L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7 mM IPTG for 4 h at 30 °C) resulted in a significant amount of L-asparaginase with a titer of 93.52 μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production.

Department of Pharmaceutical Biotechnology School of Pharmacy Shahid Beheshti University of Medical Sciences No 2660 Valiasr Niayesh Junction Vali e Asr Ave Tehran 1991953381 Iran

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Alzaeemi SA, Noman EA, Al-shaibani MM, Al-Gheethi A, Mohamed RMSR, Almoheer R, Seif M, Tay KG, Zin NM, El Enshasy HA (2023) Improvement of L-asparaginase, an anticancer agent of Aspergillus arenarioides EAN603 in submerged fermentation using a radial basis function neural network with a specific genetic algorithm (RBFNN-GA). Fermentation 9:200. https://doi.org/10.3390/fermentation9030200 DOI

Andrade KCR, Fernandes RA, Pinho DB, de Freitas MM, Filho EXF, Pessoa A, Silva JI, Magalhães PO (2021) Sequencing and characterization of an L-asparaginase gene from a new species of Penicillium section Citrina isolated from Cerrado. Sci Rep 11:17861. https://doi.org/10.1038/s41598-021-97316-1 PubMed DOI PMC

Aronson JK (2016) Asparaginase. In: Aronson JK (ed) Meyler’s side effects of drugs, sixteenth. elsevier, Oxford, pp 726–727. https://doi.org/10.1016/B978-0-444-53717-1.00333-4 DOI

Baez A, Majdalani N, Shiloach J (2014) Production of recombinant protein by a novel oxygen-induced system in Escherichia coli. Microb Cell Factories 13:1–7. https://doi.org/10.1186/1475-2859-13-50 DOI

Barros T, Brumano L, Freitas M, Pessoa A, Parachin N, Magalhães PO (2020) Development of processes for recombinant L-asparaginase II production by Escherichia coli Bl21 (De3): From shaker to bioreactors. Pharmaceutics 13:14. https://doi.org/10.3390/pharmaceutics13010014 PubMed DOI PMC

Barton RR (2013) Response surface methodology. In: Gass SI, Fu MC (eds) Encyclopedia of operations research and management science. Springer, US, Boston, MA, pp 1307–1313. https://doi.org/10.1007/978-1-4419-1153-7_1143 DOI

Behravan A, Hashemi A (2021) RSM-based model to predict optimum fermentation conditions for soluble expression of the antibody fragment derived from 4D5MOC-B humanized Mab in SHuffle™ T7 E. coli. Iran J Pharm Res 20:254. https://doi.org/10.22037/ijpr.2020.114377.14822 PubMed DOI PMC

Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA (2008) Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 76:965–977. https://doi.org/10.1016/j.talanta.2008.05.019 PubMed DOI

Biasoto HP, Hebeda CB, Farsky SHP, Pessoa A, Costa-Silva TA, Monteiro G (2023) Extracellular expression of Saccharomyces cerevisiae’s L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters. Prep Biochem Biotechnol 53:511–522. https://doi.org/10.1080/10826068.2022.2111582 PubMed DOI

Biener R, Steinkämper A, Hofmann J (2010) Calorimetric control for high cell density cultivation of a recombinant Escherichia coli strain. J Biotech 146:45–53. https://doi.org/10.1016/j.jbiotec.2010.01.004 DOI

Box GEP, Wilson KB (1992) On the experimental attainment of optimum conditions. In: Kotz S, Johnson NL (eds) Breakthroughs in statistics: methodology and distribution. Springer, New York, New York, NY, pp 270–310. https://doi.org/10.1007/978-1-4612-4380-9_23 DOI

Castro D, Marques ASC, Almeida MR, de Paiva GB, Bento HBS, Pedrolli DB, Freire MG, Tavares APM, Santos-Ebinuma VC (2021) L-asparaginase production review: bioprocess design and biochemical characteristics. Appl Microbiol Biotechnol 105:4515–4534. https://doi.org/10.1007/s00253-021-11359-y PubMed DOI

Chityala S, Venkata Dasu V, Ahmad J, Prakasham RS (2015) High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N. Bioprocess Biosyst Eng 38:2271–2284. https://doi.org/10.1007/s00449-015-1464-x PubMed DOI

Costa IM, Schultz L, de Araujo Bianchi Pedra B, Leite MS, Farsky SH, de Oliveira MA, Pessoa A, Monteiro G, (2016) Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity. Sci Rep 6:36239. https://doi.org/10.1038/srep36239 PubMed DOI PMC

El-Naggar NE-A, El-Shweihy NM (2020) Bioprocess development for L-asparaginase production by Streptomyces rochei, purification and in-vitro efficacy against various human carcinoma cell lines. Sci Rep 10:7942. https://doi.org/10.1038/s41598-020-64052-x PubMed DOI PMC

