Process development for biocatalytic reactions is a complex task due to the required interaction of several different scientific disciplines. Additionally, there is a lack of standardized procedures for guiding development and for identifying the major process limitations in these systems. This work seeks to address this problem by providing a methodology based on a simple, systematic series of experiments. Application of the methodology helps identify the major bottleneck for process implementation, whether it be enzyme activity, enzyme stability, or substrate mass transfer. In addition, the underlying mechanism behind these limitations can also be inferred. The methodology is illustrated using a simulated reaction system and is also applied to three experimental case studies. This methodology provides a set of simple experiments that may be performed at an early stage of biocatalytic process development to guide effective improvement strategies, whether they be via protein engineering or reaction engineering. Ultimately, this should afford faster and more efficient implementation of biocatalysts in industrial processes.
As the application of biocatalysis to complement conventional chemical and catalytic approaches continues to expand, an increasing number of reactions involve poorly water‐soluble substrates. At required industrial concentrations necessary for industrial implementation, this frequently leads to heterogeneous reaction mixtures composed of multiple phases. Such systems are challenging to sample, and therefore, it is problematic to measure representative component concentrations. Herein, an online method for following the progress of oxygen‐dependent reactions through accurate measurement of the oxygen mass balance in the gas phase of a reactor is demonstrated and validated. The method was successfully validated and demonstrated by using two model reactions: firstly, the oxidation of glucose by glucose oxidase and, secondly, the Baeyer–Villiger oxidation of macrocyclic ketones to lactones. Initial reaction rate constants and time‐course progressions calculated from the oxygen mass balance were validated against conventional online methods of dissolved oxygen tension and pH titration measurements. A feasible operating window and the sensitivity to dynamic changes of reaction rates were established by controlling oxygen transfer through the operating parameters of the reactor. Such kinetic data forms the basis for reaction characterisation, from which bottlenecks may be made evident and directed improvement strategies can be identified and implemented.
Previously, the biocatalytic desymmetrization of dimethyl cyclohex-4-ene-cis-1,2dicarboxylate to (1S,2R)-1-(methoxycarbonyl)cyclohex-4-ene-2-carboxylic acid, an important intermediate towards the synthesis of biologically active molecules, had been well-characterized in terms of pH and temperature optima and several aspects of process performance. Eventually this promising reaction could convert 200 mM (40 g·L -1 ) of substrate with > 99.5% e.e. using the recombinant pig liver esterase, ECS-PLE06, at a scale of 8.8 L. However, the precise influence of substrate concentration and the poorly water-soluble nature of the substrate (approximately 60 mM in water at 25 °C for structurally similar dimethyl 1,4-cyclohexanedicarboxylate) remained elusive. Therefore, this work focuses on using a recently published methodology based on reaction trajectory analysis to identify mass transfer limitations in this reaction. With the constraints of mass transfer on space-time yield considered, it was possible to evaluate and improve biocatalyst yield (mass of product per mass of biocatalyst) through the use of higher substrate concentrations.Ultimately the complete conversion of approximately 75 g·L -1 substrate was achieved in 3.65 h yielding an excellent productivity of 20 g·L -1 ·h -1 with a biocatalyst yield of 4.36 g·g biocat -1 . This work also highlights the simplicity of applying a reaction trajectory analysis methodology, importance of scale during reaction characterizations and identifies future directions for reaction improvement to address substrate supply and product inhibition/deactivation.
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