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In this study, a lignin peroxidase from Phanerochaete chrysosporium (PcLiP; GenBank accession no. X15599.1) was engineered using a semirational design strategy. Seven candidate residues were identified by molecular docking, and alanine scanning yielded three mutants (H38A, E320A, and T267A) with over 80% increased activity. Subsequent saturation mutagenesis at T267 identified T267Q as a more active variant. The triple mutant PcLiP-L4 (H38A\/T267Q\/E320A) exhibited the highest activity, corresponding to a 2.9-fold increase over the wild type. Enhanced catalytic performance was confirmed by ABTS oxidation and alkali lignin degradation assays. After expression in Pichia pastoris, PcLiP-L4 achieved a wheat straw lignin degradation rate of 12.6%, which was 3.26-fold that of the control. Molecular dynamics simulations indicate improved substrate binding stability and reduced binding free energy, contributing to enhanced catalytic efficiency.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c00180","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T06:51:46Z","timestamp":1788418306000},"page":"28124-28135","source":"Crossref","is-referenced-by-count":0,"title":["Semi-Rational Design of Lignin Peroxidase for Enhanced\nActivity and Application in Wheat Straw Lignin Degradation"],"prefix":"10.1021","volume":"74","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0202-1043","authenticated-orcid":true,"given":"Yong","family":"Chen","sequence":"first","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, P. R. China, 212013"]},{"name":"Anhui Gujing Distillery Company Limited , , ,","place":["Bozhou, Anhui, P. R. China, 236800"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Na","family":"Shi","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, P. R. China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anjun","family":"Li","sequence":"additional","affiliation":[{"name":"Anhui Gujing Distillery Company Limited , , ,","place":["Bozhou, Anhui, P. R. China, 236800"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"De","family":"Cheng","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, P. R. China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minghaonan","family":"Chen","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, P. R. 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Crops Prod."},{"key":"2026091609090541200_cit3","doi-asserted-by":"publisher","first-page":"117598","DOI":"10.1016\/j.jclepro.2019.07.073","article-title":"Sustainable Alternatives to Carbon\nIntensive Paddy Field Burning in India: A Framework for Cleaner Production\nin Agriculture, Energy, and Construction Industries","volume":"236","author":"Athira","year":"2019","journal-title":"J. Cleaner Prod."},{"key":"2026091609090541200_cit4","doi-asserted-by":"publisher","first-page":"143753","DOI":"10.1016\/j.ijbiomac.2025.143753","article-title":"Electrochemical Conversion\nof Lignocellulosic\nBiomass to Carboxymethyl Cellulose: Potential and Applications of\nInduced Electric Field","volume":"311","author":"Zhang","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"2026091609090541200_cit5","doi-asserted-by":"publisher","first-page":"107037","DOI":"10.1016\/j.jwpe.2025.107037","article-title":"A Review on the\nRecovery of Cellulose, Lignin, and\nHemicellulose Biopolymers from the Same Source of Lignocellulosic\nBiomass - Methodology, Characterization and Applications","volume":"70","author":"Manyatshe","year":"2025","journal-title":"Journal of Water Process Engineering"},{"key":"2026091609090541200_cit6","doi-asserted-by":"publisher","first-page":"121091","DOI":"10.1016\/j.indcrop.2025.121091","article-title":"Highly Efficient\nConversion of Cellulose and Hemicellulose Using an Innovative Ternary\nDeep Eutectic Solvent: Achieving the Green and Complete Use of Lignocellulose","volume":"230","author":"Ma","year":"2025","journal-title":"Ind. 