Lipase Congeners Designed by Genetic Code Engineering

Abstract

Classical enzyme optimization exploits the chemistry confined to the 20 canonical amino acids encoded by the standard genetic code. ‘Genetic code engineering’ allows the global substitution of particular residues with synthetic analogues, endowing proteins with chemical diversity not found in nature. These proteins are congeners of the parent protein because they originate from the same gene sequence, but contain a fraction of noncanonical amino acids. Global substitutions of methionine, proline, phenylalanine, and tyrosine have been carried out with related analogues in Thermoanaerobacter thermohydrosulfuricus lipase. This study represents the first extensive report of an important biocatalyst substituted with a high number of noncanonical amino acids. The generated lipase congeners displayed special features such as enhanced activation, elevated enzyme activity (by up to 25 %) and substrate tolerance (by up to 40 %), and changes in optimal temperature (by up to 20 °C) and pH (by up to 3). These emergent features achieved by genetic code engineering might be important not only for academic research, but also for numerous economical applications in the food, detergent, chemical, pharmaceutical, leather, textile, cosmetic, and paper industries.

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

“…[38][39][40] The more hydrophobic [41] analogues norleucine (Nle) and ethionine (Eth) were used to study overall effects on enzyme activity. Their incorporation in the Met-rich active site of Calmodulin lowered its activity, [42] whereas incorporation of Nle into Lipase from Thermoanaerobacter thermohydrosulfuricus (TTL) led to an enzyme highly active in the aqueous phase without the need for thermal activation [43] and enhanced stability against denaturing agents. [44] Other examples of Met analogue applications include the use of methoxinine to study the effects of Met oxidation on prion proteins, [45] of photo-methionine in protein-protein interaction studies, [46] and of homoallylglycine (Hag) for potential use in olefin metathesis.…”

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…111 The amino acids studied included fluorinated derivatives such as (3-F)Phe, (4-F)Phe, (4S-F)Pro, (4R-F)Pro, (2-F)Tyr and (3-F)Tyr. The lipase consists of 6 Pro residues, 16 Phe residues and 7 Tyr residues (Fig.…”

“…111,126 This could be explained by the results of peptide studies showing that the biased C4 ring puckering of proline due to stereoelectronic effects cannot selectively stabilize or destabilize the structure when the proline residue is involved in other interactions, e. g. aromatic-proline interactions. 116 On the other hand, the flexibility/structural rigidity of the biopolymer might be important as well.…”

Manipulation of enzyme properties by noncanonical amino acid incorporation

“…Very recently, to assess the effect of NCAA incorporation into lipase, Antranikian et al [65] globally replaced Met, Pro, Phe, and Tyr residues of lipase obtained from Thermoanaerobacter thermohydrosulfuricus (TTL) with related analogues. A total of ten TTL variants were generated by incorporating one of the ten different NCAAs, (8), (9), (10), (11), (13) (19).…”