Deciphering key residues governing thermostability in Thermomyces lanuginosus lipase through Gibbs free energy-guided engineering

Xia Xiang; Sidi Wan; Songjing Zhang; Enheng Zhu; Xuejun Lin; Nanyu Han

doi:10.1016/j.enzmictec.2025.110702

Deciphering key residues governing thermostability in Thermomyces lanuginosus lipase through Gibbs free energy-guided engineering

Enzyme Microb Technol. 2025 Jun 23:190:110702. doi: 10.1016/j.enzmictec.2025.110702. Online ahead of print.

Authors

Xia Xiang¹, Sidi Wan¹, Songjing Zhang², Enheng Zhu¹, Xuejun Lin¹, Nanyu Han³

Affiliations

¹ School of Life Sciences, Yunnan Normal University, Kunming, China.
² School of Biological Sciences, Nanyang Technological University, Singapore.
³ School of Life Sciences, Yunnan Normal University, Kunming, China; Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan Normal University, Kunming, China; Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, China. Electronic address: nyhan@ynnu.edu.cn.

PMID: 40570416
DOI: 10.1016/j.enzmictec.2025.110702

Abstract

Lipases serve as indispensable biocatalysts in many industrial applications due to their versatile catalytic abilities. To ensure their thermal resilience of the harsh biological treatment in industry, it is crucial to identify key residues which might impact thermostability. Here, computational design was adopted to decode the stability-determining residues in Thermomyces lanuginosus lipase (TLL). Systematic Gibbs free energy profiling of potent TLL single-point mutational candidates predicted proline 256 (P256) as a thermal liability hotspot. Saturation mutagenesis at P256 discovered that among nineteen P256 variants: (1) five P256 variants exhibited increased melting temperature (ΔT_m up to 2°C); (2) six variants displayed an optimum temperature with 5-10°C elevation; (3) five P256 variants retained up to 21 % higher residual activity after incubation at 80°C. Furthermore, both P256E and P256I demonstrated synergistic improvements in biodiesel conversion rates, P256I specifically exhibited long-term and recycling stability. Molecular dynamics simulations revealed that the substitutions in P256A/E/I/K with compensatory main-chain rotational freedom, facilitating hydrogen bonding network with both upstream and downstream residues, thereby preserving local structural stability. This study pioneers the identification of P256 as a critical residue governing TLL thermostability. Furthermore, our Gibbs-guided engineering strategy generates multi-property-enhanced lipase variants, directly addressing industrial demands.

Keywords: Gibbs-guided engineering; Lipase; Mutagenesis; Proline effect; Rosetta Cartesian_ddG; Site-saturation; Thermostability.