cc >> Welcome everyone to Wednesday
Nite at the Lab. I'm Tom Zinnen.
I work here at the University of Wisconsin-Madison Biotechnology
Center. I also work for UW-Extension,
Cooperative Extension. On behalf of those folks and our
other sponsors, Wisconsin Public Television,
the Wisconsin Alumni Association and the Science Alliance,
thanks for coming to Wednesday Nite at the Lab.
We do this every Wednesday night, 50 times a year.
Tonight it's my pleasure to be able to introduce to you
Raghu Yennamalli. He obtained his PhD from
Jawaharlal Nehru University in New Delhi, India,
in computational biology and bioinformatics.
His research topic at that time was designing drug molecules
to prevent the infection of dengue virus,
which was epidemic in India. Raghu did his first
post-doctoral research at Iowa State University, where he was
introduced to the field of biofuel production from
cellulosic biomass. He continues the same line of
research here at UW-Madison in the lab
of Professor George Phillips. Currently, Raghu is working on a
newly discovered enzyme that can act on the crystalline form of
cellulose. Most of us, not all of us, but
many of us would really like to have liquid fuels that can go in
to tanks of our cars and trucks. One of the big challenges we
face in biomass is how to get those kinds of liquid fuels not
just out of the kernels of corn, but out of the stover,
the leaves and stalks. I think Raghu has got a very
interesting story to tell us today.
Please join me in welcoming him to Wednesday Nite at the Lab.
[applause]
Good evening everyone. Can everyone hear me?
Okay. My name is Raghu Yennamalli, and
thanks to Tom for giving a very kind introduction.
I'm quite excited here to talk about my research to all of you.
The title of my talk today is "Crystalline Polysaccharides
to Biofuels, A Structural Perspective."
This project is part of the Great Lakes Bioenergy Research
Center, or GLBRC for short. It is funded by the Department
of Energy. It's one of the DOE bioenergy
research centers here. UW-Madison is one of the main
northern points for this. How many of you have seen this
movie, "Driving Me Crazy"? This was telecast on the TV,
I think, two or three weeks ago. I found it quite apt for my
talk, because there is this eccentric German inventor who
wants to sell a prototype car that runs only on vegetables.
The ones that kids don't eat. [laughter]
It's a comedy, and he runs into one type of problem or another.
The movie does show exactly how he made the technology, but it's
quite interesting because, for some time now, finding an
alternate fuel-source for our cars has been kind of like a
holy grail. If I'm not over-stepping that
statement. Without further adieu, I will go
into my talk now. For those who are not familiar
with the topic, a little primer. Bioenergy is any energy that is
derived from a biological source.
Biofuel is the liquid fuel that we obtain from the same
biological source. For example, like wood, charcoal
and pelletized biomass, are used and have been used for ages, to
generate heat and electricity. Methane, which is obtained from,
it is called, biogas, are obtained from manure digesters.
Since I come from India, the biogas plants in the rural areas
of the country have been quite effective in some extent
to reduce the deforestation activities.
This biogas has been used for cooking, not specifically for
running cars, but mostly for cooking and improving the health
of women there. Biodiesel, here we've seen how
many vehicles run on biodiesel that is produced from soybeans,
canola oil. Finally, ethanol.
It is obtained from plant sugars and starches.
GLBRC has been focusing more on the sustainable biofuels that
are going to be used in the near future.
Currently, we have ethanol that's being produced from corn
starch or sugar cane sugars, but these are much simpler biomasses
from which ethanol can be produced.
Apart from that they're a much larger variety of biomasses,
which are more complex than these, and developing new
technology has been one of the main areas of research.
So why do we need biofuels? Well, we want to move from a
petroleum-based bioeconomy to a plant-based bioeconomy.
We would like to be energy independent and not import fuel
from other places. And it's renewable.
The biomass that's produced is actually converted to biofuel
and then the carbon dioxide and the water that is released back,
is actually converted back into biomass by the plants.
So there is a continuous supply and demand that will be there.
Once the chain starts rolling, it's not going to stop.
It's a cleaner fuel. It's not going to leak out any
toxic gas or anything, other than carbon dioxide and water.
Basically, this part of the slide shows you that we use
microbial fermentation to ferment glucose and other
sugars, to convert it to fructose-6-phosphate.
Glucose is a six carbon molecule.
Fructose is also a six carbon molecule, but slightly different
from glucose. That is converted to pyruvate,
and further down to ethanol, which is two carbons.
So for each glucose molecule you can get three ethanol molecules.
C-6 to C-2. Current biofuel technology is
two parts. One from sugar cane and one from
corn. Sugar cane provides the glucose
gases, then you ferment it like you would go on making beer.
