{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,5]],"date-time":"2026-11-05T17:12:48Z","timestamp":1793898768015,"version":"4.8.0"},"reference-count":59,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T00:00:00Z","timestamp":1790899200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan","award":["AP23488823"],"award-info":[{"award-number":["AP23488823"]}],"id":[{"id":"https:\/\/ror.org\/05tyne317","id-type":"ROR","asserted-by":"crossref"}]},{"award":["AP23488823"],"award-info":[{"award-number":["AP23488823"]}],"id":[{"id":"https:\/\/ror.org\/05tyne317","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Phytases are widely used as feed additives because they improve phosphorus utilization from plant-derived feed ingredients and reduce phosphorus excretion into the environment. Most recombinant AppA phytases produced in Komagataella phaffii have been obtained using methanol-inducible expression systems, whereas information on constitutive methanol-free production remains limited. In this study, Escherichia coli AppA phytase was expressed in K. phaffii under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Cultivation temperature and initial glucose concentration were evaluated in shake-flask cultures, while the selected conditions were assessed in a 2 L stirred-tank bioreactor. Extracellular phytase activity reached 114.17 \u00b1 2.71 U\/mL in shake flasks at 27 \u00b0C with 3% initial glucose. The K. phaffii-produced enzyme was N-glycosylated, with an apparent molecular weight of approximately 54 kDa compared with approximately 45 kDa after deglycosylation. Compared with purified rAppA-E, the rAppA-P preparation showed a lower temperature optimum, a narrower pH\u2013activity profile, and lower apparent thermostability. In a single bioreactor run, extracellular phytase activity reached 186.81 \u00b1 0.25 U\/mL, approximately 1.6-fold higher than the maximum mean shake-flask value. These findings demonstrate constitutive, methanol-free extracellular production of AppA in K. phaffii and support further development of controlled cultivation strategies.<\/jats:p>","DOI":"10.3390\/biology15191758","type":"journal-article","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T11:21:49Z","timestamp":1790940109000},"page":"1758","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Methanol-Free Production of Recombinant AppA Phytase in Komagataella phaffii: Evaluation of Cultivation Conditions, Bioreactor Cultivation, and Biochemical Characterization"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-4471-6072","authenticated-orcid":false,"given":"Magzhan","family":"Astrakhanov","sequence":"first","affiliation":[{"name":"Laboratory for Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viktoriya","family":"Popova","sequence":"additional","affiliation":[{"name":"Laboratory for Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kairat","family":"Baltin","sequence":"additional","affiliation":[{"name":"Laboratory for Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saveliy","family":"Kirillov","sequence":"additional","affiliation":[{"name":"Laboratory for Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4572-948X","authenticated-orcid":false,"given":"Bekbolat","family":"Khassenov","sequence":"additional","affiliation":[{"name":"Laboratory for Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6346-5866","authenticated-orcid":false,"given":"Saniya","family":"Aktayeva","sequence":"additional","affiliation":[{"name":"Laboratory for Genetics and Biochemistry of Microorganisms, National Center for Biotechnology, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,10,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1186\/2191-0855-2-10","article-title":"Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168","volume":"2","author":"Boukhris","year":"2012","journal-title":"AMB Express"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"11","DOI":"10.4236\/ajmb.2017.71002","article-title":"Microbial phytases and phytate: Exploring opportunities for sustainable phosphorus management in agriculture","volume":"7","author":"Balaban","year":"2017","journal-title":"Am. J. Mol. Biol."},{"key":"ref_3","unstructured":"Nunes, C.S., and Kumar, V. (2018). General aspects of phytases. Enzymes in Human and Animal Nutrition: Principles and Perspectives, Academic Press."