Recombinant silicateins as model biocatalysts in organosiloxane chemistry

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Abstract

The family of silicatein enzymes from marine sponges (phylum Porifera) is unique in nature for catalyzing the formation of inorganic silica structures, which the organisms incorporate into their skeleton. However, the synthesis of organosiloxanes catalyzed by these enzymes has thus far remained largely unexplored. To investigate the reactivity of these enzymes in relation to this important class of compounds, their catalysis of Si-O bond hydrolysis and condensation was investigated with a range of model organosilanols and silyl ethers. The enzymes' kinetic parameters were obtained by a high-throughput colorimetric assay based on the hydrolysis of 4-nitrophenyl silyl ethers. These assays showed unambiguous catalysis with kcat/Km values on the order of 2-50 min(-1) μM(-1) Condensation reactions were also demonstrated by the generation of silyl ethers from their corresponding silanols and alcohols. Notably, when presented with a substrate bearing both aliphatic and aromatic hydroxy groups the enzyme preferentially silylates the latter group, in clear contrast to nonenzymatic silylations. Furthermore, the silicateins are able to catalyze transetherifications, where the silyl group from one silyl ether may be transferred to a recipient alcohol. Despite close sequence homology to the protease cathepsin L, the silicateins seem to exhibit no significant protease or esterase activity when tested against analogous substrates. Overall, these results suggest the silicateins are promising candidates for future elaboration into efficient and selective biocatalysts for organosiloxane chemistry.

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Directed evolution drives the next generation of biocatalysts.

N. Turner (2009)

Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.

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Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions.

Manfred Reetz (2011)

Asymmetric catalysis plays a key role in modern synthetic organic chemistry, with synthetic catalysts and enzymes being the two available options. During the latter part of the last century the use of enzymes in organic chemistry and biotechnology experienced a period of rapid growth. However, these biocatalysts have traditionally suffered from several limitations, including in many cases limited substrate scope, poor enantioselectivity, insufficient stability, and sometimes product inhibition. During the last 15 years, the genetic technique of directed evolution has been developed to such an extent that all of these long-standing problems can be addressed and solved. It is based on repeated cycles of gene mutagenesis, expression, and screening (or selection). This Review focuses on the directed evolution of enantioselective enzymes, which constitutes a fundamentally new approach to asymmetric catalysis. Emphasis is placed on the development of methods to make laboratory evolution faster and more efficient, thus providing chemists and biotechnologists with a rich and non-ending source of robust and selective catalysts for a variety of useful applications.