Abstract
L-glutaminase is a hydrolytic enzyme with wide biotechnological applications. Mostly, these enzymes are employed in the feed industry for flavor enhancement and acrylamide mitigation. Also, L-glutaminase may have antiviral and antineoplastic effects making it a good choice for pharmaceutical applications. In this study, the strain Monascus ruber URM 8542 was identified through classical and molecular taxonomy using partial sequencing of β-tubulin and calmodulin genes. Subsequently, the optimal culture conditions were evaluated by submerged fermentation (L-glutamine 10 g.L− 1) for L-glutaminase excretion. The isolate was identified as M. ruber URM 8542 which showed significant extracellular enzyme production with a yield of 11.4 times in relation to the specific activity of intracellular L-glutaminase. Regarding the optimization experiments, several factors such as L-glutamine concentration, temperature, and pH were compared using a full factorial design (23). The concentrations greater than 1% proved to be significantly better for glutaminase production (R2 = 0.9077). Additionally, the L-glutaminase was optimally active at pH 7.0 and 30 ºC. The L-glutaminase was remarkably stable across an alkaline pH range (7.0–8.0) and had a thermal stability ranging from 30 ºC to 60 ºC for 1 h. Taken together, these findings suggest that the L-glutaminase produced by M. ruber is a promising candidate for pharmacological application, although further studies need to be performed. To the best of our knowledge, this is the first report of L-glutaminase production by Monascus ruber.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
Fungal strains are deposited in the URM culture collection (Micoteca URM Profa. Maria Auxiliadora Cavalcanti) located at the Federal University of Pernambuco (UFPE), Recife, Brazil.
References
Awad MF, El-Shenawy FS, El-Gendy MMAA, El-Bondkly EAM (2021) Purification, characterization, and anticancer and antioxidant activities of l-glutaminase from Aspergillus versicolor Faesay4. Int Microbiol 24(2):169–181. https://doi.org/10.1007/s10123-020-00156-8
Barbosa RN, Leong SL, Vinnere-Pettersson O, Chen AJ, Souza-Motta CM, Frisvad JC, Samson RA, Oliveira NT, Houbraken J (2017) Phylogenetic analysis of Monascus and new species from honey, pollen and nests of stingless bees. https://doi.org/10.1016/j.simyco.2017.04.001
Barbosa RN, Bezerra JDP, Santos ACS, Melo RFR, Houbraken J, Oliveira NT, Souza-Motta CM (2020) Brazilian tropical dry forest (Caatinga) in the spotlight: an overview of species of Aspergillus, Penicillium and Talaromyces (Eurotiales) and the description of P. vascosobrinhous sp. nov. Acta Botanica Brasilica 34: 409–429. https://doi.org/10.1590/0102-33062019abb0411
Binod P, Sindhu R, Madhavan A, Abraham A, Mathew AK, Beevi US, Sukumaran RK, Singh SP, Pandey A (2017) Recent developments in L-glutaminase production and applications – An overview. In Bioresource Technology (Vol. 245). https://doi.org/10.1016/j.biortech.2017.05.059
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 72(1–2):248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Bülbül D, Karakuş E (2013) Production and optimization of L-Glutaminase enzyme from hypocrea jecorina pure culture. Prep Biochem Biotechnol 43(4). https://doi.org/10.1080/10826068.2012.741641
Dhale MA, Puttananjaiah MKH, Sukumaran UK, Govindaswamy V (2011) Production of monascus purpureus pigments; influenced by amidase and acid protease activity. J Food Biochem 35(4). https://doi.org/10.1111/j.1745-4514.2010.00447.x
Dias FFG, Ruiz ALTG, Torre della A, Sato HH (2016) Purification, characterization and antiproliferative activity of l-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity. Asian Pac J Trop Biomed 6(9):785–794. https://doi.org/10.1016/j.apjtb.2016.07.007
Durthi CP, Pola M, Rajulapati SB, Kola AK, Kamal MA (2020) Versatile and Valuable utilization of amidohydrolase L-glutaminase in Pharma and Food industries: a review. Curr Drug Metab 21(1):11–24. https://doi.org/10.2174/1574884715666200116110542
