Evolution studies

The Bacillus subtilis lipase A (BSLA) was the subject of two short directed evolution studies [19,47]. In one case systematic saturation mutagenesis at all of the ISlpositions of BSLA was performed [19]. Using meso-l,4-diacetoxy-2-cyclopentene as the substrate, reversed enantioselectivity of up to 83% ee was observed. In another study synthetic shuffling (Assembly of Designed Oligonucleotides) was tested using BSLA [47]. 

In another intriguing direded evolution study, the stereochemical course of aldol additions was significantly altered in a different sense [78] rather than evolving aldolase mutants that seledively accept stereoisomers of substrates, the... 

The initial results of an early directed evolution study are all the more significant, because no X-ray data or homology models were available then to serve as a possible guide [89]. In a model study using whole E. coU cells containing the CHMO from Adnetohacter sp. NCIM B9871,4-hydroxy-cydohexanone (3 5) was used as the substrate. The WT leads to the preferential formation of the primary product (i )-36, which spontaneously rearranges to the thermodynamically more stable lactone (R)-37. The enantiomeric excess of this desymmetrization is only 9%, and the sense of enantioselectivity (R) is opposite to the usually observed (S)-preference displayed by simple 4-alkyl-substituted cydohexanone derivatives (see Scheme 2.10) [84—87]. 

In the directed evolution study, epPCR at various mutation rates was applied (10 000 clones). Some of the hits were (R) selective, and others were (S) selective. Eight of them were sequenced [89]. Of particular interest is mutant 1-K2-F5, characterized by a single mutation F432S, because it leads to reversal of enantioselectivity (79% ee in favor of (S)-37). 

Figure 6 Model structure of CHMO. The FAD cofactor is shown as sticks. Mutations observed in a directed evolution study resulting in altered enantioselectivities are highlighted (spheres). " Note that a number of mutations were found in double/triple mutants and cannot be linked with certainty to the altered catalytic properties. Mutations in double/triple mutants that are on the...

Limits of Time-Resolved and Simultaneous Measurements. Structure evolution studies are based on the ability to carry out time-resolved scattering experiments. The power of this scattering technique is a function of the minimum cycle time during which a scattering pattern with sufficient signal-to-noise ratio can be recorded. As cycle times for anisotropic 2D SAXS patterns have fallen below a value... 

Iron-sulfur proteins are a group of enzymes and other electron carriers that contain clusters of iron and sulfide linked directly to amino-acyl side chains, usually cysteines. They are widely distributed in nature. Soon after their discovery. Hall et al. (1971) proposed that they could be used to follow the course of evolution. Studies of genome sequences have revealed that iron-sulfur cluster binding motifs are among the most commonly recognized sequences. 

A related assay was later reported which makes use of a different and more convenient indicator (bromothymol blue) 78). However, it is emphasized that all of the assays based on pH change reported so far refer to the use of isolated enzymes. In real applications, supernatants are likely to be used, as in directed evolution studies. In supernatants, however, pH variations may occur. Therefore, an optimized assay was later developed in which supernatants are employed (79). Thus, the pH of the buffer is adjusted to the acidity of the medium, enabling about 4000 samples in kinetic resolution investigations to be screened per day. 

FTIR spectroscopy to a particular pesticide, the methods have general applications to numerous compounds. Most of these utilize the high sensitivity of FTIR, and the data manipulation capability of the system. In several of the gas evolution studies, spectra were acquired at less than one-minute intervals. While this is not really "rapid scanning," the high resolution required for vapor phase spectra would not have been possible with a normal dispersive instrument. Several other techniques using FTIR show promise in the area of pesticide analysis. 

---