You can ferment the glucose and you get your ethanol out of it
after distillation. From corn you have to extract
the starch out of it. Then you have to use heat or
other enzymes which can act on the starch.
Starch is a big polymer. If you can imagine a big chain
of all different of glucose units linked to each other, in
some kind of a chemical process which can break it down or chop
it down into much smaller units, which is a single glucose
molecule. Then further, the same enzymes
that are used for converting the sugar cane to ethanol, can be
used here to convert it to ethanol.
Before I proceed, how many of you--
I want to throw a question to the audience.
Do you know how much percent mixed ethanol gasoline is
currently at a level that we can use?
[inaudible] Exactly.
It's ten percent, so in scientific parlance it's called,
E10. You see here, you add ten
percent of anhydrous ethanol to the rest of the gasoline, and
that's what is used today in most parts of the US.
In Europe, it's E5, but in Brazil, E25 is used,
25% of ethanol mix is used. These four mixes, E5, 10, 15 and
25, can be used in all regular cars.
Apart from this, you have E85, which is also a level in the US.
This can be used by flex-fuel vehicles.
The engine is slightly modified from the regular cars.
Here it's 85% of ethanol and 15% gasoline.
In Brazil, currently they have technology there that you can
use 100% ethanol in their vehicles.
It's called E100. E85 is widely available in the
US. The states that are all colored
with yellow have locations available across the state.
There are about 2,466 locations available in the country where
you can get E85. Wisconsin has 159 locations.
It's the fourth largest in terms of the number of E85 gas pumps.
The top one is Minnesota, which has about 366 E85 gas pumps.
There is a cool website called e85refueling.com.
If you go to that and look for E85 gas locations, and if you do
a road trip, if you have a flex-fuel vehicle,
you can plan it accordingly to refuel the gas.
What are the blue states? >> They do not have.
They don't? >> Yeah.
They do not have. Coming to Wisconsin, only in
2001 the ethanol production started in Wisconsin.
Until then we did not have any plants here.
Currently, there are nine plants operating.
Cumulatively they are producing 545 million gallons of ethanol
per year. This amount to Wisconsin being
the ninth largest ethanol producing state in the country.
What are the types of biomasses from which you can make ethanol
other than sugar cane and corn starch?
Just to make things clear. The sugar cane and the corn
starch are called first generation biomass.
The next ones are all second generation or the third
generation biomass, because they're more complex than the
simple sugars that are there. Agricultural crop residues
actually amount to the highest biomass that's being produced in
the US. There has been a study that's
called the Billion Ton Study, Which was carried out by DOE and
the USDA. The Billion Ton Study actually
projects, based on the amount of particles and output and other
things, what the amount of biomass that can be achieved
every year in, say, 2030, or 2050.
This document is actually public and you can have a look at it.
Basically, they divide entire biomass to be produced, making a
peak of a billion tons per year, and a majority of them are going
to be from agricultural, then some of them being from forest
residues. Like here you see logging, and
other things. The third part is urban waste,
municipal waste, from which is also a biomass source from which
biofuels can be made. When we look at, across the
entire country, we can see, based on how much tons are being
produced in each state, based on the counties, you have the
regions that are marked blue and red.
They have 250 to 500 tons, 1000 tons, per year production
of biomass. The regions in red are about
500,000 tons per year. So when it comes to Wisconsin,
you have at least a minimum of an average of, like, say,
150,000 or 200,000 tons of biomass being produced per year.
Coming to the GLBRC, or the Great Lakes Bioenergy Resource
Center, the mission is very simple.
It's by grant. It's to perform the basic
research that generates technology to sustainable
convert cellulosic biomass to ethanol and advanced biofuels.
This GLBRC is, if you look at the geographic distribution,
Wisconsin, especially in western Wisconsin, Madison is the
northern center for GLBRC. We have another partner here,
the Lucigen Corporation. And the Michigan State
University is also a partner in this.
Apart form this, you have two national labs, Oak Ridge
National Lab and Pacific Northwest National Lab, who are
also partners with us. And Iowa State, University of
Missouri, Columbia, Illinois State, Cornell, and the
University of Minnesota, University of Toledo.
This whole group forms the Great Lakes Bioenergy Resource Center.
We aren't the only one. Across the country there are
three bioenergy research centers.
They are called BRC's, the Bioenergy Research Center.
One is the DOE Joint Bioenergy Institute, which is mostly
located in the west, where the northern point is Lawrence
Berkeley National Laboratory. In the east you have the DOE
Bioenergy Science Center, where the Oak Ridge National
Laboratory is the northern point.
Then you have the Great Lakes Bioenergy Research Center
that the University of Wisconsin-Madison
is the northern point. Currently we have about 400
scientists, staff and students across sites.
