Protein Engineering by Directed Evolution

Another method used in protein engineering is directed evolution. Unlike rational enzyme design, this shategy requires neither structural information nor the knowledge of basic enzyme funchons. But there is a need for screening or selection 

In 1998, Yano et al. modified an aspartate transaminase to higher substrate specificity toward p-branched substrates and 2-oxo acids by DNA shuffling, resulting in an 10-fold higher catalytic efficiency toward 2-oxovaline, 2-oxoisoleucin, valine, 

To relieve product inhibition for higher product yields, Yun et al. created a mutant library of a transaminase from V. fluvialis by error-prone PCR [127]. The amino donor substrate was 2-aminoheptane and the inhibitory ketone was 2-butanone. In comparison with the wild-t5q e co-transaminase, the mutant had a 

Another approach in directed evolution was the evolution of an aspartate transaminase to an enz3une possessing the properhes of the closely related tyrosine transaminase [128]. Eight roxmds of DNA shuffling led to mutants with 100- to 270-fold increase of for phenylalanine and a 40- to 150-fold increase for tyrosine. 

This colorimetric assay was used in a study by Matcham et al. for detechon of mutants with higher tolerance toward (S)-methoxyisopropylamine ((S)-MOIPA) and higher thermal and chemical stability [60]. Due to a slightly increased product con-centrahon after adding higher amounts of enzyme, it was necessary to improve the product tolerance of the enzyme. Error-prone PCR and several rounds of enzyme modification created a mutant with the required properhes. At the end of the reachon the soluhon contained not only the desired product, (S)-MOIPA, but also acetone. To shift the equilibrium toward the product side by evaporahng acetone by-product, a transaminase mutant thermally stable up to 50 °C was found after five rounds of mutation and used in this process. The hnal yield of (S)-MOIPA about 2mM was obtained after 7h reaction hme with 5g/l of the transaminase at 50°C under vacuum. 

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Ligand fishing and protein engineering by directed evolution (O Neil and Hoess 1995)... 

O Neil, K.T. and Hoess, R.H., 1995. Phage display protein engineering by directed evolution, Curr. Opin. Struct. Biol., 5, pp. 443-449. 

Transaminases are important enzymes in the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter the properties of transaminases, the reaction mechanisms, and their selectivity and substrate specificity are presented. The synthesis of chiral building blocks for pharmaceutically relevant substances and fine chemicals with transaminases as biocatalysts is discussed. Enzymatic asymmetric synthesis and dynamic resolution are discussed using transaminases. Protein engineering by directed evolution as well as rational design of transaminases under process condition is presented to develop efficient bioprocesses. 

Transaminases are most powerful tools for the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter several approaches for tiie preparation of fine chemicals or building blocks for pharmaceuticals were discussed, like asymmetric synthesis or kinetic resolution. The main limitations of transaminase-catalyzed reactions are the need to shift the equihbrium to the product side and substrate and product inhibition. Some solutions to overcome such inhibition were presented here for example, multienzyme cascades or biphasic extraction of the product. Protein engineering by directed evolution or rational enzyme design is a promising option to find transaminases with different substrate specificities and enantiopreferences. This is becoming more and more important for the pharmaceutical industry. Furthermore, it is a way to alter enzyme properties known so far, like thermostability and solvent and pH stability. Protein engineering has been assisted by the recently solved structures of certain transaminases. 

The use of an LeuDH as an amino acid dehydrogenase showed a high L-enan-tiospecificity [24]. In this connection, an L-leucine dehydrogenase from Bacillus sphaericus has been applied very efficiently. The FDH from Candida boidinii is the preferred formate dehydrogenase for this process. The stability of this enzyme, which is available in technical quantities, has been remarkably improved by protein engineering and directed evolution [25], In particular the replacement of cys-... 

The development of rapid HTS assays is important to test the substrate scope, suitable amino donors/acceptors, and the stability under different reaction conditions, like temperature, pH, different solvents, and immobilization methods. Furthermore the rapid progress in protein engineering like directed evolution requires fast selection methods. This subject was extensively reviewed by Mathew et al. [148]. In the following a colorimetric, photometric, and kinetic assay for rapid transaminase activity screening is described and illustrated in Scheme 29.17. 

Fig. 13.3. Correlation of required mechanistic information, required structural information, importance of screening and number of possible enzyme variants in protein engineering by rational protein design and directed evolution.

Nakagawa Y, Hasegawa A, Hiratake J et al. (2007) Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity mechanistic implication for amide hydrolysis by serine hydrolases. Protein Eng Des Sel 20(7) 339-346 Nardini M, Lang DA, Liebeton K et al. (2000) Crystal stracture of Pseudomorms aeruginosa lipase in the open conformation. The prototype for family LI of bacterial lipases. J Biol Chem 275 31219-31225... 

Just recently, Huo et al. (2011, 2012) engineered E. coli for the protein-based higher alcohol production. This was achieved by directed evolution using chemical... 

Khurana, J., R. Singh, and J. Kaur. 2011. Engineering of Bacillus Lipase by Directed Evolution for Enhanced Thermal Stability Effect of Isoleucine to Threonine Mutation at Protein Surface. Molecular Biology Reports 38 (5) 2919-2926. 

Khurana J, Singh R, Kaur J. Engineering of Bacillus lipase by directed evolution for enhanced thermal stability effect of isoleucine to threonine mutation at protein surface. Mol Biol Rep 2010 1-8. 

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