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Pollut. Rep."},{"key":"2026091609090541200_cit13","doi-asserted-by":"publisher","first-page":"109669","DOI":"10.1016\/j.enzmictec.2020.109669","article-title":"Microbial Lignin\nPeroxidases: Applications, Production Challenges and Future Perspectives","volume":"141","author":"Biko","year":"2020","journal-title":"Enzyme Microb. Technol."},{"issue":"7","key":"2026091609090541200_cit14","doi-asserted-by":"publisher","first-page":"3753","DOI":"10.1007\/s12649-020-01275-z","article-title":"Contribution of Lignin Peroxidase,\nManganese Peroxidase, and Laccase in Lignite Degradation by Mixed\nWhite-Rot Fungi","volume":"12","author":"Shi","year":"2021","journal-title":"Waste Biomass Valorization"},{"key":"2026091609090541200_cit15","doi-asserted-by":"publisher","first-page":"139579","DOI":"10.1016\/j.ijbiomac.2025.139579","article-title":"In Silico Analysis and Gene Expression Patterns of\nLignin Peroxidase Isozymes in Phanerochaete chrysosporium","volume":"295","author":"Akbar Aly","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"2026091609090541200_cit16","doi-asserted-by":"publisher","first-page":"02036","DOI":"10.1051\/e3sconf\/202339302036","article-title":"Expanding\nthe Application of Lignin Peroxidases from Irpex lacteus in Lignin Degradation by Exploring the Effects of Small Molecules\non Enzyme Activity","volume":"393","author":"Li","year":"2023","journal-title":"E3S Web Conf."},{"issue":"10","key":"2026091609090541200_cit17","doi-asserted-by":"publisher","first-page":"6277","DOI":"10.3390\/app13106277","article-title":"Construction\nand Identification of a Food-Grade Recombinant Cyberlindnera jadinii Strain Expressing Lignin Peroxidase","volume":"13","author":"Gong","year":"2023","journal-title":"Appl. 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Food Agric."},{"key":"2026091609090541200_cit20","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1042\/bj3150015","article-title":"Expression of Lignin\nPeroxidase H8 in Escherichia coli: Folding and Activation of the Recombinant Enzyme with\nCa2+and Haem","volume":"315","author":"Doyle","year":"1996","journal-title":"Biochem. J."},{"key":"2026091609090541200_cit21","doi-asserted-by":"publisher","first-page":"849","DOI":"10.1023\/A:1005541206270","article-title":"Heterologous Expression of Lignin\nPeroxidase of Phanerochaete chrysosporium in Aspergillus niger","volume":"21","author":"Aifa","year":"1999","journal-title":"Biotechnol. Lett."},{"key":"2026091609090541200_cit22","doi-asserted-by":"publisher","first-page":"146","DOI":"10.1006\/bbrc.1998.9106","article-title":"Expression of the Lignin Peroxidase\nH2 Gene from Phanerochaete chrysosporium in Escherichia coli","volume":"249","author":"Nie","year":"1998","journal-title":"Biochem. Biophys.\nRes. Commun."},{"key":"2026091609090541200_cit23","doi-asserted-by":"publisher","first-page":"101978","DOI":"10.1016\/j.mito.2024.101978","article-title":"Impact of\nMissense Mutations on the Structure\u2013Function Relationship of\nHuman Succinyl-Coa Synthetase Using in Silico Analysis","volume":"80","author":"Elabed","year":"2025","journal-title":"Mitochondrion"},{"issue":"44","key":"2026091609090541200_cit24","doi-asserted-by":"publisher","first-page":"24620","DOI":"10.1021\/acs.jafc.4c05991","article-title":"Rational Design for Enhancing Cellobiose\nDehydrogenase Activity and Its Synergistic Role in Straw Degradation","volume":"72","author":"Wu","year":"2024","journal-title":"J. Agric. Food Chem."},{"issue":"11","key":"2026091609090541200_cit25","doi-asserted-by":"publisher","first-page":"5682","DOI":"10.1021\/acs.jafc.3c09827","article-title":"Binding Properties\nof Odorant Binding Protein 37 in Plagiodera versicolora to Host Volatile, o-Cymene","volume":"72","author":"Liu","year":"2024","journal-title":"J. Agric. Food\nChem."