How To Monitor Biocatalysis in Real Time

This is the first blog post in a 2 part series in which I will discuss the real-time monitoring of bio-based chemical synthesis.

Biocatalysis continues to evolve, with the application of recombinant organisms or isolated enzymes designed to catalyze specific chemical reactions – often with highly regiospecific and stereospecific conversions. In the optimization of any chemical synthesis reaction – by traditional or biocatalyzed routes – it is not enough to simply report the yield and the enantiameric excess (ee), the kinetics of the reaction must also be considered. Understanding how the kinetics are affected by conditions such as pH and temperature allows optimization of biocatalysis through the identification of operating conditions that can ensure a maximum yield and desired ee in a timely manner.
Many enzyme-catalyzed reactions function efficiently within relatively narrow ranges of temperature and pH. Effective screening and optimization for process development and scale-up requires accurate process monitoring and tight control of the operating conditions. Directed evolution has enhanced the ability of some organisms and enzymes to survive at higher temperatures and under a wider range of process conditions – giving greater opportunity but also increasing the complexity of optimizing reactions for satisfactory yield, high selectivity and faster conversion rates. Using Design of Experiments (DoE) with reactor technologies that offer accurate and reproducible control of process conditions can greatly reduce the time required to optimize reactions.

An earlier study of biocatalysis (ref: Dadd et al, 2000), described the use of the bacterium Rhodococcus rhodochrous LL100-21 to convert nitriles to carboxylic acids with either a one-step and two-step enzymatic pathways.

Experiments were performed with acetonitrile and benzonitrile as alternative substrates. Real-time profiles from ReactIR™ provided insight into the metabolic pathway using different substrates, and provided the basis for kinetic understanding with qualitative and quantitative validation that (a) acetonitrile conversion proceeded via the two-step pathway (as evidenced by a detection of acetamide intermediate) and (b) benzonitrile conversion proceeded via the one-step pathway (with no intermediate present).

A more recent study published in Industrial Biotechnology (Yang et al, 2006) illustrates the use of in situ ReactIR™ monitoring to track the bioconversion, and allowed calculation of reaction kinetics as a function of reactant concentration and cell density.

See this and more recently reported applications in fermentation and bioprocessing in the new white paper: Process Analytical Technology (PAT) for Biotech.