Whole cell applications

Whole Cell Applications Based on Recombinant Enzymes ( Designer Cells ). 222... 

Chiral epoxides and their corresponding vicinal diols are very important intermediates in asymmetric synthesis [163]. Chiral nonracemic epoxides can be obtained through asymmetric epoxidation using either chemical catalysts [164] or enzymes [165-167]. Biocatalytic epoxidations require sophisticated techniques and have thus far found limited application. An alternative approach is the asymmetric hydrolysis of racemic or meso-epoxides using transition-metal catalysts [168] or biocatalysts [169-174]. Epoxide hydrolases (EHs) (EC 3.3.2.3) catalyze the conversion of epoxides to their corresponding vicinal diols. EHs are cofactor-independent enzymes that are almost ubiquitous in nature. They are usually employed as whole cells or crude... 

This suite of BVMOs is available via whole-cell expression systems and represents a complementary platform of biocatalysts for diverse applications in chiral synthesis. Representatives of this collection were utilized in the enantiodivergent synthesis of the indole alkaloids alloyohimbane and antirhine from a fused bicyclic precursor (Scheme 9.19) [151]. 

The results presented in Tables 3 and 4 deserve some comments. First, a variety of enzymes, including whole-cell preparations, proved suitable for the resolution of different hydroxyalkanephosphorus compounds, giving both unreacted substrates and the products of the enzymatic transformation in good yields and, in some cases, even with full stereoselectivity. Application of both methodologies, acylation of hydroxy substrates rac-41 and rac-43 or the reverse (hydrolysis of the acylated substrates rac-42 and rac-44), enables one to obtain each desired enantiomer of the product. This turned out to be particularly important in those cases when a chemical transformation OH OAc or reverse was difficult to perform. As an example, our work is shown in Scheme 3. In this case, chemical hydrolysis of the acetyl derivative 46 proved difficult due to some side reactions and therefore an enzymatic hydrolysis, using the same enzyme as that in the acylation reaction, was applied. Not only did this provide access to the desired hydroxy derivative 45 but it also allowed to improve its enantiomeric excess. In this way. 

Conventional use has been made of the radioisotope C, and details need hardly be given here. Illustrative examples include the elucidation of pathways for the anaerobic degradation of amino acids (Chapter 7, Part 1) and purines (Chapter 10, Part 1). Some applications have used C with high-resolution Fourier transform NMR in whole-cell suspensions, and this is equally applicable to molecules containing the natural or the synthetic P nuclei. As noted later, major advances in NMR have made it possible to use natural levels of C. 

This has received widespread application, and a single example has been chosen as representative. The pathway for the degradation of morpholine by Mycobacterium aurum MOl sadMycobacterium strain RPl was examined using whole cells, and this confirmed its identity to the one that had been proposed earlier for M. chelonae (Combourieu et al. 1998 Poupin et al. 1998). 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

The utility of MALDI-FTMS analysis for use in chemotaxonomic applications has been established, but this method can be applied to other areas of interest, such as biomedical and environmental analyses. A common method used by biochemists and biologists today is recombinant overexpression of proteins using bacterial whole cells in cases where large quantity of a protein is desired. The main method presently used to determine if the overexpression was successful is the use of SDS-PAGE (sodium dodecylsulfate-poly acrylamide gel... 

---