Bacterial Enzymes as Bleach Alternatives

Laccase-like Multi-copper Oxidases (LMCOs) catalyze reduction of molecular oxygen into water. LMCOs may be used in paper pulp bleaching and laundry detergents and dishwashing powders, in lignin degradation for production of biofuel, and as green catalysts in industrial synthesis. The thesis focuses on bacterial LMCOs due to their desirable high stability. Also, bacterial enzymes are attractive due to the relative easy cloning of bacteria to form recombinant protein expression systems.

LMCOs encompass four connected copper atoms that function as a small battery, and only when the enzyme is fully charged – with four electrons – does reaction with oxygen proceed. In popular terms this makes LMCOs clean machines that drive a chemical reaction powered only by oxygen and with only water as a byproduct. This feature is highly relevant to industrial applications where oxygen is cheap and clean reactions a bonus.

In the project, a number of novel LMCOs from bacterial origin were identified and expressed and biochemically characterized.

One of these was the laccase from Bacillus clausii KSM-16, an organism known to produce a potent alkalophilic and thermostable protease. The protein was found highly salt-, alkali- and thermostable with substrate specificity similar to that of the well-characterized Bacillus subtilis CotA. The preferred pH for stability and catalytic activity was shifted about 1 unit to the more alkaline compared to Bacillus subtilis CotA. This suggests that pH adaptation of LMCOs can be deduced from the habitat of the natural host. Also, the amino acids causing increase alkaliand salt tolerance in this protein may be useful in design of new tolerant laccases.

Thermobaculum terrenum is a thermophilic bacterium cultured from a hot dirt patch in the Yellowstone National Park. Its genome codes for a LMCO, which was expressed and characterized. It was shown to be the second most thermostable LMCO characterized. A specific role for this LMCO in maturation of antibiotics is proposed. This is the first characterization of a protein from T. terrenum and the first laccase from the evolutionary distinct plylum chloroflexi. The laccase is very different from anything else characterized and this - together with its very high stability - makes it very promising as a template in future protein engineering.

Measurement of activity of LMCOs is often hampered by heat-induced increase in enzyme activity. It was observed that this activation is followed by a change of the Electron Paramagnetic Spectroscopy (EPR) signals. This feature was used to characterize the mechanism behind the process, which was found to be controlled by temperature and NaCl, while the similar transformation in B. subtilis CotA also needed a reducing agent, ascorbate, in order to take place. The discovered mechanism can most likely be expanded to also encompass other LMCOs that have previously been shown to undergo heat-activation.


Caption: Outline of the 3 domain MCOs from T.terrenum. Uniparc codes for the protein sequences are written in paranthesis.
TtMCO (D1CEU4) is a three domain LMCO. TtMCO2 D1CHB6) is a two domain typeB MCO with an extraneous cupredoxin domain fused to the n-terminal. C) TtM-CO3 (D1CH29) is a nitrite reductase with an extraneous cupredoxin domain fused to the c-terminal.