Abstract
L-asparaginase is an important therapeutic biomolecule, used to treat lymphocytic leukemia. In the present study, Iodobacter sp. PCH194 genome was mined for new L-asparaginase (Id-ASNase II). In silico analysis including amino acid composition and pI revealed its sequence-based novelty and phylogenetic closeness to the commercial bacterial L-asparaginases. Subsequently, the gene was successfully cloned and overexpressed in Escherichia coli (E. coli BL21 DE3). The monomeric molecular weight of Id-ASNase II was 38 kDa with a native size of 150 kDa. Maximum L-asparaginase activity (80 U mg− 1) was achieved in 25 mM Tris-HCl buffer (pH 8.2) at 37 °C after 10 min of incubation. The enzyme was active in wide pH (5.0–11.0) and temperature ranges (4–70 °C). The half-life (t1/2) of the enzyme at 37 °C was 13.54 h, whereas Km, Vmax, kcat, and kcat/Km were 1.2 mM, 128 µmoles min− 1, 82 s− 1, and 63.1 s− 1 mM− 1, respectively. Id-ASNase II exhibited cytotoxicity against cancer cell line K562 (IC50 value 0.4 U mL− 1) leading to cell cycle arrest in the G2/M phase after treatment. Furthermore, 10 U Id-ASNase II led to a 57% acrylamide reduction in potato chips. Thus, the study discovered and characterized Id-ASNase II with potential applications for treating leukemia and food processing.
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Abbreviations
- Id-ASNase II :
-
Iodobacter L-asparaginase type-II
- ALL:
-
Acute lymphoblastic leukemia
- PCR:
-
Polymerase chain reaction
- IPTG:
-
Isopropyl-β-D-1-thiogalactopyranoside
- CFE:
-
Cell-free extract
- DMEM:
-
Dulbecco’s modified eagle’s medium
- MTT:
-
3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-tetrazolium bromide
References
Abt E, Robin LP, McGrath S, Srinivasan J, DiNovi M, Adachi Y, Chirtel S (2019) Acrylamide levels and dietary exposure from foods in the United States, an update based on 2011–2015 data. Food Addit Contam Part Chem Anal Control Expo Risk Assess 36:1475–1490. https://doi.org/10.1080/19440049.2019.1637548
Aggarwal E, Chauhan S, Sareen D (2021) Thiopeptides encoding biosynthetic gene clusters mined from bacterial genomes. J Biosci 46:1–12. https://doi.org/10.1007/s12038-021-00158-2
Almagro AJJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, von Heijne G, Nielsen H (2019) SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol 37:420–423. https://doi.org/10.1038/s41587-019-0036-z
Atkins CA, Pate JS, Sharkey PJ (1975) Asparagine metabolism key to the nitrogen nutrition of developing legume seeds. Plant Physiol 56:807–812. https://doi.org/10.1104/pp.56.6.807
Bergmans HEN, Van Die IM, Hoekstra WPM (1981) Transformation in Escherichia coli: stages in the process. J Bacteriol 146:564–570. https://doi.org/10.1128/jb.146.2.564-570.1981
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7:248–254. https://doi.org/10.1006/abio.1976.9999
Brumano LP, da Silva FVS, Costa-Silva TA, Apolinário AC, Santos JHPM, Kleingesinds EK, Monteiro G, Rangel-Yagui C, de Benyahia O, Junior B AP (2019) Development of L-asparaginase biobetters: current research status and review of the desirable quality profiles. Front Bioeng Biotechnol 6:1–22. https://doi.org/10.3389/fbioe.2018.00212
Chi H, Chen M, Jiao L, Lu Z, Bie X, Zhao H, Lu F (2021) Characterization of a novel l-asparaginase from mycobacterium gordonae with acrylamide mitigation potential. Foods 10. https://doi.org/10.3390/foods10112819
Cory AH, Owen TC, Barltrop JA, Cory JG (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 3:207–212. https://doi.org/10.3727/095535491820873191
Darnal S, Patial V, Kumar V, Kumar S, Kumar V, Padwad YS, Singh D (2023) Biochemical characterization of extremozyme L-asparaginase from Pseudomonas sp. PCH199 for therapeutics. AMB Express 13:22. https://doi.org/10.1186/s13568-023-01521-2
Derst C, Henseling J, Röhm 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 9:2009–2017. https://doi.org/10.1110/ps.9.10.2009
