cc
Nite at the Lab. >> Welcome everyone to Wednesday
I work here at the University of I'm Tom Zinnen.
Center. WI also work for UW-Extension,y
Cooperative Extension.
other sponsors, OnWisconsin Public Television,ur
the Wisconsin Alumni Association
thanks for coming to and the Science Alliance,
We do this every Wednesday Wednesday Nite at the Lab.
Tonight it's my pleasure night, 50 times a year.
Raghu Yennamalli. to He obtained his PhD fromyou
Jawaharlal Nehru University in New Delhi, India,
in computational biology and
His research topic at that time was designing drug molecules
to prevent the infection
which was epidemic in India. of dengue virus,
post-doctoral research at Iowa Raghu did his first
introduced to the field of State University, where he was
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
cellulose. act on the crystalline form of
many of us would really like to have liquid fuels that can go in
to tanks of our cars and trucks.
face in biomass is how to get those kinds of liquid fuels not
just out of the kernels of corn,
the leaves and stalks. but out of the stover,
interesting story to tell us I think Raghu has got a very
Please join me in welcoming him to Wednesday Nite at the Lab.
[applause]
Good evening everyone.
Okay. My name is Raghu Yennamalli, and
thanks to Tom for giving a very kind introduction.
I'm quite excited here to talk
The title of my talk today is ab"Crystalline Polysaccharidesu.
to Biofuels,
This project is part of the Great Lakes Bioenergy Research
Center, or GLBRC for short. It is funded by the Department
of Energy.
research centers here. It's one of the DOE bioenergy
northern points for this. How many of you have seen this
movie, "Driving Me Crazy"?
I think, two or three weeks ago. This was telecast on the TV,
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
alternate fuel-source for our cars has been kind of like a
holy grail. If I'm not over-stepping that
statement.
into my talk now. WFor those who are not familiaro
with the topic, a little primer.
derived from a biological Bioenergy issource.ergy that is
Biofuel is the liquid fuel that we obtain from the same
biological source.
and pelletized biomass, are used For example, like wood, charcoal
generate heat and electricity. and have been used for ages, to
it is called, biogas, are obtained from manure digesters.,
Since I come from India, the
of the country have been biogas plants in the rural areas
to reduce the deforestation quite effactivities.ome extent
This biogas has been used for cooking, not specifically for
running cars, but mostly for
of women there. cBiodiesel, here we've seen howh
many vehicles run on biodiesel
canola oil. that isFinally, ethanol.ybeans,
It is obtained from plant sugars
GLBRC has been focusing more on the sustainable biofuels that
are going to be used in the near
Currently, we have ethanol future.
starch or sugar cane sugars, but that's being produced from corn
from which ethanol can be these are mproduced.er biomasses
Apart from that they're a much
which are more complex than lthese, and developing news,
technology has been one of the
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
from other places. independent and not import fuel
The biomass that's produced is And it's renewable.
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 a cleaner fuel. it's not going to stop.
toxic gas or anything, other It's not going to leak out any
Basically, this part of the tslide shows you that we user.
microbial fermentation to ferment glucose and other
sugars, to convert it to
Glucose is a six carbon fructomolecule.phate.
Fructose is also a six carbon molecule, but slightly different
from glucose. That is converted to pyruvate,
and further down to ethanol,
So for each glucose molecule you which is two carbons.
C-6 to C-2. cCurrent biofuel technology iss.
two parts.
corn. Sugar cane provides the glucosem
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
Starch is a big polymer. the starch.
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,
molecule. Then further, the same enzymes
that are used for converting the
used here to convert it to sugar cane to ethanol, can be
Before I proceed, how many of you--
I want to throw a question
Do you know how much percent to the audience.
currently at a level that we can mixed ethanol gasoline is
[inaudible] Exactly.
It's ten percent, so in scientific parlance it's called,
E10.
percent of anhydrous ethanol to the rest of the gasoline, and
that's what is used today in
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
Apart from this, you have E85, cars.
This can be used by flex-fuel which is alvehicles.l in the US.
The engine is slightly modified from the regular cars.
Here it's 85% of ethanol
In Brazil, currently they have and 15% gasoline.
use 100% ethanol in their technologyvehicles.at you can
It's called E100. E85 is widely available in the
US. The states that are all colored
with yellow have locations
There are about 2,466 locations available in the country where
you can get E85.
It's the fourth largest in terms Wisconsin has 159 locations.
