Enhancing the thermostability of lignin peroxidase: Heme as a keystone cofactor driving stability changes in heme enzymes
Joo Yeong Park,
Seunghyun Han,
Doa Kim,
Trang Vu Thien Nguyen,
Youhyun Nam,
Suk Min Kim,
Rakwoo Chang,
Yong Hwan Kim
Affiliations
Joo Yeong Park
School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea
Seunghyun Han
School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea
Doa Kim
School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea
Trang Vu Thien Nguyen
School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea
Youhyun Nam
Department of Applied Chemistry, University of Seoul, 163, Seoulsiripdae-ro, Seoul, 02504, Republic of Korea
Suk Min Kim
School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea; Corresponding author.
Rakwoo Chang
Department of Applied Chemistry, University of Seoul, 163, Seoulsiripdae-ro, Seoul, 02504, Republic of Korea; Corresponding author.
Yong Hwan Kim
School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea; Graduate School of Carbon Neutrality, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea; Corresponding author. School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), 50, UNIST-gil, Ulsan, 44919, Republic of Korea.
Heme-containing enzymes, critical across life's domains and promising for industrial use, face stability challenges. Despite the demand for robust industrial biocatalysts, the mechanisms underlying the thermal stability of heme enzymes remain poorly understood. Addressing this, our research utilizes a ‘keystone cofactor heme-interaction approach’ to enhance ligand binding and improve the stability of lignin peroxidase (LiP). We engineered mutants of the white-rot fungus PcLiP (Phanerochaete chrysosporium) to increase thermal stability by 8.66 °C and extend half-life by 29 times without losing catalytic efficiency at 60 °C, where typically, wild-type enzymes degrade. Molecular dynamics simulations reveal that an interlocked cofactor moiety contributes to enhanced structural stability in LiP variants. Additionally, a stability index developed from these simulations accurately predicts stabilizing mutations in other PcLiP isozymes. Using milled wood lignin, these mutants achieved triple the conversion yields at 40 °C compared to the wild type, offering insights for more sustainable white biotechnology through improved enzyme stability.