Dumina M, Zhgun A, Pokrovskaya M, Aleksandrova S, Zhdanov D, Sokolov N, El’darov M (2021) A novel L-asparaginase from hyperthermophilic archaeon Thermococcus sibiricus: heterologous expression and characterization for biotechnology application. Int J Mol Sci 13(18):22. https://doi.org/10.3390/ijms22189894
Farahat MG, Amr D, Galal A (2020) Molecular cloning, structural modeling and characterization of a novel glutaminase-free L-asparaginase from Cobetia Amphilecti AMI6. Int J Biol Macromol 143:685–695. https://doi.org/10.1016/j.ijbiomac.2019.10.258
Faraji M, Hamdamali M, Aryanasab F, Shabanian M (2018) 2-Naphthalenthiol derivatization followed by dispersive liquid–liquid microextraction as an efficient and sensitive method for determination of acrylamide in bread and biscuit samples using high-performance liquid chromatography. J Chromatogr A 1558:14–20. https://doi.org/10.1016/j.chroma.2018.05.021
Feng Y, Liu S, Jiao Y, Wang Y, Wang M, Du G (2019) Gene cloning and expression of the L-asparaginase from Bacillus cereus BDRD-ST26 in Bacillus subtilis WB600. J Biosci Bioeng 127:418–424. https://doi.org/10.1016/j.jbiosc.2018.09.007
Fonseca MHG, Fiúza T, da Morais S, de Souza SB, de Trevizani T R (2021) Circumventing the side effects of L-asparaginase. Biomed Pharmacother 139. https://doi.org/10.1016/j.biopha.2021.111616
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy Server. (In) John M. Walker (ed): The Proteomics Protocol Handbook, Humana Press. pp. 571–608. https://doi.org/10.1385/1592598900
Geourjon C, Deléage G (1995) Sopma: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Bioinformatics 11:681–684. https://doi.org/10.1093/bioinformatics/11.6.681
Golbabaie A, Nouri H, Moghimi H, Khaleghian A (2020) L-asparaginase production and enhancement by Sarocladium strictum: In vitro evaluation of anti-cancerous properties. J Appl Microbiol 129:356–366. https://doi.org/10.1111/jam.14623
Hatamzadeh S, Rahnama K, Nasrollahnejad S, Fotouhifar KB, Hemmati K, White JF, Taliei F (2020) Isolation and identification of L-asparaginase-producing endophytic fungi from the Asteraceae family plant species of Iran. Peer J 2020:1–16. https://doi.org/10.7717/peerj.8309
Hendriksen HV, Kornbrust BA, Østergaard PR, Stringer MA (2009) Evaluating the Potential for Enzymatic Acrylamide Mitigation in a range of Food products using an asparaginase from aspergillus oryzae. J Agric Food Chem 57:4168–4176. https://doi.org/10.1021/jf900174q
Hijiya N, Van Der Sluis IM (2016) Asparaginase-associated toxicity in children with acute lymphoblastic leukemia. Leuk Lymphoma 57:748–757. https://doi.org/10.3109/10428194.2015.1101098
Jiao L, Chi H, Lu Z, Zhang C, Chia SR, Show PL, Tao Y, Lu F (2020) Characterization of a novel type I L-asparaginase from Acinetobacter soli and its ability to inhibit acrylamide formation in potato chips. J Biosci Bioeng 129:672–678. https://doi.org/10.1016/j.jbiosc.2020.01.007b
Kotzia GA, Labrou NE (2007) L-Asparaginase from Erwinia Chrysanthemi 3937: cloning, expression, and characterization. J Biotechnol 127:657–669. https://doi.org/10.1016/j.jbiotec.2006.07.037
Krishnapura PR, Belur PD, Subramanya S (2016) A critical review on properties and applications of microbial L-asparaginases. Crit Rev Microbiol 42:720–737. https://doi.org/10.3109/1040841X.2015.1022505
Kumar V, Kumar S, Darnal S, Patial V, Singh A, Thakur V, Kumar S, Singh D (2019) Optimized chromogenic dyes-based identification and quantitative evaluation of bacterial L-asparaginase with low/no glutaminase activity bioprospected from pristine niches in Indian Trans-Himalaya. 3 Biotech 9:275. https://doi.org/10.1007/s13205-019-1810-9
Kumar V, Darnal S, Kumar S, Kumar S, Singh D (2021) Bioprocess for co-production of polyhydroxybutyrate and violacein using himalayan bacterium Iodobacter sp. PCH194. Bioresour Technol 319:124235. https://doi.org/10.1016/j.biortech.2020.124235