The top one is Minnesota, which ofhas about 366 E85 gas pumps..
There is a cool website called
If you go to that and look for e85refueling.com.
a road trip, if you have E85 gas locations, and if you do
you can plan it accordingly ato 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
Currently, there are nine plants plants here.
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.
starch are called first The generation biomass.corn
The next ones are all second
generation biomass, because they're more complex than the
simple sugars that are there. Agricultural crop residues
actually amount to the highest
the US. bThere has been a study that'sin
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
things, what the amount of particles and output and other
every year in, say, 2030, or biomass that can be achieved
This document is actually public 2050.
Basically, they divide entire biomass to be produced, making a
peak of a billion tons per year,
to be from agricultural, then asome of them being from forestg
residues. Like here you see logging, and
other things. The third part is urban waste,
municipal waste, from which is
biofuels can be made. alWhen we look at, across theich
entire country, we can see, based on how much tons are being
produced in each state, based on
regions that are marked blue and the counties, you have the
They have 250 to 500 tons, red.
of biomass. 1The regions in red are aboutn
500,000 tons per year.
you have at least a minimum of So when it comes to Wisconsin,
150,000 or 200,000 tons of an average of, like, say,
Coming to the GLBRC, or the bGreat Lakes Bioenergy Resource.
Center, the mission is very simple.
It's by grant.
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
northern center for GLBRC. Wisconsin, Madison is the
the Lucigen Corporation. We have another partner here,
University is also a partner in And the this.gan State
Apart form this, you have two national labs, Oak Ridge
National Lab and Pacific
also partners with us. Northwest National Lab, who are
Missouri, Columbia, Illinois And Iowa State, University of
University of Minnesota, State, Cornell, and the
This whole group forms the Great University of Toledo.
We aren't the only one. LaAcross the country there arer.
three bioenergy research
They are called BRC's, the centers.
One is the DOE Joint Bioenergy Bioenergy Research Center.
located in the west, where the Institute, which is mostly
Berkeley National Laboratory. In the east you have the DOE
Bioenergy Science Center, where the Oak Ridge National
Laboratory is the northern
Then you have the Great Lakes Bioenergy Research Center
that the University of
is the northern point. Wisconsin-Madison
scientists, staff and students Currently we have about 400
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
GLBRC has four main areas in part of this team.
basic research in. which they concentrate their
sustainable biofuel practices, The first one is to have
spectrum of procuring the right type of biomass, developing thee
right type of biomass to
very sustainable pipeline. ethanol, and then making it a
biomass. The second one is improved plan
where you develop new varieties Reseaof the crop, done
new varieties of the plant, that some of the hurdles
that we are facing right now
in the future. would be much easier to overcome
biomass deconstruction. That is the thing that you have
the biomass from the raw product
converting it from that point to to the basic glucose, and it's
Finally, the improved biomass conversion to fuels, which
actually encompasses the
new technologies being built for technology part, where you have
These four discovery science it.
knowledge about how to areas are going to provide us
specifically cellulosic, sustainably convert cellulosic,
transportation fuels. If you look at the cycle that we
talked about, using the plants
especially the cellulosic abiomass, they use the energy,
from the sun and then it's
they use it in the cell walls, converted to cellulose, which
recalcitrant polymer that nature and cellulose is the most
It's kind of like the hurdle for has made.
Then converting those sugars to biofuels, then biofuels to human
use.
continues back into the plant. Then finally, that cycle
Cellulose, as I just said, is part of the cell wall in a
plant.
layer, or a big wall of Ecellulose, which gives thea
structural integrity to the cell.
Animal cells do not have
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
other polysaccharides such as hemicellulose and an aromatic
compound called lignin. If you imagine this block here
which runs way across this, if
Let me walk you through from you zoom it, it looks like this.
Here you have one single unit of this part to here.
Two units are joined together by glucose.
That's the scientific term for a glycosidic 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.
stacked one on top each other, Many such polymers are actually
This is called a microfibral. aMany microfibrals are placed.
together, and each microfibral
polysaccharide called is coverehemicellulose.f another
Hemicellulose is also made up of
the same as glucose. sugars, but these sugars are not
sugars. Now all these different bundles
of cellulose, covered with
together. There needs to be some kind of
a cementing agent that holds
That's lignin. all of them together.
It's highly aromatic. LignThis helps as a cement.und.
