Going large-scale?

After having developed a plan for a potential application of our project we now plan to test our system in a real sewage treatment plant under strict safety regulations. Furthermore a lab-scale waste water system could be used to measure the enzyme activity of our immobilized laccases in active sludge and in a fixed bed reactor under realistic conditions.

Shuttle vector

The shuttle vector (pECPP11JS) <partinfo>BBa_K863204</partinfo> could be constructed and it was shown that an active protein (TVEL5) could be secreted into the medium.

The following step will be the site directed mutagenesis to eliminate the illegal XbaI restriction site in the his4 gene. Afterwards the laccases from Trametes versiculor TVEL35 and K500002 have to be integrated into the shuttle vector and afterwards into the genome of the yeast. The next step will be the optimization of the cultivation and purification of the protein. Furthermore the immobilization and measurement of the activity have to be done.

Cellulose Binding Domain

A favorable alternative to conventional immobilization and purification methods is still a key factor to the feasibility of our project. Many different BioBricks including a cellulose binding domain (CBD) could be constructed and cloned. Beneath them three different constructs to express reporter proteins, but a detectable fusion-protein with a CBD and a green fluorescent protein (GFP) to characterize the binding capacity of the CBDs in a detectable scale could not be produced. The switching from an inducible T7-promoter to a constitutive promoter (J23100/J61101) did not show a detectable expression either. Future research could focus on the linker between CBDs and the reporter GFP or the order of CBD and GFP.

Cultivation and Purification

Until now, the laccases from E. coli (ECOL), B. pumilis (BPUL), B. halodurans (BHAL) and T. thermophilus (TTHL) were produced and purified. As a first approach a batch cultivation and a quick and efficient purification procedure was chosen. The next steps will be a scale up for the B. halodurans laccase (BHAL) and a screening for cultivation conditions for the laccase from X. campestris in E. coli Rosetta-Gami 2. Due to the successful production of an eucaryotic laccase in Pichia pastoris, following cultivation experiments are planed to get a higher protein amounts. The produced eucaryotic laccase from T. versicolor (TVEL5) will be used to establish a purification procedure with a capture step based on an Ion-Exchange-Chromatography and following with an Hydrophob-Interaction-Chromatography. The purified protein could be used for characterizing the crystalline structure and the properties of the enzyme.

To improve the production and to produce a higher amount of bacterial laccases several screening approaches, like the cultivation in another medium (e.g. HSG-medium) as well as the establishment of a fed-batch cultivation with different feeding strategies to increase the durance of protein expression, are possible. Additional different fermentation condition can be proved to identify the best fermentation parameters like: pO2, agitation control, pH, temperature, air composition, etc.

For an improvement of the purification various possibilities exist. During our research the laccases were captured by a Ni-NTA resin and eluted with two elution strategies. To enhance the effectiveness of the purification procedure different elution strategies could be screened with varying elution gradients. The purification of the laccases can be expanded by 2 or 3 more steps for a better purification and a final polishing. Potential steps could be an Ion Exchange Chromatography with a following Size Exclusion Chromatography. Furthermore different buffers and so called wash buffers could be tested including different concentration of CuCl2 to prove if this has a positive influence on the purification procedure.

Activity Tests

Four different laccases have been produced via heterologous expression which showed the ability to oxidize ABTS. BPUL, ECOL, BHAL and TTHL activities have been characterized further to gain information about optimal pH or ideal CuCl2 concentration. Still there are some measurements that would help to characterize these laccases in more detail. One important aspect is the stability of our gained enzymes. This would help us to define the duration that our laccases are active in the waste water treatment plants before they have to be exchanged by new active ones.


An immobilization of Trametes versicolor- Laccase (named TVEL0), E. coli BL21- Laccase (CueO named ECOL) and Bacillus pumilus DSM 27 -Laccase (CotA named BPUL) on CPC-beads was successfully achieved. The next step would be to immobilize the laccases Lbh1 from Bacillus halodurans C-125 (named BHAL) and Ltth from Thermus thermophilus HB27 (named TTHL) and develop an optimal immobilization strategy for them. Another aspect would be to consider a method to increase the enzymatic activity of immobilized ECOL, as well as to raise the binding capacity of BPUL to the beads.

Substrate Analysis

The HPLC results showed that ECOL, BPUL, TTHL and BHAL are able to degrade estradiol in the presence and absence of ABTS. Ethinyl estradiol is not degraded by the bacterial laccases. Just TTHL showed little degradation activities on ethinyl estradiol in presence of ABTS. Due to time reasons and the decay of PAHs in Britton Robinson buffer, the analyses of the PAHs and the analgesics with the HPLC and the LC-MS methods could not been carried out. It would be interesting to analyze the produced laccases with the PAHs and analgesics. Degradation products were found after treatment of estradiol and ethinyl estradiol with TVEL0 with LC-MS. The next step would be to analyze the possible degradation of PAHs, analgesics and estrone and detect degradation products after treatment with the produced bacterial laccases and TVEL5 from Trametes versicolor.

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