Kumar V, Kumar S, Singh D (2022a) Metagenomic insights into himalayan glacial and kettle lake sediments revealed microbial community structure, function, and stress adaptation strategies. https://doi.org/10.1007/s00792-021-01252-x. Extremophiles 26
Kumar S, Darnal S, Patial V, Kumar V, Kumar V, Kumar S, Singh D (2022b) Molecular cloning, characterization, and in-silico analysis of l-asparaginase from Himalayan Pseudomonas sp. PCH44. 3 Biotech. 12:162. https://doi.org/10.1007/s13205-022-03224-0
Kumar S, Darnal S, Patial V, Kumar V, Singh D (2022c) Molecular characterization of a stable and robust L-asparaginase from Pseudomonas sp. PCH199: evaluation of cytotoxicity and acrylamide mitigation potential. Fermentation 8:568. https://doi.org/10.3390/fermentation8100568
Kumar V, Kashyap P, Kumar S, Thakur V, Kumar S, Singh D (2022d) Multiple adaptive strategies of Himalayan Iodobacter sp. PCH194 to high-altitude stresses. Front Microbiol 13:881873. https://doi.org/10.3389/fmicb.2022.881873
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Laskowski RA, Swindells MB (2011) LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 51:2778–2786. https://doi.org/10.1021/ci200227u
Lee JH, Lee J, Yhim HY, Oh D, Bang SM (2017) Venous thromboembolism following L-asparaginase treatment for lymphoid malignancies in Korea. J Thromb Haemost 15:655–661. https://doi.org/10.1111/jth.13636
Lomelino CL, Andring JT, McKenna R, Kilberg MS (2017) Asparagine synthetase: function, structure, and role in disease. J Biol Chem 292:19952–19958. https://doi.org/10.1074/jbc.R117.819060
Lubkowski J, Vanegas J, Chan WK, Lorenzi PL, Weinstein JN, Sukharev S, Fushman D, Rempe S, Anishkin A, Wlodawer A (2020) Mechanism of catalysis by L-asparaginase. Biochemistry 59:1927–1945. https://doi.org/10.1021/acs.biochem.0c00116
Maan AA, Anjum MA, Khan MKI, Nazir A, Saeed F, Afzaal M, Aadil RM (2022) Acrylamide formation and different mitigation strategies during food processing–A review. Food Rev Int 38:70–87. https://doi.org/10.1080/87559129.2020.1719505
Meghavarnam AK, Salah M, Sreepriya M, Janakiraman S (2017) Growth inhibitory and proapoptotic effects of L-asparaginase from Fusarium culmorum ASP-87 on human leukemia cells (Jurkat). Fundam Clin Pharmacol 31:292–300. https://doi.org/10.1111/fcp.12257
Mishra A (2006) Production of L-asparaginase, an anticancer agent, from Aspergillus Niger using agricultural waste in solid state fermentation. Appl Biochem Biotechnol 135:33–42. https://doi.org/10.1385/ABAB:135:1:33
Mohamed SA, Elshal MF, Kumosani TA, Aldahlawi AM (2015) Purification and Characterization of Asparaginase from Phaseolus vulgaris Seeds. Evidence-based Complement Altern Med 2015. https://doi.org/10.1155/2015/309214
Nomme J, Su Y, Konrad M, Lavie A (2012) Structures of apo and product-bound human L-asparaginase: insights into the mechanism of autoproteolysis and substrate hydrolysis. Biochem 51:6816–6826. https://doi.org/10.1021/bi300870g
Paleologos EK, Kontominas MG (2005) Determination of acrylamide and methacrylamide by normal phase high performance liquid chromatography and UV detection. J Chromatogr A 1077:128–135. https://doi.org/10.1016/j.chroma.2005.04.037
Papageorgiou AC, Posypanova GA, Andersson CS, Sokolov NN, Krasotkina J (2008) Structural and functional insights into Erwinia carotovora L-asparaginase. FEBS J 275:4306–4316. https://doi.org/10.1111/j.1742-4658.2008.06574.x
Patial V, Kumar V, Joshi R, Gupta M, Singh D (2022) Acrylamide mitigation in foods using recombinant L-asparaginase: an extremozyme from Himalayan Pseudomonas sp. PCH182. Food Res Int 162:111936. https://doi.org/10.1016/j.foodres.2022.111936
Peterson RE, Ciegler A (1969) L-asparaginase production by various bacteria. Appl Microbiol 17:929–930. https://doi.org/10.1128/am.17.6.929-930.1969
Radha R, Arumugam N, Gummadi SN (2018) Glutaminase free L-asparaginase from Vibrio cholerae: heterologous expression, purification and biochemical characterization. Int J Biol Macromol 111:129–138. https://doi.org/10.1016/j.ijbiomac.2017.12.165
