{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T04:46:42Z","timestamp":1791694002010,"version":"4.3.4"},"reference-count":136,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program","doi-asserted-by":"publisher","award":["2022YFC2105203"],"award-info":[{"award-number":["2022YFC2105203"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["52370039"],"award-info":[{"award-number":["52370039"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["52370039"],"award-info":[{"award-number":["52370039"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Science Fund of Jiangsu Province","award":["BK20230022"],"award-info":[{"award-number":["BK20230022"]}]},{"name":"Jiangsu Funding Program for Excellent Postdoctoral Talent","award":["2025ZB684"],"award-info":[{"award-number":["2025ZB684"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>High-temperature processing of carbohydrate-rich biomass, including starch liquefaction and lignocellulose saccharification, can improve sugar yields, reduce contamination risks, and facilitate integration with thermochemical pretreatments. However, the operational stability of native carbohydrate-active enzymes (CAZymes) at 60\u201390 \u00b0C remains a major bottleneck for efficient and economically viable biorefineries, and existing reviews rarely integrate multiscale molecular mechanisms with process-relevant engineering strategies. This review systematically examines the multiscale basis of thermostability in CAZymes\u2014covering core hydrophobic packing, electrostatic and metal-mediated networks, disulfide bonds, conformational dynamics, and modular architectures involving catalytic domains, linkers, and carbohydrate-binding modules\u2014and directly links these features to enzyme longevity under high-solids, inhibitor-rich conditions. We then critically compare key engineering strategies, including structure-guided rational design, semi-rational directed evolution, consensus design, ancestral sequence reconstruction, and machine learning-assisted workflows embedded in design\u2013build\u2013test\u2013learn (DBTL) cycles, highlighting their respective strengths, limitations, and complementarity. Representative case studies demonstrate that engineered thermophilic \u03b1-amylases and cellulases achieving \u0394Tm improvements of approximately 10\u201320 \u00b0C can sustain &gt;80\u201385% residual activity after prolonged exposure at process-relevant temperatures, translating into tangible benefits such as 15\u201330% reductions in enzyme dosing and measurable decreases in steam consumption, alongside higher sugar titers. By bridging molecular determinants, engineering paradigms, and quantitative process performance, this review provides a pragmatic roadmap for deploying thermostable CAZymes as robust biocatalysts in sustainable high-temperature biomass conversion.<\/jats:p>","DOI":"10.3390\/ijms27198788","type":"journal-article","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T15:20:59Z","timestamp":1790781659000},"page":"8788","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Thermostable Carbohydrate-Active Enzymes for High-Temperature Biomass Conversion: From Molecular Design to Process Integration"],"prefix":"10.3390","volume":"27","author":[{"given":"Yueli","family":"Hu","sequence":"first","affiliation":[{"name":"College of Engineering and Applied Sciences, Nanjing University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Yang","sequence":"additional","affiliation":[{"name":"School of Food and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianqi","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Engineering and Applied Sciences, Nanjing University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingshu","family":"Zheng","sequence":"additional","affiliation":[{"name":"School of Food and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenghao","family":"Jiang","sequence":"additional","affiliation":[{"name":"College of Engineering and Applied Sciences, Nanjing University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-2700-6159","authenticated-orcid":false,"given":"Xinyue","family":"Dou","sequence":"additional","affiliation":[{"name":"College of Engineering and Applied Sciences, Nanjing University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinkun","family":"Ren","sequence":"additional","affiliation":[{"name":"College of Engineering and Applied Sciences, Nanjing University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-0924-6922","authenticated-orcid":false,"given":"Jiaxin","family":"Wu","sequence":"additional","affiliation":[{"name":"College of Engineering and Applied Sciences, Nanjing University, Nanjing 210023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"122585","DOI":"10.1016\/j.biortech.2019.122585","article-title":"Biorefineries in circular bioeconomy: A comprehensive review","volume":"299","author":"Ubando","year":"2020","journal-title":"Bioresour. Technol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4497","DOI":"10.1039\/C5PY00263J","article-title":"Lignocellulosic biomass: A sustainable platform for the production of bio-based chemicals and polymers","volume":"6","author":"Isikgor","year":"2015","journal-title":"Polym. