Innovative test for asparagine screening in raw materials used for heat treated foods – prevention of acrylamide formation

The current dataset is a collection of data and metadata acquired through the project "Innovative test for asparagine screening in raw materials used for heat treated foods – prevention of acrylamide formation" . The project was funded by the Technological Agency of the Czech Republic (TACR), Contract ID: TQ03000738. In the following descriptions, specific information regarding each uploaded zip file will be provided acting as a "read me" file in each case. zip file #1 - Dataset on L-asparaginase spectrophotometric assay optimisation Last updated: 2025-11-24 ______Contact, Principal Investigator and Data manager______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ------------------------------------------------------------------------------------------------ ______About the dataset______ A comprehensive method optimisation was performed investigating the impact of several parameters that can affect the assay analytical performance. Here we report: i) the impact of incubation time on signal acquisition, ii) the inhibition by the substrate, iii) the impact of organic solvent on enzyme activity and iv) the secondary activity of L-ASNase towards L-glutamine and D-asparagine. The analysis was performed in buffer solutions using an in-house developed L-asparaginase (L-ASNase) colorimetric assay. The colour was monitored using a benchtop absorbance reader. ______Methods of data collection______ *Sample preparation and L-ASNase assay Calibration curves of L-asparagine, L-glutamine and D-asparagine were prepared in an aqueous buffer. Blank samples were also prepared to monitor the background absorbance. The recorded absorbance of a calibration point was subtracted by a blank to compensate for colour that is not related to the enzymatic catalysis. All measurements were performed at λ= 660 nm. *Methods of data processing The dataset contains only raw absorbance data. No further processing, normalization, or statistical evaluation was performed prior to deposition. ------------------------------------------------------------------------------------------------ ______File name structure_____ * The files are described in the 001_MetaData_optimisation.csv and 001_MetaData_secondary activity.csv. ______File formats______ * Tabular data - converted from XLSX to CSV *001_MetaData_optimisation.XLSX *001_MetaData_optimisation.csv *001_MetaData_secondary activity.XLSX *001_MetaData_ secondary activity.csv ______Date formats______ * YYYY-MM-DD ______Units and abbreviations______ * Absorbance is expressed as arbitrary units * Concentration is expressed as millimolar (mM) of L-asparagine in aqueous buffer ______Raw data______ The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file. zip file #2 - Dataset on the extraction methods for L-asparagine determination in cereals using an L-asparaginase spectrophotometric assay Last updated: 2025-11-24 ______Contact, Principal Investigator and Data manager______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ______About the dataset______ To investigate the most appropriate extraction protocol for the rapid screening of L-asparagine in cereal samples, three different extractants were tested. These were deionized water (dH2O), an aqueous methanol mixture and dH2O with polyvinylpolypyrrolidone (PVPP). The analysis was performed using an in-house developed L-asparaginase (L-ASNase) colorimetric assay. The colour was monitored using a benchtop absorbance reader. ______Methods of data collection______ *Sample preparation An oat sample with a L-aspargine concentration of 1200 mg/kg (concentration confirmed by liquid chromatography high resolution mass spectrometry) was used as the model sample. Upon homogenisation, an ultrasound assisted extraction was performed using different sample weights and extractants. The extracts were centrifuged and the supernatant was used for the enzymatic analysis. *L-ASNase assay To determine the amount of L-asparagine, a cereal extract was incubated with a L-ASNase solution. In the case of a control measurement, only buffer was used instead of the sample while in the case of the calibration curve, a solution containing a defined concentration of L-asparagine was used as the sample. Each tested cereal sample was accompanied by a sample blank. In this case, the cereal extract was mixed only with buffer, no enzyme solution. The recorded absorbance of a sample blank was subtracted by the raw measurement of the respective sample to compensate for colour that is not related to the enzymatic catalysis. All measurements were performed at λ= 660 nm. *Methods of data processing The dataset contains only raw absorbance data. No further processing, normalization, or statistical evaluation was performed prior to