University Place Wednesday Nite at the Lab  Crystalline Polysaccharides to Biofuel  Raghu Yennamalli  June 13, 2012 UP727   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.  So, 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]