Saeed H, Hemida A, El-Nikhely N, Abdel-Fattah M, Shalaby M, Hussein A, Eldoksh A, Ataya F, Aly N, Labrou N, Nematalla H (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
Schwartz JH, Reeves JY, Broome JD (1966) Two L-asparaginases from E. Coli and their action against tumors. Proc Natl Acad Sci USA 56:1516–1519. https://doi.org/10.1073/pnas.56.5.1516
Shakambari G, Sameer Kumar R, Ashokkumar B, Ganesh V, Vasantha VS, Varalakshmi P (2018) Cloning and expression of L-asparaginase from Bacillus tequilensis PV9W and therapeutic efficacy of solid lipid particle formulations against cancer. Sci Rep 8:1–11. https://doi.org/10.1038/s41598-018-36161-1
Sharafi Z, Barati M, Khoshayand MR, Adrangi S (2017) Screening for type II L-asparaginases: lessons from the genus Halomonas. Iran J Pharm Res 16:1565–1573. https://doi.org/10.22037/ijpr.2017.2145
Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38:3022–3027. https://doi.org/10.1093/molbev/msab120
Thakur V, Kumar V, Kumar V, Singh D (2021) Genomic insights driven statistical optimization for production of efficient cellulase by himalayan thermophilic Bacillus sp. PCH94 using agricultural waste. Waste Biomass Valori 12:6917–6929. https://doi.org/10.1007/s12649-021-01491-1
Trott O, Olson AJ (2009) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461. https://doi.org/10.1002/jcc.21334
Tyl RW, Friedman MA (2003) Effects of acrylamide on rodent reproductive performance. Reprod Toxicol 17:1–13. https://doi.org/10.1016/S0890-6238(02)00078-3
Vidya J, Vasudevan UM, Soccol CR (2011) Cloning, functional expression, and characterization of L- asparaginase II from E. Coli MTCC 739. Food Technol Biotechnol 49:286–290
Xu X, Pang M, Liu J, Wang Y, Wu X, Huang KL, Liang Z (2021) Genome mining reveals the genes of carboxypeptidase for OTA-detoxification in Bacillus subtilis CW14. Int J Biol Macromol 186:800–810. https://doi.org/10.1016/j.ijbiomac.2021.07.085
Zuo S, Zhang T, Jiang B, Mu W (2015) Reduction of acrylamide level through blanching with treatment by an extremely thermostable L-asparaginase during French fries processing. Extremophiles 19:841–851. https://doi.org/10.1007/s00792-015-0763-0
Acknowledgements
Department of Health Research (DHR) and Indian Council of Medical Research (ICMR), New Delhi is duly acknowledged for the Young Scientist award (grant number 12014/25/2020-HR) to Vr. K and Senior Research Fellowship to VP & SK. SD and VT duly acknowledge for Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), New Delhi. VK also duly acknowledges DST, New Delhi, for the Young Scientist award. Authors duly acknowledge Dr. Y S Padwad for extending animal cell culture support. DS gratefully acknowledges financial support from CSIR, New Delhi, for research grants MLP0125 & 130. This manuscript represents CSIR-IHBT publication number 5031.
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Virender Kumar: Conceived and designed the experiments, performed experiments, data curation, formal analysis, and writing-original draft. Sanyukta Darnal: Cell cytotoxicity analysis and writing. Vijeta Patial: Food application and writing, data curation. Subhash Kumar: Homology modeling and docking. Vikas Thakur: Formal analysis, data curation, writing. Vijay Kumar: Genome mining, formal analysis, editing. Dharam Singh: Conceptualization, methodology, formal analysis, supervision, resources, visualization, writing review and editing.
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Kumar, V., Darnal, S., Patial, V. et al. Biochemical characterization of genome derived L-asparaginase from eurypsychrophilic Iodobacter sp. PCH194 for therapeutic and food applications. Biologia 79, 1525–1537 (2024). https://doi.org/10.1007/s11756-024-01624-6
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DOI: https://doi.org/10.1007/s11756-024-01624-6