• Express enzymes in Saccharomyces cerevisiae
Y294 and Aspergillus niger D15
• Harvest enzymes
• Analyse paper sludge
• Hydrolysis trials
• Fermentations
Aims and objectives
Methodology
Enzyme selection
Strain preparation
Harvest enzymes
Select substrate
Hydrolysis trials
Glucose yield
Fungal cellulases
Paper sludge
Analyse
S. cerevisiae Y294[cbhI] S. cerevisiae Y294[cbhII] A. niger D15[eglA] S. cerevisiae Y294[Bgl2]
10ml working volume2% paper sludge
pH 530°C
Core Enzyme
s
Optimise for different
feedstocks and pretrements
Test new enzymes
Test accessor
y enzymes
Identify non-essential enzymes
Which enzymes to produce in a CBP organism
Which enzymes to engineer
Which enzymes to pursue by
bioprospecting
Try different ratios of enzymes
Enzyme cocktail development
Paper sludge
• Solid waste material
– Non recyclable paper fibres
• Attractive biomass for enzymatic hydrolysis
– Susceptible to enzymatic digestion
• Negative feedstock cost
– Integration of processes into an existing industrial infrastructure
• No pre-treatment neededBayer, Lamed, & Himmel, 2007
Results
Component Percentage (on dry weight)
Cellulose 34.06 ± 0.65
Hemicellulose 14.26 ± 1.51
Lignin 27.04 ± 0.93
Ash 5.68 ± 0.12
Table 1: Paper sludge analysis
Figure 1: Optimised enzyme concentrations with a four enzyme cocktail, containing one: cbhI, cbhII, EgI and Bgl enzyme𝐷𝑠𝑔𝑙𝑢𝑐𝑎𝑛 = ሾ𝑔𝑙𝑢𝑐𝑜𝑠𝑒ሿ 𝑥 0.9 [𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒] 𝑥 100%
24 48 72 96 120 1440
5
10
15
20
25
30
35
40
Cocktail (double concen-tration)
Complete cocktail (4 Enzymes)
Cocktail (without cbhI)
Cocktail (without cbhII)
Cocktail (without EgI)
Cocktail (without Bgl)
Hours
Ds g
luca
n (%
)
Looking forward
• Test enzyme cocktail with a yeast strain– Analyse for ethanol (HPLC)
• Conclusion– First report to use enzymes from
recombinant strains for the hydrolysis of paper sludge
– Important to optimise enzyme cocktails
Substrate
Enzymatic saccarification
Sugars
Microbial fermentation
Biofuels