Elshafei A (2014) Purification, Kinetic Properties and Antitumor Activity of L-Glutaminase from Penicillium brevicompactum NRC 829. Br Microbiol Res J 4(1). https://doi.org/10.9734/bmrj/2014/5098
Elzainy TA, Ali TH (2006) Detection of the antitumor glutaminase-asparaginase in the filamentous fungi. J Appl Sci 6(6). https://doi.org/10.3923/jas.2006.1389.1395
Griffiths RI, Whiteley AS, O’Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol 66(12). https://doi.org/10.1128/AEM.66.12.5488-5491.2000
Gulati R, Saxena RK, Gupta R (1997) A rapid plate assay for screening < scp > l -asparaginase producing micro‐organisms. Lett Appl Microbiol 24(1):23–26. https://doi.org/10.1046/j.1472-765X.1997.00331.x
Huang L, Liu Y, Sun Y, Yan Q, Jiang Z (2014) Biochemical characterization of a novel L-asparaginase with low glutaminase activity from Rhizomucor miehei and its application in food safety and leukemia treatment. Appl Environ Microbiol 80(5). https://doi.org/10.1128/AEM.03523-13
Husakova M, Plechata M, Branska B, Patakova P (2021) Effect of a Monascus sp. Red yeast Rice Extract on Germination of bacterial spores. Front Microbiol 12. https://doi.org/10.3389/fmicb.2021.686100
Imada A, Igarasi S, Nakahama K, Isono M (1973) Asparaginase and glutaminase activities of micro-organisms. J Gen Microbiol 76(1):85–99. https://doi.org/10.1099/00221287-76-1-85
Jambulingam K, Sudhakar S (2019) Purification and characterisation of a novel broad spectrum anti-tumor L-glutaminase enzyme from marine Bacillus subtilis strain JK-79. Afr J Microbiol Res 13(12):232–244. https://doi.org/10.5897/AJMR2017.8630
Jiang J, Srivastava S, Zhang J (2019) Starve cancer cells of glutamine: Break the spell or make a hungry monster? In Cancers (Vol. 11, Issue 6). https://doi.org/10.3390/cancers11060804
Juncheng Hu, Wang T, Xu J, Wu S, Wang L, Su H, Jiang J, Yue M, Wang J, Wang D, Li P, Zhou F, Liu Yu, Qing G, Hudan Liu (2020) WEE1 inhibition induces glutamine addiction in T-cell acute lymphoblastic leukemia. Haematologica 106(7):1816–1827. https://doi.org/10.3324/haematol.2019.231126
Kim G, Joo H, Jung J-W (2021) Recent advances in development of glutaminase inhibitors as Anticancer Agents. Yakhak Hoeji 65(5):344–348. https://doi.org/10.17480/psk.2021.65.5.344
Koli SH, Suryawanshi RK, Mohite Bv, Patil Sv (2019) Prospective of Monascus pigments as an additive to Commercial Sunscreens. Nat Prod Commun 14(12). https://doi.org/10.1177/1934578X19894095
Krebs HA (1935) Metabolism of amino-acids. Biochem J 29(8). https://doi.org/10.1042/bj0291951
Krishnan N, Muthusamy P (2011) Statistical optimization of key process variables for enhanced Lglutaminaseproduction by Penicillium brevicompactum Characterization and application of Pterin Deaminase View project Pharmacological screening of Medicinal plants View project. https://www.researchgate.net/publication/230806004
Li Y, Chen S, Zhu J, Zheng C, Wu M, Xue L, He G, Fu S, Deng X (2022) Lovastatin enhances chemosensitivity of paclitaxel-resistant prostate cancer cells through inhibition of CYP2C8. Biochemical and Biophysical Research Communications, 589. https://doi.org/10.1016/j.bbrc.2021.12.007
Liu L, Zhao J, Huang Y, Xin Q, Wang Z (2018) Diversifying of chemical structure of native Monascus pigments. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.03143
Loucif H, Dagenais-Lussier X, Avizonis D, Choinière L, Beji C, Cassin L, Routy J-P, Fritz JH, Olagnier D, van Grevenynghe J (2021) Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells. Autophagy 1–18. https://doi.org/10.1080/15548627.2021.1972403
Lücking R, Aime MC, Robbertse B, Miller AN, Ariyawansa HA, Aoki T, Cardinali G, Crous PW, Druzhinina IS, Geiser DM, Hawksworth DL, Hyde KD, Irinyi L, Jeewon R, Johnston PR, Kirk PM, Malosso E, May TW, Meyer W, …, Schoch CL (2020) Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding? IMA Fungus 11(1). https://doi.org/10.1186/s43008-020-00033-z
Mary S, Khalil MH, Moubasher, Mokhtar M, El-Zawahry, Marina M, Michel (2020) Evaluation of antitumor activity of fungal L-glutaminase produced by egyptian isolates. Lett Appl Nanobiosci 9(1):924–930. https://doi.org/10.33263/LIANBS91.924930
