Review
Online NMR for monitoring biocatalysed reactions

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Abstract

Monitoring biocatalysed reactions and metabolic pathways using NMR spectroscopy is of growing interest. As a non-invasive analytical method providing simultaneous information about intracellular and extracellular constituents, it is superior to other analytical techniques and has a wide range of applications: kinetics and stoichiometrics of metabolic events, metabolic fluxes and enzyme activities can be detected in situ or after taking a sample from the biotransformation mixture. New NMR pulse sequences provide even more valuable experiments in these fields. Research topics range from the monitoring of polymer formation to fermentations producing beverages or antibiotics. Routine monitoring of industrial fermentations by NMR seems to be imminent.

Introduction

The increasing number of biocatalysed reactions in research and development requires analytical techniques to monitor several aspects of biotransformations in situ. Most of the analytical tools used to date are invasive and can detect only certain compounds, never showing the whole picture. NMR spectroscopy is non-invasive and therefore can be performed on cultures to give insight into many intracellular reactions 1, 2. It has been used occasionally for this purpose since Shulman pioneered the use of NMR spectroscopy for biochemists in the 1970s. Nowadays it is becoming an established technique for online analysis of enzymatic and microbial reactions 3. This review shows its capability and current developments in biotechnology.

Several magnetically active nuclei in substrates, intermediates and products can be used for detection. The 13C-nucleus seems to be the first choice because it is ubiquitous in all organic molecules, but its low natural abundance (1.1%) usually requires expensive 13C-enriched starting materials. Furthermore, many complex molecules are not available in labelled form. Thus it is not suitable for large-scale applications. The 1H-nucleus is also ubiquitous in all organic substances and is of growing interest 4radical dotradical dot. This approach is still criticised 1radical dotradical dot but it is in many cases superior to use of 13C because of the natural abundance of the proton (99.98%), thus allowing analysis of unlabelled samples. The comparable sensitivities allow the use of 31P and 19F-nuclei, and in some cases 15N, in enriched samples, providing an alternative. Selective isotopic substrate labelling or addition of rarely encountered atoms, such as fluorine, assists in obtaining additional information.

A wide range of biocatalysed reactions has been covered by the NMR technique. Reactions catalysed by cell extracts and purified enyzmes in different solvents have been monitored in addition to fermentations with living cells in water, and even aerobic biotransformations using an aeration device did not pose a problem. Performing the biotransformation outside the magnet and taking samples at regular intervals for NMR analysis is surpassed these days by performing the bioreaction in the NMR-tube placed in the magnet, allowing for in situ monitoring.

Section snippets

Reactions catalysed by isolated enzymes

Online 1H-NMR monitoring of reactions catalysed by purified enzymes or crude cell extracts was used to get valuable information about kinetic data and substrate specificities 4radical dotradical dot. In most cases water was the solvent, but some problems required deuterated organic solvents.

Monitoring of microbial transformations — whole-cell systems

These kinds of conversions are more challenging to monitor compared with those using partially purified enzymes because the operating parameters are more limited. One is almost always forced to use 1H2O or 2H2O or mixtures as solvent, which necessitates the use of solvent suppression techniques with 1H-NMR. In addition, these measurements are often performed on samples with non-homogenous solutions, leading to the broadening of signals. The problems can become even more complex when aerobic

Conclusion

NMR online monitoring is nowadays an established method for obtaining information about enzyme-catalysed reactions and microbial pathways, including their regulation. The detection of 1H and 13C- nuclei allows routine use of this method to observe biotransformations in research and development. Monitoring industrial fermentations will be state-of-the-art in the future. Selectively isotopically labelled substrates or compounds with NMR-detectable nuclei that are rarely encountered in biological

Acknowledgement

The authors would like to acknowledge Douglas W Ribbons and Herbert L Holland for encouraging and helpful comments. The authors are especially grateful to AM Delort et al. for the permission to present their figure in this review. Financial support from Österreichische Nationalbank, the Austrian Science Foundation and Spezialforschungsbereich Biokatalyse is gratefully acknowledged.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • radical dot of special interest

  • radical dotradical dot of outstanding interest

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