This whole thing is part
So this is what I was just of the plant cell wall, here.
that we have from sugarcane showing and corn starch.chnology
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.
it using heat, chemicals and This biomass, if you pre-treat
cell wall material from the main pressure, you loosen up the
You want to remove the lignin, structure.
to extract only the cellulose the hemicepart of it.d you want
And then you use certain enzymes
convert that big chain of cpolymer into much smalleran
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.
actually use fermentation technology to --, which is the
next generation fuel from a more
So in a refinery, in a complex biomass.
biomass, the process will start cwith the biomass is harvested.
Say you have an agriculture land
harvested, and then the corn where the corn has been
Most of the stovers, for those stover is left.
of the plant which is left after who don't know, stover is part
leaves, the roots. harvest, like the stems, the
animal feed. This stover mostly goes as
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
chemicals to make this cellulose pretreated with heat and
integrity and apart. come out off its structural
act. Then it allows the enzymes to
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
and you can use microbes, like transport it to one of the vats,
can convert the glucose through say yeastfermentation.zymes that
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
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
These straight lines are places it's like curvy lines.
the cellulose. actually the crystalline part of
ordered. The crystalline part is very
stacked one on top of each other Each polymer chain is actually
particular order. It's like a crystal, like a
diamond or a graphite crystal.
curvy lines is what is more-- The amIt's also presentr these
in the cell wall. So you have a mixture of a
crystalline, and then amorphous,
Now these entire microfibrins aare all covered with
hemicellulose, here in green. Then you have the cementing
agent between each microfiber,
So then you do a pretreatment thprocess..
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
pre-treat the cell wall, so that does -- use ammonia to actually
expanded, or the hemicellulose the cellulose fibers are
cellulose inside is exposed for the enzymes to act on it.e
When the cellulose acts on it,
you have both the crystalline wform and the amorphous regions
all released out for the enzymes
So these enzymes that act on to act.
called cellulases. these cellulose is actually
cellulases. So how does this enzymes act on
the cellulose?
bioethanol production, these So, before this was used for
industrial importance. cellulases had a much bigger
wearing or have stonewashed Ijeans, these cellulases werey
used, and are currently used, to
jeans. make the stonewashed effect on
are exposed out from the fabric Tare more accessible for theat
enzyme to act upon, and they are
So the cloth that is most tightly present is not acted
upon by enzymes, but the loose
So you have a process where the cellulases can give the look of
stonewashed jeans.
using a stone to make that Don't imagine someone actually
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
anti-pilling, they're made of tbasically cellulases, because,
the cotton is also made up of cellulose.
When you have pilling, some of
these cellulases can act on those exposed strands and then
chop them off so you get the
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
termites. sTermites chew on wood.
Wood is from plants.
The termite has bacteria, and Ithese bacteria secrete
cellulases, which is not
and they can digest that wood, produced by termites,
So one of the other cellulose niche organisms that nature has
given us are the Leaf Cutter
You have seen this Ant System.
or the Discovery Channel, where these small ants cut these
leaves and then go in a very
There are two genus of these orderly fashion.
are the Leaf Cutter ants, the Atta and the Acromerex.
Apart from this, you have the Bark Beetle, the Wood Wasp,
and then
which are also a major source the of these cellulases,us,
and they live in cellulose.
GLBRC, specifically,
Leaf Cutter Ant System. tThis is what is calleds
bio-prospecting, like how you go about prospecting for gold,
or oil, here we go prospecting
organisms. about the cellulose-rich
is actually one of the sources And the Leaf Cutter Ant System
has been happening. So in Costa Rica, here you have
this big mound, the ant mound.
leaves, and bring them to the The ants go tmound.tree, cut the
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.
and harvest them, the ants grow this fungus and harvest them,
because the fungus, the things
fungus are actually fed to the that are harvested from the
That's their food. ant larvae.
live, and the ants need the The fufungus to live.ant to
So it's a very unique symbiotic or mutualistic relationship that
they have.
you imagine the scale of this Apaant pit, it's this big. if
This is an excavated ant mound
These are Leaf Cutter Ants. You can see the different
chambers here.
of the people how big this is. And you can see from the size
areas within the same general This is wideregion.in different
So here I'm going to move to...
This website here, Currie Lab,
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.
my feed to the Internet. Unfortunately, I did not connect
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 dirt. thEvening, as now, the nightin
vision is switched on, and you
vision camera works. can see them, like how a night
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
and then they have the area that pits, and then the fungus area,
the larvae. have It's a nice camera.y feed
You can look at it. So this ants and insects-microbe
symbiosis is actually the result
co-evolution between these of ten million years of
Now, it's not just ant and organisms.
