 I'd like to enter this next speaker. This time, Professor Matsuda talk about utilization of carbon dioxide, absorbent, and substrate for biocatalysis, so please. OK. Thank you very much for that kind introduction. I'd like to begin by thanking the organizers and the AMANA enzyme for inviting me to this first North America enzyme technology symposium. It's a tremendous honor to be here to speak and to share my study on and utilization of carbon dioxide as a substrate and absorbent for biocatalysis. As all you know, atmospheric carbon dioxide is increasing and causing an environmental problem, so it's urgent to think about how to use it. Actually, pressurized carbon dioxide is very good green solvent. It's sustainable, and it's practical. Because it's chemically inert, it's no-flammable. It has low viscosity and high diffusivity, so you can improve the reaction rate, possibly. And because it's non-popular, so it's this hydrophobic, pharmaceutical-important compounds. And the product can be easily recovered without purification step. You just have to decrease the pressure to the atmospheric pressure. Therefore, in the industrial process, they have been used since 1970s. For example, extraction process of flavor and caffeine from coffee and cholesterol from mayonnaise, and also for the dry cleaning process. When you think about carbon dioxide as a substrate, it's not good substrate, however. Although it is attractive as a C1 building block, it's challenging to use it due to its low reactivity. However, pressurized carbon dioxide is better. It has high density of carbon dioxide, which makes the reactions using carbon dioxide as a substrate favorable, and shifts the equilibrium of the carboxylation, de-carboxylation, to other carboxylation. But you need catalysts to do it. And there are enzyme and also chemical catalysts have been developed. But I use enzyme because of these merit. It's sustainable, it's selective, and it's versatile. And I think these are all have been demonstrated from this morning, so I just skip this part. As a solvent for bio-catalysis, beside aqueous solvent, there are organic solvents, ionic regin, and also pressurized carbon dioxide can be a solvent for bio-catalysis. Today, in my first topic of today, I use supercritical carbon dioxide for the lipase catalyzed reaction. And I also use the CO2 expanded liquid, which is a mixture of organic solvents, and pressurized CO2. I don't want to use conventional organic solvents due to the environmental concern. I use bio-based liquid. This is monophasic. In my second topic of today for carboxylation, I use water and pressurized carbon dioxide by phasic system. So I use carbon dioxide as both substrate and products. I mean, solvents, sorry. So this is the drying, and I'll start with lipase catalyzed transethic acidification. Lipase has been used widely due to these merits and its catalyzed hydrolysis and its catalyzed transethic acidification in organic solvents. So the replacement of conventional organic solvents to green alternatives, such as carbon dioxide, is important. This shows you the phase diagram and the image of carbon dioxide. This green square is carbon dioxide. There are solid phase, liquid and gas, and there are one more phase, supercritical fluid phase, above its critical point of 31 degree and 73 atmosphere. If you mix carbon dioxide with ordinary liquid, then you can get the CO2 expanded liquid. This shows you liquid and gas as carbon dioxide, and now it's getting to be a supercritical state when you see only monophasic system. Supercritical carbon dioxide and CO2 expanded liquid. Both have these gas-like high diffusivity and liquid-like high solverizing power. Bio-catalyzes in pressurized gas has been reported for the first time in 1985, and high enantiose reactivity of lipase catalyzed reaction around the critical points and acceleration of the reaction by using lipid-coated beta D galactosilace in supercritical carbon dioxide have been reported. But when I began this kind of research topic in 2000, there were no practical organic synthesis using supercritical carbon dioxide. So I established a flow reaction using lipase for chiral synthesis using supercritical carbon dioxide. I beautifully introduced this. So in this system, substrate, racemic substrate and carbon dioxide, supercritical carbon dioxide, is sent to a column reactor packed with lipase, and then product will coming out here. Before the back pressure regulator, the carbon dioxide is in supercritical states so that it dissolves substrate and product. But after this, it's get to be a gas. So it's escape, and the product can be easily recovered. I did a three-day operation of this system and the conversion said that 50% and E-value were 12,000. Using 1.73 gram of immobilized enzyme over time, 221 gram of racemic substrate was converted to the product of 99% EE and the remaining substrate of 99% EE. And the spacetime yield was improved by 400 times, compared to the batch system. The proton NMR spectrum of the product without any proliferation is contained no byproduct at all. So without using any organic solvent at all, racemic substrate was converted to chiral products. So after successful success in this, establishing this process, I tried to decrease the pressure. So I moved to the liquid carbon dioxide and then now I'm working using CO2 expanded liquid. It's a mixture of carbon dioxide and the liquid. So pressure and temperature can be any pressure or temperature. So the pressure was decreased. When you pressurize with carbon dioxide on the liquid, it's expanded like this. And transport property, like diffusability, are improved by dissolved carbon dioxide of just like characteristics. When I compare organic solvents and carbon dioxide expanded liquid, and the liquid or supercritical carbon dioxide in terms of viscosity, diffusability, solubility of polypone compound, and amount of CO2 waste solvents generation and working pressure. This carbon dioxide expanded liquid is just in the middle of these two and has a merit of both. When you work