Thermostable Disulfide Isomerases for Biotechnology

The TIPs project (ERA-Net) focuses on the provision of novel thermostable isomerases from thermophilic microorganisms and their biotechnological applications. Disulfide isomerases are enzymes catalyzing the interconversion of thiol-disulfide bonds within a broad spectrum of substrates including proteins. Besides catalyzing the oxidation, reduction or isomerization of disulfide bonds, they also possess chaperone activity, inhibiting aggregation of misfolded proteins and stimulating their correct folding. However their application in the improvement of folding, assembly and post-translational modification of industrial enzymes is not properly developed. Moreover, possible applications outside their in vivo function have not yet been extensively explored. We have applied comparative bioinformatic analyses of sequence data to identify different classes of thermophilic disulfide isomerases of industrial interest. Newly developed tools such as sequence similarity networks (SSN) and genome neighbourhood networks (GNN) allow protein sequences to be gathered into clusters that represent families with only a single function. Using this approach we have selected several relevant enzyme candidates from diverse sources. Synthetic codon-optimized genes have been expressed in Escherichia coli in different genetic constructs. Two fluorescence-based assays have been adapted for testing two of their potential activities. The functional characterization includes monitoring both the substrate reduction (reductase activity) as well as the recovery of the substrate functionality (chaperone/foldase activity). Enzymes will also be characterized in terms of thermostability and robustness. Strong candidates will be further upscaled, structurally and functionally characterized and further optimized towards their biotechnological application.