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1038\/s44222-024-00247-5","article-title":"Engineering strategies to optimize lignocellulosic biorefineries","volume":"3","author":"Long","year":"2025","journal-title":"Nat. Rev. Bioeng."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/1475-2859-6-9","article-title":"Potential and utilization of thermophiles and thermostable enzymes in biorefining","volume":"6","author":"Turner","year":"2007","journal-title":"Microb. Cell Fact."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"122685","DOI":"10.1016\/j.biortech.2019.122685","article-title":"Engineering aspects of hydrothermal pretreatment: From batch to continuous operation, scale-up and pilot reactor under biorefinery concept","volume":"299","author":"Ruiz","year":"2020","journal-title":"Bioresour. Technol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1007\/s10295-008-0327-8","article-title":"Bioconversion of lignocellulosic biomass: Biochemical and molecular perspectives","volume":"35","author":"Kumar","year":"2008","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5199","DOI":"10.1128\/AEM.00199-11","article-title":"High-Temperature Enzymatic Breakdown of Cellulose","volume":"77","author":"Wang","year":"2011","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1016\/j.biortech.2012.10.145","article-title":"Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes","volume":"128","author":"Bhalla","year":"2013","journal-title":"Bioresour. Technol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"D490","DOI":"10.1093\/nar\/gkt1178","article-title":"The carbohydrate-active enzymes database (CAZy) in 2013","volume":"42","author":"Lombard","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Contesini, F.J., Frandsen, R.J.N., and Damasio, A. (2021). Editorial: CAZymes in Biorefinery: From Genes to Application. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.622817"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/S0168-1656(01)00407-2","article-title":"Properties and applications of starch-converting enzymes of the \u03b1-amylase family","volume":"94","author":"Uitdehaag","year":"2002","journal-title":"J. Biotechnol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1599","DOI":"10.1016\/S0032-9592(03)00053-0","article-title":"Microbial \u03b1-amylases: A biotechnological perspective","volume":"38","author":"Gupta","year":"2003","journal-title":"Process Biochem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1308","DOI":"10.1021\/cr500351c","article-title":"Fungal Cellulases","volume":"115","author":"Payne","year":"2015","journal-title":"Chem. Rev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.biortech.2015.10.009","article-title":"Pretreatment of lignocellulose: Formation of inhibitory by-products and strategies for minimizing their effects","volume":"199","year":"2016","journal-title":"Bioresour. Technol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Humbird, D., Davis, R., Tao, L., Kinchin, C., Hsu, D., Aden, A., Schoen, P., Lukas, J., Olthof, B., and Worley, M. (2011). Process Design and Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol: Dilute-Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover, National Renewable Energy Laboratory.","DOI":"10.2172\/1013269"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1083","DOI":"10.1002\/bit.24370","article-title":"The challenge of enzyme cost in the production of lignocellulosic biofuels","volume":"109","author":"Simmons","year":"2012","journal-title":"Biotechnol. Bioeng."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"137906","DOI":"10.1016\/j.fuel.2025.137906","article-title":"Techno-economic assessment of field-scale storage for liquid organic hydrogen carriers: Dual benefits of energy storage & incremental oil recovery","volume":"410","author":"Tariq","year":"2026","journal-title":"Fuel"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"964","DOI":"10.1038\/nature06522","article-title":"Dynamic personalities of proteins","volume":"450","author":"Kern","year":"2007","journal-title":"Nature"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3310","DOI":"10.1021\/jacsau.4c00649","article-title":"Connecting Protein Millisecond Conformational Dynamics to Protein Thermal Stability","volume":"4","author":"Hou","year":"2024","journal-title":"JACS Au"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4143","DOI":"10.1002\/anie.201708408","article-title":"Directed Evolution: Bringing New Chemistry to Life","volume":"57","author":"Arnold","year":"2018","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1111\/1758-2229.12943","article-title":"Thermophilic microbial deconstruction and conversion of natural and transgenic lignocellulose","volume":"13","author":"Bing","year":"2021","journal-title":"Environ. Microbiol. Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/s00253-009-2224-0","article-title":"Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-binding module","volume":"86","author":"Koseki","year":"2010","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1126\/science.ads0018","article-title":"Simulating 500 million years of evolution with a language model","volume":"387","author":"Hayes","year":"2025","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5648","DOI":"10.1038\/s41467-025-61209-y","article-title":"A generalized platform for artificial intelligence-powered autonomous enzyme engineering","volume":"16","author":"Singh","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1042","DOI":"10.1126\/science.1219021","article-title":"The Protein-Folding Problem, 50 Years On","volume":"338","author":"Dill","year":"2012","journal-title":"Science"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3899","DOI":"10.1111\/febs.13343","article-title":"Rigidity versus flexibility: The dilemma of understanding protein thermal stability","volume":"282","author":"Karshikoff","year":"2015","journal-title":"FEBS J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1598","DOI":"10.1126\/science.1749933","article-title":"The Energy Landscapes and Motions of Proteins","volume":"254","author":"Frauenfelder","year":"1991","journal-title":"Science"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1093\/protein\/13.3.179","article-title":"Factors enhancing protein thermostability","volume":"13","author":"Kumar","year":"2000","journal-title":"Protein Eng."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1002\/tcr.20160","article-title":"From cellulosomes to cellulosomics","volume":"8","author":"Bayer","year":"2008","journal-title":"Chem. Rec."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1216","DOI":"10.1007\/PL00000935","article-title":"How do thermophilic proteins deal with heat?","volume":"58","author":"Kumar","year":"2001","journal-title":"Cell. Mol. Life Sci. CMLS"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1006\/jmbi.1998.2159","article-title":"The stability of salt bridges at high temperatures: Implications for hyperthermophilic proteins","volume":"284","author":"Elcock","year":"1998","journal-title":"J. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Lee, C.-W., Wang, H.-J., Hwang, J.-K., and Tseng, C.-P. (2014). Protein Thermal Stability Enhancement by Designing Salt Bridges: A Combined Computational and Experimental Study. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0112751"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/MMBR.65.1.1-43.2001","article-title":"Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability","volume":"65","author":"Vieille","year":"2001","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/j.jbiotec.2004.03.026","article-title":"Rational engineering of enzyme stability","volume":"113","author":"Eijsink","year":"2004","journal-title":"J. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1126\/science.1169375","article-title":"Protein Dynamism and Evolvability","volume":"324","author":"Tokuriki","year":"2009","journal-title":"Science"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.nbt.2009.04.002","article-title":"Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2","volume":"26","author":"Jun","year":"2009","journal-title":"New Biotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5869","DOI":"10.1073\/pnas.0510098103","article-title":"Protein stability promotes evolvability","volume":"103","author":"Bloom","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1042\/bj3090749","article-title":"The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel","volume":"309","author":"Hall","year":"1995","journal-title":"Biochem. J."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1042\/BJ20040892","article-title":"Carbohydrate-binding modules: Fine-tuning polysaccharide recognition","volume":"382","author":"Boraston","year":"2004","journal-title":"Biochem. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"108365","DOI":"10.1016\/j.biotechadv.2024.108365","article-title":"Carbohydrate binding modules: Compact yet potent accessories in the specific substrate binding and performance evolution of carbohydrate-active enzymes","volume":"73","author":"You","year":"2024","journal-title":"Biotechnol. Adv."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1279","DOI":"10.1126\/science.1208386","article-title":"Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface","volume":"333","author":"Igarashi","year":"2011","journal-title":"Science"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2954","DOI":"10.1007\/s00018-006-6195-3","article-title":"Recent structural studies of carbohydrate-binding modules","volume":"63","author":"Hashimoto","year":"2006","journal-title":"Cell. Mol. Life Sci. CMLS"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/j.cbpa.2021.02.012","article-title":"Computational modeling of carbohydrate processing enzymes reactions","volume":"61","author":"Mendoza","year":"2021","journal-title":"Curr. Opin. Chem. