Biocatalyst libraries

A representative set of a- and -keto esters was also tested as substrates (total 11) for each purified fusion protein (Figure 8.13b,c) [9bj. The stereoselectivities of -keto ester reductions depended both on the identity of the enzyme and the substrate stmcture, and some reductases yielded both l- and o-alcohols with high stereoselectivities. While a-keto esters were generally reduced with lower enantioselec-tivities, it was possible to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form. These results demonstrate the power of genomic fusion protein libraries to identify appropriate biocatalysts rapidly and expedite process development. 

A combinatorial approach for biocatalytic production of polyesters was demonstrated. A library of polyesters were synthesized in 96 deep-well plates from a combination of divinyl esters and glycols with lipases of different origin. In this screening, lipase CA was confirmed to be the most active biocatalyst for the polyester production. As acyl acceptor, 2,2,2-trifluoroethyl esters and vinyl esters were examined and the former produced the polymer of higher molecular weight. Various monomers such as carbohydrates, nucleic acids, and a natural steroid diol were used as acyl acceptor. 

A remaining crucial technological milestone to pass for an implanted device remains the stability of the biocatalytic fuel cell, which should be expressed in months or years rather than days or weeks. Recent reports on the use of BOD biocatalytic electrodes in serum have, for example, highlighted instabilities associated with the presence of 02, urate or metal ions [99, 100], and enzyme deactivation in its oxidized state [101]. Strategies to be considered include the use of new biocatalysts with improved thermal properties, or stability towards interferences and inhibitors, the use of nanostructured electrode surfaces and chemical coupling of films to such surfaces, to improve film stability, and the design of redox mediator libraries tailored towards both mediation and immobilization. 

As more and more enzymes become available, it is essential to develop an automated microtiter-based screening protocol, which allows the rapid identification of desired biocatalyst hits from a family of enzyme libraries using a minimal amount of substrates and enzymes. As a result of comprehensive screening, the success rate can be significantly improved and many unique enzymes or conditions can be identified, which were previously largely ignored in the synthetic community (Yazbeck et al. 2003). 

But there is still another point, not yet discussed but with considerable potential, which may also impact eventually on technical asymmetric catalysis. Even though biocatalysts are efficient, active, and selective, there still remains one big disadvantage At present, there is not yet an appropriate enzyme known or available for every given chemical reaction. It is estimated that about 25 000 enzymes exist in Nature, and 90% of these have still to be discovered [28, 29]. New biocatalysts are made available nowadays not only from screening known organism but also via screening metagenomic libraries and directed evolution techniques [30]. 

In contrast to rational approaches, the directed evolution of enzymes is based on the search of useful functionalities in libraries randomly generated and on improvement by suitable and proper selection. The directed evolution combines two powerful and independent technologies methods for the generation of random genetic libraries and strategies for the selection of variant enzymes with the specific capabilities [499-503]. This process can result in biocatalysts with non-natural proprieties, since the proteins are expressed in recombinant cells decoupled from its biological functions and evolved under unusual conditions. One additional advantage is the possibility to tailor not only individual proteins, but also the whole biosynthetic and catabohc pathways [471]. 

These enzyme libraries represent the beginning of a new set of biocatalyst tools... 

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