Mutagenesis directed evolution

During the last decade, significant advancements in biochemistry, molecular cloning, and random and site-directed mutagenesis, directed evolution of biocatalysts, metabolic engineering and fermentation technology have led us to devise methods to circumvent the disadvantages of whole-cell biotransformation discussed in Section 10.2. The applications of these methods are summarized in this section. 

There have been many attempts to improve protein stability and protein properties, utilizing methods such as random mutagenesis, directed evolution, and rational protein design approaches. In general, these methods are far from straightforward and can be time-consuming. In addition, the stabilization of proteins without loss of function is not a trivial problem. 

Just as nature uses evolution to naturally select enzyme variations that provide an advantage to the host, directed evolution of the amino acid substitution utilize techniques to screen or select for mutant enzymes that perform better than wild-type enzymes. Unlike site-directed mutagenesis, directed evolution techniques do not require a detailed understanding of the enzyme in order to identify useful variants. The techniques of applied molecular evolution however, do require a screening or selection step to identify the individual mutants of interest (Fig. 12.2). 

Biocatalysis is the study of biological catalysts with regard to their kinetics, mechanisms, specificity, and application in synthesis and analysis. In addition to the traditional study of mechanistic enzymology, biocatalysis is concerned with the use of recombinant DNA technology, site-specific mutagenesis, directed evolution, pathway engineering, substrate design, and structure-based approaches as tools for the development of novel catalysts and reactions. 

Recent advances in recombinant DNA technology, high-throughput technologies, genomics and proteomics, have fuelled the development of new biocatalysts and biocatalytic processes. In particular, site directed mutagenesis, directed evolution or metagenome approach are very valuable tools to enhance lipase properties. 

In biocatalysis research, the use of protein engineering, mutagenesis, directed evolution, and various bioinformatic... 

Technique Site-directed mutagenesis Semirandomm utagenesis Random mutagenesis Directed Evolution 1 Directed Evolution 2 pH, additives, chromophore analogs... 

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

Figure 2.9 Schematic summary of the directed evolution of enantioselective lipase variants originating from the WT PAL used as catalysts in the hydrolytic kinetic resolution of ester rac-1. CMCM = Combinatorial multiple-cassette mutagenesis [8c,22],...

The data in Figure 2.12 are the results of initial mutagenesis experiments, which does not yet constitute directed evolution. An evolutionary process was subsequently induced by combining the mutations of two improved mutants of the first round [46]. Thereby new mutants were obtained, which show an increase of activity relative to the WT by more than 2 orders of magnitude. Although enantioselectivity was not the... 

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]. 

Directed evolution of enzymes has been used to improve the reducing function of the enzymes. For example, this method was used to eliminate the cofactor requirement of B. stearothermophillus lactate dehydrogenase, which is activated in the presence of fructose 1,6-bisphosphate [12]. The activator is expensive and representative of the sort of cofactor complications that are undesirable in industrial processes. Three rounds of random mutagenesis and screening produced a mutant that is almost fully... 

Upon mutagenesis of the monoamine oxidase from Aspergillus niger (MAO-N) within several rounds of directed evolution [65], variant biocatalysts were identified with largely expanded substrate acceptance, enabling also the deracemization of tertiary amines incorporating straight-chain and cyclic structural motifs [66]. 

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

Eggert, T., Jaeger, K.-E. and Reetz, M.T. (2004) Directed evolution of random mutagenesis a critical evaluation, in Enzyme Functionality (ed. A. Svendsen), Marcel Dekker, Inc., New York, pp. 375-390. 

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