Part:BBa_K1602020

Inducible generator of Humicola insolens cutinase

This generator overexpress Humicola insolens cutinase (HIC) by using a T7 promoter system. For more information about the Humicola insolens cutinase (HIC) have a look on the coding sequence.

Activity with 4-Methylumbelliferyl butyrate (4-MUB)

To test for hydrolysis activity on aromatic esters, a fluorescence assay using 4-MUB, which was originally developed for screening lipase activity, can be used. HiC converts 4-MUB to the fluorescent product 4-methylumbelliferole (4-MU) and butyric acid. 4-MU shows fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 449 nm. As increasing fluorescence intensity directly correlates with increasing concentration of 4-MU, this assay is perfect for studying enzyme kinetics of HiC in real time and high resolution.

Figure 4 This graph show the activity of the HiC at 25°C.

Figure 5 Heat activity test with rising temperature from 45°C to 70°C..

Large polyester dergradation

Final volume of the plate was 200µl containig:

• 142µl Buffer (Na₂HPO₄ pH 7.0)
• bromothymol blue 10% (v/v)
• TES protein fraction HiC (ranging from 5µl to 30µl; concentration unknown)
• 8µl prepolymer (dissolved in Triton100 and DMSO 1:50)
• TES protein fraction (from BL21 cells ranging from 5µl to 30µl; as a control)

The negative control was done by just adding buffer to the well.

The 96 well microplate was loaded as depicted in the picture below: </p>

Figure 1 96-well microplate layout

The assay was run in a in a TECAN® Infinite 200 PRO multi plate reader for 100 kinetic cycles, each 5 mins long and with 25 photo pulses per cycle. The reader was heated to the appropriate temperature of 42° celsius. Absorbance was measured at the absorption maximum of BTB which in this case is 620nm.

Pictures of the multi plate

Figure 1 The plate at the beginning of the measurement.

Figure 2 The plate after the measurement.

Figure 3 The plate after 24 additional hours at room temperature.

The HiC is capable of degrading the prepolymer and in this process releases acidic compounds which acidify the buffer solution in the wells. This made visible via the indicator BTB. The temperature optimum was characterized as 45%deg;C. We decided on such long kinetic cycles because the metabolic rate was proven very low.