Whole cell approach

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... 

The direct whole-cell method of Holland et al. was extremely rapid, even in comparison to Lubman s MALDI analysis of fractions collected after bacterial sonnication. With the whole-cell approach bacteria were simply sampled from colonies on an agar plate, mixed with the matrix, air-dried, and introduced in batches into the mass spectrometer for analysis. In all of the spectra obtained in these and later experiments, each bacterial strain showed a few characteristic high-mass ions that were attributed to bacterial proteins. Studies demonstrating the whole cell methodology for strain-level differentiation were reported independently by Claydon et al. at almost the same time.18 Shortly thereafter a third study on whole-cell MALDI included bacteria from pathogenic and nonpathogenic strains appeared.19... 

The different whole-cell approaches are at very different stages of development. 

Table 19.4 Whole-cell approaches for the reduction of keto compounds. 

Whilst the whole-cell approach has proved invaluable, the associated problems of overmetabolism and side reactions can be encountered. Another way to counter the problems of high cost in using isolated BVMOs is to use an NADH dependent enzyme, as NADH retails at approximately one tenth of the cost of NADPH. The Type 2 DKCMOs from Pseudomonas putida ATCC 17453 (= NCIMB 10007) are NADH dependent, and Grogan et al. were successftd in applying a complement of these enzymes, termed MOl, to the transformation of bicyclo[3.2.0]hept-2-en-6-one, to yield another enantiodivergent mix of lactones enantiomeric to those obtained... 

Under appropriate conditions it is possible to produce and isolate almost completely immature DsRED from bacterial expression systems. In contrast to flow cytometry and other whole cell approaches this allows for direct measurement of the maturation rate of the protein without ongoing protein synthesis. Interestingly, it was found that the maturation of DsRED is temperature dependent with the maturation rates increasing with temperatures, Fig. (17). At 37 °C maturation is already about threefold faster and at 60 °C the wildtype matures within 1 hour. This indicates that maturation of the wildtype protein is ineffective, but not principally limited to low rates. 

Biocatalysis involves the use of enzymes, microbes, and higher organisms to carry out chemical reactions. Because the reaction conditions are often mild, water-compatible, and environmentally friendly, they offer excellent examples of green chemistry . In view of the wealth of enzymes and whole-cell approaches available and the quality of researchers, this field continues to be very dynamic and productive. Biocatalysis in polymeric materials has been reviewed periodically (/-2). It is clear from the reviews that there is no shortage of activity and creativity in this field. 

The chemotaxis protein CheY was subsequently identified by both the whole-cell and reductionist approaches as the clockwise signal, i.e., as the molecule that interacts with the switch, thereby promoting clockwise rotation. In the whole-cell approach, second-site suppression analysis implied that CheY interacts with the switch (for reviews, see [56, 459]), and overproduction of CheY in a gutted strain generated clockwise rotation ([168] for a review, see reference [212]). In the reductionist approach, purified CheY inserted into cytoplasm-free envelopes caused some of the envelopes to rotate clockwise [594]. The absence of cytoplasmic chemotaxis proteins in the intact cells and the absence of cytoplasm in the envelopes indicated that the interaction of CheY with the switch is direct. Later biochemical studies demonstrated... 

Genetically Engineered Bacteria Whole Cell Approaches... 

The whole cell approach to production of oligosaccharide has been proved to be an attractive method with high yields, time and cost effectiveness, reproducibility and simple operation. However, to reach to the industrial scale... 

The use of enzymes and whole-cell approaches in polymers is now fairly widespread. A large number of reactions and processes has been developed, and new developments continue to appear in both the open and the patent literatures. Several excellent books (1.2) and reviews (3) are available. 

Microbial biocatalysis is often employed for polymer synthesis. A successful application area is the poly(hydroxyalkanoate) (PHA). Thus, Noda et al (22) reported the development of Nodax family of copolymers, which were shown to have versatile physical properties. Srienc et al (23) engineered yeast to produce PHAs comprising 6-13 carbon monomers. Henderson (5) included whole-cell approaches and metabolic engineering in the biomass conversion program. 

Let s not forget now the whole cell approach. Which has played a key role since early times. 

Reaction cascades of groups (1) and (2) will focus on physiologically occurring P450 cascades and have not been yet explored for synthetic appHcations (or only in very few parts). Cascades of groups (3) and (4) describe examples that have been applied for synthetic purposes. Here, examples with isolated enzymes and whole-cell approaches will be discussed. 

EC 5.1.3.8). While cascade coupling of the epimerization to a NeuA-catalyzed carboligation suffers from the combination of two unfavorable equilibria [24], the alternative coupling to PEP-dependent NeuS is more productive, as demonstrated by a whole-cell approach to the production of 1 [25]. Also, KDN 3 has been produced on a 100 g scale from D-mannose (8) and 5 using a pilot-scale enzyme membrane reactor with an overall crystallized yield of 75% (Scheme 17.5) [26]. 

TABLE 36.1. Advantages and Disadvantages of a Biocatalytic Oxidation Reaction nsing an Isolated Enzyme Compared with a Whole-cell Approach... 

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