Production of self-immobilised enzyme microspheres using microfluidics

Spherezyme production in a microfluidic circuit (Schematic I). A micro emulsion of the enzyme solution in a mineral oil is formed at junction A, which is then introduced to a protein cross-linking agent at junction B downstream (microscope image II) to form self-immobilised enzyme micro particles (spherezymes). These are then applied in catalytic reactions, as demonstrated by the hydrolysis of p-nitrophenol butyrate by spherezymes of Pseudomonas fluorescens lipase, a reaction routinely used to determine the activity of lipases (Schematic III). [Display omitted] •A novel microfluidic method for producing particulate biocatalysts.•An emulsion-based process defines the shape of the final self-immobilised enzyme particles.•The particle size can be precisely controlled by adjusting the fluid flow rates.•The particles have a defined structure, with a lower porosity at the surface. Self-immobilisation of enzymes is a useful way of producing particulate biocatalysts that are easy to recover while still retaining high specific activity. The spherezyme microsphere immobilisation technique is useful in that it provides structured enzyme micro-particles within a specific size range. Herein an alternative micro-fluidics spherezyme production technology is evaluated. Spherezymes of Pseudomonas fluorescens lipase were successfully produced using a new continuous microfluidic method involving the two sequential steps of emulsion formation and subsequent protein cross-linking. The microchannel design with two flow-focussing junctions provided uniform enzyme microspheres with a particle diameter of 50 μm and a size distribution within 3% as determined by a microscopic image analysis method. The size of the particles could be finely controlled as a function of the microreactor flow rate and monitored in real time. The structure of the particles were analysed using focused ion beam scanning electron microscopy (FIB-SEM), which showed that the external structure was less porous, with pores of up to 492 nm, while internal structure had a much greater porosity with a maximum pore size of 1735 nm. The activity retention of the lipase after immobilisation was 65% on hydrolysis of p-nitrophenyl butyrate (PNPB) and the micro-particle biocatalyst could be recycled multiple times. Using microfluidics provides a superior method to scale up production of lipase spherezymes with a very narrow size distribution.