There is another organism also fungus.
bacteria. So the ants, as I said, the ants
need the fungus to live, and the
Now, the fungus has a villain in fungus needs the ants to live.
There's one organism exactly its life.
Fortunately, the ants carry a thparticular bacterium whiche.
actually grows on the ant's surface itself.
That bacteria produces
going to prevent the fungus from antibiotics that is actually
pest. So, the ants in fact act as a
kind of medicine, a moving
these drugs to the fungus and medicinekeep them alive. deliver
There are not just one kind of
colonies. There are many kinds of bacteria
there.
GLBRC for this particular niche is that you have an organism
level approach. So you isolate the bacteria that
are present in these ant
You classify them into new colonies.
You sequence their genome. species.
experiments to see which Then you do cellulose growth
grow in a cellulose-rich bacteria from this colony can
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
We can identify the genes that turned.
cellulose. get upward when grown on
going to produce a enzymes to Sdegrade the cellulose, becausey
they are present, or produce
when it's grown on cellulose. mThen you identify the proteinsm
that are secreted when grown on cellulose.
So here is a schematic view of how these enzymes act on
cellulose.
big chain of cellulose. One end of the cellulose is
called a non-reducing end.
end. The other part is the reducing
connected by this bond here. The two glucosinates are
So if you imagine a cellulose Itregion on the bottom here.d.
There is one enzyme called
Endo meaning inside, endoglucanase.
is like a glycosidic bond. and glucanase means that this
internal link in this entire Now this endoglucanase makes an
The reason is that we need some kind of place in there we can
start chewing up the cellulose
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.
the exposed area and makes a What this does, is it acts on
which consists of two glucose long chain iunits.aller parts,
This is called cellobiose.
us to make it into ethanol. NoWe need to take this furtheror
into individual glucose units.
betaglucosidase, which actually Sobreaks the single glycosidiced
bond and finally gives the
So, three enzymes: glucose.
cellobiohydrolase, endoglucanase,
This is kind of like a minimal and betaglucosidase.
At the minimum, you need three set.
polymer into glucose. eHere's where my part of these
research comes in.
a structural perspective. As iSo, how do we visualizeioned
proteins? Because all these enzymes are
basically proteins, and we know proteins are made up of amino
acids.
protein. They are the building blocks of
together to make it one big So, many protein.ids join
Then, one of the things about structural research of proteins
is that we need to visualize
It's not that we can visualize these proteins.
different abstract methods, a kind of like abstraction to
understand many different concepts.
So here I have the same enzyme
I have shown it in different in the same orientation.
Here on the top you have it in ways.
Basically, if you think of two the line representation.
bond, you can draw a line atoms, abetween them.ined by a
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.
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--
like a skeleton. You can think of it in terms of
regions, the main carbon, the alpha carbons that are connected
along the chain.
shown here, and they're And oconnected with a line.s are
This one, here it's colored in
structure. terms of its secondary
many things, but basically a I don't want to confuse you with
called a primary structure. Then when we fold it into a
globular or big, massive
basic structural units in it. Some regions can form a helix,
and some regions can be as
So here the beta strands are lines.
Here you see a helix in red. colored yellow.
these two that are shown in The green are called loops.tween
They're basically very loopy
The same representation can be regions.
the helixes, just like the helix made in this way, where you have
strands. is, and there you have the beta
arrow, so it shows the direction Here you have, it's shown as an
Here is another representation in which the chain runs.
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
like a sphere. nucleus, and you make it look
and as these two atoms are Then you have an oxygen,
joined, and certainly -- joined, then the spheres are
That is a visual representation is of how these atoms are joined
with each other.
empty space that's going to be It gives you an idea about the
bonded with each other. tThe final representation hereve
is called a surface representation.
This is something similar to
wrap it around with a cloth, and like if I have an object and I
in there a little bit if there then I allow the cloth to sink
project out where there is a is a valley, and then let it
So it looks like a continuous surface all across it.
This surface representation is
to see where these proteins can vebind with the cellulose, orave
where it is going to bind with
all around it. these water molecules which are
computer. OWhat about in reality?e
For some people, it's very
visualize the representations diffthat I talked about.and
So here we have, using the 3-D
the same protein structures in printer technology, we can print
So this looks something like 3-D form.
a printer using-- PlayHere you have the helix.ally
And here you have the beta
You can see the helix, strands here.
here. aYou can actually rotate it and
get an idea about how this
Here, this protein is colored in protein looks.
domains in it. two colors because it has two
here, which is actually this Similarly, you have this protein
proteins. If you see this part, you can
actually see the same thing on
This part is one protein and this side as well.