Ferrara MA, Severino NM, Mansure JJ, Martins AS, Oliveira EM, Siani AC, Pereira N Jr, Torres FA, Bon EP (2006) Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene. Enzyme Microb Technol 39:1457–1463. https://doi.org/10.1016/J.ENZMICTEC.2006.03.036 DOI

Ferreira SC, Bruns R, Ferreira HS, Matos GD, David J, Brandão G, da Silva EP, Portugal L, Dos Reis P, Souza A (2007) Box-Behnken design: an alternative for the optimization of analytical methods. Anal Chim Acta 597:179–186. https://doi.org/10.1016/j.aca.2007.07.011 PubMed DOI

Francis DM, Page R (2010) Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci 61(5):24 21-25.24. 29. https://doi.org/10.1002/0471140864.ps0524s61 DOI

Gilmour SG (2006) Response surface designs for experiments in bioprocessing. Biometrics 62:323–331. https://doi.org/10.1111/j.1541-0420.2005.00444.x PubMed DOI

Gomes L, Monteiro G, Mergulhão F (2020) The impact of IPTG induction on plasmid stability and heterologous protein expression by Escherichia coli biofilms. Int J Mol Sci 21:576. https://doi.org/10.3390/ijms21020576 PubMed DOI PMC

Gutiérrez-González M, Farías C, Tello S, Pérez-Etcheverry D, Romero A, Zúñiga R, Ribeiro CH, Lorenzo-Ferreiro C, Molina MC (2019) Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli. Sci Rep 9:16850. https://doi.org/10.1038/s41598-019-53200-7 PubMed DOI PMC

Hanrahan G, Lu K (2006) Application of factorial and response surface methodology in modern experimental design and optimization. Crit Rev Anal Chem 36:141–151. https://doi.org/10.1080/10408340600969478 DOI

Hien Trang NT, Thanh Hoang L, Tuyen DT (2020) Optimization of L-asparaginase production from Escherichia coli using response surface methodology. Vietnam J Biotechnol 16:767–775. https://doi.org/10.15625/1811-4989/16/4/10861 DOI

Khushoo A, Pal Y, Singh BN, Mukherjee K (2004) Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expr Purif 38:29–36. https://doi.org/10.1016/J.PEP.2004.07.009 PubMed DOI

Kim S-K, Min W-K, Park Y-C, Seo J-H (2015) Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II. Enzyme Microb Technol 79:49–54. https://doi.org/10.1016/j.enzmictec.2015.06.017 PubMed DOI

Kumar S, Venkata Dasu V, Pakshirajan K (2011) Purification and characterization of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Bioresour Technol 102:2077–2082. https://doi.org/10.1016/j.biortech.2010.07.114 PubMed DOI

Kusuma SAF, Parwati I, Rostinawati T, Yusuf M, Fadhlillah M, Ahyudanari RR, Rukayadi Y, Subroto T (2019) Optimization of culture conditions for Mpt64 synthetic gene expression in Escherichia coli BL21 (DE3) using surface response methodology. Heliyon 5:e02741. https://doi.org/10.1016/j.heliyon.2019.e02741 PubMed DOI PMC

Lopes AM, Oliveira-Nascimento Ld, Ribeiro A, Tairum CA Jr, Breyer CA, Oliveira MAd, Monteiro G, Souza-Motta CMd, Magalhães PdO, Avendaño JGF (2017) Therapeutic L-asparaginase: upstream, downstream and beyond. Crit Rev Biotechnol 37:82–99. https://doi.org/10.3109/07388551.2015.1120705 PubMed DOI

Lubkowski J, Wlodawer A (2021) Structural and biochemical properties of L-asparaginase. FEBS J 288:4183–4209. https://doi.org/10.1111/febs.16042 PubMed DOI

Mathiyalagan S, Duraisamy S, Balakrishnan S, Kumarasamy A, Raju A (2021) Statistical optimization of bioprocess parameters for improved production of L-asparaginase from Lactobacillus plantarum. Proc Natl Acad Sci India Sect B Biol Sci 91:441–453. https://doi.org/10.1007/s40011-021-01234-1 DOI

Miyake R, Kawamoto J, Wei Y-L, Kitagawa M, Kato I, Kurihara T, Esaki N (2007) Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. Strain Ac10, as the Host. Appl Environ Microbiol 73:4849–4856. https://doi.org/10.1128/AEM.00824-07 PubMed DOI PMC

Mühlmann M, Forsten E, Noack S, Büchs J (2017) Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microb Cell Factories 16:220. https://doi.org/10.1186/s12934-017-0832-4 DOI