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1016\/j.copbio.2005.08.009","article-title":"Consolidated bioprocessing of cellulosic biomass: An update","volume":"16","author":"Lynd","year":"2005","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Bisaria, V. (2024). Enzymatic Hydrolysis of Lignocellulosic Biomass at High Solids Loading. Handbook of Biorefinery Research and Technology: Biomass Logistics to Saccharification, Springer.","DOI":"10.1007\/978-94-007-6308-1"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Wolski, P., Blankenship, B.W., Umar, A., Cabrera, M., Simmons, B.A., Sale, K.L., and Achinivu, E.C. (2023). Factors that influence the activity of biomass-degrading enzymes in the presence of ionic liquids\u2014A review. Front. Energy Res., 11.","DOI":"10.3389\/fenrg.2023.1212719"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"127044","DOI":"10.1016\/j.biortech.2022.127044","article-title":"High-solids loading processing for an integrated lignocellulosic biorefinery: Effects of transport phenomena and rheology\u2014A review","volume":"351","author":"Shiva","year":"2022","journal-title":"Bioresour. Technol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1080\/07388551.2021.1973363","article-title":"Emerging trends in high-solids enzymatic saccharification of lignocellulosic feedstocks for developing an efficient and industrially deployable sugar platform","volume":"42","author":"Baral","year":"2022","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"124141","DOI":"10.1016\/j.ijbiomac.2023.124141","article-title":"Domain-wise dissection of thermal stability enhancement in multidomain proteins","volume":"237","author":"Oh","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"623","DOI":"10.1007\/s10295-020-02301-8","article-title":"Enzymatic processing of lignocellulosic biomass: Principles, recent advances and perspectives","volume":"47","author":"Hansen","year":"2020","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1016\/S0958-1669(00)00229-9","article-title":"Engineering proteins for thermostability: The use of sequence alignments versus rational design and directed evolution","volume":"12","author":"Lehmann","year":"2001","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"151068","DOI":"10.1016\/j.ijbiomac.2026.151068","article-title":"Accelerated discovery of thermophilic biocatalysts for marine polysaccharide bioprocessing via ETopt","volume":"351","author":"Liu","year":"2026","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Liu, R., Li, S., Zhang, H., Qiu, J., Liu, Y., Liu, H., Li, J., and Zheng, P. (2026). AI-guided thermal stabilization of endo-\u03b2-1,3-glucanase. Chin. Chem. Lett., 112729.","DOI":"10.1016\/j.cclet.2026.112729"},{"key":"ref_55","first-page":"290","article-title":"Xylanase carbohydrate binding module: Recent developments","volume":"26","author":"Liu","year":"2010","journal-title":"Chin. J. Biotechnol."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Yang, Y., Zhang, C., Lu, H., Wu, Q., Wu, Y., Li, W., and Li, X. (2024). Improvement of thermostability and catalytic efficiency of xylanase from Myceliophthora thermophilar by N-terminal and C-terminal truncation. Front. Microbiol., 15.","DOI":"10.3389\/fmicb.2024.1385329"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1038\/370389a0","article-title":"Rapid evolution of a protein in vitro by DNA shuffling","volume":"370","author":"Stemmer","year":"1994","journal-title":"Nature"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1021\/ar960017f","article-title":"Design by Directed Evolution","volume":"31","author":"Arnold","year":"1998","journal-title":"Acc. Chem. Res."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"288","DOI":"10.1038\/34663","article-title":"DNA shuffling of a family of genes from diverse species accelerates directed evolution","volume":"391","author":"Crameri","year":"1998","journal-title":"Nature"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/s13068-024-02457-w","article-title":"Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering","volume":"17","author":"Chen","year":"2024","journal-title":"Biotechnol. Biofuels Bioprod."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1038\/nprot.2007.72","article-title":"Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes","volume":"2","author":"Reetz","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1038\/nrg3927","article-title":"Methods for the directed evolution of proteins","volume":"16","author":"Packer","year":"2015","journal-title":"Nat. Rev. Genet."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"4011","DOI":"10.1021\/ie503060a","article-title":"High Throughput Screening and Selection Methods for Directed Enzyme Evolution","volume":"54","author":"Xiao","year":"2015","journal-title":"Ind. Eng. Chem. Res."