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Biocatalysis in the Fine Chemical and Pharmaceutical Industries
Peter W. Sutton Joseph P. Adams, Ian Archer, Daniel Auriol, Manuela Avi, Cecilia Branneby, Andrew J. Collis, Bruno Dumas, Thomas Eckrich, Ian Fotheringham, Rob ter Halle, Steven Hanlon, Marvin Hansen, K. E. Holt-Tiffin, Roger M. Howard, Gjalt W. Huisman, Hans Iding, Kurt Kiewel, Matthias Kittelmann, Ernst Kupfer, Kurt Laumen, Fabrice Lefèvre, Stephan Luetz, David P. Mangan, Van A. Martin, Hans-Peter Meyer, Thomas S. Moody, Antonio Osorio-Lozada, Karen Robins, Radka Snajdrova, Matthew D. Truppo, Andrew Wells, Beat Wirz, John W. Wong
1.1 Introduction
There are few areas of science where recent technological advances have had as great an impact as that in the area of biocatalysis and biotransformations. Arguably, in most synthetic laboratories, the biocatalysis vision of just 20 years ago extended no further than the use of a few simple hydrolases for esterification or hydrolysis to facilitate resolutions. There were certainly research groups around the world who were far more involved in this emerging science, using a much greater array of biocatalytic systems, but real industrial uptake of the work was often hindered by a single, recurring problem – availability of the enzyme(s).
More enzymes become available on a daily basis, available in greater quantities and with greater diversity than ever before. But what is the reason for this relatively recent change? The answer lies not only in consumer/scientific desire for new biocatalysts but in the advancement of three essential areas of science: bioinformatics, gene synthesis and enzyme evolution.
Much of the drive towards biocatalysis is arising from the increasing awareness that our world's resources are finite and there is a need to husband these resources. The rise in interest of biotechnology in the last decade has, in many respects, progressed with clear strategic alignment to sustainability. Many biocatalytic processes are highly aligned with Anastas and Warners1 enunciation of the twelve principles of green chemistry (Table 1.1.1).
Table 1.1.1 Biocatalysis alignment with green chemistry.
1. Prevention (of waste) | Biocatalysis can enable new, more sustainable routes to APIs effectively reducing level of waste. |
2. Atom economy | Biocatalysis often enables more efficient synthetic routes. |
3. Less hazardous (less toxic reagents and intermediates) chemical syntheses | Generally low toxicity. |
4. Designing safer (less toxic) chemicals | No impact. |
5. Safer solvents and auxiliaries | Often performed in water; when solvents are used they are generally Class I or II. |
6. Design for energy efficiency | Usually performed slightly above room temperature. |
7. Use of renewable feedstocks | Biocatalysts are renewable. |
8. Reduce derivatives (e.g., protecting groups) | Chemo-, regio-, enantio-selective nature of enzymes often obviates need for protecting groups. |
9. Catalysis (preferred over stoichiometric reagents) | Catalytic. |
10. Design for degradation (avoid environmental build-up) | No impact on design of products (although biocatalysts themselves are degradable in the environment). |
11. Real-time analysis for pollution (and hazard) prevention | No impact. |
12. Inherently safer chemistry for accident prevention | Biocatalysis is generally performed under mild conditions where risk of explosions/run-away reactions is minimal. |
A recent business report put the industrial enzyme market at ca. $3.3 billion with a prediction to grow at the rate of ca. 6% per annum ($4.4 billion in 2015).2 While these figures clearly indicate an expectation of greater biocatalysis uptake across different business sectors they do not illustrate the shear number of new biocatalysts that are emerging and do not cover the increasing number of whole cell processes that are under investigation.
The use of lipases, esterases and proteases is now widely established throughout the chemical industry with alcohol dehydrogenases (ketoreductases) starting to become increasingly recognized as the pre-eminent method of choice for asymmetric ketone reduction to chiral alcohols. Other enzyme types are starting to become more familiar as they become commercially available; nitrilases, transaminases, enoate reductases, P450 monooxygenases, monoamine oxidases and carboxylic acid reductases to name a few. The practical methods sections of this book and the first volume of “Practical Methods for Biocatalysis and Biotransformations” provide excellent examples of how these different enzyme types can be employed.
