A major goal of our project was to test whether indican could be used to dye fabric. Indican is a good candidate for dyeing because, like its industrial counterpart leuco-indigo, it is water soluble. The idea is that an enzyme called a glucosidase would cleave the sugar group from the indican leaving indoxyl. The freed indoxyl naturally oxidizes to form indigo, which would then, hopefully, be incorporated into the fabric.
First we wanted to see whether or not indican could be cleaved by a glucosidase. We suspected that a beta-glucosidase (GLU) found naturally in B. circulans would be able to recognize and cleave the beta-linked glucose moiety of indican. To test this hypothesis we cloned the gene into e. coli and expressed it with an attached 6XHis tag to allow for purification. Once we had obtained purified GLU, we set up reactions with the purified GLU and various levels of indican and visually tested for the production of a blue color - proof that indigo was being formed. The results can be seen below:
So, having confirmed we could cleave indican to make indigo, our next step was to test whether or not this process could be carried out on fabric itself. Would GLU continue to function in the presence of cotton polymers? Would the soluble indican be converted to insoluble indigo in such a way that it would strongly adhere to the cotton polymers? To test these ideas, we dunked cotton cloth into varying levels of indican dissolved in water. The cloth was then submerged in purified GLU. Over the course of 30 minutes, we could see the cloth change hues from white to varying levels of blue depending on the initial indican concentrations. See results below:
Just because the fabric turned blue however doesn’t necessarily mean that it would stay that way upon washing. The indican was converted into indigo, but was the indigo actually interacting with the cotton polymers in the same way as in a normal pair of blue jeans? To test the adherence of the indigo, we subjected the cloth to a number of different washes using water, SDS (like a laundry detergent), ethanol, and acetone. We did the same thing with a piece of actual blue jeans and compared our results. The results are below:
Remarkably, the indican dyed cloth proved to retain its color just as well as the blue jeans control. Neither showed any sign of color loss except for in the wash with acetone (an amphiphilic solvent) which is to be expected because of indigo’s higher solubility in acetone.
After confirming the activity of the beta-glucosidase (GLU) enzyme from B. circulans, we were interested in determining the kinetic activity of this enzyme on indican. We used Michaelis-Menten kinetics to model the behavior of GLU with various concentrations of indican substrate to determine the Km and Vmax of the enzyme.
From this data, we generated a Michaelis-Menten kinetics graph, and found the Vmax and Km to be 3.216*10-5 mM/s and 1.780 mM respectively. At the 95% confidence level, Vmax is in the range of [0.00002654 mM/s, 0.00003778 mM/s], while Km is in the range of [0.84 mM, 2.72 mM].
We ran this kinetic assay with purified GLU enzyme, and varying concentrations of indican dissolved in water. Absorbance measurements were taken at 620 nm with TECAN, and converted into indigo concentrations (mM) using indigo calibration data (under Characterization of Indigo Biosynthesis). Similar to the FMO kinetics assay, we assumed the oxidation step from indoxyl to indigo is instantaneous. However, we believe that because of sufficient aeration and oxygen content during our assay, the error coming from this assumption would be very limited.
We obtained further kinetic characterization for GLU on ONPG (ortho-Nitrophenyl-B-glucosidase). After generating a Michaelis-Menten kinetics graph, we found the Vmax and Km to be 2.965*10-4mM/s and 3.7035 mM respectively. At the 95% confidence level, Vmax is in the range of [0.00018959 mM/s, 0.00040333 mM/s], while Km is in the range of [-0.30 mM, 7.71 mM].
We ran this assay using pure GLU enzyme, and varying concentrations of ONPG dissolved in water. Absorbance measurements were taken at 405 nm with TECAN.
We also interested in the possibility of our GLU enzyme acting on chemicals containing beta-galactosides. Thus, we tested our enzyme for activity with X-Gal.
We can see that as we increase the X-gal concentration, the rate at which the X-gal product is made increases. This displays the definitive activity of our glucosidase on X-gal.
X-gal activity was tested by measuring absorbance at 620nm with TECAN. X-gal was dissolved in DMSO, and all assay conditions were kept at 5% DMSO of the total volume.
Paavilainen, S. "Purification, Characterization, Gene-Cloning, and Sequencing of a new-beta glucosidase from Bacillus Circulans subsp. alkaophilus." Applied and Environmental Microbiology. 59.3 (1993): 927-932. Print.
Paavilainen, Sari. "The CrystalStructure of B-glucosidase from Bacillus Circulans sp. alkalophilus: Ability to form long polymeric assemblies." Journal of Structural Biology. 129. (2000): 69-79. Print.