Characterization of a New L-Glutaminase Produced by Achromobacter xylosoxidans RSHG1, Isolated from an Expired Hydrolyzed L-Glutamine Sample
Abstract
:1. Introduction
2. Results
2.1. Screening and Isolation of Bacterial Isolates
2.2. Taxonomic Characterization of Bacterial Isolate
2.2.1. Cultural and Microscopic Characteristics
2.2.2. Biochemical Testing
2.2.3. Identification of the Bacterial Isolate
2.2.4. Phylogenetic Analysis
2.3. Microbial Production of L-Glutaminase
Optimization of Parameters for Glutaminase Production
- 1.
- Effect of incubation time
- 2.
- Effect of pH
- 3.
- Effect of Temperature
- 4.
- Effect of Carbon source
- 5.
- Effect of Nitrogen source
- 6.
- Effect of Inducers
2.4. Purification and Characterization of L-Glutaminase
2.4.1. Purification
2.4.2. Characterization
3. Discussion
4. Materials and Methods
4.1. Isolation and Screening of Bacteria
4.2. L-Glutaminase Assay
4.3. Protein Estimation
4.4. Taxonomic Characterization
Molecular Characterization
4.5. Optimization of Culture Conditions for L-Glutaminase Production
4.6. L-Glutaminase Purification
4.7. Characterization of L-Glutaminase by Achromobacter xylosoxidans RSHG1
4.8. Immobilization on Agar
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Alcántara, A.R. Biocatalysis and Pharmaceuticals: A Smart Tool for Sustainable Development. Catalysts 2019, 9, 792. [Google Scholar] [CrossRef] [Green Version]
- Abdallah, N.A.; Amer, S.K.; Habeeb, M.K. Screening of L-glutaminase produced by actinomycetes isolated from different soils in Egypt. Int. J. ChemTech Res. 2012, 4, 1451–1460. [Google Scholar]
- Nikodinovic-Runic, J.; Topakas, E. Special Issue on Environmental Biocatalysis. Catalysts 2020, 10, 490. [Google Scholar] [CrossRef]
- Ardawi, M.S.M.; Newsholme, E.A. Glutamine metabolism in lymphocytes of the rat. Biochem. J. 1983, 212, 835–842. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bülbül, D.; Karakuş, E. Production and optimization of L-glutaminase enzyme from Hypocrea jecorina pure culture. Prep. Biochem. Biotechnol. 2013, 43, 385–397. [Google Scholar] [CrossRef]
- Amobonye, A.; Singh, S.; Pillai, S. Recent advances in microbial glutaminase production and applications—a concise review. Crit. Rev. Biotechnol. 2019, 39, 944–963. [Google Scholar] [CrossRef]
- Yano, T.; Ito, M.; Tomita, K.; Kumagai, H. Purification and properties of glutaminase from Aspergillus oryzae. J. Ferment. Technol. 1988, 66, 137–143. [Google Scholar] [CrossRef]
- Singh, P.; Banik, R.M. Biochemical characterization and antitumor study of L-glutaminase from Bacillus cereus MTCC 1305. Appl. Biochem. Biotechnol. 2013, 171, 522–531. [Google Scholar] [CrossRef]
- Divya Teja, D.; Sri Devi, V.; Harsha, H.; Vishala, S.S.; Lakshmi, K.S. Production of L-glutaminase from marine ecosystems and optimal conditions for maximal production by actinomycetes. Int. J. Adv. Res. 2014, 2, 485–491. [Google Scholar]
- Yang, Y.Z.; Ding, S.; Wang, Y.; Li, C.L.; Shen, Y.; Meeley, R.; McCarty, D.R.; Tan, B.C. Small kernel2 Encodes a Glutaminase in Vitamin B6 Biosynthesis Essential for Maize Seed Development. Plant Physiol. 2017, 174, 1127–1138. [Google Scholar] [CrossRef]
- Maharem, T.M.; Emam, M.A.; Said, Y.A. Purification and characterization of L-glutaminase enzyme from camel liver: Enzymatic anticancer property. Int. J. Biol. Macromol. 2020, 150, 1213–1222. [Google Scholar] [CrossRef]
- Anbu, P.; Gopinath, S.C.B.; Chaulagain, B.P.; Tang, T.-H.; Citartan, M. Microbial enzymes and their applications in industries and medicine 2014. Biomed. Res. Int. 2015, 2015, 816419. [Google Scholar] [CrossRef]
