Development of a continuous process and dynamic modeling of the chemo-enzymatic epoxidation of α-pinene

There is a keen interest of the chemical industry in exploiting the benefits of continuous processing like consistent product quality and high space-time yield for eco-friendly biocatalysis. The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo-enzymatic epoxidation, where the intermediate peroxy acid is produced by an enzyme, are still limited as a result of the enzyme deactivation by the reactants. The aim of this work was the development of an economic process (w.r.t. space-time yield, catalytic productivity, and selectivity) for the chemo-enzymatic epoxidation of α-pinene – a renewable platform chemical and sensitive terpene – and the model based description of the multistep reaction. In peroctanoic acid formation studies, the continuous stirred tank reactor (CSTR) was identified as a suitable process to maintain enzyme activity. Moreover, the economic superiority of the continuous process over the fed-batch process was shown. Even though the enzymatic reaction was feasible using octanoic acid as both substrate and excess medium, the coupled reaction was strongly impaired as it yielded low concentrations of the product α-pinene oxide and high concentrations of by-products. In contrast, ethyl acetate was found to be a suitable substrate (less by-product formation). Therefore, the new process strategy for the in-situ chemo-enzymatic synthesis of α-pinene oxide developed in this work was based on ethyl acetate, a CSTR, and the avoidance of liquid/liquid interfaces by performing the reaction in a single-phase organic solution. Considering the numerous degrees of freedom of this continuous multistep reaction which due to enzyme deactivation additionally does not reach a steady-state, the process development is a challenging task. Therefore, the influence of operating conditions (influent concentrations and molar ratios, enzyme concentration, hydraulic residence time and temperature) on the efficiency was studied in detail for the enzymatic reaction alone and for the coupled reaction. Due to the avoidance of harsh conditions, at optimized conditions α-pinene oxide was produced at simultaneously high target values of 95% selectively, 880 gPOLd space-time yield, and 118 gPOg CalB catalytic productivity. To compensate for the reduced but still present deactivation, the performance of the organic-single-phase CSTR was enhanced by intermittent enzyme dosing. This enabled, for the first time, long-term operation (validated for 100 h) with a maintained maximum space-time yield and selectivity. In addition, after 30 d of continuous operation with enzyme dosing, a catalytic productivity of 282 gPOg CalB can be anticipated. The achieved values are well above the typical thresholds that make biocatalysis economical. Based on the experimental data, a valid and reliable deterministic model for the chemo-enzymatic epoxidation in a CSTR was developed. The model covers the different steps of the reaction cascade, and all involved kinetic constants were found to be uniquely identifiable, which is a prerequisite for trustworthy model predictions. To obtain the model structure, the goodness of fit and parameter identifiability were taken into account. Likewise, different mass action kinetics, as well as mechanistic approaches, were investigated. The developed process model matched the experimental data very accurately. Moreover, it was successfully verified and validated over a broad range of operating conditions.