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1038\/nmeth.3027","article-title":"Deep mutational scanning: A new style of protein science","volume":"11","author":"Fowler","year":"2014","journal-title":"Nat. Methods"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1016\/j.trechm.2022.02.004","article-title":"High-throughput directed evolution: A golden era for protein science","volume":"4","author":"McLure","year":"2022","journal-title":"Trends Chem."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.sbi.2015.09.002","article-title":"Protein stability: Computation, sequence statistics, and new experimental methods","volume":"33","author":"Magliery","year":"2015","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_67","first-page":"1","article-title":"Ancestral protein reconstruction: Techniques and applications","volume":"397","author":"Sterner","year":"2016","journal-title":"Biol. Chem."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1006\/jmbi.1994.1434","article-title":"Sequence Statistics Reliably Predict Stabilizing Mutations in a Protein Domain","volume":"240","author":"Steipe","year":"1994","journal-title":"J. Mol. Biol."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1016\/S0167-4838(00)00238-7","article-title":"The consensus concept for thermostability engineering of proteins","volume":"1543","author":"Lehmann","year":"2000","journal-title":"Biochim. Et. Biophys. Acta Protein Struct. Mol. Enzymol."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1016\/j.jmb.2012.04.025","article-title":"Stabilizing Proteins from Sequence Statistics: The Interplay of Conservation and Correlation in Triosephosphate Isomerase Stability","volume":"420","author":"Sullivan","year":"2012","journal-title":"J. Mol. Biol."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1038\/nature06510","article-title":"Palaeotemperature trend for Precambrian life inferred from resurrected proteins","volume":"451","author":"Gaucher","year":"2008","journal-title":"Nature"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1093\/molbev\/msr253","article-title":"On the Origin and Evolution of Thermophily: Reconstruction of Functional Precambrian Enzymes from Ancestors of Bacillus","volume":"29","author":"Hobbs","year":"2012","journal-title":"Mol. Biol. Evol."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"11067","DOI":"10.1073\/pnas.1308215110","article-title":"Experimental evidence for the thermophilicity of ancestral life","volume":"110","author":"Akanuma","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"2899","DOI":"10.1021\/ja311630a","article-title":"Hyperstability and Substrate Promiscuity in Laboratory Resurrections of Precambrian \u03b2-Lactamases","volume":"135","author":"Risso","year":"2013","journal-title":"J. Am. Chem. Soc."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1038\/s44286-023-00002-4","article-title":"Self-driving laboratories to autonomously navigate the protein fitness landscape","volume":"1","author":"Rapp","year":"2024","journal-title":"Nat. Chem. Eng."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"1553","DOI":"10.1038\/s41467-025-56751-8","article-title":"Integrating protein language models and automatic biofoundry for enhanced protein evolution","volume":"16","author":"Zhang","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1038\/s42003-018-0076-9","article-title":"An automated Design-Build-Test-Learn pipeline for enhanced microbial production of fine chemicals","volume":"1","author":"Carbonell","year":"2018","journal-title":"Commun. Biol."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1002\/mlf2.12151","article-title":"Optimizing enzyme thermostability by combining multiple mutations using protein language model","volume":"3","author":"Bian","year":"2024","journal-title":"mLife"},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"eaea1820","DOI":"10.1126\/science.aea1820","article-title":"Rapid directed evolution guided by protein language models and epistatic interactions","volume":"392","author":"Tran","year":"2026","journal-title":"Science"},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1038\/s41467-025-55987-8","article-title":"Active learning-assisted directed evolution","volume":"16","author":"Yang","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1038\/s41592-019-0496-6","article-title":"Machine-learning-guided directed evolution for protein engineering","volume":"16","author":"Yang","year":"2019","journal-title":"Nat. Methods"},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1038\/s41592-021-01100-y","article-title":"Low-N protein engineering with data-efficient deep learning","volume":"18","author":"Biswas","year":"2021","journal-title":"Nat. Methods"},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1126\/science.abj8754","article-title":"Accurate prediction of protein structures and interactions using a three-track neural