with carbon dioxide expanded liquid, you need the liquid components. And as a liquid component, I use bio-based liquid. One of them is methodative. This is one of the most attractive bio-based liquid. However, when this reaction is reported, the conversion is very low. So I decided to expand with carbon dioxide. At first, I put 1.0 millilitre of carbon dioxide, and then if I pressurize it with carbon dioxide to 5.8 megapascal, it's expanded to 8 millilitre. Other bio-based liquid, as well as conventional liquid like hexane or vinyl acetate, also expand like this. So CO2 fraction can be controlled by the pressure. And the solvent properties are tunable so that the enzymatic reaction can be controlled. At first, I tried this reaction, trans-acetic refaction of one adamantel ethanol by carbene lipase. This is difficult to substrate because it's bulky. So when I do this reaction in bio-based liquid without CO2, then conversion was low. But when I expanded this by carbon dioxide, green is bio-based liquid and blue is conventional solvent. Then conversion dramatically increased. It's also increased in the case of hexane. So hydro-phobicity, well, hexane is hydro-phobic without expansion, but it is expanded. So not only other factor other than hydro-phobicity affects this improvement. I examined this reaction in detail. I changed the temperature and pressure. More fraction corresponding to the pressure and the rate increase, but rate is affected by the temperature. So it's normalized based on the value at the zero fraction. Then it's get on to the same line because I also re-prodded this data to using polarity on the x-axis and then I normalize it. Then it's correlation between polarity and the normalized activity reaction rate is also unseen. So that end-dimetric activity are controlled by solvent property, which is controlled by temperature and pressure for the first time. I also checked other substrates using other various bulky substrates. These are all difficult substrates for the right base. When I compare method DTF and CO2-expanded method DTF, the difference is obvious. I expanded this system to also substituted one phenyl ethanol because these are important pharmaceuticals, important components. And the results was that needle disturbance afforded only low conversion where CO2-expanded liquid reaction gave higher yield. This shows user detail. It was seen using method DTF and also in Hexam. When I tried tetralol derivative, same thing happened. I mean, I use this compound because it is important pharmaceutical. I used more hindered one tetralol derivative and less hindered two tetralol derivative. For both substrates, it is improved by expanding with CO2. When I compare the rates, then in the case of one tetralol, it was 2.9 times improvement. This is more hindered. And for the case of two tetralol, it was only 1.3 times improvement. I also tested other one tetralol derivative and I changed the substituents. Then when I compare between and without carbon dioxide, it was improved up to 40 times. And when I compare between substrates, in the case where without using carbon dioxide, it was the difference between the substrates was 13 times. But when I used carbon dioxide, the difference get to be only 3.8 times. So I think about why the improvement was obtained. There are several factors we have to think about. First, hydrohobicity. For the case of methyl derivative, I think hydrohobicity affected the reaction rates. But for the case of hexane, it is, hexane is a hydrohobic, so it did not account for this. Flexibility of the endomethic is probably the most important factor to explain this. Calvary structure in the conventional sorbents has seroselective pocket. But in the liquid carbon dioxide, it's calculated it has more flexibility. So I think same thing may happen to the CO2 expanded liquid. Transport polarity of the sorbents is maybe affected by the carbon dioxide. It may be affecting a little bit because the degree of acceleration by CO2 depends on substrate. If transport property is the main factor, then all substrates will be improved. Pressure is maybe no because I did the reaction using pressurized methyl derivative and the pressurized hexane at 6 megapascals without carbon dioxide did not affect the activity. So flexibility of end time is probably the most important factor. So I so far explain the success for reaction using supercritical carbon dioxide as a sorbents. And I explained about rate acceleration of the reaction of bulky substrates using carbon dioxide expanded bio-based liquid. Next, I will talk about carbon dioxide as a substrate for carboxylation. I will introduce two reactions. They carboxylase catalyze carboxylation and isocytrate the hydrodynamic catalyze reductive carboxylation. I'll begin with the first example. In nature, carbon dioxide is used by photosynthesis by enzyme and everything necessary is produced. We should do it also using enzyme to make everything we need. From 1970s, light-based catalyze reaction was found to be reversible so that it's the hydrolysis in the presence of water but it's the esterification without carbon water. Our goal is to use the carboxylase for the reverse reaction of the carboxylation in the presence of carbon dioxide. Professor Nagasama and Yoshida in GIF University found the carboxylase from Bacillus Megathenium which catalyzes the carboxylation by law efficiently. And I wonder if equilibrium is favorable for carboxylation and the high concentration of carbon dioxide. With collaboration of their group, I tried this reaction under supercritical radical carbon dioxide and keeping pressure to be ambient. Then the yield difference was obvious. When I checked the pressure dependency, it's the yield was highest just before the critical pressure but it decreased maybe because of the carbon dioxide dissolved in the water layer decreased the pH or the activation by the carbon dioxide happening. So in this first example, high concentration of carbon dioxide was effective for the carbon dioxide but at the same time we also need