deposition. ------------------------------------------------------------------------------------------------ ______File name structure_____ * The files are described in the 001_MetaData_extraction.csv. ______File formats______ * Tabular data - converted from XSLX to CSV *001_MetaData_extraction.XLSX *001_MetaData_extraction.csv ______Date formats______ * YYYY-MM-DD ______Units and abbreviations______ * Absorbance is expressed as arbitrary units * Concentration is expressed as mg of L-asparagine per kg of homogenised cereal grains (mg/kg) ______Raw data______ The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file. zip file #3 - Dataset on the validation of the L-asparagine assay in wheat, rye and oat matrices Last updated: 2025-11-21 ______Contact, Principal Investigator and Data manager______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ______About the dataset______ Assessing the performance characteristics of an under-development method is crucial to identify whether it can be applied in real-life matrices. Here, we report the validation data for the L-asparaginase assay in wheat, rye and oat matrices. These cereals were selected as they demonstrate widely consumed food commodities with diverse free L-asparagine content. Specifically, wheat contains significantly lower L-asparagine content in comparison to rye and oat. The analysis was performed using an in-house developed L-asparaginase (L-ASNase) colorimetric assay. The developed colour was monitored using a benchtop absorbance reader. To perform the validation, wheat, rye and oat samples with diverse L-asparagine concentrations were used. In the case of wheat, 3 validation levels were tested (120, 204 and 647 mg/kg) whilst two levels were tested for rye (836 and 1339 mg/kg) and oat (654 and 1381 mg/kg). These concentrations were confirmed instrumentally using liquid chromatography high resolution mass spectrometry. For each validation level, the recovery (REC, %) and repeatability (expressed as relative standard deviation, RSD, %) were calculated from six replicate analyses (n= 6). The limit of quantification (LOQ) was estimated as the lowest calibration standard, which provided an absorbance signal higher than 0.030. A linear response was considered when the correlation coefficient (r2) was higher than 0.99 for at least 4 calibration points. The assay was validated as a quantitative method and the following performance characteristics were monitored: detectability, linearity, accuracy and repeatability. The method specificity was assured by the use of the selective L-ASNase. ______Methods of data collection______ *Sample preparation Cereal grains were homogenized and ultrasound assisted extraction was performed using a methanol-deionised water mixture as the extractant. The extracts were centrifuged and the supernatant was used for the enzymatic analysis. *L-ASNase assay To determine the amount of L-asparagine, a cereal extract was incubated with a L-ASNase solution. In the case of a control measurement, only buffer was used instead of the sample while in the case of the calibration curve, a solution containing a defined concentration of L-asparagine was used as the sample. Each tested cereal sample was accompanied by a sample blank. In this case, the cereal extract was mixed only with buffer, no enzyme solution. The recorded colour of a sample blank was subtracted by the raw measurement of the respective sample to compensate for colour that is not related to the enzymatic catalysis. *Methods of data processing Blank-corrected absorbance values were used as the analytical signal. No further processing, normalization, or statistical evaluation was performed prior to deposition. ------------------------------------------------------------------------------------------------ ______File name structure_____ * The files are described in the 001_MetaData_validation.csv. ______File formats______ * Tabular data - converted from XSLX to CSV *001_MetaData_validation.XLSX *001_MetaData_validation.csv ______Date formats______ * YYYY-MM-DD ______Units and abbreviations______ * Absorbance is expressed as arbitrary units * Concentration is expressed as mg of L-asparagine per kg of homogenised cereal grains (mg/kg) ______Raw data______ The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file. zip file #4 - Dataset on L-asparagine content in cereals using an L-asparaginase spectrophotometric assay Last updated: 2025-11-24 ______Contact, Principal Investigator and Data manager______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ------------------------------------------------------------------------------------------------ ______About the dataset______ Free L-asparagine is a key amino acid precursor involved in acrylamide formation during thermal processing of cereal-based foods. Its concentration in grains varies significantly depending on cereal species and agricultural practices, making it an important