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Gahlawat, S.K., Duhan, J.S., Salar, R.K., Siwach, P., Kumar, S., and Kaur, P. (2018). Phytase: The feed enzyme, an overview. Advances in Animal Biotechnology and Its Applications, Springer.","DOI":"10.1007\/978-981-10-4702-2"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Joye, I. (2019). Protein digestibility of cereal products. Foods, 8.","DOI":"10.3390\/foods8060199"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Selle, P.H., Macelline, S.P., Chrystal, P.V., and Liu, S.Y. (2023). The contribution of phytate-degrading enzymes to chicken-meat production. Animals, 13.","DOI":"10.3390\/ani13040603"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"129030","DOI":"10.1016\/j.biortech.2023.129030","article-title":"Advances in immobilization of phytases and their application","volume":"379","author":"Filippovich","year":"2023","journal-title":"Bioresour. Technol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1007\/s13205-021-02964-9","article-title":"Research status of Bacillus phytase","volume":"11","author":"Zhao","year":"2021","journal-title":"3 Biotech"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Mei, C., Chretien, R.L., Amaradasa, B.S., He, Y., Turner, A., and Lowman, S. (2021). Characterization of phosphate solubilizing bacterial endophytes and plant growth promotion in vitro and in greenhouse. Microorganisms, 9.","DOI":"10.3390\/microorganisms9091935"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1007\/s12010-019-03205-9","article-title":"Characteristics of an acidic phytase from Aspergillus aculeatus APF1 for dephytinization of biofortified wheat genotypes","volume":"191","author":"Saxena","year":"2020","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1007\/s42770-020-00289-y","article-title":"Fungal phytases: From genes to applications","volume":"51","year":"2020","journal-title":"Braz. J. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"9196","DOI":"10.1021\/acs.est.2c00099","article-title":"Enhancing phytate availability in soils and phytate-P acquisition by plants: A review","volume":"56","author":"Liu","year":"2022","journal-title":"Environ. Sci. Technol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"100305","DOI":"10.1016\/j.xplc.2022.100305","article-title":"Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants","volume":"3","author":"Gu","year":"2022","journal-title":"Plant Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1023\/A:1026231624543","article-title":"Intestinal phytase II: A comparison of activity and in vivo phytate hydrolysis in three teleost species with differing digestive strategies","volume":"26","author":"Ellestad","year":"2002","journal-title":"Fish. Physiol. Biochem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1577","DOI":"10.3382\/ps\/pev108","article-title":"Contribution of intestinal- and cereal-derived phytase activity on phytate degradation in young broilers","volume":"94","author":"Morgan","year":"2015","journal-title":"Poult. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1128\/AEM.65.2.367-373.1999","article-title":"Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): Catalytic properties","volume":"65","author":"Wyss","year":"1999","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1146\/annurev-animal-031412-103717","article-title":"Phytase, a new life for an \u201cold\u201d enzyme","volume":"1","author":"Lei","year":"2013","journal-title":"Annu. Rev. Anim. Biosci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"597","DOI":"10.3390\/biom3030597","article-title":"Microbial enzymes with special characteristics for biotechnological applications","volume":"3","author":"Nigam","year":"2013","journal-title":"Biomolecules"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1431","DOI":"10.1007\/s10989-018-9788-4","article-title":"Cloning and high-level expression of the enzymatic region of phytase in E. coli","volume":"25","author":"Hallaji","year":"2019","journal-title":"Int. J. Pept. Res. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5601","DOI":"10.1128\/AEM.00762-10","article-title":"Expression of a Bacillus phytase C gene in Pichia pastoris and properties of the recombinant enzyme","volume":"76","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/s12934-020-01499-7","article-title":"Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris","volume":"20","author":"Navone","year":"2021","journal-title":"Microb. Cell Fact."},{"key":"ref_22","first-page":"664","article-title":"Cloning of Bacillus subtilis phytase gene construct in Escherichia coli","volume":"13","author":"Doosti","year":"2021","journal-title":"Iran. J. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1007\/s00284-023-03471-1","article-title":"Microbial phytases: Properties and applications in the food industry","volume":"80","author":"Joudaki","year":"2023","journal-title":"Curr. