13TSB_HVC5: Green Synthesis of Dibasic Acids using Biocatalysis 2

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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.