13TSB_HVC5: Green Synthesis of Dibasic Acids using Biocatalysis 2

这项拟议的研究将分为两个工作包(WPS)。WP1--腈酶的优化。目前，由于采用了“对自然多样性进行合理抽样”的办法，从当前的技术可行性项目中已经确定了一种主要的丁腈酶(即对所选择的二腈具有活性)。由于目前正在进行的以实验室为基础的活性部位定向突变，到技术可行性项目结束时，将会有更多的主要氰化酶可用。这一合作研发工作包将涉及通过一种基于实验室的无目标随机突变方法来优化这些领先的丁腈酶。这将产生大量突变的丁腈酶(10^6-10^8)，源自当前的候选氰化铅。利用现有的内部流程，将对10^5个突变的丁腈酶进行筛选，以提高稳定性、反应速度和底物/产物浓度耐受性。在技术可行性项目之前，不可能对如此大量的酶进行筛选项目，因为不存在用于筛选腈酶粗制剂的高通量方法。然而，我们已经在技术可行性项目中成功地开发了这样一种检测方法，并将结合液体处理系统来筛选突变株。本工作包还将使用巴斯德毕赤酵母表达系统评估生产本工作包中确定的铅腈酶和任何突变的腈酶。毕赤酵母系统将被用来将腈酶定向到发酵液中，因此不需要细胞裂解，因此在下游加工方面具有潜在的成本/效率节省。WP2--腈酶的固定化。固定化酶的三个主要优点是：(A)提高了稳定性；(B)酶易于从反应介质中分离，从而降低了下游加工的成本；(C)酶的重复使用能力产生了明显的成本优势。所有这三个好处都将有助于在商业领域建立这种生物催化过程。有多种成熟的固定化技术适用于拟议项目旨在开发的生物催化过程，我们将使用在技术可行性项目期间开发的高通量筛选方法测试其中一系列技术。我们将研究共价固定化在环氧丙烯酸酯和氨基丙烯酸酯树脂上的使用，以及可控孔玻璃的使用；以及在功能化的苯乙烯树脂上的吸附固定化；以及在可生物降解的海藻酸盐珠中的固定。此外，将测试不同的颗粒大小和孔径大小的树脂，因为这些变量也已被证明影响酶的性能。交联酶聚合方法也将进行测试，因为这种固定化方法已被证明适合进行生物转化。性能最好的固定化酶将在一系列工艺中进行规模测试，以确定它们在工厂规模的潜在性能。

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.