This is the label. this is another protein.
so you can see it. So I can pass it around
a look at these models. They're quite lightweight.
Now we have this 3-D technology.
those days if they wanted to What did people used to do in
How many of you know about visualize a protein?
Wow, that's nice. So these stereograms are
nothing but you have two images.
between these two images, just There's a slight difference
They have a kind of parallax like looking at a 3-D movie.
What you would want to ideally effect.
and make the two white circles do is kind of squint your eyes
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
then you can actually get the three-dimensional view in that.d
So are you able to get it?
These stereograms of-- It's quite easy.
images that are non-proteins, Stewhen we look at protein to
structures, you can actually
So when you try to do the same make them look in stereo.
image here, when you try to move effect, and you see a central
one in the center, you can these two black dots to merge as
regions, these lines here, actually see that these loop
and some of the lines appear in actually athe back.the front,
That's how people used to view
had this technology and other 3-D monitors with which we can
view these protein structures.
Oh, it's too far apart? Probably, you need to move.
[laughter]
I kind of move my head, Wyou 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
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
So this is endoglucanase. Here you see that it uses these
two amino acids, there actually kind of like rings,
-- and residues.
an anchor. They act as kind of like
actually slide on top of this Thchain of cellulose. to
It also has an auxiliary domain,
In the structure, we don't see which is connected by a loop.
is very loopy and very floppy, the loop, because the loop
confirmation for it. and we do not have proper
actually anchor the protein onto So the aim of this domain is to
domain. So here I have a small model of
the same. Here you have the big domain,
which actually does the job.
being pulled inside. Here you can see one cellulose
the other domain which helps Then the CVM, which is actually
anchor properly and make the job this part of the protein to
I'll pass this around too, so you can get an idea about it.
Coming to the cellulose again,
cellulose are in the cell walls, mentioning how crystalline the
forms of cellulose. They're called polymorphs.
It just differs on how these
to each other. chains are arranged in respect
then they're called Polymorph I. If they're arranged in parallel,
anti-parallel, then they're called Polymorph II.
One of the polymorphs, I-Beta,
So you have this chain of cellulose arranged parallel-ly.
The way they're oriented is they're all pointing in the same
direction.
So when looking down on a cellulose chain, this is how it
looks.
You can see how flat this cellulose is when you look at it
form the side.
enzymes that can only act on the Now, there are a handful of
Previously, there was not much crystalline part of cellulose.
these enzymes act on it. information of a level about how
been happening, not only in LGLBRC, but in other places asas
well. We are trying to understand and
they are also trying to understand how these enzymes act
on these crystalline form.
cellulose, again, to re-create, is the most hard part that the
cellulases can act on.
actually the same cellulose much But as the amorphous region are
pre-treatment processing, they expanded, because of the
through them. arThe way these things act, ish
that they do not act by themselves.
Here the cellulases, the
which act on this crystalline cpart, if when in presence of
another enzyme which is shown here in blue, the rate
increases, the amount of
when these two enzymes are degradation actually increases
So this enzyme actually makes an present.
part, and then the other enzyme istarts from that created linkne
and then chews on this and makes
the cellobiose. So these cellulases, previously
thought just bind to the
to act on them and degrade them. polysaccharides, but not enough
carbohydrate binding module. So that's why they are called a
indicate they are a domain, a Moduleloop working.ord to
Then they bind
That was their original function, it was thought.
But currently, we know
to it, but they clean the thaglycosidic bond and acting
like an oxidase.
oxidation reaction that they use So they use an oxidase,
nucleotide and then act on this and break this glycosidic down.
So to visualize this reaction,
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,
You can think of something like lithat.at.
This is what these enzymes do.
structures of these enzymes at So currently there are a few
to visualize these workings and ouunderstand how these act. us
Here are the four protein IDs.
So, pdb id is a beta base that This is called pdb id.
deposited, and they are publicly these preliminary structures are
They are all funded by the NIH accessible.
So when we look at all these and NSF and other projects.
on top of the other, we can see fthat they all look very, verye
similar.
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
alignment of the helixes here. beta strands here, and very good
has an extra loop, which is Of cdifferent from the rest.ures
The reason that's shown here is the active side.
