Combinatorial biotransformations,

Manipulation of the biosynthetic pathways leading to natural compounds, so-called combinatorial biosynthesis, is presented in the third section, with particular attention paid to the opportunities arising from polyketide biosynthesis. Finally, combinatorial biotransformation of natural or synthetic compounds by means of isolated enzymes or whole microorganisms is presented in the fourth section. 

In summary, the combination of enzymes is advantageous from an enzymol-ogy and reachon engineering point of view. Reaction yields can be increased by avoiding product inhibition of single enzymatic reachons. Product decomposihon (e.g. by hydrolysis) can be overcome by further enzymatic transformahons. Tedious isolation of intermediate products is not necessary. However, both strategies - combinatorial biocatalysis and combinatorial biosynthesis - have their disadvantages. The in vitro approach needs every enzyme to be produced by recombinant techniques and purified in high amounts, which is in some cases difficult to achieve. On the other hand, product isolation from a biotransformation with permeabilized or whole host cells can be tedious and results in low yields. 

The biosynthesis of many hydroxylated natural products proceeds through regio- and enantioselective modification of polyketides, which are assembled through chain elongation via acetate or propionate units [2]. The enzymes responsible for the chain elongation and subsequent reduction, elimination, aromatiza-tion, and further modifications are classified as polyketide synthases [3]. These multifunctional enzymes have been used for whole-cell biotransformation toward unnatural metabolites that are within the scope of combinatorial biosynthesis... 

Manga, N., Duffy, J.C., Rowe, PH. and Cronin, M.T.D., A hierarchical QSAR model for urinary excretion of drugs in humans as a predictive tool for biotransformation, QSAR Combinatorial Sci., 22, 263-273, 2003. 

A wider available panel of purified enzymes, or of characterized microorganisms performing a particular biotransformation, could increase the potential of this technique. Its main limitations are the stability of the enzymes, both to organic solvents and to different temperatures, which could be significantly improved by their immobilization on a solid support, and the solubility in aqueous media required in most cases for the biotransformation substrates. Anyway, the extreme usefulness of combinatorial biocatalysis for specific classes of products (complex natural products, polyfunctionalized chiral scaffolds) has already been assessed. 

Khmelnitsky et al. (309) reported the synthesis of solution-phase libraries via combinatorial biocatalysis using a number of different substrates. One of these, bicy-clo[2.2.2]oct-5-ene-2,3-frans dimethanol (BOD, 10.102), is shown in Fig. 10.51 together with the biotransformation strategy that was applied. The selected reactions... 

Metabolism studies play an important role in dmg discovery and development. The advent of combinatorial dmg synthesis has increased the need for a fast assessment of drag metabolism [1], LC-MS is an important tool in the identification of drag metabolites [2-4], The soft ionization conditions in electrospray ionization (ESI) or atmospheric-pressure chemical ionization (APCI) facilitate the detection of metabolism in biological samples. Many common biotransformation, e.g., oxidation, hydroxy lation, hydrolysis, and reduction, can be detected from just the knowledge of the molecular mass of the metabolites. Further confirmation and/or stractnre elncidation of metabolites is possible using the variety of MS-MS platforms. 

Kurochkina VB, Nys PS (2002) Kinetic and thermodynamic approach to design of processes for enzymatic synthesis of betalactams. Biocatal Biotransform 20(1) 35-41 Lee SB, Ryu DDY (1982) Reaction kinetics and mechanism of penicillin amidase a comparative study of computer simulation. Enzyme Microb Technol 4 35-38 Lin WJ, Kuo BY, Chou CP (2001) A biochemical engineering approach for enhancing production of recombinant penicillin acylase in Escherichia coli. Bioproc Biosys Eng 24 239-247 Lindsay JP, Clark DS, Dordick JS (2004) Combinatorial formulation of biocatalyst preparation for increased activity in organic solvents salt activation of penidllin amidase. Biotechnol Bioeng 85(5) 553-560... 

Combinatorial biosynthesis can be practically realized by (i) chemical modification by biocatalysts (biotransformation), (ii) mutasynthesis, (iii) combinatorial metabolism in hybrids, and (iv) activation of silent metabolism (Figure 2.11). 

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