That picture on the right was taken three years ago and has
about 250 people in that. It has fairly increased in the
recent years. A majority of them are
undergrads and faculty who are part of this team.
GLBRC has four main areas in which they concentrate their
basic research in. The first one is to have
sustainable biofuel practices, which actually covers the entire
spectrum of procuring the right type of biomass, developing the
right type of biomass to ethanol, and then making it a
very sustainable pipeline. The second one is improved plan
biomass. Research is being done
where you develop new varieties of the crop,
new varieties of the plant, that some of the hurdles
that we are facing right now would be much easier to overcome
in the future. The third one is improved
biomass deconstruction. That is the thing that you have
the biomass from the raw product to the basic glucose, and it's
converting it from that point to this.
Finally, the improved biomass conversion to fuels, which
actually encompasses the technology part, where you have
new technologies being built for it.
These four discovery science areas are going to provide us
knowledge about how to sustainably convert cellulosic,
specifically cellulosic, biomass into liquid
transportation fuels. If you look at the cycle that we
talked about, using the plants as the main source of biomass,
especially the cellulosic biomass, they use the energy
from the sun and then it's converted to cellulose, which
they use it in the cell walls, and cellulose is the most
recalcitrant polymer that nature has made.
It's kind of like the hurdle for us, to convert it to biofuel.
Then converting those sugars to biofuels, then biofuels to human
use. Then finally, that cycle
continues back into the plant. So what is cellulose?
Cellulose, as I just said, is part of the cell wall in a
plant. Each cell of the plant has a
layer, or a big wall of cellulose, which gives the
structural integrity to the cell.
Animal cells do not have cellulose.
That's why we have tissues that are soft, whereas in plants it's
quite hard. And after some time these
celluloses are actually, once they are synthesized and made
into the structure, they are kind of re-strengthened using
other polysaccharides such as hemicellulose and an aromatic
compound called lignin. If you imagine this block here
which runs way across this, if you zoom it, it looks like this.
Let me walk you through from this part to here.
Here you have one single unit of glucose.
Two units are joined together by a glycosidic bond.
That's the scientific term for the bond.
It's basically a strong interaction between the two
carbons, and there is an oxygen molecule that there as well.
Many such glycosidic bonds make the glucose into a long chain
of -- polymer. Many such polymers are actually
stacked one on top each other, and you have many such layers.
This is called a microfibral. Many microfibrals are placed
together, and each microfibral is covered by a layer of another
polysaccharide called hemicellulose.
Hemicellulose is also made up of sugars, but these sugars are not
the same as glucose. They are different types of
sugars. Now all these different bundles
of cellulose, covered with hemicellulose, has to be held
together. There needs to be some kind of
a cementing agent that holds all of them together.
That's lignin. Lignin is an aromatic compound.
It's highly aromatic. This helps as a cement.
This whole thing is part of the plant cell wall, here.
So this is what I was just showing you about the technology
that we have from sugarcane and corn starch.
But tomorrow's technology is to use this complex biomass, which
I just described to you, that the cellulose is not as exposed,
but it's covered with a layer of hemicellulose, and then
it's next product is lignin. This biomass, if you pre-treat
it using heat, chemicals and pressure, you loosen up the
cell wall material from the main structure.
You want to remove the lignin, the hemicellulosic and you want
to extract only the cellulose part of it.
And then you use certain enzymes called cellulases, which can
convert that big chain of polymer into much smaller
pieces, and finally into, you have glucose, arabinose,
xylose. And this phenolics is where the
lignin, after it breaks down, you get -- phenolics, which is
quite toxic. Once these are formed, you can
actually use fermentation technology to --, which is the
next generation fuel from a more complex biomass.
So in a refinery, in a cellulosic technology refinery,
biomass, the process will start with the biomass is harvested.
Say you have an agriculture land where the corn has been
harvested, and then the corn stover is left.
Most of the stovers, for those who don't know, stover is part
of the plant which is left after harvest, like the stems, the
leaves, the roots. This stover mostly goes as
animal feed. Now, you can take all this
biomass that's been produced, and deliver it to a refinery
that it is cut into small-- It's chopped into smaller
and smaller pieces, and it's pretreated with heat and
chemicals to make this cellulose come out off its structural
integrity and apart. Then it allows the enzymes to
act. So here you see a chunk of
cellulose being acted upon by these enzymes that actually
convert it to like two units, and then finally you have the
simple sugars formed. Once the simple sugars are
formed, you actually can transport it to one of the vats,
and you can use microbes, like say yeast, or other enzymes that
can convert the glucose through fermentation.
Then the distillate, you purify it and then you can take it for
distribution. This is what probably is the
like the ultimate big picture of the entire research outcome.
So when I mentioned about the cellulose is being released out,
is actually you have this cellulose microfiber.