Masisi BK, Ansari E, Alfarsi R, Rakha L, Green EA, A. R., Craze ML (2020) The role of glutaminase in cancer. Histopathology 76(4):498–508. https://doi.org/10.1111/his.14014
Massidon WP, Maddison DR (2019) Mesquite: A modular system for evolutionary analysis. Version 3.6. Http://Www.Mesquiteproject.Org
Matés JM, Campos-Sandoval JA, Márquez J (2018) Glutaminase isoenzymes in the metabolic therapy of cancer. In Biochimica et Biophysica Acta - Reviews on Cancer (Vol. 1870, Issue 2). https://doi.org/10.1016/j.bbcan.2018.07.007
Miller MA, Pfeiffer W, Schwartz T (2012) The CIPRES science gateway: Enabling high-impact science for phylogenetics researchers with limited resources. ACM International Conference Proceeding Series. https://doi.org/10.1145/2335755.2335836
Moriguchi I, Hirano H, Nakagome I (1994) Comparison of reliability of log P values for drugs calculated by several methods. Chem Pharm Bull 42(4). https://doi.org/10.1248/cpb.42.976
Mosallatpour S, Aminzadeh S, Shamsara M, Hajihosseini R (2019) Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization. Sci Rep 9(1):19062. https://doi.org/10.1038/s41598-019-55587-9
Nguyen TL, Nokin M, Terés S, Tomé M, Bodineau C, Galmar O, Pasquet J, Rousseau B, Liempd S, Falcon-Perez JM, Richard E, Muzotte E, Rezvani H, Priault M, Bouchecareilh M, Redonnet‐Vernhet I, Calvo J, Uzan B, Pflumio F, …, Durán RV (2021) Downregulation of glutamine synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1‐driven acute lymphoblastic leukemia. Mol Oncol 15(5). https://doi.org/10.1002/1878-0261.12877
Orabi H, El-Fakharany E, Abdelkhalek E, Sidkey N (2020) Production, optimization, purification, characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate. Prep Biochem Biotechnol 50(4):408–418. https://doi.org/10.1080/10826068.2019.1703193
Patakova P (2013) Monascus secondary metabolites: production and biological activity. J Industrial Microbiol Biotechnol (Vol 40. https://doi.org/10.1007/s10295-012-1216-8
Patro KR (2012) Extraction, purification and characterization of L-asparaginase from Penicillium sp. by submerged fermentation. Int J Biotechnol Mol Biology Res 3(3). https://doi.org/10.5897/IJBMBR11.066
Patrovsky M, Sinovska K, Branska B, Patakova P (2019) Effect of initial pH, different nitrogen sources, and cultivation time on the production of yellow or orange Monascus purpureus pigments and the mycotoxin citrinin. Food Sci Nutr 7(11):3494–3500. https://doi.org/10.1002/fsn3.1197
Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25(7). https://doi.org/10.1093/molbev/msn083
Ramadan MEDA, Asmar F el, Greenberg DM (1964) Purification and properties of glutaminase and asparaginase from a pseudomonad. I. Purification and physical chemical properties. Archives of Biochemistry and Biophysics, 108(1). https://doi.org/10.1016/0003-9861(64)90365-0
Rambaut A, Lam TT, Carvalho LM, Pybus OG (n.d.). Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen). https://doi.org/10.1093/ve/vew007
Roberts E, Frankel S (1950) γ-Aminobutyric acid in brain: its formation from glutamic acid. J Biol Chem 187(1):55–63
Rocha WRV, Costa-Silva TA, Agamez‐Montalvo GS, Feitosa VA, Machado SEF, Lima S, Pessoa‐ GM, A., Alves HS (2019) Screening and optimizing fermentation production of < scp > l ‐asparaginase by aspergillus terreus strain S‐18 isolated from the brazilian Caatinga Biome. J Appl Microbiol 126(5):1426–1437. https://doi.org/10.1111/jam.14221
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) Mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3). https://doi.org/10.1093/sysbio/sys029
Sakhaei M, Alemzadeh I (2017) Enzymatic synthesis of theanine in the presence of L-glutaminase produced by Trichoderma koningii. Appl Food Biotechnol 4(2). https://doi.org/10.22037/afb.v4i2.15713
Santos AC, dos, Land MGP, Silva NP da, Santos KO, Lima-Dellamora E (2017) da C. Reactions related to asparaginase infusion in a 10-year retrospective cohort. Revista Brasileira de Hematologia e Hemoterapia, 39(4). https://doi.org/10.1016/j.bjhh.2017.08.002
Schulze E, Bosshard E (1883) Ueber das Glutamin. Ber Dtsch Chem Ges 16(1):312–315. https://doi.org/10.1002/cber.18830160173
