{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T08:06:47Z","timestamp":1779696407599,"version":"3.53.1"},"reference-count":51,"publisher":"Springer Science and Business Media LLC","license":[{"start":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T00:00:00Z","timestamp":1779667200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T00:00:00Z","timestamp":1779667200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100012530","name":"Gebze Technical University","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100012530","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Folia Microbiol"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    L-Asparaginase (L-ASNase) is an enzyme of significant therapeutic value within the pharmaceutical industry, primarily due to its clinical efficacy in treating acute lymphoblastic leukemia (ALL). Despite its benefits, the clinical application of commercially available bacterial-derived L-ASNases is frequently impeded by severe hypersensitivity reactions and immunogenicity. This limitation has necessitated the exploration of novel L-ASNase variants and the development of alternative heterologous expression systems that minimize adverse immune responses. As unicellular eukaryotic models, yeast-based expression platforms represent a robust alternative for the production of therapeutic proteins. In the present study, the\n                    <jats:italic>asnase<\/jats:italic>\n                    gene from\n                    <jats:italic>Lachancea thermotolerans<\/jats:italic>\n                    (\n                    <jats:italic>L. thermotolerans<\/jats:italic>\n                    ,\n                    <jats:italic>Lt<\/jats:italic>\n                    ASNase), along with its Lys99Ala mutant variant was heterologously expressed in\n                    <jats:italic>Pichia pastoris<\/jats:italic>\n                    (\n                    <jats:italic>P. pastoris<\/jats:italic>\n                    ). To maximize yield, cultivation parameters were systematically optimized, followed by scale-up in a bioreactor under stabilized conditions. Initial shake-flask cultivations of the wild-type and mutant\n                    <jats:italic>Lt<\/jats:italic>\n                    ASNase yielded maximum whole-cell activities of 191 U g\n                    <jats:sub>DCW<\/jats:sub>\n                    <jats:sup>\u22121<\/jats:sup>\n                    (628 U L\n                    <jats:sup>\u2212\u20091<\/jats:sup>\n                    ) and 57 U gdcw\n                    <jats:sup>\u2212\u20091<\/jats:sup>\n                    (174 U L\u207b\u00b9) over induction periods of three and two days, respectively. Process optimization identified the ideal conditions as pH 6.0 and 20\u00a0\u00b0C for the wild-type enzyme, and pH 7.5 and 16\u00a0\u00b0C for the mutant. Notably, bioreactor-scale expressions facilitated 4- and 7-fold increase in volumetric whole-cell activity for the wild-type and mutant enzymes, compared to shake-flask results. These findings suggest that\n                    <jats:italic>P. pastoris<\/jats:italic>\n                    is a viable host for microbial\n                    <jats:italic>Lt<\/jats:italic>\n                    ASNase production, achieving yields higher than established\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    (\n                    <jats:italic>E. coli<\/jats:italic>\n                    ) systems; however, further investigation into secretory expression pathways is required to provide cost-effective production.