So in proteins, you can basically divide them into two
regions.
the function. That is where the main reaction
happens.
and stay and then carry out this So some residues come with it
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.
gives support to this is called Thea structural function.ich
You can kind of divide the
basically. protein into two parts,
family of cellulases called Thereglycosyl hydrolases.ss or
Glycosyl hydrolases do
what the previous function was hydrolases function, which is
This belongs to a family called 61, so it's called GH61.
Currently, there are more than a
They all do act on cellulose and hundred GH families.
which they can act on cellulose. they have different functions in
cellobiohydrolases, which actually chews up the bigger
chunks into two glucosinates. Some of the GH families are
bigger glucosidases, which
single glucosinate. actuaSome of them areo the
endoglucidases. Some of them are
exoglucosidases.
combined within them, and they Some of them have two functions
as well. have different specificity
families, GH61 specifically, So here when we look at the GH
structures which are labeled again here I took these three
one on top of the other and see that they do match,
very similar, and they
Then you have this loop that is different in all of these.
So if you recall the earlier
slide, then you can see that the slide, and if you look at this
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.
takes. The shape that the protein
kind of gives us an idea about The fold of a protein actually
protein. the structural function of the
though they are different, they have the same, very similarn
structures. And the active site also is
quite --, within them,
because when we overlay these structures and then when we look
at these amino acids that really
breaking down the cellulose, carry out this function of
of each other really nice. they actually overlay one on top
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
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,
It needs a divalent metal using links, we can act on this
cellulose.
look at both the proteins, So the active site area, when we
in a surface representation, so here is the protein
the real place where the and then the active site,
here. acSo if we take this moleculered
and look at it going from a side view here to a top view.
So that's where the active
Here we can see that this part site is.
flat. of Not exactly flat, but it'site
quite flat when compared to like
The active site, it is where this region.
relating to protein surfaces, is this kind of like a flat area,
Then we look at the other family present.
shown in the side view. oThis is where the active sites
is. You can see looking at the
grooves here, that this is
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
able to bind to, the flat plasurface of the cellulose. is
It can bind to it, and instead
like the model that I showed of pulling one chain out just
chain, it can actually may not you, instead of pulling one
travel across the surface and chop, chop, chop, and then make
some internal links all over the
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
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
They are commonly spaced in such the active site residues.
and the water molecule, they a way that along with the metal
region. aSo it can be visualized liker
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,
this is the exact place where othis copper metal is present,at
and then the water molecule is
So when we look at the chemistry present.
mentioned these enzymes are ooxidators, so they use anI
oxidation reaction to actually
hydrolyzing effect, which is clear it rather than using a
for the amorphous regions. what the other cellulases use
mechanisms that are currently being used, where you have this
copper ion allowing the water molecule, and that uses a
hydroxylation step to actually
copper to this part of the bind this oxygen bound to the
you have the electron transfer cellulose, and then finally,
oxygens, and then finally giving happening between the metal and
like how you see here. rise to a cleave product,
whether it's the first carbon or Depending on which carbon acts,
two different products. tSo you have to go to go theet
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
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
So here so far, the structures the cellulose.
the protein in isolation. thSo if we have the proteinly
allowing the substrate bound to it, then we can make an idea, an
educated kind of like an exact
carbon for, on which it's going to act, and this is all that's
going to come of it.
can actually make models. BuWe can make models using thee
three-dimensional shape of the
three-dimensional of the protein, using the
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
in the structure, they are on thesthe planar surface. are,
And if you see that they are
terms, like more parallel to the actually kind of in layman
This can be that, you know, the protein is able to bind to the
cellulose.
shown here is the GH61, Similone representativetein
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
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
of these structures, can we widentify some features or some
characteristics of these
identify another set of new prenzymes and make a good to
educated guess or prediction
cleave a cellulose or some other about whether they can also
So using structural bioinfomatics, that is what I do
basically, is I use protein structures and I use the
computational power to model
and then understand the tmechanism of how these enzymes
can act.
but it's kind of like making So, in a way it's a prediction,
evaluating it with further an educated guess and you know,
the real or exact mechanism experimentsor action.identifying
So, with that, this is my last
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,
and Cameron Currie, myand two other post-docsx,
with whom I'm currently working on this project, and
John Greenler, he's the person
for Great Lakes Bioenergy in Research Center,ch
and Bacter for funding. Thank you.
[applause]