},{"issue":"9","key":"2026091609090541200_cit26","doi-asserted-by":"publisher","first-page":"8337","DOI":"10.3390\/ijms24098337","article-title":"Rational Design of Daunorubicin C-14 Hydroxylase Based\non the Understanding of Its Substrate-Binding Mechanism","volume":"24","author":"Zhang","year":"2023","journal-title":"Int. J. Mol. Sci."},{"issue":"3","key":"2026091609090541200_cit27","doi-asserted-by":"publisher","first-page":"1841","DOI":"10.1021\/acscatal.4c06840","article-title":"Simultaneous\nEngineering of the Thermostability\nand Activity of a Novel Aldehyde Dehydrogenase","volume":"15","author":"Xu","year":"2025","journal-title":"ACS Catal."},{"issue":"17","key":"2026091609090541200_cit28","doi-asserted-by":"publisher","first-page":"8012","DOI":"10.3390\/app14178012","article-title":"Biodegradation\nand Detoxification of Some Dyes by Crude Lignin Peroxidase Complex\nProduced by Escherichia coli Accession No: LR0250096.1\nand Pseudomonas aeruginosa Accession No: CP031449.2","volume":"14","author":"Dube","year":"2024","journal-title":"Appl. Sci."},{"issue":"9","key":"2026091609090541200_cit29","doi-asserted-by":"publisher","first-page":"1446","DOI":"10.3390\/antiox10091446","article-title":"Agaricales Mushroom\nLignin Peroxidase: From Structure\u2013Function to Degradative Capabilities","volume":"10","author":"Sa\u0301nchez-Ruiz","year":"2021","journal-title":"Antioxidants"},{"key":"2026091609090541200_cit30","doi-asserted-by":"publisher","first-page":"15331","DOI":"10.1021\/jacs.6c03371","article-title":"Rethinking\nthe Terms \u201c\u03c0-Stacking\u201d\nand \u201c\u03c0-\u03c0 Stacking\u201d Again: A Proposal to\nClarify the Language of Aromatic Interactions","volume":"148","author":"Xiao","year":"2026","journal-title":"J. Am. Chem. Soc."},{"key":"2026091609090541200_cit31","doi-asserted-by":"publisher","first-page":"123881","DOI":"10.1016\/j.ijbiomac.2023.123881","article-title":"Characterization and\n3D Printing of a Biodegradable Polylactic Acid\/Thermoplastic Polyurethane\nBlend with Laccase-Modified Lignin as a Nucleating Agent","volume":"236","author":"Murillo-Morales","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"issue":"1","key":"2026091609090541200_cit32","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1007\/s10529-022-03325-z","article-title":"Straw Lignin\nDegradation by Lignin\nPeroxidase from Irpex lacteus Cooperated with Enzymes\nand Small Molecules","volume":"45","author":"Chen","year":"2023","journal-title":"Biotechnol. Lett."},{"issue":"45","key":"2026091609090541200_cit33","doi-asserted-by":"publisher","first-page":"24928","DOI":"10.1021\/acs.jafc.4c08453","article-title":"Comprehensive Understanding of Laboratory\nEvolution for Food Enzymes:\nFrom Design to Screening Innovations","volume":"72","author":"Wu","year":"2024","journal-title":"J. Agric.\nFood Chem."},{"key":"2026091609090541200_cit34","doi-asserted-by":"publisher","first-page":"110188","DOI":"10.1016\/j.enzmictec.2022.110188","article-title":"Improving the Catalytic\nActivity of \u03b2-Glucosidase from Coniophora puteana via Semi-Rational Design for Efficient Biomass Cellulose Degradation","volume":"164","author":"Zhou","year":"2023","journal-title":"Enzyme Microb. Technol."},{"issue":"12","key":"2026091609090541200_cit35","first-page":"4601","article-title":"Improving\nthe Activity of Creatinase From Alcaligenes sp. Ks-85 through Semi-Rational Design","volume":"38","author":"Bian","year":"2022","journal-title":"Chinese Journal of Biotechnology"},{"issue":"2","key":"2026091609090541200_cit36","doi-asserted-by":"publisher","first-page":"871","DOI":"10.1021\/acs.jcim.8b00926","article-title":"Alanine Scanning\nEffects on the Biochemical and Biophysical\nProperties of Intrinsically Disordered Proteins: A Case Study of the\nHistidine to Alanine Mutations in Amyloid-\u03b242","volume":"59","author":"Coskuner-Weber","year":"2019","journal-title":"J. Chem. Inf. Model."