Part:BBa_K2043007

bpuI laccase codon optimized for E. coli

This part corresponds to Bacillus pumilus laccase cloned by the Paris Bettencourt team in 2016 in the context of the (Frank&Stain project). This enzymes originally comes from Bacillus pumilus, which we codon optimised for E. coli.
In order to facilitate working with this enzyme, we added a His-tag at the C-terminal. This tag allows for purification in an easier way.
This gene has already been registered in the Biobrick Registry (part number:BBa_K863000.) All the results presented here for bpul have been tested in pCOLA vector.
There are several reasons why we have chosen this enzyme:

1. It already exists in the registry of Standard Biological Parts, and it is one of the most well documented BioBricks.
2. Apart from the registry, a good biochemical analysis and methods can be found in the paper by Reiss et. al 2011.
3. The laccasse comes from bacteria (Bacillus pumilus) which makes it potentially easier to express and study in E. coli than fungal lacasses.
4. To our knowledge, bpul hasn't been shown to degrade indigo. On the other hand Reiss et. Al 2011. have shown that it successfully degrades indigo carmine which is structurally similar to indigo dye (figure 4A).
5. Due to a large number of substrates laccases can potentially degrade compounds from wine making it a good basis for collaboration with the Enzyme Group.

Over-expression of bpul

SDS-PAGE electrophoresis was performed on cell extracts to confirm the protein expression.

Figure 1 Over-expression of bpul. The BL21(DE3)-lacZ is used as a negative control. This strain expresses alpha subunit of lacZ gene instead of Bpul. The left side of the figure is showing the results of the electrophoresis of the cell extracts at the moment of adding IPTG. The right side are the results 5h after the addition of IPTG. We can see the successful expression of bpul and the leakiness of the promoter.

Figure 2 The over-expression of bpul alongside the other two enzymes used in Frank&Stain project.

Testing the Activity

We tested our cell extract for BpuI activity in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used as substrate, as recommended in the literature.
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.

https://static.igem.org/mediawiki/parts/d/d2/Paris_Bettencourt_notebook_bpuI_good.jpg As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product at 420nm, which results from the oxidation of ABTS. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional.

The transformant BL21(DE3)-bpul, and the negative control BL21(DE3) have been induced with IPTG. The cell extracts of both strains were tested for the ability to degrade indigo carmine (figure 4C) and indigo (figure 4D). Additionally, 10µM acetosyringone (ACS) was added to the reaction mixture to facilitate the oxidation of the substrates. ACS is a known mediator of the laccases and it is often used and needed in the studies of the laccase activitiy As expected, the 1mM indigo carmine solution is entirely degraded within the 13 hours of the experiment in the cell extract which contains overexpressed bpul. Additionally, we have shown that more than 60% of indigo can be degraded by bpul laccase in the 13 hours of the experiment. It is important to notice, that in longer experiments would probably lead to even more degradation since the degradation curve didn't reach the saturation.

Figure 4 (A) Structural formulas of two substrates (indigo carmine, and indigo) of bpul laccase are showing structural similarity. (B) SDS-PAGE electrophoresis gel with samples from left to right: BIO-RAD kaleidoscope protein ladder; BL21(DE3) cell extract not induced; BL21(DE3) cell extract induced with 0.5 mM IPTG; BL21(DE3)-bpul cell extract not induced; BL21(DE3)-bpul cell extract induced with 0.5mM IPTG. Electrophoresis was performed in BIO-RAD's Mini-PROTEAN Tetra cell electrophoresis gear on 10% BIO-RAD mini-PROTEAN precast TGX gel. (C and D) Indigo carmine and indigo degradation by cell extract containing bpul are shown over a period of 13h. The degradation was observed in 96-well plate at the λ=650 for indigo carmine, and λ=680 nm for indigo, at room temperature. 1 µL of cell extract was added to the reaction mixture which consisted of substrate dissolved in 0.1 mM of potassium phosphate buffer (pH 7.8), and 10 µM ACS added to the reaction mix.

Sequence and Features BBa_K2043007 SequenceAndFeatures Cho, E. A., Seo, J., Lee, D. W., & Pan, J. G. (2011). Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores. Enzyme and microbial technology, 49(1), 100-104.

Reiss, R., Ihssen, J., & Thöny-Meyer, L. (2011). Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC biotechnology, 11(1), 1. We tested the activity of BpuI using cell extract of cells expressing our protein.