If you look at this microfiber, at certain places it's like
straight lines and certain places it's like curvy lines.
These straight lines are actually the crystalline part of
the cellulose. The crystalline part is very
ordered. Each polymer chain is actually
stacked one on top of each other exactly, and they have a
particular order. It's like a crystal, like a
diamond or a graphite crystal. The amorphous region, or these
curvy lines is what is more-- It's also present
in the cell wall. So you have a mixture of a
crystalline, and then amorphous, and then crystalline.
Now these entire microfibrins are all covered with
hemicellulose, here in green. Then you have the cementing
agent between each microfiber, the lignin.
So then you do a pretreatment process.
The pretreatment process can be under high heat, high pressure,
or the one other pretreatment process that's currently used is
called Ammonia Fiber Expansion, AFEX.
This Ammonia Fiber Expansion does -- use ammonia to actually
pre-treat the cell wall, so that the cellulose fibers are
expanded, or the hemicellulose is broken down so that the
cellulose inside is exposed for the enzymes to act on it.
When the cellulose acts on it, when the cellulose is released,
you have both the crystalline form and the amorphous regions
all released out for the enzymes to act.
So these enzymes that act on these cellulose is actually
called cellulases. There are different types of
cellulases. So how does this enzymes act on
the cellulose? So, before this was used for
bioethanol production, these cellulases had a much bigger
industrial importance. If any one of you are actually
wearing or have stonewashed jeans, these cellulases were
used, and are currently used, to make the stonewashed effect on
jeans. The reason is, the fibers that
are exposed out from the fabric are more accessible for the
enzyme to act upon, and they are chewed upon.
So the cloth that is most tightly present is not acted
upon by enzymes, but the loose ones are.
So you have a process where the cellulases can give the look of
stonewashed jeans. Don't imagine someone actually
using a stone to make that effect on your jeans.
It's mostly the cellulases. It's also used in detergents to
remove pilling. So if you have those pillings
from like old sweaters, not wool, but the cotton sweaters,
then if you find a detergent that says enzymes that can act,
anti-pilling, they're made of basically cellulases, because
the cotton is also made up of cellulose.
When you have pilling, some of the strands are exposed, and
these cellulases can act on those exposed strands and then
chop them off so you get the anti-pilling effect.
Nature has provided us with answers on where to find these
cellulases. So there are many niches,
ecological niches where you can actually find the cellulases.
One common example that is not shown in this slide are
termites. Termites chew on wood.
Wood is from plants. It's rich in cellulose.
The termite has bacteria, and these bacteria secrete
cellulases, which is not produced by termites,
and they can digest that wood, the hard wood.
So one of the other cellulose niche organisms that nature has
given us are the Leaf Cutter Ant System.
You have seen this on National Geographic
or the Discovery Channel, where these small ants cut these
leaves and then go in a very orderly fashion.
There are two genus of these ants which actually
are the Leaf Cutter ants, the Atta and the Acromerex.
Apart from this, you have the Bark Beetle, the Wood Wasp,
and then the Free-livingwood Fungus,
which are also a major source of these cellulases,
and they live in cellulose.
GLBRC, specifically, they are working on this
Leaf Cutter Ant System. This is what is called
bio-prospecting, like how you go about prospecting for gold,
or oil, here we go prospecting about the cellulose-rich
organisms. And the Leaf Cutter Ant System
is actually one of the sources in which the current research
has been happening. So in Costa Rica, here you have
this big mound, the ant mound. The ants go to the tree, cut the
leaves, and bring them to the mound.
Then, here you have different chambers in the mound.
One of those chambers, which is marked "D", has a fungus.
The ants grow this fungus. Just like how we grow crops
and harvest them, the ants grow this fungus and harvest them,
because the fungus, the things that are harvested from the
fungus are actually fed to the ant larvae.
That's their food. The fungus needs the ant to
live, and the ants need the fungus to live.
So it's a very unique symbiotic or mutualistic relationship that
they have. Apart from this fungus, so if
you imagine the scale of this ant pit, it's this big.
This is an excavated ant mound in Costa Rica.
These are Leaf Cutter Ants. You can see the different
chambers here. And you can see from the size
of the people how big this is. This is widespread in different
areas within the same general region.
So here I'm going to move to...
This website here, Currie Lab, they're co-collaborators.
They have an ant camera here in the Microbial Sciences building
that's right across the street. So, if you go to this ant cam,
you can actually see them. Unfortunately, I did not connect
my feed to the Internet. But you can see live images of
the ants feeding on the fungus. You can actually, depending on
when you are looking at it, like in the daytime, the lights
are switched on so you can see the ants in real colors and in
the dirt. Evening, as now, the night
vision is switched on, and you can see them, like how a night
vision camera works. You can see the ants crawling
all across this fungus. If you go there, you can see the
different chambers, the place where they collect the leaves,
and then they dump it into the pits, and then the fungus area,
and then they have the area that have the larvae, and they feed
the larvae. It's a nice camera.