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Bustos, C., Quezada, J., Veas, R., Altamirano, C., Braun-Galleani, S., Fickers, P., and Berrios, J. (2022). Advances in cell engineering of the Komagataella phaffii platform for recombinant protein production. Metabolites, 12.","DOI":"10.3390\/metabo12040346"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"foaf010","DOI":"10.1093\/femsyr\/foaf010","article-title":"Advancing recombinant protein expression in Komagataella phaffii: Opportunities and challenges","volume":"25","author":"Lv","year":"2025","journal-title":"FEMS Yeast Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"26502","DOI":"10.1038\/s41598-025-11833-x","article-title":"Comparative biochemical properties of recombinant goat and calf chymosins and their implications in dairy processing","volume":"15","author":"Akishev","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Akishev, Z., Aktayeva, S., Kiribayeva, A., Abdullayeva, A., Baltin, K., Mussakhmetov, A., Tursunbekova, A., Ramankulov, Y., and Khassenov, B. (2022). Obtaining of recombinant camel chymosin and testing its milk-clotting activity on cow\u2019s, goat\u2019s, ewes\u2019, camel\u2019s and mare\u2019s milk. Biology, 11.","DOI":"10.3390\/biology11111545"},{"key":"ref_28","first-page":"78","article-title":"Overexpression of Escherchia coli phytase with high specific activity","volume":"20","author":"Luo","year":"2004","journal-title":"Sheng Wu Gong Cheng Xue Bao"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1016\/S1872-2075(06)60041-1","article-title":"Improving phytase expression by increasing the gene copy number of appA-m in Pichia pastoris","volume":"22","author":"Luo","year":"2006","journal-title":"Sheng Wu Gong Cheng Xue Bao"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e27553","DOI":"10.5812\/jjm.27553","article-title":"Optimization of recombinant expression of synthetic bacterial phytase in Pichia pastoris using response surface methodology","volume":"8","author":"Akbarzadeh","year":"2015","journal-title":"Jundishapur J. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6007","DOI":"10.1021\/jf401853b","article-title":"Overexpression of Escherichia coli phytase in Pichia pastoris and its biochemical properties","volume":"61","author":"Tai","year":"2013","journal-title":"J. Agric. Food Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3276","DOI":"10.1002\/bit.28510","article-title":"Improving phytase production in Pichia pastoris fermentations through de-repression and methanol induction optimization","volume":"120","author":"Weng","year":"2023","journal-title":"Biotechnol. Bioeng."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5463","DOI":"10.1073\/pnas.74.12.5463","article-title":"DNA sequencing with chain-terminating inhibitors","volume":"74","author":"Sanger","year":"1977","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e07137","DOI":"10.1016\/j.heliyon.2021.e07137","article-title":"Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris","volume":"7","author":"Akishev","year":"2021","journal-title":"Heliyon"},{"key":"ref_35","first-page":"310","article-title":"Prediction of glycosylation across the human proteome and the correlation to protein function","volume":"7","author":"Gupta","year":"2002","journal-title":"Pac. Symp. Biocomput."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1038\/s41587-019-0036-z","article-title":"SignalP 5.0 improves signal peptide predictions using deep neural networks","volume":"37","author":"Tsirigos","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/msb.2011.75","article-title":"Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega","volume":"7","author":"Sievers","year":"2011","journal-title":"Mol. Syst. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"W320","DOI":"10.1093\/nar\/gku316","article-title":"Deciphering key features in protein structures with the new ENDscript server","volume":"42","author":"Robert","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1007\/s00253-002-1033-5","article-title":"Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis","volume":"59","author":"Tye","year":"2002","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1186\/s12934-024-02362-9","article-title":"Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella phaffii","volume":"23","author":"Albacar","year":"2024","journal-title":"Microb. Cell Fact."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.enzmictec.2004.05.007","article-title":"A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase","volume":"35","author":"Chen","year":"2004","journal-title":"Enzym. Microb. Technol."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Barone, G.D., Emmerstorfer-Augustin, A., Biundo, A., Pisano, I., Coccetti, P., Mapelli, V., and Camattari, A. (2023). Industrial production of proteins with Pichia pastoris\u2014Komagataella phaffii. Biomolecules, 13.","DOI":"10.3390\/biom13030441"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Unver, Y., and Dagci, I. (2024). Komagataella phaffii (Pichia pastoris) as a powerful yeast expression system for biologics production. Front. Biosci. (Elite Ed.), 16.","DOI":"10.31083\/j.fbe1602019"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1186\/s12934-026-02988-x","article-title":"Enhanced secretion of thermostable phytases from Myceliophthora thermophila by Komagataella phaffii","volume":"25","author":"Gratzer","year":"2026","journal-title":"Microb. Cell Fact."