We describe a protocol for the light-catalyzed generation of hydrogen peroxide — a cofactor for oxidative transformations.
The overall goal of this experiment is to drive enzymatic reactions using visible light. In this approach, light is used to convert fatty acids to terminal alkynes under mild reaction conditions. Decarboxylation requires the breaking of CC bonds.
And this bond is very stable, which makes this a very difficult reaction. The use of light is a sustainable approach to achieve such a difficult reaction. We first had the idea for this method when we tried to add hydrogen peroxide directly to the reaction, which lead to inactivation of the enzymes.
The main advantage of this technique is that it supplies a steady amount of hydrogen peroxide. First we used purified OleT for light-driven biocatalysis to characterize our enzyme. Later we also tested crude extract which would be important for the development of industrial applications.
After cloning the synthetic OleT gene into the expression vector and transforming the plasmid into E.Coli as described in the text protocol, inoculate the cells in two test tubes containing three milliliters of LB medium with 100 micrograms per milliliter of ampicillin. Incubate the pre-cultures in a shaker for 15 hours. Dilute two milliliters of the culture into 200 milliliters of LB with ampicillin in two one liter flasks.
Incubate the flasks in a shaker at 37 degree celsius and 180 RPM until the cultures reach an optical density at 600 nanometers between 0.6 and 0.8. Next, add 0.5 millimolar of delta aminolevulinic acid to the cultures. And then induce the cells by adding tetracycline at 0.2 micrograms per milliliter to the flasks.
Incubate the cultures for 15 hours at 20 degree celsius and 180 RPM. After induction, decant the cultures into centrifuge tubes and centrifuge the tubes at 12000 times G and four degree celsius for 20 minutes. Discard the supernatant.
Resuspend the pellets by pipetting in 50 milliliters of ice cold Tris buffer and transfer the suspension to a conical centrifuge tube. Continue by centrifuging at 4000 times G and four degree celsius for 15 minutes. Discard the supernatant and resuspend each pellet in three milliliters of buffer by repeat pipetting.
Lyse the cells by sonicating with three 30 second cycles pausing for one minute between cycles. Next, centrifuge the lysate at 15000 times G and four degree Celsius for 20 minutes, and then carefully pipette the cell free extract into conical centrifuge tubes. For preparation of the crude extract for the light-driven biocatalysis transfer three milliliters of one cell free extract to a 10 kilodalton centrifuge filter and centrifuge at 4000 times G at four degree Celsius to remove small molecules.
Resuspend the protein in three milliliters of Tris. To characterize the activity of OleT in the light-driven biocatalysis, the protein needs to be purified. To start protein purification, load three milliliters of the cell free extract on to equilibrated nickel NTA spin columns.
Seal the columns with bottom plugs and screw caps. And shake them lightly until the resin is evenly distributed. Continue by incubating the columns in an overhead shaker.
Then centrifuge the column. Next wash the columns by adding one milliliter of washing buffer and centrifuging them at 700 times G for two minutes. Repeat the wash twice more.
After washing, place the columns into conical centrifuge tubes and add one milliliter of elution buffer to each column. Centrifuge the tubes at 700 times G for two minutes and repeat the elution twice more. Add the combined column extracts to a 10 kilodalton centrifuge filter and centrifuge it at 4000 times G and four degree celsius to separate the imidazole used for the elution from the enzyme.
Finally determine protein purity and concentration as indicated in the text protocol. To start the bio transformation procedure, first add 10%of a surfactant such as tergitol and 28.4 milligrams of stearic acid to distilled water to make 10 milliliters of a 10 millimolar stock solution. Heat the solution in a heating chamber, at 60 degrees celsius until the stearic acid is completely dissolved.
Use this solution to prepare a reaction mixture according to the next protocol. Add FMN, EDTA, buffer, and stearic acid to two clear glass tubes and stir in a water bath at 25 degree Celsius. Continue by adding 200 micrograms per milliliter of the purified enzyme or crude extract to the tubes.
And illuminate them with a clear LED light bulb at a distance of two centimeters. Next, take 200 microliter samples at specific time points in tubes prefilled with 20 microliters of 37%hydrochloric acid to stop the reactions. Then add five microliters of a 10 millimolar myristic acid solution to each sample as an internal standard.
To analyze the reaction products, twice add 500 microliters of ethyl acetate to the tubes. Invert the tubes to mix them and centrifuge for one minute at 13, 000 times G.Next, transfer 400 microliters of the supernatants to micro centrifuge tubes and completely evaporate them by gently blowing on the tubes with compressed air. Add 200 microliters of MSTFA to the tubes.
And incubate the mixture at 60 degree celsius for 30 minutes to derivatize the samples before analysis. Analyze the reaction products by injecting a four microliter sample into a gas chromatograph fitted with a flame ionization detector using the temperature profile described in the text protocol. Next, determine the ratio of one heptadecane and beta hydroxy acid formed in each reaction sample with a gas chromatograph coupled to a mass spectrometer.
Use oven temperature and mass spectrometer settings as described in the text protocol. Inject one microliter of each sample into the gas chromatograph and record the chromatogram. Finally, monitor the GCMS chromatograms for each sample and analyze them according to the manufacturer's protocol.
The molecular weights of the products from the enzymatic reactions were determined by using a gas chromatograph coupled to a mass spectrometer. For fatty acids with longer acyl chains, the enzyme OleT, preferentially catalyzes their decarboxylation to olefins of different lengths. Samples from the biotransformation reaction were analyzed by a gas chromatograph fitted with a flame ionization detector.
Decarboxylation of the stearic acid was shown to occur three times faster than the hydroxylation reaction with 99%of the stearic acid converted to a 3.3:1 mixture of one heptadecane to 2-hydroxy stearic acid. Once mastered, the light-driven biocatalysis can be done in a few hours if say I perform properly. In light catalysis, the challenge is to link useful reactions to the harvest of light.
In our case, we have the light within the molecule FMN as photosensitizer and link them to our enzyme via transfer molecule which is hydrogen peroxide.