One particular area that has seen considerable growth is that associated with P450 oxidations. Remote hydroxylations of a desired molecule can be difficult to achieve using ‘traditional’ chemical methods, often requiring an entirely new route to provide the desired molecules. This is particularly true for those molecules that need to be synthesized as a consequence of being first-pass metabolites. However, hydroxylation is a common consequence of cytochrome P450 catalyzed metabolism, and so there has been a growing interest and demand for P450s that can be used as scientific tools (catalysts). Recombinant versions of these enzymes (particularly when made self-sufficient by fusion to a reductase domain) is a growing research topic, and enzyme kits to allow rapid evaluation are now readily available.
Many of the enzymes which are starting to become popular research tools are often best applied in a host cell. These whole-cell approaches are increasingly being utilized as any co-factors which are required, e.g., ATP (adenosine triphosphate) or SAM (S-adenosylmethionine) are already prepared within the cell as part of its normal operation. This makes whole cell approaches highly competitive from a cost perspective. The two key arguments against the use of whole cells are that the processes are generally dilute (low throughput) and can result in poor purity profiles due to the potentially large number of by-products and impurities that can arise. However, modern molecular biology allows the scientist to overexpress the desired enzymes to such a degree that the desired transformations are often very clean and although the processes are generally more dilute, the waste itself is typically an aqueous solution which can be easily and cheaply treated before disposal.
Handling the aqueous waste is often sited as a concern with biocatalytic approaches and waste treatment of aqueous waste prior to disposal is clearly essential. In some instances incineration is seen as expedient and this entails significant energy consumption – as higher aqueous volumes are often used in biocatalytic approaches the energy consumption, and carbon footprint, is likewise increased. Downstream processing often involves extraction of products from the aqueous stream using organic solvents. At this point the processing and issues of using organic solvents are similar to those encountered in ‘chemical’ approaches. Where infrastructure is in place for solvent recovery this can be a relatively green process but where this infrastructure is missing incineration is again a common form of disposal (for more detail, see Section 1.3.1).
Whole cell processes will become increasingly common moving forward as an inevitable consequence of the rise of synthetic biology, most particularly that aspect seeking to use multiple enzymes within a given cell to enable a cascade of reactions to occur (much like telescoping a traditional chemical process). There are a growing number of researchers seeking to achieve this aim and as the genes associated with new (either recently discovered or ‘designer’) enzymes become known and understood so the potential number of reactions that can be performed within an organism will also grow.
Chemistry drove much of the growth in the life-sciences in the last century but our world is changing. It is the biosciences which will spur innovation in the coming century and this includes synthetic approaches to small molecules.
1.2 Biotrans Outsourcing – AstraZeneca
The successful design, development and execution of a synthetic route containing a biotransformation is an area that requires a truly interdisciplinary effort between organic chemistry, analytical, (bio)/chemical engineering, fermentation, molecular biology, etc. A few companies have all of these scientific skills and plant capabilities in house, but increasingly, many outsource part or all of this complex exercise.
A starting enzyme may be obtained from a propriety collection, or from an external public culture collection. Genomes can now rapidly be searched for novel enzymes using sequence homology. If a likely protein is identified, a gene can be rapidly and cheaply obtained through gene synthesis companies, cloned and over-expressed into a suitable producer host. A number of companies now offer screening services in this area, to identify a known or likely lead candidate enzyme.
For those who have access to a chemical processing plant but not fermentation capacity, many service companies exist who can produce biocatalysts from plasmids or cultures and can supply solid enzyme, solutions or whole cells which can be purchased and used in-house. If an enzyme cannot be identified that gives the correct stereoselectivity and purity, or good enough perfo...