13TSB_HVC5: Green Synthesis of Dibasic Acids using Biocatalysis 2

13TSB_HVC5：利用生物催化绿色合成二元酸2

• 批准号: BB/M004821/1
• 负责人: Gary Black
• 金额: $ 17.66万
• 依托单位: Northumbria University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM004821%2F1
• 关键词: 13TSB_HVC5; Green; Synthesis; Dibasic; Acids

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.

这项拟议的研究将分为两个工作包（wp）。WP1 -腈酶的优化。目前，由于采用了“合理的自然多样性抽样”方法，已经从目前的技术可行性项目中确定了一种铅腈酶（即对所选的二腈有活性）。由于目前正在进行的基于实验室的活性位点靶向诱变，在技术可行性项目结束时，将有更多的先导腈酶可用。该合作研发工作包将涉及通过基于实验室的非靶向随机诱变方法优化这些先导腈酶。这将产生一个大的突变体nitrilases（10^6 - 10^8）衍生自目前的先导nitrilase候选者。使用现有的内部流程，将筛选10^5的突变腈酶，以提高稳定性、反应速率和底物/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，因为不存在筛选硝化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，并将与液体处理系统结合使用来筛选突变体。该工作包还将评估使用毕赤酵母表达系统生产该工作包中鉴定的先导腈酶和任何突变腈酶。毕赤酵母系统将用于引导硝化酶进入发酵液，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/效率节约。WP2 -腈酶的固定化。固定化酶的三个主要优点是：(a)增强稳定性；(b)易于从反应介质中分离酶，从而降低下游处理的成本；(c)酶的重复使用能力，从而产生明显的成本优势。所有这三个好处将有助于在商业领域建立这种生物催化过程。有许多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，我们将使用技术可行性项目期间开发的高通量筛选方法对其中的一系列技术进行测试。我们将研究共价固定在环氧丙烯酸酯和氨基丙烯酸酯树脂上的使用，以及控制孔隙玻璃；功能化苯乙烯树脂吸附固定化；并在可生物降解的海藻酸珠中进行隔离。此外，不同的粒度和孔径树脂将被测试，因为这些变量也被证明会影响酶的性能。交联酶聚合体方法也将被测试，因为这种固定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过程中进行大规模测试，以确定它们在工厂规模上的潜在性能。