13TSB_HVC5: Green Synthesis of Dibasic Acids using Biocatalysis 2

This proposed research will be divided up into two work packages (WPs).
WP1 - Optimisation of nitrilases. Currently one lead nitrilase (i.e. active against the dinitrile of choice) has been identified from the current Technical Feasibility project as a result of the "rational sampling of natural diversity" approach used. There will be further lead nitrilases available by the end of the Technical Feasibility project as a result of the laboratory-based active site targeted mutagenesis that is currently underway. This Collaborative R&D work package will involve the optimisation of these lead nitrilases via an untargeted random laboratory-based mutagenesis approach. This will produce a large panel of mutant nitrilases (10^6 - 10^8) derived from the current lead nitrilase candidates. Using existing in-house processes, 10^5 of the mutant nitrilases will be screened for increased stability, rate of reaction and substrate/product concentration tolerance. Prior to the Technical Feasibility project it would not have been possible to undertake a screening project on such a large number of enzymes, as a high-throughput method for screening crude preparations of nitrilase did not exist. However, we have successfully developed such an assay during the Technical Feasibility project and will use it in combination with a liquid handling system to screen the mutants. This work package will also evaluate producing the lead nitrilases and any mutant nitrilases identified in this work package using a Pichia pastoris expression system. The Pichia system will be used to direct the nitrilases to the fermentation broth so that no cell lysis is required and therefore have potential cost/efficiency savings with respect to downstream processing.
WP2 - Immobilisation of nitrilases. The three main benefits of immobilized enzymes are (a) enhanced stability (b) ease of separation of the enzyme from the reaction medium thereby reducing the costs of downstream processing, and (c) the capacity to reuse the enzyme yielding an obvious cost advantage. All three benefits would aid in establishing this biocatalytic process in the commercial arena. There are a wide range of mature immobilisation technologies which are applicable to the biocatalytic process that the proposed project aims to develop, and we will test a range of them using the high-throughput screening method developed during the Technical Feasibility project. We will investigate the use of covalent immobilisation onto epoxy acrylate and amino acrylate resins, and controlled pore glass; and adsorption immobilisation onto functionalized styrene resins; and sequestration within biodegradable alginate beads. Additionally, different particle sizes and pore sizes resins will be tested as these variables have also been shown to affect enzyme performance. The cross-linked enzyme aggregate approach will also be tested as this method of immobilisation has been shown to be suitable for performing biotransformations. The best performing immobilised enzymes will be tested at scale in a range of processes to ascertain their potential performance at plant scale.

This proposed research will be divided up into two work packages (WPs).
WP1: Optimisation of Nitrilases. A number of lead nitrilases will be available from the previous project. WP1 will involve the optimisation of these lead nitrilases via a untargetted random laboratory-based mutagenesis approach. This will produce a large panel of mutant nitrilases (10^6-10^8) of which 10^5 will be screened for increased stability, rate of reaction and substrate/product concentration tolerance using a high throughput screening assay developed in the previous project. This work package will also evaluate producing the lead nitrilases and any mutant nitrilases identified in this work package using Biocatalysts' proprietary (license-free) Pichia pastoris expression system. The Pichia system will be used to direct the nitrilases to the fermentation broth so that no cell lysis is required and therefore have potential cost/efficiency savings with respect to downstream processing and increased enzyme yields.
WP2: Immobilisation of Nitrilases. Immobilised enzymes offer (a) enhanced stability (b) ease of separation of the enzyme from the reaction medium, and (c) reuse of the enzyme (cost advantage). All three benefits would aid in establishing this biocatalytic process in the commercial arena. We will test a range of immobilisation techinques using a high-throughput screening method. We will investigate different carriers, particle sizes and pore sizes. The cross-linked enzyme aggregate approach will also be tested as this method of immobilisation has been shown to be suitable for performing biotransformations. The best performing immobilised enzymes will be tested at scale in a range of processes informed by WP4 to ascertain their potential performance at plant scale.

Economically, the market size for the final product (dibasic ester) that is to be produced using the lead nitrilases(s) produced in this research is ca 100,000 tonnes per annum, with a market value of £150 million. The market is expanding at 6% per annum, whilst the key feedstock for production is a by-product of a mature and declining market. Therefore the development of a new process using biocatalysis and therefore a different feedstock will have significant economic benefits for the SME Chemoxy International Ltd, the lead partner on this TSB project application, and therefore the economic competitiveness of the UK. Additionally, Biocatalysts Ltd, another project partner, could benefit as there may be a potential for some of the developed nitrilases becoming strategic products for the company. Also, the High Value Manufacturing Catapult, the Centre for Process Innovation, another project partner, will develop skills and experience with regards to scale-up of a biotransformation expanding their knowledge base and proven capabilities leading to further paid development work. Furthermore, the global industrial coatings company Beckers, the end user project partner, may benefit from the potential of replacing and augmenting the existing source of dibasic ester with the sustainable version produced by the biocatalytic process that this proposed project aims to develop. Similarly, Nzomics Biocatalysis, the business-facing unit of the Biocatalysis Research Group at Northumbria University, will benefit from sales associated with the nitrilases generated in this project, which are surplus to requirement for dibasic ester production by Chemoxy International Ltd. It will also be a useful exemplar project involving a HEI, 2 SMEs, a multi-national and a Catapult Centre, converting university-based research to an industrial process operating at scale. As such, whilst Nzomics Biocatalysis generates revenue for Northumbria University through its enzymes sales through third parties, this project would more explicitly link the conversion of academic knowledge to an identifiable commercial output. From an environmental, health and safety standpoint, the process which would be employed using the nitrilases developed in this research avoids the use of hazardous chemicals and elevated temperatures, as well as making use of a current waste stream which is currently disposed of at high energy cost and with high carbon dioxide generation. Also, production of biocatalysts for this process via fermentation is a low energy sustainable process, with far less material waste and process costs than the manufacture of traditional catalysts. Therefore, throughout the proposed manufacturing process there are reductions in carbon emissions, waste production and process cost vs. the proposed chemical route to manufacture. It should also be noted that the biotransformation will be performed under lower temperatures and with fewer hazardous chemicals, thus reducing any health and safety risk to operators. With respect to job creation, if the process is successful, significant employment opportunities will be created for scientists, engineers and process technicians in areas of high structural unemployment, i.e. Teesside, where Chemoxy International Ltd and the Centre for Process Innovation are based, and Cardiff, where Biocatalysts Ltd is based. In addition, this will secure the intellectual property of this development inside the UK, thus further protecting those jobs.