Integrated lipase-catalyzed isoamyl acetate synthesis in a miniaturized system with enzyme and ionic liquid recycle

Abstract

Isoamyl acetate synthesis employing aqueous Candida antarctica lipase B (CaLB) solution was performed in a two-phase solvent system comprising hydrophilic ionic liquid 1-butyl-3-methylpyridinium dicyanamide and n-heptane. An X-junction glass microfluidic chip was used to obtain uniform microdroplets of n-heptane within a continuous phase of ionic liquid with dissolved enzyme and reactants, namely, isoamyl alcohol and acetic anhydride. A developed flow pattern resulted in a very large specific interfacial area for the reaction with amphiphilic CaLB and simultaneous extraction of isoamyl acetate in n-heptane. Biotransformation was performed within a continuously operated microfluidic system consisting of an X-junction chip and a silanized tube of a submillimeter diameter, which was further integrated with a microseparator based on a hydrophobic membrane. Efficient separation of n-heptane with product from ionic liquid phase containing enzyme and remaining substrates and products was achieved, enabling reuse of CaLB together with ionic liquid phase. More than 80% of productivity was preserved in each of the eight consecutive biotransformations within the integrated microfluidic system with reused lipase B in the ionic liquid phase. Subsequent ionic liquid regeneration accomplished by vacuum distillation enabled efficient reuse of this solvent in esterification with fresh enzyme.

Microscale technology and biocatalytic processes: opportunities and challenges for synthesis

“…Typically very low bioproduct concentrations represent a huge challenge resulting in up to 90% of production costs needed for product isolation. Studies considering liquid-liquid extraction of bioproducts within microfluidic systems clearly confirm the benefit of taking advantage of the high surface-to-volume area and short diffusion paths in either microflow systems with parallel flow enabling phase separation at the exit [25] or droplet or segmented flow, where further phase separation is needed [36].…”

“…Efficient lipase-catalyzed synthesis of isoamyl acetate in a two-liquid-phase system enabling the reuse of solvent with the dissolved enzyme was developed using commercially available microfluidic units. A miniaturized process system comprising an X-junction glass microfluidic chip provided droplet flow with a very large interfacial area for the reaction and simultaneous product separation in a silanized polymeric microchannel and a membrane-based phase microseparator [36]. The fully integrated continuous two-step synthesis of Aliskiren at a nominal throughput of 41 g/h illustrates a process configuration comprising tubular reactors and membrane-based separators that were scaled up from microfluidic designs [30].…”

“…Concluding remarks and future perspectives The development of integrated reaction-separation systems at the microscale leading to process intensification of biocatalytic processes [36,65], as well as the increased capability of using mathematical models to predict phenomena in microscale devices, are some of the most significant advances in this field. Development of a microfluidic platform for directed evolution, using water-in-oil-droplet compartments for screening [37], plays a significant role in faster process development, while multi-inlet microfluidic reactors for controlled addition of inhibitory/toxic substrates [22] and microfluidic devices with entrapped biocatalysts using a magnetic field [69] indicate new possibilities for the establishment of highly efficient biocatalytic processes with long-term stability.…”

The Promises and the Challenges of Biotransformations in Microflow

“…Depending on biocatalyst surface properties and solvents, enzymes can be homogeneously dissolved in a liquid phase (Figure a,b) or may be adsorbed on the surface between two immiscible fluids either in a parallel (Figure c) or in a droplet‐flow (Figure d). The large interfacial area provided by droplet‐flow in a microfluidic droplet generator, as well as in much larger commercial mesoscale reactor was very useful for the biotransformation, where in situ product removal in an organic phase also contributed to very high volumetric productivities. Separation of highly inhibitory product from the reaction mixture of penicillin acylase‐catalyzed production of 6‐aminopenicillanic acid was recently performed by anaqueous two‐phase system (ATPS) within microfluidic reactor‐separator …”

Biotechnical Micro-Flow Processing at the EDGE – Lessons to be learnt for a Young Discipline

“…This allowed to generate high interfacial area for continuous product removal, and further recovery of the enzyme in solvent when coupled with a phase separator. 60 …”