Real time monitoring of commercial biocatalysis processes using near and mid infrared spectroscopies

这项建议的目的是调查近红外和中红外光谱(NIRS和MIRS)在监测商业生物催化过程中的使用。这两种红外光谱已被证明在生物过程中提供了更好的监测能力，特别是在生物过程(发酵)监测中。在发酵中，NIR/MIR用于实时(近)实时监测分析物水平的实用价值是显而易见的，但很少有研究关注这种光谱技术在生物催化中的应用。那些这样做的人，专注于定性方法来表征产品，而不是量化它们或监控有限商业兴趣的反应。这是令人惊讶的，因为发酵液通常是复杂的，最近在将光谱技术应用于这些具有挑战性的过程方面的进展清楚地表明了此类技术在生物催化中的潜力，在生物催化中，基质往往更简单。在生物催化中使用定量红外光谱可以开发此类反应的实时分析，从而实现过程中的控制。科学案例Ingenza一直在开发利用爱丁堡大学建立的技术中的生物催化来制造胺和氨基酸。这项技术代表了一种强有力的方法来制造对映体，非天然手性胺和氨基酸，这是一种高价值的医药中间体。然而，技术发展的一个限制因素是对生物催化过程的分析，因为目前没有一种方法允许实时监测，因此，使用现有分析方法改进过程缓慢。IR通过加强监控和过程中的控制，显著改进了生物催化过程。Ingenza和Strathclyde大学进行的初步可行性研究表明，近红外/MIR在监测L-氨基丁酸(L-阿BA)稳健、经济的生产过程方面具有相当大的潜力。该过程包括动力学拆分，在该拆分过程中，DL-脱落酸的外消旋混合物被转化为L-脱落酸和酮丁酸(KBA)。不需要的DL-ABA的D-对映体被D-氨基酸氧化酶氧化为亚氨基丁酸，随后亚氨基丁酸迅速水解为KBA和氨。L-脱落酸易于从反应混合物中分离出来，产率高，对映体过量(e.e)高。两个关键成分(ABA和KBA)的消失和出现，对于理解这一生物过程中的反应动力学、化学效率和体积生产率至关重要。在两种红外光谱中都可以很容易地检测到每个分析物的不同光谱特征。在此基础上，建立能够预测ABA和KBA浓度的模型是可能的。由于对映选择性酶促氧化是一种主要的化学物质，即胺、氨基酸和醇的生产路线，因此红外监测可能在其应用中得到广泛的应用。此外，所有这些类别的化合物都可能有很强的红外吸收，这是由于从结构上明显的强偶极矩所致。这意味着原位红外监测在生物催化监测方面具有深远的潜力。因此，我们希望进一步研究这些技术在工业上重要的生物催化过程中的使用，包括上述氨基酸氧化物型反应。这项研究具有广泛的工艺应用潜力，因为Ingenza将动力学拆分和去消旋化过程作为一种涵盖多种氨基酸的平台技术进行操作。它将通过研究关键反应参数(如温度、pH、底物/酶负载量等)对整体效率和生产率的影响来促进工艺开发。

The aim of this proposal is to investigate the use of Near and Mid Infrared Spectroscopy (NIRS and MIRS) in monitoring commercial biocatalysis processes. Both IR spectroscopies have been shown to offer improved monitoring capabilities in bioprocessing, especially in bioprocess(fermentation) monitoring. In fermentations, the practical utility of NIR/MIR for monitoring analyte levels in (near) real time is clear, but few studies focus on such spectroscopic techniques in biocatalysis. Those that do, concentrate on qualitative methods to characterise products rather than quantify them or monitor reactions of limited commercial interest. This is surprising, since fermentation fluids are often complex, the recent advances in applying spectroscopic techniques to these challenging processes clearly points to the potential of such techniques in biocatalysis where the matrix tends to be simpler. The use of quantitative IR in biocatalysis could allow development of real time analysis for such reactions, permitting in-process control. Scientific Case Ingenza has been developing amine and amino acid manufacture using biocatalysis from technology established at Edinburgh University. This technology represents a powerful approach to manufacture enantiopure unnatural chiral amines and amino acids, which are high value pharmaceutical intermediates. However, one limiting aspect of the technology development is the analysis of the biocatalytic process, as no current method allows real time monitoring, thus, process improvements are slow using current analytical methods. IR offers significant improvement in biocatalytic processes via enhanced monitoring and in-process control. Initial feasibility studies carried out by Ingenza and Strathclyde University have shown the considerable potential of NIR/MIR in monitoring a robust, economical manufacturing process for L-aminobutyric acid ( L-ABA). The process comprises of a kinetic resolution in which a racemic mixture of DL-ABA is converted to L-ABA and ketobutyric acid (KBA). The unwanted D-enantiomer of DL-ABA is oxidised to imino-butyric acid by a D-amino acid oxidase, subsequently imino-butyric acid rapidly hydrolyses to KBA and ammonia. The L-ABA is easily isolated from the reaction mixture in high yield and excellent entantiomeric excess (e.e). The disappearance and appearance of the two key components (ABA and KBA), is vital to understanding the reaction kinetics, chemical efficiency, and volumetric productivity in this bioprocess. Distinct spectral signatures for each analyte could readily be detected in both IRS. On this basis, the formulation of models capable of predicting the concentrations of ABA and KBA should be possible. Since enantioselective enzymatic oxidation is a route of manufacture for major classes of chemicals, namely amines, amino acids and alcohols IR monitoring is likely to be broad reaching in its application. In addition, all of these classes of compounds are likely to have a strong IR absorbance, due to strong dipole moments that are apparent from the structure. This means in-situ IR monitoring has far reaching potential for biocatalysis monitoring. Accordingly, we wish to investigate the use of such techniques further in industrially important biocatalytic processes, including the amino acid oxidase type reactions described above. The investigation has the potential to be wide ranging in process application since Ingenza operate the kinetic resolution and deracemisation processes as a platform technology across a broad range of amino acids. It will enhance process development by examining what effect critical reaction parameters (e.g. temp, pH, substrate/enzyme loading, etc) have on the overall efficiency and productivity.