Immobilised whole-cell recombinant monoamine oxidase biocatalysis

Abstract

This work demonstrates the first example of the immobilisation of MAO-N whole cells to produce a biocatalyst that remained suitable for repetitive use after 11 months of storage and stable up to 15 months after immobilisation. The production of Escherichia coli expressing recombinant MAO-N was scaled up to bioreactors under regulated, previously optimised conditions (10% DO, pH 7), and the amount of biomass was almost doubled compared to flask cultivation. Subsequently, pilot immobilisation of the whole-cell biocatalyst using LentiKats technology was performed. The amount of the immobilised biomass was optimised and the process was scaled up to a production level by immobilising 15 g of dry cell weight per litre of polyvinyl alcohol to produce 3 kg of whole-cell ready-to-use biocatalyst. The immobilised biocatalyst retained its initial activity over six consecutive biotransformations of the secondary amine model compound 3-azabicylo [3,3,0]octane, a building block of the hepatitis C drug telaprevir. Consecutive cultivation cycles in growth conditions not only increased the initial specific activity of biocatalyst produced on the industrial plant by more than 30%, but also significantly increased the rate of the biotransformation compared to the non-propagated biocatalyst.

High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

“…Final biomass concentrations during the initial fermentations were also similar at 2.1, 1.8 and 1.7 g DCW l −1 for E. coli DE3 gold, plyss and plyse respectively. These levels of MAO-N-D5 specific activity are in agreement with literature results [22,25]. Therefore E. coli BL21 DE3 Gold was selected as the host strain for all further work due to the slightly higher cell concentrations produced during fermentation.…”

“…In terms of progressing to a first full kinetic model for the MAO-N-D5 enzyme compound 7 was selected since this model substrate has been used in previous works where partial kinetic data has been gathered and is available for comparison [22,23]. Given the novelty of the products obtained from compounds 13 and 14, kinetic models were also established for these two additional substrates.…”

“…MAO-N-D5 has previously been found to possess a high catalytic rate and selectivity with many aliphatic and aromatic amines [19,20]. The particular enzyme variant studied here has been subject to in vitro evolution, and the mutant Ile246Met/Asn336Ser/Met348Lys/Thr384Asn has been shown to facilitate the selective oxidation of secondary and tertiary amines [21][22][23]. The aim of this work is to further examine the substrate selectivity of the MAO-N-D5 mutant and to establish the first full kinetic models of the enzyme for conversion of a range of substrates.…”

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

“…A recent in depth characterization of Lentikat particles unveiled a pronounced physical strength of the lenses [51], making them well suited for stirred tank reactor setups. Of further importance, Lentikats including E. coli whole cell catalyst were proven storable for up to 15 month [52], enable almost identical reaction rates as free cells, and maintain their catalytic activity much better than freely suspended catalyst [53]. Another stabilizing technology is the silicone coating of recombinant E. coli adhesively grown on ion exchange resin.…”

Development of in situ product removal strategies in biocatalysis applying scaled‐down unit operations

“…The lentil shaped PVA particles, which are recognized to have low diffusional limitations (Neto et al, 2015) are cheap, long term stable, biologically inert, and non-toxic immobilization material (Zajkoska et al, 2014). The lentil shaped PVA particles, which are recognized to have low diffusional limitations (Neto et al, 2015) are cheap, long term stable, biologically inert, and non-toxic immobilization material (Zajkoska et al, 2014).…”