Team:Bielefeld-Germany/Outlook

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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 dirty 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 culitvation 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-Chromatographe 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. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 ECOL], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 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.

Immobilization

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

Anthracene, lindane and diclofenac are to be detected with the HPLC. Ibuprofen and naproxen need an improved calibration curve. For naphthalene, acenaphthene and phenantrene we have to test different buffers or lower the temperature too keep it more stable. We have only tested BPUL and ECOL so far. "Team Activity Test" showed that TTHL is active so we can test this laccase for the different substrates. Also there will be the Trametis versicolor laccases TVEL5, TVEL10, TVEL13 and TVEL20. Additional we want to determine k_cat/k_m for the degradable substances. On the LC-MS we did not measure our substrates with our self produced laccases. If we get more time granted, we would test our self produced laccases with the substrates. Besides we did not measure all substrates that needed to be measured. Furter we want to identify degradation products.