UK Catalysis Hub


Biocatalysis Scientific Progress

The Biocatalysis Theme of the Catalysis Hub is now underway with five core projects which address a range of contemporary themes in biocatalysis, namely (i) computational moedelling and mechanistic studies of enzymes; (ii) intergration of chemo- and bio-catalytic reactions; (iii) application of flow sysyems to biocatalysis; (iv) the discovery of biocatalysts with novel reaction chemistry; (v) microsacle process development in biocatalysis.

Biocatalysis in Flow: Synthesis of Sesquiterpenes

The aim of the project is to use a flow system for scale up of enzyme synthesis. The system has been tested on terpenes with much improved reaction rates and yields being observed. We have applied this flow system to two different terpene cyclases as well as to the synthesis of analogues. Sesquiterpene cyclases have been used in the synthesis of important natural products, and the flow system has allowed the integration of enzyme reactions with chemical steps for a continuous production system.

Biocatalysis in Flow synthesis of sesquiterpenes figSpacer 640px

Novel Olfactory Ligands via Terpene Synthases, S. Touchet, K. Chamberlain, C. M. Woodcock, D. J. Miller, M. A. Birkett, J. A. Pickett and R. K. Allemann Chem. Commun., 51, 7550-7553 (2015). DOI:10.1039/C5CC01814E

Fundamental & Underpinning Mechanistic Studies on Enzymes

This project has investigated the use of Baeyer-Villiger Monooxygenases (BVMO) for their biocatalytic ability to generate useful lactone monomers for the production of valuable polymers. We initially aim to produce polymenthide, polycarvomenthide and polyhydrocarvide as renewable thermoplastic elastomers and components of pressure sensitive adhesives.
  We have identified candidates from the BVMO family catalysing selectively the production of the targeted monoterpene lactone monomers. The lactones were identified & analysed. We have also studied the regio-selectivity of CHMO and switched successfully the regio-selectivity toward the normal lactone production. Because BVMOs require NADPH as coenzyme, we have generated coenzyme self-sufficient versions of the targeted BVMOs by fusing the BVMO with a coenzyme regenerating enzyme (CRE) to yield CRE/BVMO. CRE is a thermostable variant of phosphite dehydrogenase (PTDH), which regenerates the NADPH making the biotransformation process more efficient and cheaper.  We have produced the targeted lactone monomers in a laboratory-scale production and optimised the method toward larger scales. We are currently working toward the polymerisation reaction and aim to explore the use of mild catalysts to produce the targeted polymers thus improving the current polymerisation methods. To expand our targets, we have screened for other monoterpenoid substrates (17 substrates) for lactone formation to create a library of new lactones for potential application in synthesising novel polymers.

Flow Biocatalysis

Fig bio theme flow biocat

Terpenes are important natural products with many applications in the pharmaceutical and agricultural industry. The synthesis of such structurally complicated compounds is challenging, but enzyme catalysts such as terpene cyclases can largely facilitate the access. However, the low-stability and slow turnover rate of these enzymes make the current bench-based synthesis of terpenes very challenging to scale-up. We have addressed this problem by developing an operational simple and highly efficient segmented flow system. Our system now enables the continuous synthesis of terpenes with much improved reaction rates and yields. We have applied this flow system to two different terpene cyclase as well as to the synthesis of analogues.

Integration of Bio- and Chemo-Catalysis into Telescoped Processes

Our research programme oscillates around the identification of carbon-carbon bond-forming chemical reactions that can be implemented in a cascade process with an enzymatic transformation. The studies are aimed at achieving a chemical process that is highly biocompatible i.e. minimise the need for compartmentalisation, thus creating a process amenable to industrial application. To this end, we have been carrying out work in two areas of chemistry: The photochemical activation of tetrahydroisoquinoline (THIQ) with ruthenium photoredox catalysis, and the water-tolerant multicomponent Petasis-borono Mannich (PMB) reaction Ru(II) photoredox approach. This method has been developed to be water-tolerant with the intermediate iminium formed up to 50 % from THIQ. Several approaches have been undertaken to find a suitable C-nucleophile. However, work is still ongoing towards the optimisation of this cascade and development of the one-pot process with the amine oxidase.
  Multicomponent PMB reaction offers a very effective way of assembling biologically-active diarylmethanamines. The PMB reaction has a great advantage in its modularity meaning that a variety diarylamethanamines can be accessed from the commercially available building blocks: amine, salicylaldehyde and a boronic acid. Work is underway towards combining the chemical process with the biotransformation and engineer more promiscuous amine oxidases.

Development of the Next Generation of Biocatalysts

The target start date is 1st October 2016. UCL CoIlaborators have attended Hub meetings to discuss with other theme leaders regarding potential enzyme targets for study. An initial approach has been made to the Protein Production Facility at Harwell to start to understand how this facility can contribute to the rapid development of biocatalytic processes. Through related funding the UCL high throughput bioprocessing platforms have also been enhanced (e.g. Rios-Solis, 2015 a,b).

New Modes of Symmetrical Heterocycle Synthesis in Biocatalysis

Fig bio theme heterocycle synthesis

18F labelled trehalose can be used as a diagnosis agent for virulence in a number of pathogenic organisms such as mycobacterium tuberculosis (mtb), a causative agent for TB in humans and infected one-third of the world’s population. The OtsA-OtsB pathway plays an important role in the synthesis of trehalose. LPS free OtsA-OtsB system has been obtained from a LPS free host such as an insect cell expression system. The expressions of these two proteins (OtsA and OtsB) have been investigated in this system. The P1, P2 and P3 baculovirus of OtsA and OtsB were successfully generated from their bacmid DNA, and the protein expressions and purifications were also confirmed by western blot and LCMS. The P2 and P3 virus of these two proteins are enough made for scale up and protein purification. This project is designed to be used for new modes of symmetrical heterocycle synthesis in biocatalysis

De novo Design of Next Generation Palladozyme for Use in Programmed in vivo Hormone Manipulation

Ex vivo Investigation of Suzuki-Miyaura Reaction on Proteins. The Pd catalysed Suzuki-Miyaura reaction was investigated on thyroglobulin (Tg) protein under biological conditions. Based on western blot results, thresholds of substrate (boronic acid) and catalyst (Pd complex) were determined. Further MSMS analysis double confirmed the happening of Suzuki-Miyaura reaction under in vitro and ex vivo conditions and the positions of modified residues (by Suzuki-Miyaura reaction) in Tg protein have been identified.  Based on such modification, bio-resin can be applied to capture and purify the target iodinated protein in biological conditions. Further optimizations of such reaction under in vivo conditions (animal model) are still under investigation.

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