Summary

Initially some trials of shaking flask cultivations were made with different parameters to identify the best conditions for the production of the His-tagged laccase LttH from [http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzreso Thermus thermophilus HB27] named TTHL. Due to the absence of enzyme activity of the enzyme in the cell lysate a purification method was established (using Ni-NTA-His tag resin). Using E. coli KRX containing BioBrick <partinfo>BBa_K863010</partinfo>, TTHL could not be detected by SDS-PAGE (molecular weight of 53 kDa) or by activity test. Therefore a new BioBrick <partinfo>BBa_K863012</partinfo> was constructed and expressed in E. coli Rosetta-Gami 2. With this expression system the TTHL could be detected by SDS-PAGE and purified by using a small scale Ni-NTA column. The fractionated samples were tested regarding their activity. TTHL was shown to oxidize ABTS. After measuring activity of TTHL a scale up of the fermentation was successfully implemented up to 6 L. A further scale up to 12 L with a optimized medium (HSG) and a labscale Ni-NTA-Purification were implemented to enable additional experiments to characterize TTHL.

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivation

• flask design: shaking flask without baffles
• medium: LB-Medium
• antibiotics: 60 µg mL^-1 chloramphenicol and 300 µg mL^-1 ampicillin
• temperature: 37 °C
• cultivation time: 24 h

The reproducibility of the measured data and results were investigated for the shaking flask cultivation, but not yet for the bioreactor cultivation.

Fermentation of E. coli KRX with <partinfo>BBa_K863012</partinfo>

Figure 1: Fermentation of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863012</partinfo> (TTHL) in a Bioengineering NFL22. Conditions: 6 L of autoinduction medium + 60 µg/mL chloramphenicol at 37 °C, pH 7. Agitation increased when pO₂ was below 30 % and OD₆₀₀ was measured each hour.

Purification of TTHL

The cells were harvested by centrifugation and resuspended in Ni-NTA-equilibrationbuffer, mechanically disrupted by high pressure homogenization and centrifuged. After preparing the cell paste the TTHL could not be purified with the 15 mL column, due to a not available column. For this reason a small scale purification (6 mL) of the supernatant of the homogenisation was made with a 1 mL Ni-NTA-column.

SDS-PAGE of purified TTHL

Figure 2: SDS-PAGE of purified E. coli Rosetta-Gami 2 containing <partinfo>BBa_K863012</partinfo> lysate (fermented in 6 L Bioengineering NFL22). The flow-through, wash and elution fraction 1 to 5 are shown. The arrow marks the TTHL band with a molecular weight of 53 kDa.

Figure 2 shows the SDS-PAGE of the purified E. coli Rosetta-Gami 2 lysates fermented in 6 L Bioengineering NFL22 fermenter. Additionally the flow-through, wash and all elution fractions (1 to 5) are shown. TTHL has a molecular weight of 53 kDa and the corresponding band is marked with a red arrow. The TTHL band can be found in fractions 1 to 3, but not in the other two elution fractions. Furthermore there are some other non-specific bands, which could not be identified. To improve the purification an 15 mL column was implemented.

Since Regionals: 12 L Fermentation of E. coli Rosetta Gami 2 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012]

Figure 3: Fermentation of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863012</partinfo> (TTHL) in a Bioengineering NFL22. Conditions: 12 L of HSG autoinduction medium + 60 µg/mL chloramphenicol at 37 °C, pH 7. Agitation increased when pO₂ was below 50 % and OD₆₀₀ was measured each hour.

Since Regionals: Purification of TTHL since Regionals

The harvested cells were resuspended in Ni-NTA- equilibration buffer and mechanically disrupted by homogenization. The cell debris were removed by centrifugation and microfiltration via [http://www.millipore.com/catalogue/module/C7493 Millipore Pellicon XL 50]. The supernatant of the cell lysate was concentrated with [http://www.millipore.com/catalogue/module/C7493 Millipore Pellicon XL 50] with 10 kDa and loaded on the Ni-NTA column (15 mL Ni-NTA resin) with a flow rate of 1 mL min^-1 cm^-2. Then the column was washed with 10 column volumes (CV) Ni-NTA equilibration buffer. The bound proteins were eluted by an increasing Ni-NTA elution buffer step elution from 5 % (equates to 25 mM imidazol) with a length of 50 mL, to 50 % (equates to 250 mM imidazol) with a length of 80 mL and finally to 100 % (equates to 500 mM imidazol) with a length of 80 mL. This strategy was chosen to improve the purification caused by a step by step increasing Ni-NTA-elution buffer concentration. The elution was collected in 10 mL fractions. In figure 3 only the UV-detection signal of the wash step and the elution are shown, this is because of the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak. A typical chromatogram of purified laccases is illustrated here. The chromatogram of the TTHL elution is shown in Figure 5:

Figure 3: Chromatogram of wash and elution fractions from FLPC Ni-NTA His-tag Purification of TTHL produced by 12 L fermentation of E. coli Rosetta Gami 2 with <partinfo>BBa_K863022</partinfo>. TTHL was eluted by a concentration of 50 % (equates to 250 mM imidazol) with a maximal UV-detection signal of 123 mAU.