biochemical indicator for both food quality and safety assessment. This dataset contains results of free asparagine determination in wheat, rye and oat samples collected from two consecutive harvest years (2023 and 2024). The analysis was performed using an in-house developed L-asparaginase (L-ASNase) colorimetric assay. The colour was monitored using a benchtop absorbance reader. ______Methods of data collection______ *Sample preparation Cereal grains were homogenized and ultrasound assisted extraction was performed using a methanol-deionised water mixture as the extractant. The extracts were centrifuged and the supernatant was used for the enzymatic analysis. *L-ASNase assay To determine the amount of L-asparagine, a cereal extract was incubated with a L-ASNase solution. In the case of a control measurement, only buffer was used instead of a sample while in the case of a calibration curve, a solution containing a defined concentration of L-asparagine was used. Each tested cereal sample was accompanied by a sample blank. In this case, the cereal extract was mixed only with buffer, no enzyme solution. The recorded colour of a sample blank was subtracted by the raw measurement of the respective sample to compensate for colour that is not related to the enzymatic catalysis. All measurements were performed at λ= 660 nm. *Methods of data processing The dataset contains only raw absorbance data. No further processing, normalization, or statistical evaluation was performed prior to deposition. ------------------------------------------------------------------------------------------------ ______File name structure_____ * The files are described in the 001_MetaData_assay.csv. ______File formats______ * Tabular data - converted from XSLX to CSV *001_MetaData_assay.XLSX *001_MetaData_assay.csv ______Date formats______ * YYYY-MM-DD ______Units and abbreviations______ * Absorbance is expressed as arbitrary units * Concentration is expressed as mg of L-asparagine per kg of homogenised cereal grains (mg/kg) ______Raw data______ The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file. zip file #5 - Dataset on L-asparagine content in cereals using an L-asparaginase colorimetric assay coupled with a smartphone as the analytical detector Last updated: 2025-11-21 ______Contact, Principal Investigator and Data manager______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ------------------------------------------------------------------------------------------------ ______About the dataset______ Free L-asparagine is a key amino acid precursor involved in acrylamide formation during thermal processing of cereal-based foods. Its concentration in grains varies significantly depending on cereal species and agricultural practices, making it an important biochemical indicator for both food quality and safety assessment. This dataset contains results of free asparagine determination in wheat, rye and oat samples collected from two consecutive harvest years (2023 and 2024). The analysis was performed using an in-house developed L-asparaginase (L-ASNase) colorimetric assay. The colour was monitored using the camera of a smartphone as the analytical detector. ______Methods of data collection______ *Sample preparation Cereal grains were homogenized and ultrasound assisted extraction was performed using a methanol-deionised water mixture as the extractant. The extracts were centrifuged and the supernatant was used for the enzymatic analysis. *L-ASNase assay To determine the amount of L-asparagine, a cereal extract was incubated with a L-ASNase solution. In the case of a control measurement, only buffer was used instead of the sample while in the case of the calibration curve, a solution containing a defined concentration of L-asparagine was used as the sample. Each tested cereal sample was accompanied by a sample blank. In this case, the cereal extract was mixed only with buffer, no enzyme solution. The recorded colour of a sample blank was subtracted by the raw measurement of the respective sample to compensate for colour that is not related to the enzymatic catalysis. *Methods of data processing The dataset was acquired based on image data analysis. To record a picture, a standardised set-up was used assuring controlled ambient light conditions. Following recording, a picture was uploaded to an online cloud and processed off-line using the ImageJ 1.53a free software. A region of interest (ROI) was chosen for each microwell and the RGB numerical values were exported. A correction factor, based on white balancing, was applied. The corrected R channel values modulated the best towards the blue colour development and for this reason were used as the analytical signal. No further processing, normalization, or statistical evaluation was performed prior to deposition. ------------------------------------------------------------------------------------------------ ______File