Is the first screening kit in the market that comprises a selection of evolved fungal unspecific peroxygenases (UPOs), and it has been designed to achieve scalable production of chemicals and human drug metabolites. Coming from different fungal species (short and long-type UPO families), our exclusive collection of evolved UPOs can perform an extensive range of selective oxyfunctionalizations on pharmacological compounds, emulating the role of human liver P450 monoxygenases. Unlike P450s, UPOs simply use H2O2 as an oxidant, making laboratory manipulations easier and more robust. The catalytic repertoire of the selected UPOs in EVOkit are relevant to both the pharmaceutical and chemical sector, and its portfolio includes alkyl and aromatic hydroxylations, aliphatic and aromatic epoxidations, O-dealkylations, N-dealkylations (including ester and ether cleavage), N-oxidations, S-oxidations and brominations.
EvoEnzyme’s fungal peroxygenase kit is presented in glass vials designed to carry out efficient and directed screening, following a step-by-step protocol. EVOkit contains 12 evolved UPO variants carefully selected for their catalytic properties, encompassing a wide array of biotransformations and selectivity. To make the assay easier, EVOkit contains three accessory vials with reaction buffer, hydrogen peroxide and a UPO substrate to be used as a positive control. We also offer constant support to our customers to assist them with any specific reactions they wish to carry out with the EVOkit (https://evoenzyme.com/contact/).
EVOkit Case Study
Synthesis of human drug metabolites
Two selected engineered UPO variants in the EVOkit have been tested for their capacity to synthesize authentic human drug metabolites (HDMs) from three pharmaceutical compounds of interest: dextromethorphan, naproxen and tolbutamide. It was seen that both evolved UPOs converted dextromethorphan and naproxen to their corresponding HDMs by demethylation, with different degrees of conversion. When testing tolbutamide, the hydroxylation mediated by the peroxygenases produced 4-hydroxymethyl-tolbutamide.
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Scale-up production of evolved peroxygenases
Larger quantities of our evolved UPO enzymes are available upon request. A Pichia pastoris expression system has been adapted, and production protocols established, for the bio-reactor secretion of soluble, very stable, extracellular peroxygenases. EvoEnzyme also has in place downstream protocols and handling instructions for the secure supply of any desired products.
EvoEnzyme can prepare kits with alternative formats adjusted to specific testing scenarios. In this sense, we can adapt EVOKIT to the screening of multiple chemical entities.
In-house testing and analysis
EvoEnzyme has considerable experience in adapted biocatalysis, especially in setting-up peroxygenase reactions. Our clients may also be interested in undertaking an exploratory assay to test the EVOkit for a specific biotransformation or challenge at our company’s facilities. After careful analysis, a detailed report will be provided that details the best candidate enzymes and our team of experts will also advise you as to how you can optimize your process to achieve your objectives.
Peroxygenase customization: Directed Evolution
The main expertise of EvoEnzyme is to identify the best customized biocatalyst for the costumer’s process. Once the EVOkit has been tested and the best candidates chosen, the company offers the possibility of further development through ad-hoc directed evolution campaigns that aim to meet the long-term goals of our customers. These projects may include the design of specific high-throughput screening assays and library creation methods (based on the company’s EvoShuffler yeast technology), as well as mutant analysis and scale-up production.
Late-stage oxyfunctionalization services
The EVOkit tool-box of evolved peroxygenases can be particularly useful in the drug discovery pipeline for late-stage functionalization processes. Based on our experience in UPO mediated transformations, new molecules can be obtained by biocatalysis through the introduction of functional groups and the screening of the chemical libraries constructed.