Fructo-oligosaccharides (FOS) are linear fructans comprising 2–5 fructose units linked to a terminal glucose residue. They are widely used as food and feed additives due to their sweetness, low calorific value, and prebiotic properties. Here we describe the synthesis of FOS catalyzed by a cell-free crude enzyme solution containing recombinant fructosyltransferase (1-FFT) produced in the yeast Kluyveromyces lactis. During the enzyme catalysis, glucose accumulates as a by-product and eventually inhibits FOS production. We therefore used an enzyme membrane reactor (EMR) to achieve the continuous removal of glucose and the simultaneous replenishment of sucrose. We observed a loss of flux during the reaction with the characteristics of complete pore blocking, probably caused by a combination of proteins (enzyme molecules) and polysaccharides (FOS). Such complex fouling mechanisms must be overcome to achieve the efficient production of FOS using EMR systems.

The antibiotic resistance crisis has prompted research into alternative candidates such as antimicrobial peptides (AMPs). However, the demand for such molecules can only be met by continuous production processes, which achieve high product yields and offer compatibility with the Quality-by-Design initiative by implementing process analytical technologies such as turbidimetry and dielectric spectroscopy. We developed batch and perfusion processes at the 2-L scale for the production of BR033, a cecropin-like AMP from Lucilia sericata, in stably-transformed polyclonal Sf-9 cells. This is the first time that BR033 has been expressed as a recombinant peptide. Process analytical technology facilitated the online monitoring and control of cell growth, viability and concentration. The perfusion process increased productivity by ~ 180% compared to the batch process and achieved a viable cell concentration of 1.1 × 107 cells/mL. Acoustic separation enabled the consistent retention of 98.5–100% of the cells, viability was > 90.5%. The recombinant AMP was recovered from the culture broth by immobilized metal affinity chromatography and gel filtration and was able to inhibit the growth of Escherichia coli K12. These results demonstrate a successful, integrated approach for the development and intensification of a process from cloning to activity testing for the production of new biopharmaceutical candidates.