name structure_____ * The files are described in the 001_MetaData_smartphone.csv. ______File formats______ * Tabular data - converted from XSLX to CSV *001_MetaData_smartphone.XLSX *001_MetaData_smartphone.csv ______Date formats______ * YYYY-MM-DD ______Units and abbreviations______ * Red channel intensity is expressed as counts * Concentration is expressed as mg of L-asparagine per kg of homogenised cereal grains (mg/kg) ______Raw data______ The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file. zipf ile #6 - Dataset on L-asparagine content in cereals using liquid chromatography mass spectrometry (LC-MS) Last updated: 2025-11-06 ______Contact, Principal Investigator and Data manager______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ------------------------------------------------------------------------------------------------ ______About the dataset______ Free L-asparagine is a key amino acid precursor involved in acrylamide formation during thermal processing of cereal-based foods. Its concentration in grains varies significantly depending on cereal species and agricultural practices, making it an important biochemical indicator for both food quality and safety assessment. This dataset contains results of free asparagine determination in four cereal species — winter wheat, winter rye, naked oat, and common oat — collected from two consecutive harvest years (2023 and 2024) and cultivated under conventional and organic farming systems. The study aimed to evaluate the natural variability of free asparagine content in cereals grown under different agricultural regimes and environmental conditions. The analysis was performed using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) operated in full-scan positive electrospray ionization (ESI⁺) mode. Chromatographic separation was achieved on a BEH Amide column under hydrophilic interaction chromatography (HILIC) conditions. This dataset supports studies focused on acrylamide precursor monitoring and provides reference data for assessing the influence of cereal species, harvest year, and farming system on the biochemical composition of cereal grains. ______Methods of data collection______ *Sample preparation Ground cereal grain samples (winter wheat, winter rye, naked oat, and common oat) were extracted with deionised water under ultrasonic conditions. The extracts were filtered through 0.45 µm microfilters and diluted with methanol to a final methanol content of 50 %. Samples were stored at −28 °C until LC-MS analysis. *UHPLC-HRMS analysis Chromatographic separation of free asparagine was achieved on a BEH Amide column operated under hydrophilic interaction conditions (HILIC). Detection was performed using an Orbitrap-based UHPLC-HRMS system (Exactive, Thermo Fisher Scientific) in full-scan positive ESI mode, providing high mass accuracy and resolving power suitable for quantitative determination of amino acids. *Methods of data processing The dataset contains only raw LC-MS data. No further processing, normalization, or statistical evaluation was performed prior to deposition. ------------------------------------------------------------------------------------------------ ______File name structure_____ * The files are described in the 001_MetaData.csv. ______File formats______ * Tabular data - converted from XSLX to CSV *001_MetaData_LCMS.XLSX *001_MetaData_LCMS.csv ______Date formats______ * YYYY-MM-DD ______Units and abbreviations______ * Area is expressed as arbitrary units (a.u) * Concentration is expressed as mg of L-asparagine per kg of homogenised cereal grains (mg/kg) ______Raw data______ The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file. zip file #7 - Dataset on production and characterization of L-asparaginase from P. thiaminolyticus Last updated: 2025-12-11 ______Contact and Data manager______ * Eva Benesova * [e-mail] * [phone] * ORCID: 0000-0002-3696-3517 * Dept. of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technická 5, 166 28, Prague 6, Czech Republic ______Principal Investigator ______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ------------------------------------------------------------------------------------------------ ______About the dataset______ This dataset contains the results from production and characterization of Paenibacillus thiaminolyticus iso 1 l-asparaginase. ______Methods of data collection______ Methods used for l-asparaginase from Paenibacillus thiaminolyticus (production and biochemical characterization) a) Production and purification l-Asparaginase was produced in E. coli BL21 (DE3) (using LBA medium) and E. coli Lemo21 (DE3) (using LBACm medium supplemented with 50, 100 or 500 µM rhamnose). Overnight cultures were used to inoculate 200 mL fresh medium (1% v/v) and grown at 16, or 22 °C to OD600 ≈ 0.5. Recombinant protein expression was then induced either by