network","volume":"373","author":"Baek","year":"2021","journal-title":"Science"},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"e2016239118","DOI":"10.1073\/pnas.2016239118","article-title":"Biological structure and function emerge from scaling unsupervised learning to 250 million protein sequences","volume":"118","author":"Rives","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1126\/science.ade2574","article-title":"Evolutionary-scale prediction of atomic-level protein structure with a language model","volume":"379","author":"Lin","year":"2023","journal-title":"Science"},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"4348","DOI":"10.1038\/s41467-022-32007-7","article-title":"ProtGPT2 is a deep unsupervised language model for protein design","volume":"13","author":"Ferruz","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"1089","DOI":"10.1038\/s41586-023-06415-8","article-title":"De novo design of protein structure and function with RFdiffusion","volume":"620","author":"Watson","year":"2023","journal-title":"Nature"},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"1680","DOI":"10.1038\/s41467-026-68384-6","article-title":"Sequence-based generative AI design of versatile tryptophan synthases","volume":"17","author":"Lambert","year":"2026","journal-title":"Nat. Commun."},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1126\/science.abd7331","article-title":"Learning the language of viral evolution and escape","volume":"371","author":"Hie","year":"2021","journal-title":"Science"},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1002\/fbe2.70029","article-title":"Accelerating Enzyme Engineering With Artificial Intelligence in Biocatalysis: Challenges and Opportunities","volume":"4","author":"Lu","year":"2025","journal-title":"Food Bioeng."},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"100005","DOI":"10.1016\/j.bidere.2025.100005","article-title":"Recent advances on protein engineering for improved stability","volume":"7","author":"Shi","year":"2025","journal-title":"BioDesign Res."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"100044","DOI":"10.1016\/j.bidere.2025.100044","article-title":"From machine learning to multimodal models: The AI revolution in enzyme engineering","volume":"8","author":"Shi","year":"2026","journal-title":"BioDesign Res."},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1038\/nrmicro3239","article-title":"A brief history of synthetic biology","volume":"12","author":"Cameron","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1038\/s41929-018-0212-4","article-title":"A comprehensive metabolic map for production of bio-based chemicals","volume":"2","author":"Lee","year":"2019","journal-title":"Nat. Catal."},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.1016\/j.cell.2016.02.004","article-title":"Engineering Cellular Metabolism","volume":"164","author":"Nielsen","year":"2016","journal-title":"Cell"},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"1755","DOI":"10.1002\/bit.26590","article-title":"Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously","volume":"115","author":"Xiong","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"1355","DOI":"10.1126\/science.1193990","article-title":"Manufacturing Molecules Through Metabolic Engineering","volume":"330","author":"Keasling","year":"2010","journal-title":"Science"},{"key":"ref_99","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1016\/j.tim.2011.05.003","article-title":"Biosensors and their applications in microbial metabolic engineering","volume":"19","author":"Zhang","year":"2011","journal-title":"Trends Microbiol."},{"key":"ref_100","doi-asserted-by":"crossref","first-page":"15114","DOI":"10.1021\/acssuschemeng.4c04974","article-title":"Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design","volume":"12","author":"Lu","year":"2024","journal-title":"ACS Sustain. Chem. Eng."},{"key":"ref_101","doi-asserted-by":"crossref","unstructured":"Zhou, X., Xu, Z., Li, Y., He, J., and Zhu, H. (2022). Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.815990"},{"key":"ref_102","doi-asserted-by":"crossref","first-page":"5391","DOI":"10.1021\/acs.jafc.0c01817","article-title":"Engineering of \u03b2-Glucosidase Bgl15 with Simultaneously Enhanced Glucose Tolerance and Thermostability To Improve Its Performance in High-Solid Cellulose Hydrolysis","volume":"68","author":"Cao","year":"2020","journal-title":"J. Agric. Food Chem."},{"key":"ref_103","doi-asserted-by":"crossref","first-page":"12388","DOI":"10.1021\/acssuschemeng.0c02465","article-title":"KnowVolution of a GH5 Cellulase from Penicillium verruculosum to Improve Thermal Stability for Biomass Degradation","volume":"8","author":"Contreras","year":"2020","journal-title":"ACS Sustain. Chem. Eng."},{"key":"ref_104","doi-asserted-by":"crossref","first-page":"1874","DOI":"10.1002\/bit.24864","article-title":"Engineered thermostable fungal Cel6A and Cel7A Cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures","volume":"110","author":"Wu","year":"2013","journal-title":"Biotechnol. Bioeng."