Combinatorial biocatalysis

Keywords Directed evolution. Random mutagenesis, DNA shuffling. Biocatalysis, Combinatorial libraries, Enantioselectivity. 

Apart from the traditional organic and combinatorial/high-throughput synthesis protocols covered in this book, more recent applications of microwave chemistry include biochemical processes such as high-speed polymerase chain reaction (PCR) [2], rapid enzyme-mediated protein mapping [3], and general enzyme-mediated organic transformations (biocatalysis) [4], Furthermore, microwaves have been used in conjunction with electrochemical [5] and photochemical processes [6], and are also heavily employed in polymer chemistry [7] and material science applications [8], such as in the fabrication and modification of carbon nanotubes or nanowires [9]. 

Michels PC, Khmelnitsky YL, Dordick JS, et al. Combinatorial biocatalysis a natural approach to drug discovery. Trends Biotechnol 1998 16 210-15. 

Combinatorial biocatalysis, where redox enzymes are used in mulit-component systems for new molecule discovery. 

In vitro multi-enzyme systems are set up by the combination of enzyme modules including pathway and even pathway-unrelated enzymes. Also, the synthesis of saccharides in combination with de novo enzymatic sugar synthesis can be accomplished. This so-called combinatorial biocatalysis can be performed in sequential reactors or in a one-pot reaction vessel which challenges further reaction engineering for optimization. Even the combination of an enzyme module with a chemical... 

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. 

Table 5.1 Multi-enzyme systems for the synthesis of glycoconjugates by the combination of enzyme modules in combinatorial biocatalysis and combinatorial biosynthesis.

UDP-a-D-Gal UDP-a-D-GlcNAc UDP-a-D-GalNAc GDP-a-D-Man GDP-P-l-Fuc CMP-Neu5Ac dTDP-a-D-Glc, dTDP-P-L-Rha combinatorial biocatalysis [32]... 

In order to provide dTDP-deoxy sugars by combinatorial biocatalysis we have utiHzed the enzymes for the dTDP- 3-L-rhamnose pathway. The successful combination of pathway enzymes with optimized enzyme productivities (amount of product per unit of enzyme) needs a concise kinetic and inhibition analysis. Scheme 5.1 depicts the biosynthetic pathway of dTDP- 3-L-rhamnose with important km and Ki constants. The enzymes RmlA and RmlB are highly controlled by the intermediate, dTDP-4-keto-6-deoxy-a-D-glucose 3, the product 5 or by... 

Figure 5.2 Enzyme modules in combinatorial biocatalysis of nucleotide sugars. See Table 5.2 for enzymes.

I 5 Multi-Enzyme Systems for the Synthesis of Clycoconjugates Table 5.2 Enzymes used in modules for combinatorial biocatalysis of nucleotide sugars. 

In summary, the synthesis and in situ regeneration of nucleotide sugars by combinatorial biocatalysis suffers from the main disadvantage that each enzyme has to be produced in sufficient amounts. This affords efficient recombinant protein produchon hosts being a bottleneck for some genes [25]. However, once a multi-enzyme system has been developed, the productivity can be improved by repetitive use of the biocatalysts as demonstrated for repetitive batch syntheses with soluble enzymes [25, 38] or with immobilized enzymes [48]. The advantage... 

Scheme 5.6 Poly-LacNAc-derived oligosaccharides synthesized by combinatorial biocatalysis [80].

Natural polyols have been used as substrates for the so-called combinatorial biocatalysis , a proposed approach to drug discovery [33]. For instance, complementary enzymatic regioselectivity was applied to produce a combinatorial library of 167 distinct selectively acylated derivatives of the flavonoid bergenin (11) on a robotic workstation [34]. Another lead compound, the antitumoral paclitaxel (12, a molecule with very low water solubility) has been similarly derivatized, initially exploiting the selectivity of the protease thermolysin for its side-chain C-2 OH. 

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 YL, Michels PC, Dordick JS, Clark DS, Generation of solution-phase libraries of organic molecules by combinatorial biocatalysis, in Molecular Diversity and Combinatorial Chemistry (Eds. I.W. Chaiken, K.D. Janda), pp. 144-157, 1996, American Chemical Society, Washington. 

Figure 4.21 Structure of a combinatorial biocatalysis BOD-derived library L32.

---