adding 0.3 mM IPTG or by parallel cultivation in autoinduction medium. Following induction, cells were harvested by centrifugation (10,000 g, 10 min, 4 °C). Cells were harvested by centrifugation (10,000 g, 10 min, 4 °C) at a specified time point following induction, or at an appropriate time for cultures grown in autoinduction medium. The protein was then purified using Ni-NTA agarose and desalted with a PD-10 column. Protein concentration was determined using the BCA assay. Absorbance values (corrected for blank) for the calibration curves and samples were derived as the mean of triplicate measurements, while sample absorbance values represent the mean of triplicate measurements. b) Biochemical characterization Measurement of the activity of l-asparaginase was based on the cleavage of l-asparagine to l-aspartic acid and ammonia. The amount of produced ammonia was determined by Nessler´s method. Absorbance values (corrected for blank) were derived as the mean of triplicate or tetraplicate measurements. pH optimum of l-asparaginase was determined by carrying out the reactions at different pH ranging from 8.1 to 11.9 and measuring the activity as mentioned above. Temperature profile of l-asparaginase was determined by carrying out the reactions at different temperatures ranging from 15 to 60 oC and measuring the activity as mentioned above. Michaelis-Menten kinetics was determined within the substrate concentrations ranging from 1.9 to 145 mM l-asparagine and by measuring the activity as mentioned above. The temperature stability of l-asparaginase was determined by long-term incubation of the enzyme at 25, 37 and 55 oC. Then, the activity of the enzyme was measured as mentioned above. The reaction mixture was the same for both substrates as for l-asparagine and contained 30 and 24 mmol L−1 of l-glutamine or urea, respectively, dissolved in 100 mM Tris pH 9.0. The reaction with l-glutamine was carried out for 90 h at 37oC. l-glutaminase activity was determined by thin layer chromatography (TLC) on silica gel/TLC-card (Fluka). The samples were separated on the TLC card using a phenol:water solution (2:1, v/v), stained with 0.2% ninhydrin in acetone, and the color was developed by heating with a hair dryer. l-Glutamine (Roth) and l-glutamic acid (Merck) were used as standards (see picture 06_Figure_P_thiam_TLC). Lane 1 - sample after reaction, lane 2 - reaction mixture without enzyme, lane 3 - l-glutamine standard (12 nmol), lane 4 - l-glutamate standard (12 nmol). The oligomeric state of the protein was determined using size exclusion chromatography HiPrep 16/60 Sephacryl S-100 HR (GE Healthcare) and the automated NGC 10 Discover Chromatography Systems (Bio-Rad, USA), with standard proteins utilized for calibration. The effect of NaCl concentration on l-asparaginase activity was observed in the range of 2.5 to 250 mM NaCl and the activity was measured as mentioned above. Glutaminase activity was determined with l-glutamine as a substrate. The presence of potential product of the reaction, l-glutamic acid, was analyzed by thin layer chromatography. Detailed information is available in the publication on https://doi.org/10.1007/s12223-025-01296-y ______File formats______01_Data_P_th_recombinant_production.xlsx01_Data_P_th_recombinant_production.csv03_Data_P_th_biochemical characteristics.xlsx03_Data_P_th_biochemical characteristics.csv06_Figure_P_thiam_TLC.png ______Date formats______* YYYY-MM-DD ______Units and abbreviations______* Absorbance is expressed as arbitrary units* Concentration is expressed as mg of protein per mL of fraction. Concentration is expressed as mmol of L-Asn, L-Gln, NaCl or NH4+ per L of solution.Elution volume is expressed as mL. Temperature is expressed as degree CelsiusAPTES (3-aminopropyl)triethoxysilaneLBACm medium Luria-Bertani medium supplemented with ampicillin (100 μg mL-1) and chloramphenicol (34 μg mL-1) ______Raw data______The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file. zip file #8 - Dataset on production, characterization and immobilization of L-asparaginase from A. polaris Last updated: 2025-12-11 ______Contact and Data manager______ * Eva Benesova * [e-mail] * [phone] * ORCID: 0000-0002-3696-3517 * Dept. of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technická 5, 166 28, Prague 6, Czech Republic ______Principal Investigator ______ * Aristeidis Tsagkaris * [e-mail] * [phone] * ORCID: 0000-0003-4450-2611 * Dept. of Food Analysis and Nutrition. Faculty of Food and Biochemical Technology, University of chemistry and Technology, Prague * Technicka 5, 166 28, Prague 6, Czech Republic ______Data availability______ * With respect to IP right protection and/or commercial interest protection, the dataset is not published openly. However, access to the dataset may be requested through the contact person listed above. NDA might be