},{"key":"ref_105","doi-asserted-by":"crossref","unstructured":"Yoav, S., Stern, J., Salama-Alber, O., Frolow, F., Anbar, M., Karpol, A., Hadar, Y., Morag, E., and Bayer, E.A. (2019). Directed Evolution of Clostridium thermocellum \u03b2-Glucosidase A Towards Enhanced Thermostability. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20194701"},{"key":"ref_106","doi-asserted-by":"crossref","first-page":"3458","DOI":"10.1128\/AEM.07985-11","article-title":"Improved Thermostability of Clostridium thermocellum Endoglucanase Cel8A by Using Consensus-Guided Mutagenesis","volume":"78","author":"Anbar","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_107","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1186\/s13068-019-1386-y","article-title":"Creation of a functional hyperthermostable designer cellulosome","volume":"12","author":"Kahn","year":"2019","journal-title":"Biotechnol. Biofuels"},{"key":"ref_108","doi-asserted-by":"crossref","first-page":"22429","DOI":"10.1038\/s41598-024-73541-2","article-title":"Combinatorial optimization of the hybrid cellulase complex structure designed from modular libraries","volume":"14","author":"Nakazawa","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_109","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/S0167-7799(97)01082-2","article-title":"Enzymes involved in the processing of starch to sugars","volume":"15","author":"Mitchinson","year":"1997","journal-title":"Trends Biotechnol."},{"key":"ref_110","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1590\/S1517-83822010000400004","article-title":"Application of Microbial \u03b1-Amylase In Industry\u2014A Review","volume":"41","author":"Souza","year":"2010","journal-title":"Braz. J. Microbiol."},{"key":"ref_111","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1111\/j.1365-2672.1981.tb00875.x","article-title":"Characterization of a Thermostable \u03b1-Amylase from Bacillus licheniformis NCIB 6346","volume":"50","author":"Morgan","year":"1981","journal-title":"J. Appl. Bacteriol."},{"key":"ref_112","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/S0167-4838(00)00240-5","article-title":"Protein engineering of bacterial \u03b1-amylases","volume":"1543","author":"Nielsen","year":"2000","journal-title":"Biochim. Et. Biophys. Acta Protein Struct. Mol. Enzymol."},{"key":"ref_113","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.1006\/jmbi.2000.4025","article-title":"Probing structural determinants specifying high thermostability in Bacillus licheniformis \u03b1-amylase","volume":"301","author":"Declerck","year":"2000","journal-title":"J. Mol. Biol."},{"key":"ref_114","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1006\/jmbi.1994.0106","article-title":"Crystal Structure of Calcium-depleted Bacillus licheniformis\u03b1-amylase at 2.2 \u00c5 Resolution","volume":"246","author":"Machius","year":"1995","journal-title":"J. Mol. Biol."},{"key":"ref_115","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1093\/oxfordjournals.jbchem.a002878","article-title":"Crystal Structure of Bacillus stearothermophilus a-Amylase: Possible Factors Determining the Thermostability","volume":"129","author":"Suvd","year":"2001","journal-title":"J. Biochem."},{"key":"ref_116","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/S1016-8478(23)13289-4","article-title":"Crystal Structure of Thermostable \u03b1-Amylase from Bacillus licheniformis Refined at 1.7 \u00c5 Resolution","volume":"7","author":"Hwang","year":"1997","journal-title":"Mol. Cells"},{"key":"ref_117","doi-asserted-by":"crossref","first-page":"18928","DOI":"10.1021\/acs.jafc.3c06761","article-title":"Enhanced Thermostability of Geobacillus stearothermophilus \u03b1-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement","volume":"71","author":"Zhu","year":"2023","journal-title":"J. Agric. Food Chem."},{"key":"ref_118","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.bbrc.2023.02.064","article-title":"Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations","volume":"653","author":"Yuan","year":"2023","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_119","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1016\/j.csbj.2024.02.005","article-title":"Computational design of \u03b1-amylase from Bacillus licheniformis to increase its activity and stability at high temperatures","volume":"23","author":"Fan","year":"2024","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_120","doi-asserted-by":"crossref","first-page":"1839","DOI":"10.1007\/s00449-022-02790-0","article-title":"Simultaneously improving the specific activity and thermostability of \u03b1-amylase BLA by rational design","volume":"45","author":"Cui","year":"2022","journal-title":"Bioprocess Biosyst. Eng."