You can look at it. So this ants and insects-microbe
symbiosis is actually the result of ten million years of
co-evolution between these organisms.
Now, it's not just ant and fungus.
There is another organism also present, and that's the
bacteria. So the ants, as I said, the ants
need the fungus to live, and the fungus needs the ants to live.
Now, the fungus has a villain in its life.
There's one organism exactly that causes the fungus to die.
Fortunately, the ants carry a particular bacterium which
actually grows on the ant's surface itself.
That bacteria produces antibiotics that is actually
going to prevent the fungus from getting that disease from the
pest. So, the ants in fact act as a
kind of medicine, a moving medicine chest, and they deliver
these drugs to the fungus and keep them alive.
There are not just one kind of bacteria present in these ant
colonies. There are many kinds of bacteria
there. So the discovery strategy of
GLBRC for this particular niche is that you have an organism
level approach. So you isolate the bacteria that
are present in these ant colonies.
You classify them into new species.
You sequence their genome. Then you do cellulose growth
experiments to see which bacteria from this colony can
grow in a cellulose-rich environment.
You do microarray analysis. So microarray is nothing but a
technique in which we can see which genes are upward turned
and which genes are downwardly turned.
We can identify the genes that get upward when grown on
cellulose. So those genes are most probably
going to produce a enzymes to degrade the cellulose, because
they are present, or produce more in number than the organism
when it's grown on cellulose. Then you identify the proteins
that are secreted when grown on cellulose.
So here is a schematic view of how these enzymes act on
cellulose. Here, as I said, it's one
big chain of cellulose. One end of the cellulose is
called a non-reducing end. The other part is the reducing
end. The two glucosinates are
connected by this bond here. It's called a glycosidic bond.
So if you imagine a cellulose region on the bottom here.
There is one enzyme called endoglucanase.
Endo meaning inside, and glucanase means that this
is like a glycosidic bond. Now this endoglucanase makes an
internal link in this entire cellulose chain.
The reason is that we need some kind of place in there we can
start chewing up the cellulose to make it into simple sugars.
So it makes an internal link, and you can see this glycosidic
bond here in red. Then you have, now this part is
exposed for another enzyme to act, which is called
cellobiohydrolase. What this does, is it acts on
the exposed area and makes a long chain into smaller parts,
which consists of two glucose units.
This is called cellobiose. Now, this also is not enough for
us to make it into ethanol. We need to take this further
into individual glucose units. So we have another enzyme called
betaglucosidase, which actually breaks the single glycosidic
bond and finally gives the glucose.
So, three enzymes: endoglucanase,
cellobiohydrolase, and betaglucosidase.
This is kind of like a minimal set.
At the minimum, you need three enzymes to convert a cellulose
polymer into glucose. Here's where my part of the
research comes in. As in the topic, I had mentioned
a structural perspective. So, how do we visualize
proteins? Because all these enzymes are
basically proteins, and we know proteins are made up of amino
acids. They are the building blocks of
protein. So, many amino acids join
together to make it one big protein.
Then, one of the things about structural research of proteins
is that we need to visualize these proteins.
It's not that we can visualize it in one way, we need many
different abstract methods, a kind of like abstraction to
understand many different concepts.
So here I have the same enzyme in the same orientation.
I have shown it in different ways.
Here on the top you have it in the line representation.
Basically, if you think of two atoms, and they're joined by a
bond, you can draw a line between them.
That's what is seen here. All atoms that are joined, that
make a bond, are actually connected.
It's kind of like a wire frame. In fact, this representation is
called a wire frame. The second representation is
sticks. Here it's very similar to wire
frame, but then imagine if we had made a model with ice cream
sticks. So this would probably look like
that. This representation helps us to
understand the way in which the protein has been folded.
This is called the ribbon or a trace.
So every-- You can think of it in terms of
like a skeleton. So this part is only the C-alpha
regions, the main carbon, the alpha carbons that are connected
along the chain. And only those alpha carbons are
shown here, and they're connected with a line.
This one, here it's colored in terms of its secondary
structure. I don't want to confuse you with
many things, but basically a single amino acid chain is
called a primary structure. Then when we fold it into a
globular or big, massive protein, then it forms some
basic structural units in it. Some regions can form a helix,
and some regions can be as lines.
So here the beta strands are colored yellow.
Here you see a helix in red. The regions that connect between
these two that are shown in green are called loops.
They're basically very loopy regions.