required. ------------------------------------------------------------------------------------------------ ______About the dataset______ This dataset contains the results from production, characterization and immobilization of Arthrobacter polaris iso 1 l-asparaginase. Methods used for l-asparaginase from Arthrobacter polaris (production, biochemical characterization, and immobilization) a) l-Asparaginase was produced in E. coli BL21 (DE3) (using LBA medium) and E. coli Lemo21 (DE3) (using LBACm medium supplemented with 50, 100, 500, and 750 or 1000 µM rhamnose). Overnight cultures were used to inoculate 200 mL fresh medium (1% v/v) and grown at 16, or 22 °C to OD600 ≈ 0.5. Recombinant protein expression was then induced either by adding 0.3 mM IPTG or by parallel cultivation in autoinduction medium. Cells were harvested by centrifugation (10,000 g, 10 min, 4 °C) at a specified time point following induction, or at an appropriate time for cultures grown in autoinduction medium. The protein was then purified using Ni-NTA agarose and desalted with a PD-10 column. Protein concentration was determined using the BCA assay. Absorbance values (corrected for blank) for the calibration curves were derived as the mean of duplicate measurements, while sample absorbance values represent the mean of triplicate measurements. b) Enzyme activity was determined using the Nessler method, which measures the ammonia released from the l-asparaginase-catalyzed breakdown of l-asparagine (l-Asn). To accurately quantify the released ammonia, a calibration curve was constructed using the Nessler method with various known concentrations of ammonium sulphate as the standard. Absorbance values (corrected for blank) were derived as the mean of triplicate or tetraplicate measurements. The pH optimum for activity was evaluated within the broad range of 6.6 to 12.5. To identify the temperature profile, activity was measured across a range of 10 to 80 °C. Michaelis-Menten kinetics were determined by measuring the enzyme activity dependency on the substrate (l-Asn) concentration, ranging from 0.157 mM to 80.555 mM. Thermal stability was assessed by measuring residual activity after incubation at 37, 55, 60, and 65 °C. Long-term stability was measured on samples stored at −20 °C, with their activity assessed at 50 °C. The effect of sodium chloride (NaCl) on enzyme activity was investigated across a concentration range of 2.5 mM to 2500 mM. The oligomeric state of the protein was determined using size exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (Cytiva, formerly GE Healthcare, USA) and the automated NGC 10 Discover Chromatography Systems (Bio-Rad, USA), with standard proteins utilized for calibration. Glutaminase activity was determined using thin-layer chromatography (TLC). The chromatogram displays the separation of the substrate l-glutamine (l-Gln) and the product l-glutamate (l-Glu). R represents the test reaction with l-ASNase iso 1 from A. polaris incubated at 37 °C for 90 h with 20 mM l-Gln. SR is the substrate reaction blank under identical conditions but without the enzyme. Positive controls utilizing l-glutaminase from E. coli cell lysate were run: C1 (150 μL supernatant) and C2 (50 μL supernatant). Both controls were incubated for 4 h 40 min at 37 °C with 20 mM l-Gln; SC1 and SC2 are their respective blanks without the enzyme supernatant. The final lanes serve as concentration standards for the substrate, Q12, Q6, Q3 (12 mM, 6 mM, and 3 mM l-Gln, respectively), and the product, E12, E6, E3 (12 mM, 6 mM, and 3 mM l-Glu, respectively) (see picture 06_Figure_A_pol_TLC). c) For enzyme immobilization, APTES, glutaraldehyde, and plasma-treated microtiter plates were utilized. The reaction on the microplates was conducted for 5 min at room temperature, and activity was subsequently determined using the Nessler method directly within the plates. ------------------------------------------------------------------------------------------------ ______File formats______02_Data_A_pol_recombinant_production.xlsx02_Data_A_pol_recombinant_production.csv04_Data A_pol_biochemical characteristics.xlsx04_Data A_pol_biochemical characteristics.csv05_Data_A_pol_immobilization.xlsx05_Data_A_pol_immobilization.csv06_Figure_A_pol_TLC.png ______Date formats______* YYYY-MM-DD ______Units and abbreviations______* Absorbance is expressed as arbitrary units* Concentration is expressed as mg of protein per mL of fraction. Concentration is expressed as mmol of L-Asn, L-Gln, NaCl or NH4+ per L of solution.Elution volume is expressed as mL. Temperature is expressed as degree CelsiusAPTES (3-aminopropyl)triethoxysilaneLBACm medium Luria-Bertani medium supplemented with ampicillin (100 μg mL-1) and chloramphenicol (34 μg mL-1) ______Raw data______The raw data are available upon reasonable request. They are not included here as they do not provide any further insight than what is already available in the metadata file.