Contrary to our expectations, the chromatogram shows one distinguished peak. This peak was detected at a Ni-NTA-equilibration buffer concentration of 100 % (equates to 500 mM imidazol) and resulted from the elution of bound protein. Earlier measurements of other bacterial laccases showed that the elution of these laccases begins with a elution buffer concentration of 50 %(equates to 250 mM imidazol). One explanation of this result could be a low concentration of the produced TTHL. Consequently all elution fractions were analyzed by SDS-PAGE to detect TTHL. In the chromatogram no further peaks were detected. The following increasing UV detection signal by increasing concentration of the eltutionbuffer results from the rising imidazol concentration of the Ni-NTA elution buffer. The corresponding SDS-PAGES are shown in Figure 4.

Activity analysis of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL]

Initial activity tests of purified fractions

There was no activity measurable after cultivation and purification of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BBa_K863022] under the control of a T7 promoter. Activity tests of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL] under a constitutive promoter did reveal TTHL laccases capable of oxidizing ABTS. Fractions 1 to 5 of the purification above were rebuffered with deionized H₂O and incubated with 0.4 mM CuCl₂ for 2 hours. Activity measurements were performed using 140 µL sample, 0.1 mM ABTS and 100 mM sodium acetate buffer (pH 5) to a final volume of 200 µL. The change in optical density at 420 nm was detected, reporting the oxidization of ABTS through laccases. Fractions 2 to 5 show activity (Figure 3). Fraction 2 seems to contain most of TTHL showing the highest activity compared to the other fractions: 40% of the used ABTS has been oxidized after 2 hours. Based on these results protein concentrations have to be determined and the activity of the TTHL laccase can be characterized in further experiments including pH optimum and activity in regard of temperature shifts.

Figure 3: Activity test of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL] fractions resulting from the purification. Reaction setup includes 140 µL fraction sample (CuCl₂ incubated), 0.1 mM ABTS and 100 mM sodium actetate buffer (pH 5) to a final volume of 200 µL. Measurements were done at 25°C and over a time period of 5 hours. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL] shows activity in oxidizing ABTS except fractions 1 seems to have no active [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL]. (n=4)

Since Regionals: Initial activity tests of purified fractions

The purificated fractions of the cultivation after the Regional Jamborees in Amsterdam were tested concerning their protein concentration. After re-buffering the protein concentration was determined again and all fraction were incubated with 0.4 mM CuCl₂. For the initial activity test the protein amount was adjusted for comparison. The fractions were measured in Britton-Robinson buffer at pH 5 with 0.1 mM ABTS. Fraction 50% 1 showed the highest activity (Fig. x). Regarding the protein amount of this fraction and the statement, that 90 % of this are TTHL laccase, fraction 50% 1 contains 4,03 µg mL^-1. To ensure enough protein for further experiments, the second best fraction, which is fraction 5% 3 was added to fraction 50% 1. In total, both fraction contain 4,4 µg mL^-1.

Activity assay of each purified fraction of the new cultivation with TTHL. Samples were re-buffered into H₂O and the protein amount in each fraction has been adjusted. The measurements were done using the standard activity assay protocol over night.

Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL] pH optimum

Microtiter plate of the measurements for pH optimum determination. The more intensive the blue color the more ABTS got oxidized. At pH 5 the darkest colour has been reached.

Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL] activity at different temperatures

Standard activity test for TTHL measured at 10°C and 25°C resulting in a decreased activity at 10°C. As a negative control the impact of 0.4 mM CuCl2 in oxidizing ABTS at 10°C and 25°C were analyzed.

Deriving from the obtained values of oxidized ABTS in time at 10°C and 25°C the specific enzyme activity was calculated. For the temperatures a difference of 2 U/mg could be detected.

To investigate the activity of TTHL at temperatures that will apply at a waste water treatment plant throughout the year, activity tests as described above were performed at 10°C and 25°C. The measurements were conducted for 30 minutes. The obtained results reveal a decreased activity of TTHL at 10°C in comparison to 25°C (see Fig. X). The obtained results were used to calculate the specific enzyme activity which was at 13 and 15 U/mg, respectively (see Figure X). The negative control without TTHL laccase but 0.4 mM CuCl₂ at 10°C and 25°C show a negligible oxidation of ABTS. The low difference observed between the two samples is great news for the possible application in waste water treatment plants.

Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 TTHL] activity depending on different ABTS concentrations