Biocatalyst Selection

Multicopper oxidases. Owing to their relatively high non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications. We have screened in microbial genome databases the presence of hyperthermophilic laccase-like enzymes since extremophilic enzymes, as a result of their superior stability, offers new opportunities for biocatalysis. As a result, the genes encoding for the multicopper oxidases McoA from Aquifex aeolicus (Fernandes et al, 2007, 2009) and McoP from Pyrobaculum aerophilum (Fernandes et al. 2010), were cloned and overexpressed in Escherichia coli and the proteins purified and characterized. Several remarkable features were found: a notable cuprous and ferrous oxidase activity, an extremely intrinsic thermostability (mid-point of unfolding above 100°C as measured by DSC), and for McoP, the first multicopper oxidase from an Archaea to be characterized, the use of nitrous oxide as well as dioxygen as oxidising substrate. Other multicopper oxidases from bacterial origin have also been studied namely, multicopper oxidases from Myxococcus xanthus (Sánchez-Sutil et al. 2007) and Campylobacter jejuni (Silva et al. 2012).

FMN-dependent NADDH:dye (quinone) oxidoreductase. We have created a small collection of soil microorganisms (around 150) harvested from a variety of sources. The screening of this resource allowed for the selection of a bacterial strain Pseudomonas putida MET94, able to decolourise a wide range of structurally different azo dyes (Mendes et al. 2011a). An azoreductase was cloned and expressed in E. coli and the recombinant enzyme was purified and characterized, showing a typical signature of a FMN-dependent flavoprotein.

DyP-type peroxidases. These constitute a novel family of heme-containing peroxidases, capable of efficient synthetic dyes decolourisation, including antraquinonic-based and azo dyes. They are unrelated to other known peroxidases with respect to primary sequence, catalytic properties, and tertiary structure and therefore are very interesting under the fundamental point of view. We have cloned and characterized two new Dye decolourising-type-peroxidases from P. putida and Bacillus subtilis(Santos et al. 2013). Our results show that these bacterial enzymes are promising candidates for the direct oxidation of non phenolic lignin units, with potential to replace the high-redox fungal LiP and VP peroxidises.


MET | Microbial & Enzyme Technology 2013