Recent Advances in Enzyme Engineering through Incorporation of Unnatural Amino Acids (original) (raw)
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Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids
Journal of the American Chemical Society, 2011
The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions. Figure 7. Comparison of reaction velocities of wild-type arPTE and variants for hydrolysis of paraoxon at pH 7.5 and 8.5.
Strategies for Improving Enzymes for Efficient Biocatalysis
2004
Biocatalytic processes are finding increasingly widespread application not only in academia, but also in industry. This is particularly true in the pharmaceutical and agrochemical industries where the need for optically pure molecules is critical. Biocatalysis is also receiving a major thrust from the generation of new and novel biocatalysts via microbial screening, developments in the biocatalytic processes themselves (e.g. use of nonaqueous solvents for synthetic purposes) and improvements in the activation of the enzymes by numerous mechanisms (solvent and enzyme modifications), including protein engineering. The most dramatic results have been achieved by targeting the gene encoding a particular enzyme in order to undertake protein engineering by rational and non-rational methods. This review discusses various means of improving enzyme properties or creating new activities.
Advances in Enzyme Development and Applied Industrial Biocatalysis
ACS Symposium Series, 2001
Biocatalysts play important roles in various biotechnology products and processes in the food and beverage industries and have already been recognized as valuable catalysts for various organic transformations and production of fine chemicals and pharmaceuticals. At present, the most commonly used biocatalysts in biotechnology are hydrolytic enzymes which catalyze the breakdown of larger biopolymers into smaller units. Enzymes catalyze reactions in a selective manner, not only regio-but also stereoselectively and have been used both for asymmetric synthesis and racemic resolutions. The chiral selectivity of enzymes has been employed to prepare enantiomerically pure pharmaceuticals, agrochemicals and food additives. Biocatalytic methods have already replaced some conventional chemical processes. Biocatalytic routes, in combination with chemical synthesis, are finding increased use in the synthesis of novel polymeric materials. The present global market for enzymes is estimated to be more than US $1.5 billion. The discovery of new and improved enzymes and their use in various processes and products will create new market opportunities for biocatalysts and helps solve environmental problems. Applied biocatalysis can be defined as the application of biocatalysts to achieve a desired conversion under controlled conditions in a bioreactor (/). A biocatalyst can be an enzyme, an enzyme complex, a cell organelle or whole cells. The source of biocatalyst can be of microbial, plant or animal origin. Catalysis by an enzyme offers 2
Improving Nature’s Enzyme Active Site with Genetically Encoded Unnatural Amino Acids
The ability to site-specifically incorporate a diverse set of unnatural amino acids (>30) into proteins and quickly add new structures of interest has recently changed our approach to protein use and study. One important question yet unaddressed with unnatural amino acids (UAAs) is whether they can improve the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the >30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active site and a >2.3-fold improvement over the best possible natural amino acid. Because immense structural and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to improve enzyme active sites and tune a site to multiple substrates.
The search for the ideal biocatalyst
Nature Biotechnology, 2002
While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.
Advances in Enzymatic Synthesis of D-Amino Acids
International Journal of Molecular Sciences, 2020
In nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these “unnatural” molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-...
International Journal of Molecular Sciences, 2013
Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.