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9). https://doi.org/10.1093/bioinformatics/btu033
Sun Y, Gan Y, Zhang L, Shi Y, Yue T, Yuan Y (2022) Isolation and identification of Monascus and evaluation of its selenium accumulation. LWT, 154. https://doi.org/10.1016/j.lwt.2021.112887
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12). https://doi.org/10.1093/molbev/mst197
Tan H, Xing Z, Chen G, Tian X, Wu Z (2018) Evaluating antitumor and antioxidant activities of yellow monascus pigments from monascus ruber fermentation. Molecules 23(12). https://doi.org/10.3390/molecules23123242
Tork SE, Aly MM, Elsemin O (2018) A new l-glutaminase from Streptomyces pratensis NRC 10: gene identification, enzyme purification, and characterization. Int J Biol Macromol 113:550–557. https://doi.org/10.1016/j.ijbiomac.2018.02.080
Upadhyaya S, Tiwari S, Arora N, Singh DP (2016) Microbial protein: a Valuable Component for Future Food Security. Microbes and Environmental Management, January
van Tieghem M, Ph (1884) Monascus, Genre Nouveau De L’Ordre Des Ascomycétes. Bull de La Société Botanique de France 31(5):226–231. https://doi.org/10.1080/00378941.1884.10828230
Wu HC, Chen JJ, Wu M, Cheng MJ, Chang HS (2020) Identification of new pigments produced by the fermented rice of the fungus Monascus pilosus and their anti-inflammatory activity. Phytochemistry Letters, 40. https://doi.org/10.1016/j.phytol.2020.04.014
Xiong Z, Cao X, Wen Q, Chen Z, Cheng Z, Huang X, Zhang Y, Long C, Zhang Y, Huang Z (2019) An overview of the bioactivity of monacolin K / lovastatin. Food and Chemical Toxicology, 131. https://doi.org/10.1016/j.fct.2019.110585
Yang P, Huang K, Zhang Y, Li S, Cao H, Song H, Zhang Y, Guan X (2022) Biotransformation of quinoa phenolic compounds with Monascus anka to enhance the antioxidant capacity and digestive enzyme inhibitory activity. Food Bioscience, 46. https://doi.org/10.1016/j.fbio.2022.101568
Yano T, Ito M, Tomita K, Kumagai H (1988) Purification and properties of glutaminase from aspergillus oryzae. J Ferment Technol 66(2). https://doi.org/10.1016/0385-6380(88)90039-8
Yongxia Z, Jian X, Suyuan H, Aixin N, Lihong Z (2020) Isolation and characterization of ergosterol from Monascus anka for anti-lipid peroxidation properties. J de Mycol Medicale 30(4). https://doi.org/10.1016/j.mycmed.2020.101038
Yoon S-M, Pyo Y-H (2022) The in vitro antioxidant and antidiabetic activity of various fractions from Monascus-Fermented Ginger Extract. J Korean Soc Food Sci Nutr 51(2). https://doi.org/10.3746/jkfn.2022.51.2.125
Acknowledgements
We would like to thank “Micoteca URM” Federal University de Pernambuco, Department of Mycology of the Federal University of Pernambuco for the support for the identification of the microorganism and Microorganism Biology Laboratory (BIOMICRO) of the Federal University of Paraiba for providing equipment and assistance in the enzymatic characterization analyses.
Funding
This study received financial support from the Fundação de Amparo a Ciência e Tecnologia do Estado de Pernambuco – FACEPE (FACEPE-IBPG-1034-2.12/17; APQ-0350-2.12/19), Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Processo: 310298/2018-0) and Financiadora de Estudos e Projetos – FINEP (nº 01.19.0171.00).
Author information
Authors and Affiliations
Contributions
Sarah S. do Nascimento: methodology, formal analysis and investigation, writing (preparation of the original draft); Emanuella M. da Conceição: classic morphological identification; Renan do N. Barbosa: molecular identification ande writing-review; Wellma de Oliveira Silva: writing-review; Leonor A. de O. da Silva: methodology, formal analysis and investigation, supervision; Cristina M. de Souza-Motta and Neiva T. de Oliveira: conceptualization, funding acquisition, supervision. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
do Nascimento, S.S., Barbosa, R.d.N., de Oliveira Silva, W. et al. Optimization of L-glutaminase production by Monascus ruber URM 8542 isolated from ice cream industrial effluent. World J Microbiol Biotechnol 39, 288 (2023). https://doi.org/10.1007/s11274-023-03733-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-023-03733-x