(582bl) Secretory Expression of Laccase From Geobacillus

Castagnaro, T. - Presenter, University of New Hampshire
Wu, K., University of New Hampshire

Laccases are multi-copper oxidases that can oxidize a disparate range of organic substrates using oxygen and producing water as a byproduct without requirement for additional reactive compounds, making them excellent candidates for industrial oxidants. Compared to fungal laccases which are difficult to produce in active forms in heterologous hosts due to incorrect glycosylation, bacterial laccases are much easier to express heterologously and are more active and stable at high temperatures, pH and salt concentrations. However, most bacterial laccases studied are intracellular enzymes or periplasmic proteins. Purification of these proteins compared to extracellular enzymes can considerably increase the downstream processing costs.

Geobacillus is a genus of Gram-positive thermophilic bacteria, many of which have been found to naturally secrete proteins at high levels (Unpublished data). Genome analysis of several Geobacillus species revealed the presence of laccase with a signal peptide for secretion. In this work, we screened a collection of 170 Geobacillus species for their functional secretion of laccase and heterologously overexpressed these secretory laccases as well as intracellular laccase from Bacillus subtilis in Geobacillus thermoglucosidasius. Signal peptides of these laccases were engineered to improve the secretion efficiency. We also investigated the secretion pathways and tuned the expression of copper chaperons of the host to enhance the functional expression and secretion of laccase. These results will aid in the understanding of the mechanisms of copper binding, laccase folding and secretion. In addition, using laccase as an example, we demonstrated functional secretion of otherwise intracellular enzymes by signal peptide engineering.