Summary

First some trials of shaking flask cultivations were made with various parameters to identify the best conditions for production of the His tagged laccase Lbh1 from Bacillus halodurans C-125 named BHAL. Due to inactivity of the enzyme in the cell lysate a purification method was established (using Ni-NTA-Histag resin). BHAL could not be detected by SDS-PAGE (theoretical molecular weight of 56 kDa) or activity test by using the BioBrick <partinfo>BBa_K863020</partinfo> and E. coli KRX as expression system. Due to this results the new BioBrick <partinfo>BBa_K863022</partinfo> was constructed and expressed E. coli Rossetta-Gami 2. With this expression system the laccase could be produced and analysed via SDS-PAGE. A small scale Ni-NTA-column was used to purify the laccase. The fractionated samples were tested regarding their activity with ABTS and showed ability in oxidizing ABTS. A scale up to 12 L with a optimized medium (HSG) and a labscale Ni-NTA-Purification were implemented to enable additional experiments to characterize BHAL.

Cultivation, Purification and SDS-PAGE

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

Purification

SDS-PAGE

Figure 1:SDS-PAGE of purified lysate derived from a flask cultivation of E. coli Rosetta-Gami 2 carrying <partinfo>BBa_K863022</partinfo>. Lanes 2 to 7 show the flow-through, the wash and the elution fractions 1 to 4. BHAL has a molecular weight of 56 kDa and is marked with an arrow.

In figure 1 the different fractions of the purified cell lysate of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863022</partinfo> are shown in a SDS-PAGE. BHAL has a molecular weight of 56 kDa. In lane 5, which corresponds to the elution fraction 2, a faint band of 56 kDa is visible. Therefore the fractions were further analysed by activity test and MALDI-TOF.

Since Regionals: 12L Fermentation of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863022</partinfo>

Figure 2: Fermentation of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863022</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 BHAL

Activity Analysis of BHAL

Initial activity tests of purified fractions

The resulting fractions of the cultivation and purification of BHAL (fraction 1 to 5) were analysed with activity tests. After rebuffering into deionized H₂O and incubation with 0.4 mM CuCl₂ for 2 hours, the samples were measured with 140 µL sample, 0.1 mM ABTS, 100 mM sodium acetate buffer to a final volume of 200 µL. The change in optical density was measured at 420 nm, reporting the oxidation of ABTS for 5 hours at 25°C. An increase in ABTS_ox can be seen (Figure 2), indicating produced BHAL laccase in each fraction. Fraction 2 shows the highest amount of ABTS_ox (55%) reaching saturation after 3 hours. Similar to BPUL laccase, BHAL is capable to reach saturation after 3 hours with approximately oxidizing 55% of the supplied ABTS. Therefore BHAL is going to be characterized further.

Figure 2: Activity test of BHAL fractions after 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. Each fraction shows activity, especially fraction 2, which therefore contains most BHAL laccase. (n=4)

To determine the substrate saturation with ABTS as a substrate, different concentrations of ABTS were used for activity measurements. 616 ng of BHAL laccase were used for measurements in Britton-Robinson buffer (pH 5) and ABTS concentrations ranging from 0.1 mM to 5 mM ABTS, 308 ng of BHAL laccase for measurements ranging from 5 mM to 8 mM.

Since Regionals: Initial activity tests of purified fractions

Different fractions of the purification of a new cultivation since the Regional Jamborees in Amsterdam were tested regarding their activity of the produced BHAL enzyme. Before re-buffering, the protein concentration was determined and again after re-buffering. The initial activity tests were done in Britton-Robinson buffer (pH 5) with 0.1 mM ABTS at 25 °C. The protein amount was adjusted in each sample for comparison. One distinct fraction showed the highest activity: fraction 5% 3 (Fig. 3). The contained laccase amount was calculated by assuming, that the most active fraction contains 90 % laccase. This leads to a BHAL laccase concentration 10,9 ng mL^-1.

Figure 3: Activity assay of each purified fraction of the new cultivation with BHAL. 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: BHAL pH optimum

Figure x: Microtiter plate of the measurements for pH optimum determination. The more intensive the blue color the more ABTS got oxidized. At pH 4 and pH 5 the darkest colour has been reached.

To determine the optimal experimental setup for BHAL activity measurements the best pH has to be determined. Using Britton-Robinson buffer pHs between pH 4 and pH 9 had been adjusted. 308 ng BHAL laccase per well had been tested under these pH conditions using 7 mM ABTS. The CuCl₂ incubated and therefor activated BHAL laccases showed a high activity at pH 4 and pH 5, where most of ABTS has been oxidized (compare to Fig. x and y). The calculated specific enzyme activity of BHAL shows the high activity at both mentioned pHs (Fig. x). While BHAL has an activity of ~8 U mg^-1 at pH 4 and pH 5, the enzyme activity decreases at higher pHs. At a pH of 6 only 1/3 of enzyme activity can be detected compared to the activity at pH 4 and pH 5. While still active at pH 7, the BHAL laccase is not as suitable as thought for an application at a waste water treatment plant because of its high activity in acidic environments.

Figure x: Oxidzed ABTS by BHAL laccases at different pH adjustments. The experimental setup included CuCl₂ incubated BHAL laccase (308 ng), Britton Robinson buffer adjusted to the tested pHs and 5 mM ABTS. Measurements were done at 25 °C for 30 minutes. The most amount of oxidzed ABTS can be detected at pH 4 and pH 5.

Figure x: Calculated specific enzyme activity of BHAL at different pH conditions. The highest specific enzyme activity for ABTS is under pH 4 and pH 5 conditions. The higher the pH, the less ABTS gets oxidzed.

Since Regionals: BHAL activity at different temperatures

Since Regionals: BHAL activity depending on different ABTS concentrations

To be able to calculate the activity in Units mg^-1, measurments have to be dne under substrate saturation. This allows the comparison of Units mg^-1 with other laccase activities and the literature. For this purpose ABTS concentrations ranging from 0.1 mM to 8 mM were applied in an experimental setup contain Britton-Robinson buffer (pH) and tmeperature conditions of 25 °C. For measurements with 0.1 mM to 5 mM ABTS 616 ng BHAL laccase were used (Fig. x). Measurements with 5 mM to 8 mM ABTS only 308 ng BHAL laccase were applied (Fig. y). Applying less then 7 mM ABTS a static increase in oxidized ABTS is given (compare with Figure x and y). Measurements with 8 mM ABTS show a slower increase in oxidized ABTS as with 7 mM ABTS (Fig. y). This may be due to a substrate toxication. The most compromising ABTS concentration is 7 mM with the highest increase in oxidized ABTS. Therefor a substrate saturation is reached with 7 mM ABTS.

Figure x: Activity assay to determine the substrate saturation with ABTS as a substrate. Measurements were done with 616 ng BHAL laccase in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 0.1 mM to 5 mM.

Figure x: Activity assay to determine the substrate saturation with ABTS as a substrate. Measurements were done with 308 ng BHAL laccase in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 5 mM to 8 mM. An ABTS concentration of 7 mM was determined as substrate saturated.