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. The Cutinase as an α/β-hydrolase posseses two very interesting properties. It can connect as well as cleave ester bonds which makes it possible to conduct composition as well as degradation of a varaiety of substrates. HiC is a monomeric enzyme with its active site at its surface. Due to this unique feature HiC is capable to hydrolyze large polymers. The protons released by this hydrolization can be detected by using a pH indicator in an assay, making the reaction visible even to the bare eye. We characterized that fact by designing some assays showing this activity using our own prepolymer (poly(PEG-itaconate)) as a substrate and bromthymol blue as the pH indicator. The occurance of the change in color for BTB is at a pH of 6.2 as it changes from blue to yellow for a rise in acidity in the solution.

Figure 1 X-Ray structure of HiC with a resolution of 3 Angström (PDB: 4OYY).

Production of HiC in E.coli

You can easily produce HiC by inoculation of 20 mL LB 100 mL flask with E.coli BL21(DE3) containing Bba_K1602020. After incubation at 180 repulsion per minute (rpm) at 37°C until an OD of 0.6 is reached you can start induction with IPTG (conc. 0.5 mM). The final incubation of the cell suspension take place over night at 180 rpm at 28°C. The SDS-Page reveals protein expression before and after induction. HiC can be detected with an molecular mass of 22-24 kDa.

Figure 2 SDS Page of HiC expression.	Figure 3 Evaluation of SDS Page by contrast analysis

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 1 mechanism for the reaction of 4-Methylumbelliferyl butyrate

Results

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 degradation

Figure 1 reaction pathway of the degradation of poly(peg-200 itaconate

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:

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.