},{"issue":"6","key":"2026091609090541200_cit37","doi-asserted-by":"publisher","first-page":"4109","DOI":"10.1007\/s12010-025-05223-2","article-title":"Enhanced Thermal Stability\/Activity of Geobacillus jurassicus Esterase by Rational Design and Application in\nthe Synthesis of Cinnamyl Acetate","volume":"197","author":"Li","year":"2025","journal-title":"Appl. Biochem.\nBiotechnol."},{"issue":"1","key":"2026091609090541200_cit38","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1007\/s11274-016-2185-2","article-title":"Recent Advances\nof Molecular Toolbox Construction Expand Pichia pastoris in Synthetic Biology Applications","volume":"33","author":"Kang","year":"2017","journal-title":"World J.\nMicrobiol. Biotechnol."},{"key":"2026091609090541200_cit39","doi-asserted-by":"publisher","first-page":"140755","DOI":"10.1016\/j.ijbiomac.2025.140755","article-title":"Effects of N-Linked\nGlycosylation on the Enzymatic Properties of Gh12 Bifunctional Enzymes\nfrom Aspergillus terreus Expressed in Pichia pastoris","volume":"304","author":"Zuo","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"issue":"24","key":"2026091609090541200_cit40","doi-asserted-by":"publisher","first-page":"e01615","DOI":"10.1128\/aem.01615-19","article-title":"Role of BGS13 in the Secretory Mechanism\nof Pichia pastoris","volume":"85","author":"Naranjo","year":"2019","journal-title":"Appl. Environ.\nMicrobiol."},{"issue":"26","key":"2026091609090541200_cit41","doi-asserted-by":"publisher","first-page":"14912","DOI":"10.1021\/acs.jafc.4c02623","article-title":"Enhancing the Thermostability and Catalytic Activity\nof the Lipase from Rhizopus oryzae via Introducing\nN-Glycosylation","volume":"72","author":"Wang","year":"2024","journal-title":"J. Agric. Food Chem."},{"issue":"5","key":"2026091609090541200_cit42","doi-asserted-by":"publisher","first-page":"2400098","DOI":"10.1002\/biot.202400098","article-title":"High-Activity\nRecombinant Human Carboxypeptidase\nB Expression in Pichia pastoris Through Rational\nProtein Engineering and Enhancing Secretion From the Golgi Apparatus\nto the Plasma Membrane","volume":"19","author":"Li","year":"2024","journal-title":"Biotechnol. J."},{"issue":"4","key":"2026091609090541200_cit43","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2417343122","article-title":"The Pseudomonas Ligninolytic Catalytic\nNetwork Reveals the Importance\nof Auxiliary Enzymes in Lignin Biocatalysts","volume":"122","author":"Liang","year":"2025","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026091609090541200_cit44","doi-asserted-by":"publisher","first-page":"112069","DOI":"10.1016\/j.rser.2021.112069","article-title":"Extremophiles\nand Extremozymes in Lignin Bioprocessing","volume":"157","author":"Zhu","year":"2022","journal-title":"Renewable\nand Sustainable Energy Reviews"},{"issue":"13","key":"2026091609090541200_cit45","doi-asserted-by":"publisher","first-page":"6161","DOI":"10.3390\/app11136161","article-title":"Mushroom ligninolytic\nenzymes\u2015Features and Application of Potential Enzymes for Conversion\nof Lignin into Bio-Based Chemicals and Materials","volume":"11","author":"Kim","year":"2021","journal-title":"Appl. Sci."},{"issue":"12","key":"2026091609090541200_cit46","doi-asserted-by":"publisher","first-page":"6668","DOI":"10.1021\/acssuschemeng.6b01540","article-title":"Relative Significance\nof the Negative\nImpacts of Hemicelluloses on Enzymatic Cellulose Hydrolysis is Dependent\non Lignin Content: Evidence From Substrate Structural Features and\nProtein Adsorption","volume":"4","author":"Chen","year":"2016","journal-title":"ACS Sustainable Chem. 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