\n                  <\/jats:p>","DOI":"10.1007\/s12223-026-01514-1","type":"journal-article","created":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T07:47:00Z","timestamp":1779695220000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Heterologous expression of wild-type and Lys99Ala mutant L-asparaginase from Lachancea thermotolerans in Pichia pastoris: culture optimization and laboratory-scale bioreactor production"],"prefix":"10.1007","author":[{"given":"Buse","family":"\u00c7alo\u011flu Susamaz","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katja","family":"Koschorreck","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vlada B.","family":"Urlacher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bar\u0131\u015f","family":"Binay","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,25]]},"reference":[{"key":"1514_CR1","doi-asserted-by":"publisher","first-page":"e202400507","DOI":"10.1002\/biot.202400507","volume":"19","author":"BY Aktar","year":"2024","unstructured":"Aktar BY, Aysan A, Turunen O et al (2024) L-Asparaginase from Lachancea Thermotolerans: Effect of Lys99Ala on Enzyme Performance and in vitro Antileukemic Efficacy. Biotechnol J 19:e202400507","journal-title":"Biotechnol J"},{"key":"1514_CR2","doi-asserted-by":"crossref","unstructured":"Alexander PA, He Y, Chen Y et al (2009) A minimal sequence code for switching protein structure and function. Proc Natl Acad Sci 106:21149\u201321154","DOI":"10.1073\/pnas.0906408106"},{"key":"1514_CR6","doi-asserted-by":"publisher","first-page":"108854","DOI":"10.1016\/j.bej.2023.108854","volume":"193","author":"B \u00c7alo\u011flu","year":"2023","unstructured":"\u00c7alo\u011flu B, Binay B (2023) Utilization potential of agro-industrial by-products and waste sources: Laccase production in bioreactor with Pichia pastoris. Biochem Eng J 193:108854","journal-title":"Biochem Eng J"},{"key":"1514_CR3","first-page":"27","volume":"21","author":"FF Bauer","year":"2000","unstructured":"Bauer FF, Pretorius IS (2000) Yeast stress response and fermentation efficiency: how to survive the making of wine-a review. S Afr J Enol Vitic 21:27\u201351","journal-title":"S Afr J Enol Vitic"},{"key":"1514_CR4","doi-asserted-by":"publisher","first-page":"511","DOI":"10.1080\/10826068.2022.2111582","volume":"53","author":"HP Biasoto","year":"2023","unstructured":"Biasoto HP, Hebeda CB, Farsky SHP et al (2023) Extracellular expression of Saccharomyces cerevisiae\u2019s L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters. Prep Biochem Biotechnol 53:511\u2013522","journal-title":"Prep Biochem Biotechnol"},{"key":"1514_CR5","doi-asserted-by":"publisher","first-page":"1285","DOI":"10.2217\/fon-2021-1288","volume":"18","author":"MJ Burke","year":"2022","unstructured":"Burke MJ, Zalewska-Szewczyk B (2022) Hypersensitivity Reactions to Asparaginase Therapy in Acute Lymphoblastic Leukemia: Immunology and Clinical Consequences. Future Oncol 18:1285\u20131299","journal-title":"Future Oncol"},{"key":"1514_CR7","doi-asserted-by":"publisher","first-page":"4515","DOI":"10.1007\/s00253-021-11359-y","volume":"105","author":"D Castro","year":"2021","unstructured":"Castro D, Marques ASC, Almeida MR et al (2021) L-asparaginase production review: bioprocess design and biochemical characteristics. Appl Microbiol Biotechnol 105:4515\u20134534","journal-title":"Appl Microbiol Biotechnol"},{"key":"1514_CR8","doi-asserted-by":"publisher","first-page":"619","DOI":"10.1002\/bab.1888","volume":"67","author":"S Chand","year":"2020","unstructured":"Chand S, Mahajan RV, Prasad JP et al (2020) A comprehensive review on microbial l-asparaginase: Bioprocessing, characterization, and industrial applications. Biotechnol Appl Biochem 67:619\u2013647","journal-title":"Biotechnol Appl Biochem"},{"key":"1514_CR9","first-page":"22","volume":"1","author":"AY Chang","year":"2017","unstructured":"Chang AY, Chau V, Landas JA, Pang Y (2017) Preparation of calcium competent Escherichia coli and heat-shock transformation. JEMI methods 1:22\u201325","journal-title":"JEMI methods"},{"key":"1514_CR10","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1016\/j.jbiosc.2013.02.020","volume":"116","author":"T Charoenrat","year":"2013","unstructured":"Charoenrat T, Khumruaengsri N, Promdonkoy P et al (2013) Improvement of recombinant endoglucanase produced in Pichia pastoris KM71 through the use of synthetic