Hello all! 🙂
Tokyo is getting colder and colder but it is still not that cold and it’s almost sunny everyday! ^^
Now I’m gonna tell you a bit of the research that i will do in Tokyo Tech. As i have told you, I am now in Tomoko Matsuda Laboratory in Bioengineering Department of Tokyo Tech. The big topic of all the researches conducted in my laboratory is about green chemistry.
At first i really did not get what green chemistry is. But actually green chemistry in my laboratory is the usage of microorganisms enzyme to catalyze bioorganic chemical reaction.
During the first month in my lab i was being introduced to all of the topic conducted in my lab. Basically we’re working on those 3 sub topics :
- Baeyer Villiger Oxygenases from Fusarium sp.
- Asymmetric Reduction of Ketones Derivatives by Reductases from Geotrichum candidum
- Reaction of Isocitric Acid (i’m really unsure about this one)
At first i was pretty much shocked by the fact that it all involve so many organic chemistry (i’m very lame at organic chemistry, really lame, not forget to mention my organic chemistry score was only B). The first thing that i’ve learned is a whole cell reaction and analyze the product using Gas Chromatography. And then Silica Gel Chromatography, Evaporation, HPLC, I was like… ok, dear God, am i in the wrong place??
But then now i’ve already in the acceptance phase and ready to wrap this research up! It’s also because my sensei picked some sub-sub-topic that is still related a bit to microbiology. And i’m working into some cool stuffs which is MANIPULATING ENZYMES! This is a combination of enzymology (which was my favorite object) and Molecular Genetics (although i am actually not so into Progenmol)
Remember my old hope of being a food microbiologist? Well, let’s forget it for a while, because not everything in the world can be happening just like what you want to be! Be mature! 🙂
So my research topic is….
RANDOM MUTAGENESIS OF ACETOPHENONE REDUCTASE (APRD) GENE OF Geotrichum candidum
What is this all about?
So basically, coming back to the green chemistry idea, many kind of reduction reaction of organic chemistry still involves chemical catalyst. And we have known that reaction using chemical catalyst sometimes has to be done in a harsh condition and can give some unnecessary residues and by products. This is definitely not a green process.
Meanwhile, we still have a thing called enzyme! 🙂 Enzyme from microorganisms can be isolated and generally preferred because it is a natural thing. The reaction can proceed in a mild condition, the solvent can be water, etc. etc. something much greenier than the chemical catalyst.
So now we have this cute fungi called Geotrichum candidum and it is already known that this fungi has a NADH Dependent Acetophenone Reductase Enzyme (known for APRD for short) that can promotes asymmetric reduction of acetophenone and its other derivatives! This enzyme is somehow really excellent 🙂
The enzyme only catalyze the reduction reaction from ketones into (S) alcohol (ok, remember this stereoselective properties from organic chemistry lesson?) with enantioselectivity of >99,99% (almost perfect!) and the yield is also very high, >99%.
The current enzyme is actually already very excellent. It has good thermal stability, pH stability, and also quite good stability in organic solvent. Why Organic Solvent is so important? The reason is we want to operate it inside a cofactor regeneration system (with excess 2-propanol). You can see the system in this figure :
But still, my sensei want to make the enzyme super excellent. So one of my senpai is doing a genetical approach to make better enzyme by doing directed mutagenesis and i am told to do a random mutagenesis.
I guess one more question must appear in your mind? What is random mutagenesis?
Random mutagenesis is way to alter genetic code thus later on will alter the amino acid sequence and it is also expected to change the properties of a protein or enzyme. It differs than the directed mutagenesis than we don’t exactly have to know about the crystal structure of the enzyme. We only need to have the access to the genetic code and do random mutagenesis. The method that i will use is error prone PCR (kind of PCR that generates error rather than preventing error). Later on we will transform the plasmid with the altered gene into E. coli and we have to find the successful mutant that has the better properties than the wild type enzyme.
Right now i’m still in a big dilemma of finding a screening method for a successful mutant. In a random mutagenesis we will find thousands of colony and if i check the activity one by one it will waste so much time. So i have to do some quick screening on the agar plate itself, and the only option i think is colorimetric screening.
Colorimetric screening usually using the oxidation reduction reaction. So the color compounds will later on be reduced or oxidized to produce color that can be seen by naked eyes. Or the color formation will differ according to the NADH level in the reaction. That seems to be simple but actually it is not!
Right now i’m working with formazan salt colorimetric reagent. The type that i’m using right now is MTT (or her full name is 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The color change depend on the NADH level on the environment. In a rich NADH condition it is purple and as the NADH is used up it will change into yellow. It’s quite suitable with my enzyme reaction because it is using NADH and in the end of the reaction it is expected that the NADH is used up and the color will change from purple to yellow.
and although i’ve several times adjusting the substrate concentration, reagent concentration, enzyme concentration, etc. the color still cannot change 😥 and i’ve been only using the cell free extract (the crude extract of the enzyme) still not using a whole cell system.
and here is what i’m gonna do next!
Oh what else that can i say rather than..
May God bless you all (all YSEP participant and their sotsuron) and also my friends back in Indonesia whose struggling with their final project!
Loads of love,
Afifa Ayu Koesoema