Directed evolution for Si-C bond cleavage of volatile siloxanes in glass bioreactors

Volatile methylsiloxanes (VMS) are a class of non-biodegradable anthropogenic compounds with propensity for long-range transport and potential for bioaccumulation in the environment. As a proof-of-principle for biological degradation of these compounds, we engineered P450 enzymes to oxidatively cleave Si-C bonds in linear and cyclic VMS. Enzymatic reactions with VMS are challenging to screen with conventional tools, however, due to their volatility, poor aqueous solubility, and tendency to extract polypropylene from standard 96-well deep-well plates. To address these challenges, we developed a new biocatalytic reactor consisting of individual 2-mL glass shells assembled in conventional 96-well plate format. In this chapter, we provide a detailed account of the assembly and use of the 96-well glass shell reactors for screening biocatalytic reactions. Additionally, we discuss the application of GC/MS analysis techniques for VMS oxidase reactions and modified procedures for validating improved variants. This protocol can be adopted broadly for biocatalytic reactions with substrates that are volatile or not suitable for polypropylene plates.