The same representation can be made in this way, where you have
the helixes, just like the helix is, and there you have the beta
strands. Here you have, it's shown as an
arrow, so it shows the direction in which the chain runs.
Here is another representation or model.
Here, you actually imagine each atom has a center and then, say
the hydrogen atom, and you draw the radius around which the
electrons will be from the nucleus, and you make it look
like a sphere. Then you have an oxygen,
and as these two atoms are joined, then the spheres are
joined, and certainly -- in other parts.
That is a visual representation is of how these atoms are joined
with each other. It gives you an idea about the
empty space that's going to be there after these molecules have
bonded with each other. The final representation here
is called a surface representation.
This is something similar to like if I have an object and I
wrap it around with a cloth, and then I allow the cloth to sink
in there a little bit if there is a valley, and then let it
project out where there is a hill.
So it looks like a continuous surface all across it.
This surface representation is very helpful for us when we have
to see where these proteins can bind with the cellulose, or
where it is going to bind with these water molecules which are
all around it. Okay, so this was on the
computer. What about in reality?
For some people, it's very difficult to understand and
visualize the representations that I talked about.
So here we have, using the 3-D printer technology, we can print
the same protein structures in 3-D form.
So this looks something like Play-doh, but then it's actually
a printer using-- Here you have the helix.
And here you have the beta strands here.
You can see the helix, and they're connected by a loop
here. You can actually rotate it and
get an idea about how this protein looks.
Here, this protein is colored in two colors because it has two
domains in it. Similarly, you have this protein
here, which is actually this model is made up of two
proteins. If you see this part, you can
actually see the same thing on this side as well.
This part is one protein and this is another protein.
This is the label. So I can pass it around
so you can see it. Feel free to come and have
a look at these models. They're quite lightweight.
Now we have this 3-D technology. What did people used to do in
those days if they wanted to visualize a protein?
How many of you know about stereograms?
Wow, that's nice. So these stereograms are
nothing but you have two images. There's a slight difference
between these two images, just like looking at a 3-D movie.
They have a kind of parallax effect.
What you would want to ideally do is kind of squint your eyes
and make the two white circles on the top, make it appear here
as one. So if you're not able to get it
once, you can actually move front and back, and then
you know, you can get it. Finally, you see the picture
that comes in the middle has these valleys and mountains, and
then you can actually get the three-dimensional view in that.
So are you able to get it? It's quite easy.
These stereograms of-- Stereograms, when it comes to
images that are non-proteins, when we look at protein
structures, you can actually make them look in stereo.
So when you try to do the same effect, and you see a central
image here, when you try to move these two black dots to merge as
one in the center, you can actually see that these loop
regions, these lines here, actually appear in the front,
and some of the lines appear in the back.
That's how people used to view protein structures before we
had this technology and other 3-D monitors with which we can
view these protein structures. Was someone successful?
Oh, it's too far apart? Probably, you need to move.
[laughter] When I see it on my screen,
I kind of move my head, you know, front and back.
You can try this at home. It's quite easy.
So, coming back to cellulose, the structural perspective of
the talk. So if you have this cellulose
here, then extraction of a single chain out from this
cellulose, one polymer, and making an internal link at any
part of the polymer is actually kind of like the way to make --.
The enzyme has to work really hard to actually pull out one
area of the polymer and then just chop it off.
So this is endoglucanase. Here you see that it uses these
two amino acids, there actually kind of like rings,
-- and residues. They act as kind of like
an anchor. That helps the enzyme to
actually slide on top of this chain of cellulose.
It also has an auxiliary domain, which is connected by a loop.
In the structure, we don't see the loop, because the loop
is very loopy and very floppy, and we do not have proper
confirmation for it. So the aim of this domain is to
actually anchor the protein onto the cellulose and help this
domain. So here I have a small model of
the same. Here you have the big domain,
which actually does the job. Here you can see one cellulose
being pulled inside. Then the CVM, which is actually
the other domain which helps this part of the protein to
anchor properly and make the job successful.
I'll pass this around too, so you can get an idea about it.
Coming to the cellulose again, mentioning how crystalline the
cellulose are in the cell walls, there are many different
forms of cellulose. They're called polymorphs.
It just differs on how these chains are arranged in respect
to each other. If they're arranged in parallel,
then they're called Polymorph I. If they're arranged
anti-parallel, then they're called Polymorph II.
One of the polymorphs, I-Beta, looks like this.
So you have this chain of cellulose arranged parallel-ly.
The way they're oriented is they're all pointing in the same
direction. This is a view from the top.
So when looking down on a cellulose chain, this is how it
looks. The side view looks like this.
You can see how flat this cellulose is when you look at it
form the side. Now, there are a handful of
enzymes that can only act on the crystalline part of cellulose.
