Abstract
Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme’s activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.
Highlights
The selective oxyfunctionalization of organic molecules is fundamental for the synthesis of chemicals, building blocks, specialized and functionalized polymers, as well as pharmacological compounds
We have resolved the first need by subjecting the Unspecific peroxygenases (UPOs) from the edible mushroom Agrocybe aegerita (AaeUPO) to five rounds of directed evolution for functional expression in Saccharomyces cerevisiae [10]
Directed Unique-point Covalent Immobilization (DUCI) relies on the engineering of specific Cys-mutants, establishing selective covalent interactions between the enzyme and the support
Summary
The selective oxyfunctionalization of organic molecules is fundamental for the synthesis of chemicals, building blocks, specialized and functionalized polymers, as well as pharmacological compounds. We have resolved the first need by subjecting the UPO from the edible mushroom Agrocybe aegerita (AaeUPO) to five rounds of directed evolution for functional expression in Saccharomyces cerevisiae [10] For this purpose, we combined different library creation methods including random mutagenesis with in vivo DNA shuffling, as well as focused mutagenesis at the signal peptide to give rise to a readily secreted UPO variant (named PaDa-I). When dealing with selective transformations, it is necessary to precisely orient the enzyme during immobilization, such as in the construction of nanobiodevices and for flow biocatalysis [18] In these cases, controlled/oriented immobilization can be achieved by using antibodies, histidine tags, biotin/avidin systems or disulfide bonds [19]. The biocatalysts were inspected by fluorescence confocal microscopy and characterized in terms of their immobilization yield, activity and stability in response to temperature, organic solvents and changes in pH
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