American Chemical Society
sc2c04531_si_001.pdf (1.2 MB)

Engineering β‑agarase Aga0917 from Pseudoalteromonas fuliginea YTW1-15‑1 to Improve the Thermostability and Enzyme Activity

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journal contribution
posted on 2022-11-11, 18:40 authored by Tao Li, Ning Cui, Jiwu Fan, Xinqi Liu, Jianyi Guan, Wenbo Zhang, Yang Li, Qiyu Gao, Yihan Liu, Yongtao Xu, Lei Wang, Changjiang Guo, Yan Wang
High thermostability and activity are vital to agarases due to the higher accessibility of sol state agarose to enzymes. Glu128 and Glu133 were proven to be the two essential catalytic residues of Aga0917 through site-directed mutation. To improve the thermostability of Aga0917, a proline was introduced into the loop Asp138-Gln142 at the site of V140 after rational analyses. The half-life of V140P at 50 °C is 48.4 min, 1.7-fold longer than that of the wildtype enzyme (28.4 min), demonstrating a significantly enhanced thermostability. After incubation at 50 °C for 45 min, the V140P still demonstrated a neoagarotetraose productivity of 1.38 mg/mg/h, which was 2.76 folds that of wildtype Aga0917 (0.50 mg/mg/h), displaying improved thermostability and hydrolysis activity at a high temperature. Molecular dynamics simulations revealed that a stable combination formed between loop Asp138-Gln142 and its adjacent loop Pro240-Pro246 with the aid of increased hydrogen bonds and salt bridges in V140P. Moreover, a shrunken substrate-binding cleft appeared for the shortened distance between loops Pro240-Pro246 and Pro160-Lys167. As a result, a decreased substrate fluctuation in the binding cleft, an increased number of hydrogen bonds, enhanced binding free energy, and a decreasing nucleophilic attack distance between the catalytic residues and substrate were observed, which should be responsible for the improved thermostability and activity. Our results demonstrated a new perspective on accelerating protein evolvability by modulating the flexibility of the loop away from the active center, which ultimately remodeled the active site in a remote-control manner.