Engineering fungal antifreeze proteins through “EKylation” and its mechanism analysis

CUI Zhongxin; WANG Yi; ZHANG Lei; QI Haishan

doi:10.12211/2096-8280.2025-019

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Engineering fungal antifreeze proteins through “EKylation” and its mechanism analysis

Research Article | 更新时间：2026-01-15

- Engineering fungal antifreeze proteins through “EKylation” and its mechanism analysis
- Synthetic Biology Journal Vol. 6, Issue 6, Pages: 1435-1447(2025)
- 作者机构：
  
  1.天津大学合成生物与生物制造学院，天津 300072
  2.天津大学合成生物技术全国重点实验室，教育部合成生物学前沿科学中心，天津 300072
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2025-019
  CLC： Q81
- Received：19 March 2025，
  
  Revised：2025-07-18，
  
  Published：31 December 2025
- 稿件说明：
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崔忠信，王怡，张雷，齐海山. “EKylation”策略改造真菌抗冻蛋白及机制解析［J］. 合成生物学， 2025， 6（6）： 1435-1447

CUI Zhongxin， WANG Yi， ZHANG Lei， QI Haishan. Engineering fungal antifreeze proteins through “EKylation” and its mechanism analysis［J］. Synthetic Biology Journal， 2025， 6（6）： 1435-1447
崔忠信，王怡，张雷，齐海山. “EKylation”策略改造真菌抗冻蛋白及机制解析［J］. 合成生物学， 2025， 6（6）： 1435-1447 DOI： 10.12211/2096-8280.2025-019.

CUI Zhongxin， WANG Yi， ZHANG Lei， QI Haishan. Engineering fungal antifreeze proteins through “EKylation” and its mechanism analysis［J］. Synthetic Biology Journal， 2025， 6（6）： 1435-1447 DOI： 10.12211/2096-8280.2025-019.

摘要

抗冻蛋白（antifreeze protein， AFP）是一类通过非依数性机制降低冰点并有效抑制冰晶生长的生物大分子，广泛分布于极地鱼类、昆虫及耐寒微生物等生物体内以维持其低温适应性，在食品冷冻保存、低温医学及低温生物技术等领域具有广泛应用，开发高活性AFP具有重要研究价值。本研究利用“EKylation”策略对雪腐病核瑚菌（

Typhula ishikariensis

）来源的AFP（RCSB ID：5B5H）开展分子改造研究。基于两性离子多肽的电荷可调特性与蛋白质稳定化机制，将两性离子多肽（EK）

定点偶联至5B5H的N端结构域，经两性离子改造的重组蛋白5B5H-EK高级结构保持稳定，热滞活性较野生型提升27.8%。分子动力学模拟进一步揭示5B5H-EK的冰晶结合面并未发生改变，且具有更强的抑制冰晶生长能力和冰晶结合能力，促使水分子形成短程有序的“类冰水”结构。该研究为理性设计高效抗冻蛋白及环境适应性抗冻材料提供了新策略。

Abstract

Antifreeze proteins (AFPs)

a functionally unique and diverse class of

biomolecules

possess the ability to depress the freezing point of aqueous solutions non-colligatively and inhibit the damaging process of ice recrystallization. These critical activities stem from their surface-adsorption mechanism at the ice-water interface

preventing ice crystal growth and maturation. Consequently

the strategic development of novel AFP analogues exhibiting significantly enhanced activity and stability represents an area of substantial research interest and practical value. Herein

we introduce and implement an “EKylation” strategy designed to rationally engineer the AFP (RCSB ID:5B5H) derived from the freeze-tolerant fungus

Typhula ishikariensis

. This approach specifically leverages the charge-tunable properties inherent to zwitterionic peptides to modify protein surfaces. We chemically coupled the synthetic zwitterionic polypeptide (EK)

comprising alternating glutamate (E

negatively charged) and lysine (K

positively charged) residues

to the N-terminal structural domain of the wild-type 5B5H

which yielded the recombinant conjugate

designated 5B5H-EK. Comprehensive biophysical characterization revealed that the 5B5H-EK conjugate exhibited markedly enhanced structural stability compared to its unmodified counterpart. Crucially

this conjugation strategy led to a significant functional improvement

with the modified protein demonstrating a thermal hysteresis (TH) activity of 27.8%

representing a substantial enhancement over the wild-type AFP. Further insight into the molecular basis of this activity boost was gained through molecular dynamics (MD) simulations. These simulations indicated that the core ice crystal binding surface architecture of 5B5H-EK was largely preserved. Therefore

the conjugated zwitterionic chain significantly augmented the protein’s inherent ability to inhibit ice crystal growth and bind tenaciously to the ice surface. Analysis of water dynamics near the modified protein surface suggested that the (EK)

chain promotes the

formation of an extensive

short-range ordered hydration shell

effectively structuring interfacial water molecules into an “ice-water-like” layer distinct from bulk water. This engineered interfacial hydration likely contributes synergistically to the enhanced TH performance by reinforcing the AFP’s anchoring to the quasi-liquid layer and facilitating greater surface coverage. In conclusion

this study proposes a novel and effective protein engineering strategy

utilizing zwitterionic peptide conjugation (“EKylation”)

for generating highly efficient antifreeze proteins and informs the rational design of next-generation

environmentally adaptable antifreeze materials.

关键词

Keywords

references

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Related Author

CUI Zhongxin

WANG Yi

ZHANG Lei

QI Haishan

Related Institution

School of Synthetic Biology and Biomanufacturing， Tianjin University

State Key Laboratory of Synthetic Biology， Frontiers Science Center for Synthetic Biology， Tianjin University

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