The colors of peroxygenase activity: Colorimetric high-throughput screening assays for directed evolution.

Fungal unspecific peroxygenases (UPOs) are arising as versatile biocatalysts for C-H oxyfunctionalization reactions. In recent years, several directed evolution studies have been conducted to design improved UPO variants. An essential part of this protein engineering strategy is the design of reliable colorimetric high-throughput screening (HTS) assays for mutant library exploration. Here, we present a palette of 12 colorimetric HTS assays along with their step-by-step protocols, which have been validated for directed UPO evolution campaigns. This array of colorimetric assays will pave the way for the discovery and design of new UPO variants.

Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs.

Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % ß-sheet, 19.4 % ß-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/ß hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.