medium for inoculum and pH control of proteolysis. J Biosci Bioeng 116:193\u2013198","journal-title":"J Biosci Bioeng"},{"key":"1514_CR11","doi-asserted-by":"crossref","unstructured":"Ciesarov\u00e1 Z, Kukurov\u00e1 K (2024) Chap. 23 - Impact of l-asparaginase on acrylamide content in fried potato and bakery products. In: G\u00f6kmen V, Mogol BABT-A in F (Second E (eds). Academic Press, pp 473\u2013491","DOI":"10.1016\/B978-0-323-99119-3.00021-7"},{"key":"1514_CR12","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1007\/s43393-026-00433-z","volume":"6","author":"F da Costa Alves","year":"2026","unstructured":"da Costa Alves F, da Silva Rodrigues-Junior V, Cibulski SP (2026) L-Asparaginase bioprospection from Bacillus species. Syst Microbiol Biomanufacturing 6:40","journal-title":"Syst Microbiol Biomanufacturing"},{"key":"1514_CR13","doi-asserted-by":"publisher","first-page":"2009","DOI":"10.1110\/ps.9.10.2009","volume":"9","author":"C Derst","year":"2000","unstructured":"Derst C, Henseling J, R\u00f6hm K-H (2000) Engineering the substrate specificity of Escherichia coli asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248. Protein Sci 9:2009\u20132017","journal-title":"Protein Sci"},{"key":"1514_CR14","doi-asserted-by":"publisher","first-page":"1380","DOI":"10.1021\/pr8007623","volume":"8","author":"M Dragosits","year":"2009","unstructured":"Dragosits M, Stadlmann J, Albiol J et al (2009) The effect of temperature on the proteome of recombinant Pichia pastoris. J Proteome Res 8:1380\u20131392","journal-title":"J Proteome Res"},{"key":"1514_CR15","doi-asserted-by":"publisher","first-page":"679","DOI":"10.1080\/10826068.2019.1599396","volume":"49","author":"B Effer","year":"2019","unstructured":"Effer B, Lima GM, Cabarca S et al (2019) L-Asparaginase from E. chrysanthemi expressed in glycoswitch\u00ae: effect of His-Tag fusion on the extracellular expression. Prep Biochem Biotechnol 49:679\u2013685","journal-title":"Prep Biochem Biotechnol"},{"key":"1514_CR16","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/s13205-023-03600-4","volume":"13","author":"F Erden-Karao\u011flan","year":"2023","unstructured":"Erden-Karao\u011flan F, Karao\u011flan M (2023) Improvement of recombinant L-Asparaginase production in Pichia pastoris. 3 Biotech 13:1\u20139","journal-title":"3 Biotech"},{"key":"1514_CR17","doi-asserted-by":"publisher","first-page":"150","DOI":"10.1186\/s13068-022-02234-7","volume":"15","author":"BG Erg\u00fcn","year":"2022","unstructured":"Erg\u00fcn BG, La\u00e7\u0131n K, \u00c7alo\u011flu B, Binay B (2022) Second generation Pichia pastoris strain and bioprocess designs. Biotechnol Biofuels Bioprod 15:150","journal-title":"Biotechnol Biofuels Bioprod"},{"key":"1514_CR18","doi-asserted-by":"publisher","first-page":"1457","DOI":"10.1016\/j.enzmictec.2006.03.036","volume":"39","author":"MA Ferrara","year":"2006","unstructured":"Ferrara MA, Severino NMB, Mansure JJ et al (2006) Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene. Enzyme Microb Technol 39:1457\u20131463","journal-title":"Enzyme Microb Technol"},{"key":"1514_CR19","doi-asserted-by":"publisher","first-page":"746","DOI":"10.3390\/ph15060746","volume":"15","author":"M Freitas","year":"2022","unstructured":"Freitas M, Souza P, Homem-De-mello M et al (2022) L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain. Pharmaceuticals 15:746","journal-title":"Pharmaceuticals"},{"key":"1514_CR20","doi-asserted-by":"crossref","unstructured":"Gupta R, Brunak S (2001) Prediction of glycosylation across the human proteome and the correlation to protein function. In: Altman RB, Dunker AK, Hunter L, Lauderdale K, Klein TE (eds) Biocomputing 2002. World Scientific, pp 310\u2013322","DOI":"10.1142\/9789812799623_0029"},{"key":"1514_CR21","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1038\/s41392-024-01886-1","volume":"9","author":"M He","year":"2024","unstructured":"He M, Zhou X, Wang X (2024) Glycosylation: mechanisms, biological functions