},{"key":"ref_121","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.ijbiomac.2021.12.057","article-title":"Enhancing thermostabilization of a newly discovered \u03b1-amylase from Bacillus cereus GL96 by combining computer-aided directed evolution and site-directed mutagenesis","volume":"197","author":"Pouyan","year":"2022","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_122","doi-asserted-by":"crossref","first-page":"804","DOI":"10.1126\/science.1137016","article-title":"Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production","volume":"315","author":"Himmel","year":"2007","journal-title":"Science"},{"key":"ref_123","doi-asserted-by":"crossref","first-page":"506","DOI":"10.1128\/MMBR.66.3.506-577.2002","article-title":"Microbial Cellulose Utilization: Fundamentals and Biotechnology","volume":"66","year":"2002","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_124","doi-asserted-by":"crossref","first-page":"1186","DOI":"10.1038\/s41467-018-03501-8","article-title":"Engineering enhanced cellobiohydrolase activity","volume":"9","author":"Taylor","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_125","doi-asserted-by":"crossref","first-page":"2991","DOI":"10.1007\/s00253-013-5177-2","article-title":"Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae","volume":"98","author":"Voutilainen","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_126","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1186\/1754-6834-5-45","article-title":"Novel enzymes for the degradation of cellulose","volume":"5","author":"Horn","year":"2012","journal-title":"Biotechnol. Biofuels"},{"key":"ref_127","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1007\/s00203-026-04845-2","article-title":"Fungal cellulases: A comprehensive review on production, innovations, and applications","volume":"208","author":"Mahima","year":"2026","journal-title":"Arch. Microbiol."},{"key":"ref_128","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1038\/nrmicro.2016.164","article-title":"Cellulosomes: Bacterial nanomachines for dismantling plant polysaccharides","volume":"15","author":"Artzi","year":"2017","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_129","doi-asserted-by":"crossref","unstructured":"Mora\u00efs, S., Barak, Y., Caspi, J., Hadar, Y., Lamed, R., Shoham, Y., and Wilson, D. (2010). Cellulase-Xylanase Synergy in Designer Cellulosomes for Enhanced Degradation of a Complex Cellulosic Substrate. MBio, 1.","DOI":"10.1128\/mBio.00285-10"},{"key":"ref_130","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1021\/sb300047u","article-title":"Functional Display of Complex Cellulosomes on the Yeast Surface via Adaptive Assembly","volume":"2","author":"Tsai","year":"2013","journal-title":"ACS Synth. Biol."},{"key":"ref_131","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1186\/1754-6834-6-182","article-title":"A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates","volume":"6","author":"Vazana","year":"2013","journal-title":"Biotechnol. Biofuels"},{"key":"ref_132","doi-asserted-by":"crossref","first-page":"4370","DOI":"10.1111\/febs.15251","article-title":"Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis","volume":"287","author":"Kahn","year":"2020","journal-title":"FEBS J."},{"key":"ref_133","doi-asserted-by":"crossref","unstructured":"L\u00f3pez-L\u00f3pez, A., Santiago-Hern\u00e1ndez, A., Cayetano-Cruz, M., Garc\u00eda-Huante, Y., Campos, J.E., Bustos-Jaimes, I., Marsch-Moreno, R., Cano-Ram\u00edrez, C., Benitez-Cardoza, C.G., and Hidalgo-Lara, M.E. (2023). TtCel7A: A Native Thermophilic Bifunctional Cellulose\/Xylanase Exogluclanase from the Thermophilic Biomass-Degrading Fungus Thielavia terrestris Co3Bag1, and Its Application in Enzymatic Hydrolysis of Agroindustrial Derivatives. J. Fungi, 9.","DOI":"10.3390\/jof9020152"},{"key":"ref_134","doi-asserted-by":"crossref","unstructured":"Nemmaru, B., Douglass, J., Yarbrough, J.M., DeChellis, A., Shankar, S., Thokkadam, A., Wang, A., and Chundawat, S.P.S. (2024). Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose. Front. Energy Res., 12.","DOI":"10.3389\/fenrg.2024.1372916"},{"key":"ref_135","doi-asserted-by":"crossref","first-page":"e02079-18","DOI":"10.1128\/AEM.02079-18","article-title":"Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents","volume":"85","author":"Zheng","year":"2019","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_136","doi-asserted-by":"crossref","unstructured":"Zhang, J., Shi, H., Xu, L., Zhu, X., and Li, X. (2015). Site-Directed Mutagenesis of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima Based on Rational Design. PLoS ONE, 10, Correction in PLoS ONE\u00a02015, 10, e0141937.","DOI":"10.1371\/journal.pone.0141937"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/19\/8788\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T04:10:38Z","timestamp":1791691838000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/19\/8788"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,30]]},"references-count":136,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["ijms27198788"],"URL":"https:\/\/doi.org\/10.3390\/ijms27198788","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,30]]}}}