Previously, there was not much information of a level about how
these enzymes act on it. Like recently, more research has
been happening, not only in GLBRC, but in other places as
well. We are trying to understand and
they are also trying to understand how these enzymes act
on these crystalline form. The crystalline part of the
cellulose, again, to re-create, is the most hard part that the
cellulases can act on. But as the amorphous region are
actually the same cellulose much expanded, because of the
pre-treatment processing, they are more easily, they can reach
through them. The way these things act, is
that they do not act by themselves.
Here the cellulases, the cellulases here shown in red,
which act on this crystalline part, if when in presence of
another enzyme which is shown here in blue, the rate
increases, the amount of degradation actually increases
when these two enzymes are present.
So this enzyme actually makes an internal link on the crystalline
part, and then the other enzyme starts from that created link
and then chews on this and makes smaller products here, like
the cellobiose. So these cellulases, previously
thought just bind to the polysaccharides, but not enough
to act on them and degrade them. So that's why they are called a
carbohydrate binding module. Module is another word to
indicate they are a domain, a loop working.
Then they bind to carbohydrates.
That was their original function, it was thought.
But currently, we know that they're not just binding
to it, but they clean the glycosidic bond and act
like an oxidase. So they use an oxidase,
oxidation reaction that they use the water molecule as a
nucleotide and then act on this and break this glycosidic down.
So to visualize this reaction, you can think of something like
on the cellulosic face, just an enzyme binds to it and then just
randomly chops off with its two razor sharp teeth.
Then it chops, chops, chops, like that.
You can think of something like that.
This is what these enzymes do. So currently there are a few
structures of these enzymes at our level, fortunately, for us
to visualize these workings and understand how these act.
Here are the four protein IDs. This is called pdb id.
So, pdb id is a beta base that these preliminary structures are
deposited, and they are publicly accessible.
They are all funded by the NIH and NSF and other projects.
So when we look at all these four structures and overlay one
on top of the other, we can see that they all look very, very
similar. That is, this is again a
representation of the C-alpha trace, so only the alpha carbons
are shown here. You can see that it is a very
good alignment or overlay of the beta strands here, and very good
alignment of the helixes here. Of course, one of the structures
has an extra loop, which is different from the rest.
The reason that's shown here is the active side.
So in proteins, you can basically divide them into two
regions. One region which is involved in
the function. That is where the main reaction
happens. So some residues come with it
and stay and then carry out this reaction.
But for them to carry it out, you need a basic structure or a
framework which can support them.
So this active side region is the functional part of the
protein. The rest of the region which
gives support to this is called a structural function.
You can kind of divide the protein into two parts,
basically. There is also another class or
family of cellulases called glycosyl hydrolases.
Glycosyl hydrolases do hydrolases function, which is
what the previous function was talking about.
This belongs to a family called 61, so it's called GH61.
Currently, there are more than a hundred GH families.
They all do act on cellulose and they have different functions in
which they can act on cellulose. Some of the GH families are
cellobiohydrolases, which actually chews up the bigger
chunks into two glucosinates. Some of the GH families are
bigger glucosidases, which actually gives rise to the
single glucosinate. Some of them are
endoglucidases. Some of them are
exoglucosidases. Some of them have two functions
combined within them, and they have different specificity
as well. So here when we look at the GH
families, GH61 specifically, again here I took these three
structures which are labeled here, and then you overlay
one on top of the other and see that they do match,
very similar, and they look very similar.
Then you have this loop that is different in all of these.
So if you recall the earlier slide, and if you look at this
slide, then you can see that the main core of these two families,
they look the same. They have basically what we call
as the same fold. The fold of the protein is
actually how the protein is going to be in its functional
shape. The shape that the protein
takes. The fold of a protein actually
kind of gives us an idea about the structural function of the
protein. So both these families, even
though they are different, they have the same, very similar
structures. And the active site also is
quite --, within them, structurally, what we call,
because when we overlay these structures and then when we look
at these amino acids that really carry out this function of
breaking down the cellulose, they actually overlay one on top
of each other really nice. With this, we can actually
kind of extrapolate and say that these two proteins have
a very similar mechanism of action, even though there are
slight differences within them. They both do that same oxidation
kind of mechanism, and they're made of metal.
Here we have one enzyme which is bound to zinc.
The other enzyme is bound to a nickel or a copper.
So all the three metals are actually divalent,
so they are all of two-plys, and are two-plys.
It needs a divalent metal using links, we can act on this
cellulose. So the active site area, when we
look at both the proteins, so here is the protein
in a surface representation, and then the active site,
the real place where the activity happens is shown in red
here. So if we take this molecule
and look at it going from a side view here to a top view.
So that's where the active site is.
