(R)-citronellal is an important intermediate for aroma compounds and pharmaceuticals. Herein, we engineered the choline oxidase, AcCO6, from Arthrobacter chlorophenolicus, to enhance enantioselective oxidation of citronellol. Through site-directed and combinatorial mutagenesis, a quadruple mutant, AcCO6_M4a (W332V/A89S/W331F/N463E) was obtained, exhibiting 336% of the wild-type activity and a 10.2-fold improvement in enantioselectivity (E = 24.2). Structural analysis and MD simulations revealed that the mutations expanded the binding pocket, promoting favorable interactions with (R)-citronellol and enhancing hydride transfer, while disfavoring the (S)-enantiomer. Consequently, the catalytic efficiency (kcat/KM) for (R)-citronellol was 12.5-fold higher than that for (S)-citronellol. Kinetic resolution of racemic citronellol using AcCO6_M4a achieved approximately 50% conversion, yielding (R)-citronellal (86.7% ee) and (S)-citronellol (97.9% ee) with 39.1% and 42.0% yield, respectively. This work demonstrates the potential of engineered AcCO6 for enantioselective biocatalysis, offering an efficient route to synthesize chiral alcohols and aldehydes as intermediates for fragrances and pharmaceuticals.
Peng-Cen Han, Die Lu, Xiaomei Wu et al.· Journal of Agricultural and...· 0 citations
Aspartase from Escherichia coli (AspA) catalyzes the direct conversion of acrylic acid to β-alanine; however, its substrate specificity and low catalytic efficiency limit its broader application. We engineered an AspA mutant capable of efficiently catalyzing the amination of acrylic acid for β-alanine synthesis, using Rosetta Enzyme Design to computationally redesign the Cβ-binding region of the acrylic acid binding site in AspA. Based on energy scores, structural configurations, and hydrogen bonding networks, 51 candidate variants with penalty scores below 30 were selected for mutant construction and performance testing; >70% of these variants exhibited enhanced catalytic activity in acrylic acid’s hydrogen amination. Four mutants achieved over 3-fold improved activity. The optimal mutant, M1 (T190I-M324I-K327L-N329C), demonstrated a 7.3-fold increased specific enzyme activity and a 13.0-fold improved kcat/Km compared with the wild type. Conformational changes in the S-loop and enhanced hydrophobic interactions near the active site contributed significantly to M1’s enhanced activity. Upon reaction optimization, the conversion of β-alanine synthesis using M1 in whole-cell catalysis increased from 5% with the wild type to 90% with M1. This study provides a reference for the biocatalytic synthesis of β-alanine, significantly enhancing the conversion of acrylic acid and demonstrating the enzyme’s potential for industrial applications.
Long-Xian Li, Baodi Ma, Yi Xu· Catalysts· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
