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Computational-guided semi-rational design to reprogram phospholipase D into industrially robust enzyme.

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Τίτλος: Computational-guided semi-rational design to reprogram phospholipase D into industrially robust enzyme.
Συγγραφείς: Kong XL; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: 3171281291@qq.com., Zhang P; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A&F University, Hangzhou 311300, PR China. Electronic address: 2652726703@qq.com., Qin ZQ; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: 76674326@qq.com., Su C; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: 174020590@qq.com., Li H; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: liheng@jiangnan.edu.cn., Lu ZM; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: zmlu@jiangnan.edu.cn., Gong JS; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China; Shandong Kunda Biotechnology Co., Ltd, Linyi 276400, PR China. Electronic address: jinsonggong.bio@hotmail.com., Xu ZH; Institute of Future Food Technology, JITRI, Yixing 214200, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China; Innovation Center for Advanced Brewing Science and Technology, College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, PR China. Electronic address: zhenghxu@jiangnan.edu.cn., Shi JS; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: shijs@163.com.
Πηγή: Bioresource technology [Bioresour Technol] 2026 Jul; Vol. 451, pp. 134528. Date of Electronic Publication: 2026 Mar 29.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Elsevier Applied Science Country of Publication: England NLM ID: 9889523 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-2976 (Electronic) Linking ISSN: 09608524 NLM ISO Abbreviation: Bioresour Technol Subsets: MEDLINE
Imprint Name(s): Original Publication: Barking, Essex, England : New York, N.Y. : Elsevier Applied Science ; Elsevier Science Pub. Co., 1991-
Ιατρικοί όροι (MeSH): Phospholipase D*/chemistry , Phospholipase D*/genetics , Phospholipase D*/metabolism , Protein Engineering*/methods , Computer-Aided Design*, Mutation/genetics ; Molecular Dynamics Simulation ; Enzyme Stability ; Temperature ; Thermodynamics
Περίληψη: Phospholipase D (PLD) catalyzes the synthesis of rare phospholipid derivatives from phospholipids, and these derivatives possess significant application value and market potential. However, most reported PLDs show poor thermal stability, with melting temperatures below 60 °C, limiting their industrial application. This study employed a computer-aided design strategy integrating ancestral sequence reconstruction (ASR) and Gibbs folding free energy (ΔΔG) calculation to enhance PLD's thermal stability. Through iterative accumulation mutagenesis, a combinatorial mutant PLDM5 was generated. Its melting temperature and half-life at 50 °C increased by 13.4 °C and 415-fold, respectively. Meanwhile, the mutant retained effective catalytic performance, achieving a phosphatidylserine conversion of 78.8% with a productivity of 39.4 g/L/h. Structural and dynamics simulations revealed that the ASR and ΔΔG mutations synergistically enhanced structural rigidity through distinct mechanisms. This work expands the industrial application scope of PLD and enables high-temperature catalysis of phospholipids.
(Copyright © 2026 Elsevier Ltd. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Ancestral enzyme residue substitution; Energy calculation; Molecular dynamics simulation; Phosphatidylserine; Thermal stability modification
Substance Nomenclature: EC 3.1.4.4 (Phospholipase D)
Entry Date(s): Date Created: 20260331 Date Completed: 20260714 Latest Revision: 20260714
Update Code: 20260714
DOI: 10.1016/j.biortech.2026.134528
PMID: 41916458
Βάση Δεδομένων: MEDLINE
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  Data: Computational-guided semi-rational design to reprogram phospholipase D into industrially robust enzyme.
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  Data: <searchLink fieldCode="AU" term="%22Kong+XL%22">Kong XL</searchLink>; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: 3171281291@qq.com.<br /><searchLink fieldCode="AU" term="%22Zhang+P%22">Zhang P</searchLink>; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A&F University, Hangzhou 311300, PR China. Electronic address: 2652726703@qq.com.<br /><searchLink fieldCode="AU" term="%22Qin+ZQ%22">Qin ZQ</searchLink>; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: 76674326@qq.com.<br /><searchLink fieldCode="AU" term="%22Su+C%22">Su C</searchLink>; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: 174020590@qq.com.<br /><searchLink fieldCode="AU" term="%22Li+H%22">Li H</searchLink>; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: liheng@jiangnan.edu.cn.<br /><searchLink fieldCode="AU" term="%22Lu+ZM%22">Lu ZM</searchLink>; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: zmlu@jiangnan.edu.cn.<br /><searchLink fieldCode="AU" term="%22Gong+JS%22">Gong JS</searchLink>; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China; Shandong Kunda Biotechnology Co., Ltd, Linyi 276400, PR China. Electronic address: jinsonggong.bio@hotmail.com.<br /><searchLink fieldCode="AU" term="%22Xu+ZH%22">Xu ZH</searchLink>; Institute of Future Food Technology, JITRI, Yixing 214200, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China; Innovation Center for Advanced Brewing Science and Technology, College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, PR China. Electronic address: zhenghxu@jiangnan.edu.cn.<br /><searchLink fieldCode="AU" term="%22Shi+JS%22">Shi JS</searchLink>; Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: shijs@163.com.
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  Data: Phospholipase D (PLD) catalyzes the synthesis of rare phospholipid derivatives from phospholipids, and these derivatives possess significant application value and market potential. However, most reported PLDs show poor thermal stability, with melting temperatures below 60 °C, limiting their industrial application. This study employed a computer-aided design strategy integrating ancestral sequence reconstruction (ASR) and Gibbs folding free energy (ΔΔG) calculation to enhance PLD's thermal stability. Through iterative accumulation mutagenesis, a combinatorial mutant PLD<subscript>M5</subscript> was generated. Its melting temperature and half-life at 50 °C increased by 13.4 °C and 415-fold, respectively. Meanwhile, the mutant retained effective catalytic performance, achieving a phosphatidylserine conversion of 78.8% with a productivity of 39.4 g/L/h. Structural and dynamics simulations revealed that the ASR and ΔΔG mutations synergistically enhanced structural rigidity through distinct mechanisms. This work expands the industrial application scope of PLD and enables high-temperature catalysis of phospholipids.<br /> (Copyright © 2026 Elsevier Ltd. All rights reserved.)
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  Data: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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  Data: <i>Keywords: </i>Ancestral enzyme residue substitution; Energy calculation; Molecular dynamics simulation; Phosphatidylserine; Thermal stability modification
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