and clinical implications. Signal Transduct Target Ther 9:194","journal-title":"Signal Transduct Target Ther"},{"key":"1514_CR22","unstructured":"Invitrogen (2014) Pichia Expression Kit User Guide. Life Technologies Date Accessed: 03.06.2021"},{"key":"1514_CR23","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1016\/S0168-1656(03)00003-8","volume":"102","author":"M Jahic","year":"2003","unstructured":"Jahic M, Gustavsson M, Jansen A-K et al (2003) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J Biotechnol 102:45\u201353. https:\/\/doi.org\/10.1016\/S0168-1656(03)00003-8","journal-title":"J Biotechnol"},{"key":"1514_CR24","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1016\/j.bej.2010.07.009","volume":"52","author":"H Jin","year":"2010","unstructured":"Jin H, Liu G, Ye X et al (2010) Enhanced porcine interferon-\u03b1 production by recombinant Pichia pastoris with a combinational control strategy of low induction temperature and high dissolved oxygen concentration. Biochem Eng J 52:91\u201398","journal-title":"Biochem Eng J"},{"key":"1514_CR25","doi-asserted-by":"publisher","first-page":"397","DOI":"10.1093\/protein\/14.6.397","volume":"14","author":"PH Jonson","year":"2001","unstructured":"Jonson PH, Petersen SB (2001) A critical view on conservative mutations. Protein Eng 14:397\u2013402","journal-title":"Protein Eng"},{"key":"1514_CR26","doi-asserted-by":"publisher","first-page":"1438","DOI":"10.3390\/foods11101438","volume":"11","author":"JA Joseph","year":"2022","unstructured":"Joseph JA, Akkermans S, Van Impe JFM (2022) Effects of temperature and pH on recombinant Thaumatin II production by Pichia pastoris. Foods 11:1438","journal-title":"Foods"},{"key":"1514_CR27","unstructured":"Josuran R (2026) Prot pi: Protein tool. https:\/\/www.protpi.ch. Accessed 14 Apr 2026"},{"key":"1514_CR28","doi-asserted-by":"publisher","first-page":"5867","DOI":"10.1002\/jcp.29583","volume":"235","author":"M Karbalaei","year":"2020","unstructured":"Karbalaei M, Rezaee SA, Farsiani H (2020) Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol 235:5867\u20135881","journal-title":"J Cell Physiol"},{"key":"1514_CR29","doi-asserted-by":"publisher","first-page":"e2971","DOI":"10.1002\/btpr.2971","volume":"36","author":"S Katla","year":"2020","unstructured":"Katla S, Pavan SS, Mohan N, Sivaprakasam S (2020) Biocalorimetric monitoring of glycoengineered. cultivation for the production of recombinant huIFN\u03b12b: A quantitative study based on mixed feeding strategies. Biotechnol Prog 36:e2971","journal-title":"Biotechnol Prog"},{"key":"1514_CR30","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1016\/j.procbio.2023.06.006","volume":"131","author":"EK Kleingesinds","year":"2023","unstructured":"Kleingesinds EK, de Almeida Parizotto L, Effer B et al (2023) Downstream process and evaluation of the concomitant impact of a recombinant glycosylated L-asparaginase on leukemic cancer cells and the bone marrow tumor microenvironment. Process Biochem 131:41\u201351","journal-title":"Process Biochem"},{"key":"1514_CR32","doi-asserted-by":"publisher","first-page":"107516","DOI":"10.1016\/j.bej.2020.107516","volume":"156","author":"GM Lima","year":"2020","unstructured":"Lima GM, Effer B, Biasoto HP et al (2020) Glycosylation of L-asparaginase from E. coli through yeast expression and site-directed mutagenesis. Biochem Eng J 156:107516","journal-title":"Biochem Eng J"},{"key":"1514_CR33","doi-asserted-by":"publisher","first-page":"106","DOI":"10.1186\/s12934-017-0724-7","volume":"16","author":"B Lin","year":"2017","unstructured":"Lin B, Tao Y (2017) Whole-cell biocatalysts by design. Microb Cell Fact 16:106","journal-title":"Microb Cell Fact"},{"key":"1514_CR34","doi-asserted-by":"publisher","first-page":"621","DOI":"10.1021\/bp0603199","volume":"23","author":"H Lin","year":"2007","unstructured":"Lin H, Kim T, Xiong F, Yang X (2007) Enhancing the Production of Fc Fusion Protein in Fed-Batch Fermentation of Pichia pastoris by