Rapid Screening of Biocatalysts

The ideal assay system for HTS would include automated manipulation of single wells and each should be tested for a variety of activities simultaneously. One of the main challenges is to develop a simple assay sensitive enough to pick up enzyme activities that might be below their optimal level under the assay conditions. Nowadays novel colorimetric, luminescence, and fluorescence methods have been established in HTS with automated multiplex compound testing (typically 10-20 compounds per well) (Winson, 1997). 

Another strategy exploits the nucleic acids from diverse organisms where it is not necessary to isolate the bacteria themselves. This approach has particular benefits because only a small fraction of organisms in a particular habitat is culturable (Table 3.4). 

Preparation of Condensed Plates from Source Exprekssion Library 

Schleifer, Phylogenetic identification and in-situ detection of individual microbial cells without cultivation, 

Hafenbradl, H. W. Jannasch, and K. O. Stetter, I yrolohus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113°C, Extremophiles 1997, 

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The reports mentioned above provide a systematic coverage of the nonimmobi-lized enzymatic reactors used in biocatalytic reactions under continuous flow operation. Results from microreactor experiments were comparatively higher than conventionally mixed batch reactors in terms of conversion rate and improvement of product yield as demonstrated for hydrolysis [140], dehalogenation [141], oxidation [142], esteriflcation [143], synthesis of isoamyl acetate [144,145], synthesis of cyanohydrins [147,148], synthesis of chiral metabolites [153], reduction [151], and bioluminescent reaction [149]. The small volumes involved and the favorable mass transfer inherent to these devices make them particularly useful for the screening of biocatalysts and rapid characterization of bioconversion systems. The remarkable results of such studies revealed that the product yield could be enhanced significantly in comparison with the conventional batch runs. 

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). 

Winson MK, Todd M, Rudd BAM, Jones A, Alsberg BK, Woodward AM, Goodacre R, Rowland JJ, Kell DB (1998) A DRASTIC (Diffuse Reflectance Absorbance Spectroscopy Taking in Chemometrics) approach for the rapid analysis of microbial fermentation products quantification of aristeromycin and neplanocin A in Streptomyces citricolor broths. In Kieslich, K (ed) New Frontiers in Screening for Microbial Biocatalysts. Elsevier Science B.V., The Netherlands, p 185... 

The problem of low enantioselectivity of whole-cell reduction possessing more than two enzymes with opposite enantioselectivities has been solved by overexpressing the individual enzymes in E. coli. Now, 18 key reductases from baker s yeast have been overproduced in E. coli as glutathione S-transferase (GST) fusion proteins. Therefore, rapid identification of synthetically useful biocatalysts is possible. A set of fusion proteins consisting of GST hnked to the N-terminus of putative dehydrogenases produced by baker s yeast was screened for the reduction of various substrates. For example, ethyl 2-oxo-4-phenylbutyrate was reduced... 

Even considering only the example of the proline family of aldol catalysts, it is dear that there will soon be hundreds of cases of organocatalysts described in the literature. Direct, organocatalytic aldol reactions do not yet have the generality of traditional stoichiometric methods, which can offer predictable results for a wide variety of substrates. However, companies already offer to screen substrates against panels of up to 200 enzymes to find the optimum biocatalyst for a reaction, and the same approach could be applied to identify rapidly the best organocatalyst for a process. 

These investigations also showed that the conversion of ECB to ECB nucleus would proceed more rapidly if ECB were first solubilized in a suitable solvent such as methanol or acetone. However, if the concentration of solvent was too high, the enzyme activity was reduced. Ideally, the enzyme itself could be tailored to suit the industrially preferred conditions (e.g., to make it more resistant to solvent or active at a different pH). One method for achieving this is to use directed evolution [42], whereby genes encoding the enzyme are mutated, screened and then recombined in vitro. Although the contributions of individual amino acid mutations are small, the accumulation of multiple mutations by directed evolution allows significant improvement in the biocatalyst for reactions on substrates or under conditions not already optimized in nature. This approach was used by Arnold and Moore [43] to make a 150-fold improvement in the activity of a -nitrobenzyl esterase in the presence of 15% DMSO. 

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