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Kiribayeva, A., Mukanov, B., Silayev, D., Akishev, Z., Ramankulov, Y., and Khassenov, B. (2022). Cloning, expression, and characterization of a recombinant xylanase from Bacillus sonorensis T6. PLoS ONE, 17.","DOI":"10.1371\/journal.pone.0265647"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e28064","DOI":"10.1016\/j.heliyon.2024.e28064","article-title":"An impact of N-glycosylation on biochemical properties of a recombinant \u03b1-amylase from Bacillus licheniformis","volume":"10","author":"Kiribayeva","year":"2024","journal-title":"Heliyon"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1016\/j.enzmictec.2007.10.013","article-title":"Effect of glycosylation on biochemical characterization of recombinant phytase expressed in Pichia pastoris","volume":"42","author":"Guo","year":"2008","journal-title":"Enzym. Microb. Technol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/s13036-024-00453-0","article-title":"Avoiding overflow metabolite formation in Komagataella phaffii fermentations to enhance recombinant protein production","volume":"18","author":"Steimann","year":"2024","journal-title":"J. Biol. Eng."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1186\/s12934-026-02948-5","article-title":"Systematic perturbation of cultures of K. phaffii by carbon co-feeding show gene signatures associated with production of recombinant proteins","volume":"25","author":"Acharya","year":"2026","journal-title":"Microb. Cell Fact."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2057","DOI":"10.1080\/10408398.2021.1970508","article-title":"Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges","volume":"63","author":"Wu","year":"2023","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.bbagen.2005.07.003","article-title":"Controlling N-linked glycan site occupancy","volume":"1726","author":"Jones","year":"2005","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1223","DOI":"10.1002\/jps.21504","article-title":"Effects of glycosylation on the stability of protein pharmaceuticals","volume":"98","author":"Griebenow","year":"2009","journal-title":"J. Pharm. Sci."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"foab059","DOI":"10.1093\/femsyr\/foab059","article-title":"What makes Komagataella phaffii non-conventional?","volume":"21","author":"Ata","year":"2021","journal-title":"FEMS Yeast Res."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1186\/s12934-014-0163-7","article-title":"High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10","volume":"13","author":"Zhong","year":"2014","journal-title":"Microb. Cell Fact."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Pan, Y., Yang, J., Wu, J., Yang, L., and Fang, H. (2022). Current advances of Pichia pastoris as cell factories for production of recombinant proteins. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.1059777"},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Gasset, A., Garcia-Ortega, X., Garrig\u00f3s-Mart\u00ednez, J., Valero, F., and Montesinos-Segu\u00ed, J.L. (2022). Innovative bioprocess strategies combining physiological control and strain engineering of Pichia pastoris to improve recombinant protein production. Front. Bioeng. Biotechnol., 10.","DOI":"10.3389\/fbioe.2022.818434"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"1380","DOI":"10.1021\/pr8007623","article-title":"The effect of temperature on the proteome of recombinant Pichia pastoris","volume":"8","author":"Dragosits","year":"2009","journal-title":"J. Proteome Res."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"e70106","DOI":"10.1111\/1751-7915.70106","article-title":"Impact of oxygen availability on the organelle-specific redox potentials and stress in recombinant protein producing Komagataella phaffii","volume":"18","author":"Kostopoulou","year":"2025","journal-title":"Microb. Biotechnol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1002\/bit.20318","article-title":"Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: Case study with recombinant ovine interferon-tau","volume":"89","author":"Sinha","year":"2005","journal-title":"Biotechnol. Bioeng."}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/19\/1758\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T11:27:40Z","timestamp":1790940460000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/19\/1758"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,2]]},"references-count":59,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["biology15191758"],"URL":"https:\/\/doi.org\/10.3390\/biology15191758","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,2]]}}}