Semisynthetic Enzymes

Abstract

Natural enzymes play essential roles in the living cell by accelerating the rate of chemical processes. High catalytic efficiency, substrate specificity, stereoselectivity, and lack of toxicity are properties of enzymes that make them valuable in a multitude of applications, such as the production of fine chemicals [1, 2] and pharmaceuticals [3,4], food processing [5], bioremediation [6], and mining of metals [7,8]. Nevertheless, major obstacles in the use of natural enzymes in biotechnology are limitations in solubility, stability, and efficiency under nonphysiological conditions, including the presence of organic solvents or oxidants, extremes of temperature, pressure, or pH. Conventional genetic engineering of enzymes has provided solutions for these obstacles in some cases [9][10][11]. However, the repertoire of functional groups provided by the genetic code is restricted to the assortment of 20 canonical proteinogenic amino acids [phenylalanine (Phe), leucine (Leu), isoleucine (Ile), methionine (Met), valine (Val), serine (Ser), proline (Pro), threonine (Thr), alanine (Ala), tyrosine (Tyr), histidine (His), glutamine (Gln), lysine (Lys), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), cysteine (Cys), tryptophan (Trp), arginine (Arg), and glycine (Gly)], plus selenocysteine (Sec) [12] and pyrrolysine (Pyl) [13]. For many applications it could be advantageous to go beyond the conventional building blocks and incorporate novel chemical groups in available enzymes. In the cell, enzyme functionalities can be expanded by post-translational chemical modifications and incorporation of metal ions or organic cofactors. In protein engineering, semisynthetic enzymes are derived from natural protein scaffolds, which are chemically modified in ways that mimic or go beyond what cellular systems provide. We define a semisynthetic enzyme as an entity that has been procured by insertion of novel chemical groups via genetic engineering or chemical methods in the laboratory. A rigorous demarcation between natural posttranslational modification and semisynthesis is difficult to define, but semisynthesis generally implies that the product is not naturally occurring.The first known attempt to construct active and intact semisynthetic proteins was described by Cowie and Cohen in 1957 [14]. They replaced the amino acid Met in

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“…(25) have designed methods to overcome the limitations set by the genetic code and added ϳ70 novel amino acids with diverse side chains to enrich protein functions. Numerous related procedures have been developed, but each of them is hampered by particular limitations (26). Obstacles include amino acids that are incompatible with ribosomal translation or have structures toxic to cells.…”

The quest for molecular quasi‐species in ligand‐activity space and its application to directed enzyme evolution

“…Recent advances in gene synthesis have enabled the synthesis of mutant libraries to meet specified requirements. It is even possible to create proteins with unnatural amino acids and thereby go beyond the limitations of the genetic code [5].…”