Design of Experiments. Biotechnol Prog 23:621\u2013625","journal-title":"Biotechnol Prog"},{"key":"1514_CR31","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1007\/s12010-007-0003-x","volume":"142","author":"P Li","year":"2007","unstructured":"Li P, Anumanthan A, Gao X-G et al (2007) Expression of Recombinant Proteins in Pichia Pastoris. Appl Biochem Biotechnol 142:105\u2013124","journal-title":"Appl Biochem Biotechnol"},{"key":"1514_CR35","doi-asserted-by":"publisher","first-page":"4183","DOI":"10.1111\/febs.16042","volume":"288","author":"J Lubkowski","year":"2021","unstructured":"Lubkowski J, Wlodawer A (2021) Structural and biochemical properties of L-asparaginase. FEBS J 288:4183\u20134209","journal-title":"FEBS J"},{"key":"1514_CR36","doi-asserted-by":"publisher","first-page":"921","DOI":"10.1042\/bj3020921","volume":"302","author":"ZB Moola","year":"1994","unstructured":"Moola ZB, Scawen MD, Atkinson T, Nicholls DJ (1994) Erwinia chrysanthemil-asparaginase: epitope mapping and production of antigenically modified enzymes. Biochem J 302:921\u2013927","journal-title":"Biochem J"},{"key":"1514_CR37","doi-asserted-by":"publisher","first-page":"282","DOI":"10.1006\/prep.2001.1509","volume":"23","author":"A Murasugi","year":"2001","unstructured":"Murasugi A, Asami Y, Mera-Kikuchi Y (2001) Production of Recombinant Human Bile Salt-Stimulated Lipase in Pichia pastoris. Protein Expr Purif 23:282\u2013288","journal-title":"Protein Expr Purif"},{"key":"1514_CR38","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1002\/jctb.6540","volume":"96","author":"O Pillaca-Pullo","year":"2021","unstructured":"Pillaca-Pullo O, Rodrigues D, S\u00e1nchez-Moguel I et al (2021) Recombinant l-asparaginase production using Pichia pastoris (MUTs strain): establishment of conditions for growth and induction phases. J Chem Technol Biotechnol 96:283\u2013292. https:\/\/doi.org\/10.1002\/jctb.6540","journal-title":"J Chem Technol Biotechnol"},{"key":"1514_CR39","doi-asserted-by":"publisher","first-page":"157","DOI":"10.2217\/fmb-2023-0131","volume":"19","author":"KC Rodrigues Andrade","year":"2024","unstructured":"Rodrigues Andrade KC, Cordeiro de Abreu JA, Guimar\u00e3es MB et al (2024) Heterologous Expression of Fungal L-Asparaginase: A Systematic Review. Future Microbiol 19:157\u2013171. https:\/\/doi.org\/10.2217\/fmb-2023-0131","journal-title":"Future Microbiol"},{"key":"1514_CR40","doi-asserted-by":"publisher","unstructured":"Rodrigues D, Pillaca-Pullo O, Torres-Obreque K et al (2019) Fed-batch production of Saccharomyces cerevisiae L-Asparaginase II by recombinant Pichia pastoris MUTs strain. Front Bioeng Biotechnol 7. https:\/\/doi.org\/10.3389\/fbioe.2019.00016","DOI":"10.3389\/fbioe.2019.00016"},{"key":"1514_CR41","doi-asserted-by":"publisher","first-page":"3677","DOI":"10.3390\/ijms22073677","volume":"22","author":"Z Rosenbergov\u00e1","year":"2021","unstructured":"Rosenbergov\u00e1 Z, K\u00e1ntorov\u00e1 K, \u0160imkovi\u010d M et al (2021) Optimisation of recombinant myrosinase production in Pichia pastoris. Int J Mol Sci 22:3677","journal-title":"Int J Mol Sci"},{"key":"1514_CR42","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1080\/10242422.2016.1212843","volume":"34","author":"MG Savvidou","year":"2016","unstructured":"Savvidou MG, Sotiroudis TG, Kolisis FN (2016) Cell surface and cellular debris-associated heat-stable lipolytic enzyme activities of the marine alga Nannochloropsis oceanica. Biocatal Biotransform 34:24\u201332. https:\/\/doi.org\/10.1080\/10242422.2016.1212843","journal-title":"Biocatal Biotransform"},{"key":"1514_CR44","doi-asserted-by":"publisher","unstructured":"Shishparenok AN, Gladilina YA, Zhdanov DD (2023) Engineering and expression strategies for optimization of L-Asparaginase development and production. Int J Mol Sci 24. https:\/\/doi.org\/10.3390\/ijms242015220","DOI":"10.3390\/ijms242015220"},{"key":"1514_CR43","doi-asserted-by":"publisher","first-page":"321","DOI":"10.1016\/S1046-5928(02)00706-4","volume":"28","author":"X