Rowan McDonough1*; Charlotte C. Williams2 ; Carol Hartley3 ; Nigel French3 ; Colin Scott3 and David A. Lewis1

 

1 Centre for Nanoscale Science and Technology, Flinders University, Adelaide, Australia.

2 CSIRO Manufacturing, Melbourne, Australia

3 CSIRO Land and Water, Canberra, Australia

 

Synthetic biology utilises enzyme catalyzed reactions to form a range of valuable chemicals through environmentally friendly, highly specific and efficient routes.  Enzymatic biocatalysis is however limited by product inhibition and long-term instability of the biocatalysts, as well as the consumption of small, diffusible and expensive cofactors that must be regenerated after each catalytic cycle in several critical enzyme transformations.

Flow systems, in which biocatalytic components are specifically tethered to a solid support, allow in situ regeneration, recovery, re-use, stabilization and simplified product separation, overcoming some of these limitations. The main challenge, however, is retention of the native interactions, function and activity of the tethered enzymes and cofactors.

In order to address the challenges of immobilising the biocatalysts, in this work, the cofactor β-nicotinamide adenine dinucleotide  (NAD) has been tethered to the surface of silica nanoparticles (SiNPs) via a simple thiol-ene ‘click’ reaction. This was achieved through functionalisation of NAD with a tether arm at the N6 position allowing the key chemical functionality to be retained. The length and flexibility of the tether linkage coupled to the Brownian motion driven mobility of the SiNPs along with the high attachment density of tethered NAD (up to 0.5 attachments/nm2) and hence high NAD concentrations localised to the nanoparticle surface has led to high activities at low enzyme and cofactor concentrations, surpassing that of untethered NAD. This is thought to be due to surface bound NAD saturating enzymes as they approach the surface interface of the heterogeneous/homogeneous system, driving the enzyme towards its maximum rate (Vmax) at low overall cofactor concentrations. Tethered NAD has also been successfully regenerated over 1000 times in a multi-enzyme biocatalytic reaction and was found to have drastically enhanced heat stabilty, retaining 85% activity after heating at 100ºC for 12 hours

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