Replacement of immobilised cell bioreactors by smaller immobilised enzyme bioreactors: unique-outcome predictability for cytochromes P450 isoforms?

Abstract

Both immobilized enzymes (IME) and immobilized cells (IMC) are acceptable as the biocatalysts essential for the attainment of rapid rates of bioconversion in bioreactors. IMC can display higher than expected cellular permeability whilst IME can exhibit high catalytic constant (kcat/Km) despite limitations on substrate utilisation due to an unstired diffusion layer of solvent. Scale-down switching from IMC to IME involves the replacement of high-volume biotechnology by low-volume biotechnology, sometimes using IME mimics in partially non-aqueous solvent systems. Highly purified IME systems covalently immobilised to particles of, for instance, microcrystalline cellulose or porous glass, can retain both the hydrophilic and hydrophobic intermediate products in situ of the chosen sequence of enzyme reactions. These bioconversions, therefore, are as efficient as those with IMC where enzymes are often particle- or membrane-bound so that even hydrophilic intermediates are not released rapidly into solution. This mimicry of in vivo biosynthetic pathways that are compartmentalised in vivo (e.g. of lysosomes, mitochondria and endoplasmic reticulum) can replace larger IMC by IME especially in application of up to 2700 cytochromes P450 isoforms in bioprocessing. In silico investigation of appropriate model IME systems, in comparison with IMC systems, will be needed to define the optimal bioreactor configuration and parameters of operation, such as pH, T and oxygen mass transfer rate (OTR). The application solely of hazop (applied hazard and operability concepts) may, nevertheless, not be recommended to replace fully the in silico and real-lab pilot-scale and scale studies. Here, food-safe bioprocessing has to be achieved without incorporation of recognised biohazards; especially in the form of unacceptable levels of toxic metals that promote a risk-analysis uncertainty.