- Anbu, P.; Gopinath, S.C.B.; Chaulagain, B.P.; Lakshmipriya, T. Microbial Enzymes and Their Applications in Industries and Medicine 2016. Biomed. Res. Int. 2017, 2017, 2195808. [Google Scholar] [CrossRef] [PubMed]
- Hmidet, N.; Nawani, N.; Ghorbel, S. Recent Development in Production and Biotechnological Application of Microbial Enzymes. Biomed. Res. Int. 2015, 2015, 280518. [Google Scholar] [CrossRef]
- Sabu, A.; Keerthi, T.R.; Kumar, S.R. Chandrasekaran M Glutaminase production by marine Beauveria sp. under solid state fermentation. Process. Biochem. 2000, 35, 705–710. [Google Scholar] [CrossRef]
- Orabi, H.; El-Fakharany, E.; Abdelkhalek, E.; Sidke, N. Production, optimization, purification, characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate. Prep. Biochem. Biotechnol. 2020, 50, 408–418. [Google Scholar] [CrossRef] [PubMed]
- Dube, M.; Schafer, C.; Neidhart, S.; Carle, C. Texturisation and modification of vegetable proteins for food applications using microbial transglutaminase. Eur. Food Res. Technol. 2021, 225, 287–299. [Google Scholar] [CrossRef]
- Bhandari, S.; Poudel, D.K.; Marahatha, R.; Dawadi, S.; Khadayat, K.; Phuyal, S.; Shrestha, S.; Gaire, S.; Basnet, K.; Khadka, U.; et al. Microbial Enzymes Used in Bioremediation. J. Chem. 2021, 2021, 8849512. [Google Scholar] [CrossRef]
- Viniegra-Gonzalez, G.; Favela-Torres, E.; Aguilar, C.N.; De Jesus Rómero-Gomez, S.; Dıaz-Godınez, G.; Augur, C. Advantages of fungal enzyme production in solid-state over liquid fermentation systems. Biochem. Eng. J. 2003, 13, 157–167. [Google Scholar] [CrossRef]
- Vo, T.D.; Sulaiman, C.; Tafazoli, S.; Lynch, B.; Roberts, A.; Chikamatsu, G. Safety assessment of glutaminase from Aspergillus niger. Food Sci. Nutr. 2020, 8, 1433–1450. [Google Scholar] [CrossRef] [PubMed]
- Awad, H.M.; El-Deen, A.M.N.; El-Sayed, E.M.; Hassabo, A.A. Biochemical studies and biological activities on L-glutaminase from rhizosphere soil Streptomyces rochei SAH2_CWMSG. Egypt. Pharm. J. 2019, 18, 27–41. [Google Scholar] [CrossRef]
- Bagriyanik, D.B.; Karakuş, E. Glutaminase Enzyme Biosensor for Determination of Glutamine in Cerebrospinal Fluid, Human Serum and L-Glutamine Capsule. J. Chem. Soc. Pak. 2014, 36, 728–735. [Google Scholar]
- Kumar, S.R.; Chandrasekaran, M. Continuous production of L-glutaminase by an immobilized marine Pseudomonas sp. BTMS-51 in a packed bed reactor. Process. Biochem. 2003, 38, 1431–1436. [Google Scholar] [CrossRef]
- Desai, S.S.; Chopra, S.J.; Hungund, B.S. Production, purification and characterization of L-Glutaminase from Streptomyces sp. isolated from soil. J. Appl. Pharm. Sci. 2016, 6, 100–105. [Google Scholar] [CrossRef] [Green Version]
- Klimberg, V.; McClellan, J. Glutamine, cancer, and its therapy. Am. J. Surg. 1996, 172, 418–424. [Google Scholar] [CrossRef]
- Wise, D.R.; Thompson, C.B. Glutamine addiction: A new therapeutic target in cancer. Trends Biochem. Sci. 2010, 35, 427–433. [Google Scholar] [CrossRef] [Green Version]
- Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406–425. [Google Scholar] [CrossRef]
- Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985, 39, 783–791. [Google Scholar] [CrossRef]
- Tamura, K.; Nei, M.; Kumar, S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 2004, 101, 11030–11035. [Google Scholar] [CrossRef] [Green Version]
- Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
- Irajie, C.; Mohkam, M.; Nezafat, N.; Hosseinzadeh, S.; Aminlari, M.; Ghasemi, Y. In silico analysis of Glutaminase from Different Species of Escherichia and Bacillus. Iran. J. Med Sci. 2016, 41, 406–414. [Google Scholar]
- Karim, G.F.; Thalij, K.M. Determination of L-Glutaminase Activity by Some Bacterial Species. Int. J. Curr. Microbiol. Appl. Sci. 2016, 5, 218–225. [Google Scholar] [CrossRef]
