Abstract
l-Glutaminase is an amidohydrolase which can act as a vital chemotherapeutic agent against various malignancies. In the present work, l-glutaminase productivity from Aspergillus versicolor Faesay4 was significantly increased by 7.72-fold (from 12.33 ± 0.47 to 95.15 ± 0.89 U/mL) by optimizing submerged fermentation parameters in Czapek’s Dox (CZD) medium including an incubation period from 3 (12.33 ± 0.47 U/mL) to 6 days (23.36 ± 0.58 U/mL), an incubation temperature from 30 °C (23.36 ± 0.49 U/mL) to 25 °C (31.08 ± 0.60 U/mL), initial pH from pH 5.0 (8.49 ± 0.21 U/mL) to pH 7.0 (32.18 ± 0.57 U/mL), replacement of glucose (30.19 ± 0.52 U/mL) by sucrose (48.97 ± 0.67 U/mL) as the carbon source at a concentration of 2.0% (w/v), increasing glutamine concentration as the nitrogen source from 1.0% (w/v, 48.54 ± 0.48 U/mL) to 1.5% (w/v, 63.01 ± 0.60 U/mL), and addition of a mixture of KH2PO4 and NaCl (0.5% w/v for both) to SZD as the metal supplementation (95.15 ± 0.89 U/mL). Faesay4 l-glutaminase was purified to yield total activity 13,160 ± 22.76 (U), specific activity 398.79 ± 9.81 (U/mg of protein), and purification fold 2.1 ± 3.18 with final enzyme recovery 57.22 ± 2.17%. The pure enzyme showed a molecular weight of 61.80 kDa, and it was stable and retained 100.0% of its activity at a temperature ranged from 10 to 40 °C and pH 7.0. In our trials, to increase the enzyme activity by optimizing the assay conditions (which were temperature 60 °C, pH 7.0, substrate glutamine, substrate concentration 1.0%, and reaction time 60 min), the enzyme activity increased by 358.8% after changing the assay temperature from 60 to 30 °C and then increased by 138% after decreasing the reaction time from 60 to 40 min. However, both pH 7.0 and glutamine as the substrate remain the best assay parameters for the l-glutaminase activity. When the glutamine in the assay as the reaction substrate was replaced by asparagine, lysine, proline, methionine, cysteine, glycine, valine, phenylalanine, l-alanine, aspartic acid, tyrosine, and serine, the enzyme lost 23.86%, 29.0%, 31.0%, 48.3%, 50.0%, 73.6%, 74.51%, 80.42%, 82.5%, 83.43%, 88.36%, and 89.78% of its activity with glutamine, respectively. Furthermore, Mn2+, K+, Na+, and Fe3+ were enzymatic activators that increased the l-glutaminase activity by 25.0%, 18.05%, 10.97%, and 8.0%, respectively. Faesay4 l-glutaminase was characterized as a serine protease enzyme as a result of complete inhibition by all serine protease inhibitors (PMSF, benzamidine, and TLCK). Purified l-glutaminase isolated from Aspergillus versicolor Faesay4 showed potent DPPH scavenging activities with IC50 = 50 μg/mL and anticancer activities against human liver (HepG-2), colon (HCT-116), breast (MCF-7), lung (A-549), and cervical (Hela) cancer cell lines with IC50 39.61, 12.8, 6.18, 11.48, and 7.25 μg/mL, respectively.
Similar content being viewed by others
References
Abu-Tahon MA, Isaac GS (2019) Purification, characterization and anticancer efficiency of L-glutaminase from Aspergillus flavus. J Gen Appl Microbiol 65:284–292
Amobonye A, Singh S, Pillai S (2019) Recent advances in microbial glutaminase production and applications-a concise review. Crit Rev Biotechnol 39(7):944–963
Diba K, Rezaie S, Mahmoudi M, Kordbacheh P, Diba CK (2007) Identification of Aspergillus species using morphological characteristics. Pak J Med Sci 23:867–872
Domsch KH, Gams W, Anderson TH (1980) Compendium of soil fungi, vol 1. Academic Press Ltd, London, pp viii + 860pp
El-Bondkly AMA (2006) Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases. Appl Biochem Biotechnol 135(2):117–132
El-Bondkly AMA (2012) Molecular identification using ITS sequences and genome shuffling to improve 2-deoxyglucose tolerance and xylanase activity of marine-derived fungus, Aspergillus sp. NRCF5. Appl Biochem Biotechnol 167:2160–2173
El-Bondkly AMA, El-Gendy MMA (2012) Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol. Antonie Van Leeuwenhoek 101:331–346
El-Gendy MMAA, Taher MT, Nageh FA, Fareed HSM (2016) Process optimization of L-glutaminase production; a tumour inhibitor from marine endophytic isolate Aspergillus sp. ALAA-2000. Int J PharmTech Res 9(8):256–267
El-Gendy MMAA, Al-Zahrani SHM, El-Bondkly AMA (2017) Construction of potent recombinant strain through intergeneric protoplast fusion in endophytic fungi for anticancerous enzymes production using rice straw. Appl Biochem Biotechnol 183:30–50
El-Gendy MMAA, Yahya SMM, Hamed AR, Soltan MM, El-Bondkly AMA (2018) Phylogenetic analysis and biological evaluation of marine endophytic fungi derived from Red Sea sponge Hyrtios erectus. Appl Biochem Biotechnol 185:755–777
El-Sayed ASA (2009) L-Glutaminase production by Trichoderma koningii under solid-state fermentation. Indian J Microbiol 49:243–250
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791
Fraga ME, Santana DMN, Gatti MJ, Direito GM, Cavaglieri LR, Rosa CAR (2008) Characterization of Aspergillus species based on fatty acid profiles. Mem Inst Oswaldo Cruz Rio de Janeiro 103(6):540–544
Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA (2007) The current status of species recognition and identification in Aspergillus. Stud Mycol 59:1–10
Hamed SR, Al-wasify RS (2016) Production and optimization of L-glutaminase from a terrestrial fungal Fusarium oxysporum. Int J PharmTech Res 9(4):233–241
Hemalatha V, Kalyani P, Chandana VK, Hemalatha KPJ (2018) Isolation and identification of L-glutaminase producing fungi from agricultural soil. Inter J Curr Adv Res 07(12):16712–16717
Hiyagh EM, Ravasan SM, Darvishi F, Mokhtarzadeh A, Baradaran B (2019) Anti-cancer effect of L-glutaminase on acute lymphoblastic leukemia (Raji), breast cancer (MCF7) and colorectal cancer (A549) cell lines. Biochem Mol biol J 5:54
Imada A, Igarasi S, Nakahama K, Isono M (1973) Asparaginase and glutaminase activities of microorganisms. J Gen Microbiol 76:85–99
Khalil MS, Moubasher MH, El-Zawahry MM, Miche MM (2020) Evaluation of antitumor activity of fungal L-glutaminase produced by Egyptian isolates. Lett Appl NanoBioSci 9(1):924–930
Klich MA (2002) Identification of common Aspergillus species. CBS, Netherlands
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
López de la Oliva AR, Campos-Sandoval JA, Gómez-García MC, Cardona C, Martín-Rufián M, Sialana FJ, Castilla L, Bae N, Lobo C, Peñalver A, García-Frutos M, Carro D, Enrique V, Paz JC, Mirmira RG, Gutiérrez A, Alonso FJ, Segura JA, Matés JM, Lubec G, Márquez J (2020) Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation. Sci Rep 10:2259
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
Masisi BK, El Ansari R, Alfarsi L, Rakha EA, Green AR, Craze ML (2020) The role of glutaminase in cancer. Histopathology 76(4):498–508
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63
Nanjundaswamy A, Okeke BC (2020) Comprehensive optimization of culture conditions for production of biomass-hydrolyzing enzymes of Trichoderma SG2 in submerged and solid-state fermentation. Appl Biochem Biotechnol 191:444–462
Nemec T, Jernec K, Cimerman A (1997) Sterols and fatty acids of different Aspergillus species. FEMS Microbiol Lett 149:201–205
Olarewaju MO, Nzelibe HC (2019) Statistical optimization of L–glutaminase production by Trichoderma species under solid state fermentation using African locust beans as substrate. Afr J Biochem Res 13(6):73–81
Raczka AM, Reynolds PA (2019) Glutaminase inhibition in renal cell carcinoma therapy. Cancer Drug Resist 2:356–364
Rani SA, Lalitha S, Pravesh BV (2011) In vitro antioxidant and anti-cancer activity of L-asparaginase from Aspergillus flavus (KUFS 20). Asian J Pharm Clin Res 4(2):174–177
Raper KB, Fennell DI (1973) The genus Aspergillus. Robert E. Krieger, New York, p 686
Ren L, Ruiz-Rodado V, Dowdy T, Huang S, Issaq SH, Beck J, Wang H, Tran Hoang C, Lita A, Larion M, LeBlanc AK (2020) Glutaminase-1 (GLS1) inhibition limits metastatic progression in osteosarcoma. Cancer Metab 8:4
Rodrigues P, Soares C, Kozakiewicz Z, Paterson RRM, Lima N (2007) Identification and characterization of Aspergillus flavus and aflatoxins. In: Méndez-Vilas A (ed) Communicating current research and educational topics and trends in applied microbiology. FORMATEX, Badajoz, pp 527–534
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425
Sajitha N, Vasuki S, Suja M (2014) Antibacterial and antioxidant activities of L-glutaminase from seaweed endophytic fungi Penicillium citrinum. World J Pharm Pharm Sci 3(4):682–695
Stahl PD, Klug MJ (1996) Characterization and differentiation of filamentous fungi based on fatty acid composition. Appl Environ Microbiol 62(11):4136–4146
Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighborjoining method. Proc Natl Acad Sci 101(30):11030–11035
Tang R, Sun-Waterhouse D, Xiong J, Cui C, Wang W (2020) Feasibility of synthesizing γ-[Glu](n≥1)-Gln using high solid concentrations and glutaminase from Bacillus amyloliquefaciens as the catalyst. Food Chem 310:125920
Unissa R, Sudhakar M, Reddy ASK, Sravanthi KN (2014) A review on biochemical and therapeutic aspects of glutaminase. Int J Pharm Sci Res (IJPSR) 5(11):4617–4634
Vo TD, Sulaiman C, Tafazoli S, Lynch B, Roberts A, Chikamatsu G (2020) Safety assessment of glutaminase from Aspergillus niger. Food Sci Nutr 8:1433–1450
Warcup J (1950) The soil-plate method for isolation of fungi from soil. Nature 166:117–118
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, New York, pp 315–322
Acknowledgements
Authors are grateful to Taif University, Saudi Arabia for funding this work through Taif University Researchers Supporting Project.
Funding
The current work was funded by Taif University Researchers Supporting Project number TURSP-2020/111, Taif University, Taif, Saudi Arabia.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there are no conflicts of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Awad, M.F., El-Shenawy, F.S., El-Gendy, M.M.A.A. et al. Purification, characterization, and anticancer and antioxidant activities of l-glutaminase from Aspergillus versicolor Faesay4. Int Microbiol 24, 169–181 (2021). https://doi.org/10.1007/s10123-020-00156-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10123-020-00156-8