Iterative enhancement of cutinase thermostability by multiple strategies based on combined directed evolution and computationally assisted design

Shiheng Chen; Lei Wang; Demin Kong; Zhisheng Wei; Xiaoqi Ling; Zhaohong Deng; Fengshan Zhang; Lingqia Su; Zhanzhi Liu; Jing Wu; Sheng Chen

doi:10.1016/j.biortech.2025.132882

Iterative enhancement of cutinase thermostability by multiple strategies based on combined directed evolution and computationally assisted design

Bioresour Technol. 2025 Jun 23:435:132882. doi: 10.1016/j.biortech.2025.132882. Online ahead of print.

Authors

Shiheng Chen¹, Lei Wang¹, Demin Kong¹, Zhisheng Wei¹, Xiaoqi Ling², Zhaohong Deng², Fengshan Zhang³, Lingqia Su¹, Zhanzhi Liu⁴, Jing Wu⁴, Sheng Chen⁵

Affiliations

¹ Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
² The School of Artificial Intelligence and Computer Science, Jiangnan University, Wuxi 214122, China.
³ Shandong Huatai Paper Co., Ltd. and Shandong Yellow Triangle Biotechnology Industry Research Institute Co. Ltd, Dongying 257335, China.
⁴ Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Shandong Huatai Paper Co., Ltd. and Shandong Yellow Triangle Biotechnology Industry Research Institute Co. Ltd, Dongying 257335, China; State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
⁵ Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: chensheng@jiangnan.edu.cn.

PMID: 40562246
DOI: 10.1016/j.biortech.2025.132882

Abstract

Cutinase exhibits versatile biocatalytic potential in polymer degradation, textile processing, and industrial biocatalysis, where enhancing the thermal stability under extreme conditions is essential for practical applications. To enhance the thermal stability ofHumicola insolens cutinase (HiC), a combination of strategies approach integrating error-prone PCR, computational design, and machine learning were implemented.Through systematic iterative recombination, an octuple mutant (M8) was developed with thermal tolerance and preserved catalytic activity. The engineered mutant demonstrated exceptional stability enhancements, exhibiting 8110- and 3982-fold increases in half-life at 65 °C and 70 °C, respectively, compared with wild-type HiC. Differential scanning calorimetry revealed a 13.0 °C elevation in melting temperature (T_m), while thermal inactivation analysis showed a 26.2 °C improvement in T (temperature causing 50 % activity loss in 60 min). Notably, M8 retained full activity at standard assay temperature and exhibited a broad pH profile despite incorporating eight stabilizing mutations. Molecular dynamics simulations and structural analyses revealed that the redistribution of surface electrostatic charges and a more compact overall structure were key factors in enhancing thermal stability. In summary, this study established a framework for rational thermostabilization of cutinases while providing molecular level insights into thermal adaptation mechanisms, with methodological implications for α/β-hydrolase family enzyme optimization. The engineered HiC mutant present significant potential for industrial processes requiring high-temperature operations.

Keywords: Humicola insolens cutinase (HiC); Iterative combination; Molecular dynamics simulation; Multi-strategy combination; Thermostability.