Biocatalysis whole-cell biotransformations

For a brief survey of the background to (and nomenclature in) whole-cell biotransformations see Introduction to Biocatalysis using Enzymes and Micro-organisms by Roberts, S.M., Turner, N.J., Willetts, A.J. and Turner, M.K. Cambridge University Press, New York, 1995, Chapter 2, p. 34-78. 

Lam KS (2010) Application of whole-cell biotransformation in the pharmaceutical industry. In Tao J, Lin G-Q, Liese A (eds) Biocatalysis for the pharmaceutical industry discovery, development, and manufacturing. Wiley, New York, pp 213-227... 

The term biotransformation or biocatalysis is used for processes in which a starting material (precursor) is converted into the desired product in just one step. This can be done by use either of whole cells or of (partially) purified enzymes. Product examples range from bulk chemicals (such as acrylamide) to fine chemicals and chiral synthons (chiral amines or alcohols, for example). There are several books and reviews dealing with the use of bio transformations either at laboratory or at industrial scales [1, 10-13]. 

Many reported biotransformations are initially only demonstrated on a very small scale, the substrates or products may be subject to competing reactions if other enzymes are present (this can be a serious issue in whole-cell biocatalysis), or the desired enzyme is insufficiently active or produced in low levels. For many biotransformations a little care and attention is needed in the growth of the microbe to achieve the desired results. Production of a specific enzyme from a microbe can often be increased by growing the cells in the presence of a very small concentration (typically micromolar) of an inducer. The inducer could be a natural enzyme substrate, a substrate mimic or a molecule which is in some way associated with a substrate s availability or role in metabolism. This process is called induction and represents a genetic switch which cells use to respond... 

A strain of Pseudomonas aeruginosa has been recently described, which shows the opposite enantioselectivity, converting racemic arylaminonitriles efficientiy into the D-amino acids. Again, whole-cell biocatalysis worked well, the cells being entrapped in alginate beads. It is unclear whether this biotransformation involves an amide intermediate. 

Poppe L, Novak L (1992) Selective biocatalysis. A synthetic approach. VCH, Weinheim Roberts SM, Wiggins K, Casy G (1992) Preparative biotransformations. Whole cell and isolated enzymes in organic synthesis. WUey, Chichester Flohe L (1979) CIBA Foundation Symposium 65 95... 

Biocatalysis. Biocatalysis, also termed biotransformation and bioconversion, makes use of natural or modified isolated enzymes, enzyme extracts, or whole-cell systems for the production of small molecules. A starting material is converted by the biocatalyst in the desired product. Enzymes are differentiated from chemical catalysts particularly with regard to stereoselectivity. 

The whole-cell biocatalysis approach is typically used when a specific biotransformation requires multiple enzymes or when it is difficult to isolate the enzyme. A whole-cell system has an advantage over isolated enzymes in that it is not necessary to recycle the cofactors (nonprotein components involved in enzyme catalysis). In addition, it can carry out selective synthesis using cheap and abundant raw materials such as cornstarches. However, whole-cell systems require expensive equipment and tedious work-up because of large volumes, and have low productivity. More importantly, uncontrolled metabolic processes may result in undesirable side reactions during cell growth. The accumulation of these undesirable products as well as desirable products may be toxic to the cell, and these products can be difficult to separate from the rest of the cell culture. Another drawback to whole-cell systems is that the cell membrane may act as a mass transport barrier between the substrates and the enzymes. 

Biotransformations for the synthesis of asymmetric compounds can be divided into two types of reactions those where an achiral precursor is converted into a chiral product (true asymmetric synthesis) and those involving the resolution of a racemic mixture. Both types of reaction are used at Lonza, which is a leading producer of intermediates for the life science industry. Lonza also uses biocatalysis for the synthesis of achiral molecules, for example, an immobilized whole-cell biocatalyst is used for the nitrile hydratase-catalyzed synthesis of thousands of tons per year of nicotinamide from 3-cyanopyridine. 

Biocatalysis or biotransformation encompasses the use of biological systems, whether whole cells, cellular extracts or isolated enzymes, to catalyze the conversion of one compound to another. 

For cost reasons, if ever possible, whole-cell biocatalysis is used to perform biotransformations. This is possible when the following criteria are met (i) no diffusion limitations for substrate(s) and/or product(s) and (ii) no side or follow-up reactions due to the presence of other cellular enzymes. If these conditions are not fulfilled, the use of isolated enzymes - or in special cases of permeabilized cells - is indicated. 

Apart from the usually low activity and sometimes insufficient selectivity of P450s towards steroids (which can be improved by means of protein engineering), the low solubility of steroid compounds in water (1-100 pM [334]) represents a challenging problem for the establishment of whole-cell biocatalysis. Consequently, several promising reaction-engineering techruques that were applied for biotransformations of other hydrophobic compoimds have also been tested with steroid substrates. Among these are (1) biphasic reaction setups with an organic phase, which serves as substrate reservoir, (2) surfactant-... 

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