Peebo K, Valgepea K, Nahku R, Riis G, Õun M, Adamberg K, Vilu R (2014) Coordinated activation of PTA-ACS and TCA cycles strongly reduces overflow metabolism of acetate in Escherichia coli. Appl Microbiol Biotechnol 98:5131–5143. https://doi.org/10.1007/s00253-014-5613-y PubMed DOI

Peternel Š (2013) Bacterial cell disruption: a crucial step in protein production. N Biotechnol 30:250–254. https://doi.org/10.1016/j.nbt.2011.09.005 PubMed DOI

Pucci F, Rooman M (2017) Physical and molecular bases of protein thermal stability and cold adaptation. Curr Opin Struct Biol 42:117–128. https://doi.org/10.1016/j.sbi.2016.12.007 PubMed DOI

Radmard M, Hashemi A (2024) Response surface methodology approach to optimize the expression of Thioredoxin-MOG fusion protein. Pharm Sci. https://doi.org/10.34172/ps.2024.1 DOI

Rodrigues D, Pillaca-Pullo O, Torres-Obreque K, Flores-Santos J, Sánchez-Moguel I, Pimenta MV, Basi T, Converti A, Lopes AM, Monteiro G (2019) Fed-batch production of Saccharomyces cerevisiae L-Asparaginase II by recombinant Pichia pastoris MUT s strain. Front Bioeng Biotechnol 7:16. https://doi.org/10.3389/fbioe.2019.00016 PubMed DOI PMC

Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. https://doi.org/10.3389/fmicb.2014.00172 PubMed DOI PMC

Rostami N, Goharrizi LY (2023) Cloning, expression, and purification of the human synthetic survivin protein in Escherichia Coli using response surface methodology (RSM). Mol Biotechnol 65:326–336. https://doi.org/10.1007/s12033-021-00399-4 PubMed DOI

Saeed H, Hemida A, El-Nikhely N, Abdel-Fattah M, Shalaby M, Hussein A, Eldoksh A, Ataya F, Aly N, Labrou N (2020) Highly efficient Pyrococcus furiosus recombinant L-asparaginase with no glutaminase activity: expression, purification, functional characterization, and cytotoxicity on THP-1, A549 and Caco-2 cell lines. Int J Biol Macromol 156:812–828. https://doi.org/10.1016/j.ijbiomac.2020.04.080 PubMed DOI

Shafqat I, Shahzad S, Yasmin A, Chaudhry MT, Ahmed S, Javed A, Afzal I, Bibi M (2023) Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9. PLoS ONE 18:e0288620. https://doi.org/10.1371/journal.pone.0288620 PubMed DOI PMC

Shojaei S, Shojaei S, Band SS, Farizhandi AAK, Ghoroqi M, Mosavi A (2021) Application of Taguchi method and response surface methodology into the removal of malachite green and auramine-O by NaX nanozeolites. Sci Rep 11:16054. https://doi.org/10.1038/s41598-021-95649-5 PubMed DOI PMC

Sinclair K, Warner JP, Bonthron DT (1994) The ASP1 gene of Saccharomyces cerevisiae, encoding the intracellular isozyme of L-asparaginase. Gene 144:37–43. https://doi.org/10.1016/0378-1119(94)90200-3 PubMed DOI

Soares AL, Guimaraes GM, Polakiewicz B, de Moraes Pitombo RN, Abrahão-Neto J (2002) Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli L-asparaginase. Int J Pharm 237:163–170. https://doi.org/10.1016/S0378-5173(02)00046-7 PubMed DOI

Sørensen HP, Mortensen KK (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Factories 4:1–8. https://doi.org/10.1186/1475-2859-4-1 DOI

Upadhyay AK, Singh A, Mukherjee K, Panda AK (2014) Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein. Front Microbiol 5:486. https://doi.org/10.3389/fmicb.2014.00486 PubMed DOI PMC

Vasina JA, Baneyx F (1996) Recombinant protein expression at low temperatures under the transcriptional control of the major Escherichia coli cold shock promoter cspA. Appl Environ Microbiol 62:1444–1447. https://doi.org/10.1128/aem.62.4.1444-1447.1996 PubMed DOI PMC

Vidya J, Vasudevan UM, Soccol CR, Pandey A (2011) Cloning, functional expression and characterization of L-asparaginase II from E coli MTCC 739. Food Technol Biotechnol 49:286

Vimal A, Kumar A (2022) Optimized production of medically significant enzyme L-asparaginase under submerged and solid-state fermentation from agricultural wastes. Curr Microbiol 79:394. https://doi.org/10.1007/s00284-022-03095-x PubMed DOI

Xie M, Li Y, Xu L, Zhang S, Ye H, Sun F, Mei R, Su X (2021) Optimization of bacterial cytokine protein production by response surface methodology for environmental bioremediation. RSC Adv 11:36105–36115. https://doi.org/10.1039/D1RA03565G PubMed DOI PMC