Shi","year":"2003","unstructured":"Shi X, Karkut T, Chamankhah M et al (2003) Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 28:321\u2013330","journal-title":"Protein Expr Purif"},{"key":"1514_CR48","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1007\/s12033-020-00281-9","volume":"63","author":"A T\u00fclek","year":"2021","unstructured":"T\u00fclek A, Karata\u015f E, \u00c7akar MM et al (2021) Optimisation of the production and bleaching process for a new laccase from Madurella mycetomatis, expressed in Pichia pastoris: from secretion to yielding prominent. Mol Biotechnol 63:24\u201339","journal-title":"Mol Biotechnol"},{"key":"1514_CR45","doi-asserted-by":"publisher","first-page":"224","DOI":"10.1111\/1751-7915.12871","volume":"11","author":"M Tom\u00e0s-Gamisans","year":"2018","unstructured":"Tom\u00e0s-Gamisans M, Ferrer P, Albiol J (2018) Fine-tuning the P. pastoris iMT1026 genome-scale metabolic model for improved prediction of growth on methanol or glycerol as sole carbon sources. Microb Biotechnol 11:224\u2013237. https:\/\/doi.org\/10.1111\/1751-7915.12871","journal-title":"Microb Biotechnol"},{"key":"1514_CR46","doi-asserted-by":"publisher","unstructured":"Tripathy RK, Anakha J, Pande AH (2024) Towards development of biobetter: L-asparaginase a case study. Biochim Biophys Acta (BBA) - Gen Subj 1868:130499. https:\/\/doi.org\/10.1016\/j.bbagen.2023.130499","DOI":"10.1016\/j.bbagen.2023.130499"},{"key":"1514_CR47","doi-asserted-by":"publisher","first-page":"1269282","DOI":"10.3389\/fmicb.2024.1269282","volume":"15","author":"K Tsegaye","year":"2024","unstructured":"Tsegaye K, Tsehai BA, Getie B (2024) Desirable L-asparaginases for treating cancer and current research trends. Front Microbiol 15:1269282","journal-title":"Front Microbiol"},{"key":"1514_CR49","doi-asserted-by":"publisher","first-page":"949","DOI":"10.1016\/j.procbio.2009.04.019","volume":"44","author":"Y Wang","year":"2009","unstructured":"Wang Y, Wang Z, Xu Q et al (2009) Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris. Process Biochem 44:949\u2013954. https:\/\/doi.org\/10.1016\/j.procbio.2009.04.019","journal-title":"Process Biochem"},{"key":"1514_CR50","doi-asserted-by":"publisher","first-page":"108806","DOI":"10.1016\/j.bej.2023.108806","volume":"191","author":"B Yilmazer Aktar","year":"2023","unstructured":"Yilmazer Aktar B, Georgakis N, Labrou N et al (2023) Comparative structural and kinetic study for development of a novel candidate L-asparaginase based pharmaceutical. Biochem Eng J 191:108806. https:\/\/doi.org\/10.1016\/j.bej.2023.108806","journal-title":"Biochem Eng J"},{"key":"1514_CR51","doi-asserted-by":"publisher","first-page":"2495","DOI":"10.1038\/s41467-024-46332-6","volume":"15","author":"J Zheng","year":"2024","unstructured":"Zheng J, Guo N, Huang Y et al (2024) High temperature delays and low temperature accelerates evolution of a new protein phenotype. Nat Commun 15:2495","journal-title":"Nat Commun"}],"container-title":["Folia Microbiologica"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s12223-026-01514-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s12223-026-01514-1","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s12223-026-01514-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T07:47:04Z","timestamp":1779695224000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s12223-026-01514-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,25]]},"references-count":51,"alternative-id":["1514"],"URL":"https:\/\/doi.org\/10.1007\/s12223-026-01514-1","relation":{},"ISSN":["0015-5632","1874-9356"],"issn-type":[{"value":"0015-5632","type":"print"},{"value":"1874-9356","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,25]]},"assertion":[{"value":"3 February 2026","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"15 May 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"25 May 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}]}}