Directed evolution of a novelN-carbamylase/D-hydantoinase fusion enzyme for functional expression with enhanced stability

Abstract

Bifunctional enzymes find a wide application as a monitoring facility and a potential biocatalyst in molecular biology and biotechnology. Recombination of natural enzymes to a bifunctional fusion offers valuable tools, but the functional and structural instability of artificial fusion enzymes remains to be solved. Based on structural traits of microbial D-hydantoinase, we attempted to construct a bifunctional N-carbamylase/Dhydantoinase fusion enzyme that would be useful for the synthesis of nonnatural D-amino acids in a concerted fashion. The bifunctional ability of D-hydantoinase, as a fusion partner, was noticeable, but the resulting fusion enzyme was subjected to serious proteolysis in vivo, as generally encountered in the expression of large the multidomain polypeptide in E. coli. In an effort to improve the structural instability imposed by artificial linear fusion, directed evolution of the fusion enzyme was performed using DNA shuffling with a consensus primer to maintain a crucial domain for the enzyme activity. The evolved fusion enzyme, F11, was selected after repeated rounds, and this enzyme was found to show sixfold increased performance in the production of D-amino acid compared with the parent fusion enzyme, which was mainly due to the enhanced structural stability of the evolved fusion enzyme. This result is an example showing that directed evolution of the linearly fused polypeptide may broaden the opportunity to generate a fusion enzyme with greater potential.

Directed Evolution and Biocatalysis

“…However, the fusion enzyme was unstable as a result of extensive proteolysis, and DNA shuffling was used subsequently to enhance the stability of the fusion enzyme. An evolved enzyme was identified that was six times as active as the parent fusion enzyme in converting the hydantoin into the D ‐amino acid 41. A direct comparison of the performance of the evolved fusion enzyme with the best available co‐expression system has yet to be reported.…”

Crystal Structure of d-Hydantoinase from Bacillus stearothermophilus: Insight into the Stereochemistry of Enantioselectivity,

“…Hydantoinase was also subjected to directed evolution for enhanced stereochemical specificity and thermostability ( , ). Because of the potential commercial value of the structure-based design of the novel properties of these enzymes, both the l - and d -enantiomer specific hydantoinases were subjected to structural studies.…”

Evolutionary relationship and application of a superfamily of cyclic amidohydrolase enzymes

“…However, it exhibited structural instability resulting in extensive proteolysis in vivo probably due to the low stability of the N‐terminal fusion partner, N‐carbamoylase. This unfavorable property of a fusion enzyme was improved by using DNA shuffling 58. Through three rounds of directed evolution, an evolved fusion enzyme with nine amino acid substitutions was obtained.…”