An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency

Abstract

Laccases are widely used in industrial production due to their broad substrate availability and environmentally friendly nature. However, the pursuit of laccases with superior stability and increased heterogeneous expression to meet industry demands appears to be an ongoing challenge. To address this challenge, we resurrected five ancestral sequences of laccase BsCotA and their homologues. All five variants were successfully expressed in soluble and functional forms with improved expression levels in Escherichia coli. Among the five variants, three exhibited higher catalytic rates, thermal stabilities, and acidic stabilities. Notably, AncCotA2, the best-performing variant, displayed a kcat/KM of 7.5 × 105 M−1·s−1, 5.2-fold higher than that of the wild-type BsCotA, an improved thermo- and acidic stability, and better dye decolorization ability. This study provides a laccase variant with high application potential and presents a new starting point for future enzyme engineering.

Improving the Quality of Wheat Flour Bread by a Thermophilic Xylanase with Ultra Activity and Stability Reconstructed by Ancestral Sequence and Computational-Aided Analysis

“…ancestral sequence showed higher thermal and acid stability [16]. Moreover, ancestral enzymes may have a broader substrate spectrum than the original enzymes.…”

“…By constructing a phylogenetic tree and inferring the amino acids of ancestral protein nodes, it is possible to find more stable proteins among these ancestral sequences. Reconstructed ancestral proteins, like the xylanase AncXyl09, have been shown to have improved stability, with a half-life of 65.08 h at 50 • C. Similarly, the designed AncCotA2 based on the ancestral sequence showed higher thermal and acid stability [16]. Moreover, ancestral enzymes may have a broader substrate spectrum than the original enzymes.…”

“…AliView software (Vesion 1.28) was used for multi-alignment [35], and 10,000 iterations were performed to create the phylogeny tree. Using the FastML (version 3.11) with a high posterior probability [36,37], we determined the most likely sequences from the same branch, and the ancestral sequences were obtained from the nodes as described before [16].…”

“…Ancestral sequence reconstruction has emerged as a useful methodology for engineering proteins with enhanced properties [14]. Previous studies on enzymes such as alcohol dehydrogenase KpADH [15], laccase AnCotA2 [16], and xylanase [17] have all recorded improved stability and a broader substrate spectrum. However, care should always be taken since reconstructed sequences represent historical proteins.…”