Cellulase enzyme activity

Report
Powering the Future: Biofuels
Activity: Cellulase enzymes
• Describe the breakdown of cellulose by cellulases
and cellulose producing microbes
• Carry out quantitative assays of enzyme activity
• Assess the relative merits of immobilised
cellulases and microbe produced cellulases
Facts and Figures
• To help combat climate change the UK has a target to reduce carbon
emissions by 80% by 2050.
• 30% of the UK renewable energy could come from biomass heat and
electricity by 2020.
• To meet the European Renewable Energy Directive, the UK is aiming for
10% of transport energy to be from renewable sources by 2020.
• By 2020, 8% of our petrol and 5% of our diesel could come from crops
grown in the UK.
© Rothamsted Research
Bioenergy is the energy derived from
harvesting biomass such as crops, trees or
agricultural waste and using it to generate
heat, electricity or transport fuels.
© Rothamsted Research Ltd
Optimising the yield of fast growing energy crops that are not part of the
food chain is one way scientists aim to make sustainable, green bioenergy
replacements for fossil fuels a reality.
© University of Cambridge
Scanning Electron Microscope cross-section of straw: Non-edible waste from agriculture, such as
straw, could be used in the future as a secure, green source of fuel without taking up land needed
for growing food. Scientists will be looking at this as one possible way to provide sustainable,
environmentally friendly bioenergy replacements for fossil fuels.
Improving barley straw for bioenergy production and transferring the new knowledge to
other crops: Our challenge is to discover how the properties of lignin in barley straw
can be changed, to make it easier to produce biofuel (or bioenergy) from this waste
material without having any detrimental effects on the yield or quality of the crop.
© University of Dundee at SCRI
Plant Cell Walls: Lignin is a strengthening and waterproofing material
that encrusts the sugar based polymers in plant cell walls making them
hard to access for biofuel production.
© University of Dundee at SCRI
Plant Cell Walls: Woody plants, such as miscanthus and willow, convert much of the
carbon that they capture into lignocellulosic polymers, which are not a readily
fermentable form of carbohydrate.
© University of Dundee at SCRI
Plant Cell Walls: Feedstocks rich in lignocellulose require treatment with acids,
alkalis or steam explosion methods to hydrolyse hemicellulose and break
down lignin, enabling access to the cellulose by enzymes.
© University of York
The Gribble: This tiny seawater pest can destroy wooden boats and piers but
remarkably the gut enzymes that allow it to eat wood are being harnessed by
scientists to break down wood for conversion into green, sustainable bioenergy.
© University of York
The Gribble: By examining genes that are expressed in the guts of gribble,
researchers have demonstrated that its digestive system contains enzymes
which could hold the key to converting wood and straw into liquid biofuels.
© University of York
Bioenergy from sea pests: Gribbles are voracious consumers of lignocellulose and have
all the enzymes needed for digestion of wood and straw. Scientists have already
sequenced the genes that are expressed in the gribble gut and will study the enzymes
and digestive process
© Richard Gribble University of York
Bioenergy from sea pests: Remarkably the little marine wood borer, or Gribble, that caused
this damage could hold the secret to sustainable energy for us all. The gut enzymes that
allow the bug to damage wooden sea structures such as piers will be harnessed by
scientists to break down wood for sustainable bioenergy production
Bioenergy from sea pests: The gribble
digestive tract is dominated by enzymes
that attack the polymers that make up
wood. One of the most abundant
enzymes is a cellulose degrading
enzyme never before seen in animals.
© University of York
© G. Watson University of York
Bioenergy from sea pests: Unlike termites and other wood-eating animals, gribble have no
helpful microbes in their digestive system. This means that they must possess all of the
enzymes needed to convert wood into sugars themselves.
© Institute of Food Research
Steam explosion unit: We need to optimise the release of sugars from agricultural and woodindustry wastes to produce a fermentable feedstock that microorganisms can use to produce
fuels. Pretreatment of feedstocks with steam opens up the structures in plant cell walls to
enable access by cellulase enzymes.
© Institute of Food Research
Food Waste: Scientists at the Institute of Food Research are investigating
how our waste problem can be turned into an energy solution.
© University of Nottingham
To harness the potential of lignocellulosic (plant cell wall) materials, we need to optimise the
release of sugars from agricultural and wood-industry wastes to produce a fermentable
feedstock that microorganisms can use to produce fuels. Developing robust microbial
strains that can use these feedstocks will enable sustainable production of bioethanol.
Professor Katherine Smart
BSBEC LACE Programme
School of Biosciences
University of Nottingham
© National Collection of Yeast Cultures
Scanning Electron Microscope image of Yeast: Researchers are developing
novel yeast strains and fermentation processes that optimise bioethanol
production.
© University of Nottingham
Researchers are using synthetic biology approaches to generate bacterial strains that
can convert lignocellulose to fermentable sugars efficiently to maximise butanol
productivity.
Method: Gel diffusion
1. Prepare an agar gel containing 1.7% agar and 0.5% CMC (carboxymethylcellulose). Pour this gel
into petri dishes and allow it to set.
2. Prepare the fruit extracts by mashing a variety of ripe fruits in a mortar and pestle.
3. If testing enzymes or microbes using paper discs, prepare the paper discs by cutting them out of
filter paper using a narrow cork borer or using a hole punch. Sterilise the discs by autoclaving
wrapped in foil.
4. If testing enzymes or microbes immobilised in sodium alginate, make up the alginate the night
before to allow it to fully dissolve.
5. After the agar gel has set, use a narrow cork borer to punch small cylinders in the gel. Then, using
a mounted needle, remove each of these cylinders to create a series of similar sized wells in the
agar. Four or more wells can be put in a single dish, provided they are spaced apart.
6. Place similar volumes of extracts of fruits in each of the wells. In one well, place some distilled
water, as a control.
7. Mix the enzyme or microbes with the alginate shortly before adding it to the wells in the agar
using a syringe.
8. Incubate the dishes for at least 24 hours at 30 °C.
9. After the incubation period is finished, use tap water to rinse out the contents of the wells, and
then flood the dishes with Congo red solution for 15 minutes. Then rinse the dishes with 1 M
sodium chloride solution for at least 10-15 minutes.
Method: Viscosity reduction
1. Make up a 2% (w/v) wallpaper paste solution, sufficient to provide 25 ml for each
sample to be tested.
2. Place 25 ml of the paste in a boiling tube and add 2 to 5 ml of fruit extract. Mix
thoroughly.
3. Then pour the mixture into the barrel of a syringe, held in a retort stand, pointing
downwards into a small beaker. Note the time taken for all the mixture to drain
through the syringe nozzle into the beaker.
4. Incubate the fruit or enzyme-wallpaper paste mixture at different temperatures, such
as in a water bath at 30°C, allow to return to room temperature and repeat the
investigation, checking the change in viscosity.
The more active the enzyme the greater the reduction in viscosity, and so the shorter
the drainage times.
Activity: Cellulase enzymes
• Describe the breakdown of cellulose by cellulases
and cellulose producing microbes
• Carry out quantitative assays of enzyme activity
• Assess the relative merits of immobilised
cellulases and microbe produced cellulases
Contributors

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