Engineering Bacterial Laccase with Improved Catalytic Activity and Thermostability by Rational Design

Laccases (benzenediol:oxygen oxidoreductases) are important multi-copper oxidases with widespread applications in industry. Here, Bacillus subtilis laccase CotA that has been widely studied was engineered to improve catalytic activity and thermostability via rational design. After iterative mutation of beneficial mutation sites, a triple mutant of CotA laccase (DTA) was obtained, whose catalytic activity and thermostability were improved by 2.7-fold and 1.4-fold compared with the wild-type (WT) CotA, respectively. The enhanced activity of DTA is primarily due to strengthened intermolecular forces in the active site, while its improved thermostability is attributed to increased hydrophobic residues, augmented protein surface flexibility, collectively rendering DTA a more active and stable enzyme with potential industrial applications. Compared to WT, DTA can degrade mycotoxins aflatoxin B1 (55.09% vs 44.51%) and ZEN (zearalenone) (71.59% vs 41.09%) more efficiently, and DTA can also better pretreat lignocellulose, promoting the hydrolysis of cellulose by cellulase. All these indicate that DTA has the potential to be used in industry.