Here we can see that this part of the protein is actually quite
flat. Not exactly flat, but it's
quite flat when compared to like this region.
The active site, it is where this kind of like a flat area,
relating to protein surfaces, is present.
Then we look at the other family of enzymes, the GH61, here it's
shown in the side view. This is where the active site
is. You can see looking at the
grooves here, that this is basically that's where the flat
surface is. It has been hypothesized and we
think that this is the region that this enzyme binds to, so it
has an inherent flat area or a planar subface with which it is
able to bind to, the flat surface of the cellulose.
It can bind to it, and instead of pulling one chain out just
like the model that I showed you, instead of pulling one
chain, it can actually may not pull it out, it just has to
travel across the surface and chop, chop, chop, and then make
some internal links all over the place so that the next enzyme
can actually come and start chewing up, and then breaking it
into much smaller pieces. This is what the hypothesis is,
and the main area of research that's been driving us.
That's the active site. So the active site mechanism,
just to show you the detail, you have this divalent metal,
here in this case the copper, you have a water molecule, and
then you have one amino acid, the histamine,
another amino acid histamine, and the third amino acid.
So these three amino acids are the active site residues.
They are commonly spaced in such a way that along with the metal
and the water molecule, they actually make kind of a planar
region. So it can be visualized like
those atoms are arranged on a plane.
So this plane is actually going to be parallel onto the surface
of the cellulose. It can travel back and forth and
then identify one random area that it can make a cleavage.
So you have the different, other experiments that show that
this is the exact place where this copper metal is present,
and then the water molecule is present.
So when we look at the chemistry of these reactions, so as I
mentioned these enzymes are oxidators, so they use an
oxidation reaction to actually clear it rather than using a
hydrolyzing effect, which is what the other cellulases use
for the amorphous regions. So here we have different
mechanisms that are currently being used, where you have this
copper ion allowing the water molecule, and that uses a
hydroxylation step to actually bind this oxygen bound to the
copper to this part of the cellulose, and then finally,
you have the electron transfer happening between the metal and
oxygens, and then finally giving rise to a cleave product,
like how you see here. Depending on which carbon acts,
whether it's the first carbon or the fourth carbon, you can get
two different products. So you have to go to go the
first carbon, then you have the mechanism that the oxygen and
the electron and then the hydrogen gives rise to one
water molecule, then you have the nucleic that happened
to break this bond. If it was acting on that fourth
carbon, then you can have this kind of a product from there.
We still have no correct, or we do not have an exact idea of how
this happens, because we do not have a structure of the enzyme
allowing the substrate, which is the cellulose.
So here so far, the structures that have come out, have only
the protein in isolation. So if we have the protein
allowing the substrate bound to it, then we can make an idea, an
educated kind of like an exact way of saying yes, this is the
carbon for, on which it's going to act, and this is all that's
going to come of it. But since we do not have it, we
can actually make models. We can make models using the
three-dimensional shape of the protein, using the
three-dimensional of the cellulose that's available.
Some of the models that a current or recent paper came out
from the group in Lawrence-Berkeley, is that you
have this protein and you have these amino acids which are,
in the structure, they are on the planar surface.
And if you see that they are actually kind of in layman
terms, like more parallel to the surface of the cellulose.
This can be that, you know, the protein is able to bind to the
cellulose. Similarly, this one protein
shown here is the GH61, one representative
of the GH61 family. Here is another CVM-- family
protein, which is usually you see this, which is quite planar
in terms of binding to the cellulose.
This is the active site, with links that help it to cleave it.
So these two histamines act on the planar and then make
cleavages on the cellulose. So, kind of questions that we
want to ask is that, using whatever existing data, because
of these structures, can we identify some features or some
characteristics of these proteins that can be used to
identify another set of new enzymes and make a good
educated guess or prediction about whether they can also
cleave a cellulose or some other complex polysaccharides.
So using structural bioinfomatics, that is what I do
basically, is I use protein structures and I use the
computational power to model them and then make simulations,
and then understand the mechanism of how these enzymes
can act. So, in a way it's a prediction,
but it's kind of like making an educated guess and you know,
evaluating it with further experiments and then identifying
the real or exact mechanism or action.
So, with that, this is my last slide.
I'd like to point out that GLBRC has a website:
GLBRC.org and you can subscribe to their email and newsletter.
The newsletter contains all the updated research about what's
happening in GLBRC with all the four different areas that we are
currently working on. With that, I'd like to thank
my supervisor George Phillips, my collaborators Brian Fox,
and Cameron Currie, and two other post-docs
with whom I'm currently working on this project, and
John Greenler, he's the person in charge of outreach
for Great Lakes Bioenergy Research Center,
and Bacter for funding. Thank you.
[applause]