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Updated: Apr 4, 2026
A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Engineering of β-Agarase with Enhanced Thermostability via Multitool Consensus Prediction and Structure-Guided
Qianqian Diao1,2,3, Xingfei Li1,2,3, Yuxiang Bai1,2
1The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
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β-Agarase plays a crucial role in preparing bioactive agar oligosaccharides, but its insufficient thermostability limits industrial application. In this study, we developed a stepwise design strategy integrating multitool consensus prediction, structure-based rational screening, and greedy algorithm-based optimization to enhance AgaDcat's thermostability. This yielded mutant M3 (N120S-D243N-Q246A-S287E-A335D), which exhibits an 11 °C higher melting temperature (Tm) and 14-fold longer half-life (t1/2) at 50 °C than the wild-type. Molecular dynamics simulations showed that mutations strengthened hydrophobic interactions, salt bridges, and hydrogen bond networks, while optimizing surface charge. The M3 variant performed well in high-temperature agarose hydrolysis, mainly producing neoagarotetraose (NA4) and neoagarohexaose (NA6), showing great industrial potential. This framework improves the efficiency of enzyme thermostability engineering and provides a general approach for industrial enzymes modification.