better enzyme. Then I moved to the second example. Group of enzyme including are acetylate dehydrogenase and the malate dehydrogenase catalyzes the carboxylation and reduction. I used some of plasma acetophenome acetylate dehydrogenase and glucose dehydrogenase to do carboxylation reaction using gaseous carbon dioxide. In the reference, it is reported that organic solvent tolerance of the enzyme is related to the thermostability. So thermostable enzyme is also tolerance to organic solvents. I wonder if thermostable enzyme is also has high stability to add high pressure carbon dioxide. So I used the thermoplasmastophenome enzyme. At first I tried using commercially available enzyme and when I checked the stability, CO2 stability as well as thermostability, it decreased, it was very weak. And then I started using thermoplasma acetophenome because the DNA is commercially available over expression of the enzyme, these three enzymes were successfully done by using E. coli. And then I examined the thermostability and carbon dioxide stability of these two enzymes. Then we found out that it was very stable in CO, under high pressure 10 megaspascal of CO2. And then I did the reaction using at first the IDH. But the reaction unfortunately didn't proceed smoothly and only 4% of the product was obtained. Then I added glucose and glucose dehydrogenase for the recycling of the coenzyme. Then it improved dramatically to 67% from 4%. After that, we tried to further improve the versatility of these two enzymes. To make it more stable and useful, I immobilized these two enzymes by forming enzyme in organic hybrid nanocrystal. This immobilization method is reported by in 2012 and I use this method because it is a very simple method. You just have to mix the enzyme solution in phosphate baffled saring and the metal ion solution. In this case, we use manganese. Well, we tried several and we found out manganese was the best. After mixing these two solutions, you just have to incubate, then you can get precipitated enzyme. So that you just have to do the centrifugation to get this immobilized enzyme. When I compare the activity, reminding activity of the enzyme, then activity of the IDH shown in blue improved to around 200%. However, TA-ZDH activity showing orange decrease, but this is slightly get better using higher ratio of TA-IDH. Then I did the carboxylation reaction using a core immobilized enzyme with an optimized ratio. Then carboxylation yield was obtained here, was better than free enzyme or separately immobilized enzyme. In this case, I use only one megapascal of carbon dioxide. So to conclude my presentation, I utilize carbon dioxide for biocatarsis. First of all, investigated the solvents engineering. Overlipase catalyzed reaction. Flow reaction is successful and CO2 expanded by a very sticky is also useful to expand the substrate. Secondary, I need the carboxylation reaction. And for this reaction, carbon dioxide pressure was necessary to shift the reaction equilibrium to our carboxylation. But for this reaction of reductive carboxylation, NADPH recycling was necessary. So at last, I'd like to acknowledge the individual who did this work. I appreciate my students and staff in our laboratory since 2004 and the core average for dedication and hard work. And I also appreciate my supervisor in graduate school and professor Tadah O'Hara there. He was my boss where I was an assistant professor for guidance and support. And also I appreciate the funding I got before. And thank you very much for your attention and I'm happy to answer your questions. Thank you for a very nice presentation. I'm intrigued when you tested your hypothesis about the supercritical CO2 influencing the flexibility of your enzyme. Is there a way, and maybe this is a naive question but please bear with me, is there a way of changing the concentration? Basically, can you change the liquid dynamics or the fluid dynamics of the solvent by changing the ratio of the supercritical CO2 with a co-solvent in order to see whether there's a concentration dependency that you can demonstrate as far as enzyme flexibility or the consequences of enzyme flexibility is concerned? Well, conversion increased according to the CO2 pressure but for the flexibility of the enzyme is actually impossible to actually measure because it's high pressure. And I only can do it in the simulation. And I haven't done the simulation using a different concentration of carbon dioxide yet. I need to do it, but when it's obvious that hexane and carbon dioxide is different, so yeah, I will do it. Well, actually I have to ask my collaborator to do it. I mean by changing the concentration of CO2 to see the fluctuation, I mean the how root mean squared, well, how much the amino acid, each amino acid, well, some of the amino acid fluctuate, move very much after changing from hexane to liquid carbon dioxide. So I hope I can see the increase gradually when I change the carbon dioxide concentration. Yeah, thank you very much for your good suggestion. So flexibility enhance the activity, but sometimes it increases the selectivity. So if you apply the chiral synthesis or something's needs, the reaction needs specificity, is it some negative effect or not? Well, in this case, we didn't have any negative consequence. In terms of E-value, it stays high, but another example using supercritical carbon dioxide, I'm sorry, it's only in Japanese, I'm very sorry, it's Japanese, but when I use this substrate with low enantio-selectivity, this is pressure and this is enantio-selectivity. When I did the reaction in the three temperature, according to the pressure, it's decreased. Around here, it has high density, and here it has low density, so that when the density is lower, so which means the flexibility is higher, enantio-selectivity is higher, so it's opposite of what we expect, but enantio-selectivity changes sometimes, in this case, especially. Very interesting, okay, thank you. Any other questions or comments? Okay, so I'd like to close your talk. Thank you very much. Thank you again.