- Aly, M.M.; Kadi, R.H.; Alkhatib, M.H.; Wali, A.N. Production of the antitumor l-glutaminase enzyme from thermotolerant Streptomyces sp. D214, under submerged fermentation conditions. J. Exp. Biol. Agric. Sci. 2017, 5, 878–885. [Google Scholar] [CrossRef]
- Emelda, J. Isolation and Screening of L-asparaginase and L-glutaminase Producing Bacteria and their Antimicrobial Potential from Environmental Sources. J. Pharm. Biol. Sci. 2016, 11, 47–53. [Google Scholar] [CrossRef]
- Katikala, P.k.; Bobbarala, V.; Tadimalla, P.; Guntuku, G.S. Screening of L-glutaminase producing marine bacterial cultures for extracellular production of L-glutaminase. Int. J. ChemTech Res. 2009, 1, 1232–1235. [Google Scholar]
- Dubey, R.; Paul, A.; Prity, N. Isolation, production and screening of anticancer enzyme L-Glutaminase from Bacillus Subtilis. Int. J. Pharm. Biol. Sci. 2015, 5, 96–105. [Google Scholar]
- Kiruthika, J.; Murugesan, S. Studies on optimization of L-glutaminase production under submerged fermentation from marine Bacillus subtilis JK-79. Afr. J. Microbiol. Res. 2020, 14, 16–24. [Google Scholar] [CrossRef] [Green Version]
- Pandian, S.R.K.; Deepak, V.; Sivasubramaniam, V.S.; Nellaiah, H.; Sundar, K. Optimization and purification of anticancer enzyme L-glutaminase from Alcaligenes faecalis KLU102. Biologia 2014, 69, 1644–1651. [Google Scholar] [CrossRef]
- Blazek, R.; Benbough, J.E. Improvement in the persistence of microbial asparaginase and glutaminase in the circulation of the rat by chemical modifications. Biochim. Biophys. Acta 1981, 677, 220–224. [Google Scholar] [CrossRef]
- Zhang, X.; Xu, Z.; Liu, S.; Qian, K.; Xu, M.; Yang, T.; Xu, J.; Rao, Z. Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168. Molecules 2019, 24, 592. [Google Scholar] [CrossRef] [Green Version]
- Jyothi, H.; Kumar, S.; Vandana, R. Production of L-Glutaminase by Pseudomonas VJ-6. Res. J. Biotechnol. 2011, 6, 42–49. [Google Scholar]
- Nagaraju, K.; Raghu Ram, M. Studies on optimization of l-glutaminase from Bacillus endophyticus RN-7. Int. J. Pharm. Biol. Sci. 2018, 8, 1146–1153. [Google Scholar]
- Kiruthika, J.S.; Saraswathy, N. Selective isolation and molecular identification of L-glutaminase producing bacteria from marine sediments. Res. J. Biotechnol. 2013, 8, 64–69. [Google Scholar]
- Kiruthika, J.S.; Saraswathy, N.; Murugesan, S. Maximizing L-glutaminase production from marine Bacillus subtilis JK-79 under solid state fermentation. Afr. J. Biotechnol. 2018, 17, 288–305. [Google Scholar] [CrossRef] [Green Version]
- Al-Zahrani, N.; Al-Qahtani, S.; Al-Sehri, W. Microbial genetics studies on L-glutaminase producer Psuedomonas NS16 isolated from eye contact lenses. J. Am. Sci. 2020, 16, 34–39. [Google Scholar] [CrossRef]
- Kiruthika, J.; Nachimuthu, S. Isolation and characterization of a novel L-glutaminase producing marine Bacillus subtilis strain JK-79. Asian J. Microbiol. Biotechnol. Environ. Sci. 2014, 16, 601–610. [Google Scholar]
- Sinha, S.; Nigam, V.K. Production and characterization of L-glutaminase by Bacillus sp. 1620. Int. J. Pharm. Sci. Res. 2016, 7, 1620–1626. [Google Scholar] [CrossRef]
- Abdallah, N.A.; Amer, S.K.; Habeeb, M.K. Production, purification and characterization of L-glutaminase enzyme from Streptomyces avermitilis. Afr. J. Microbiol. Res. 2013, 7, 1184–1190. [Google Scholar] [CrossRef]
- Mostafa, Y.S.; Alamri, S.A.; Alfaifi, M.Y.; Alrumman, S.A.; Elbehairi, S.; Taha, T.H.; Hashem, M. L-Glutaminase Synthesis by Marine Halomonas meridiana Isolated from the Red Sea and Its Efficiency against Colorectal Cancer Cell Lines. Molecules 2021, 26, 1963. [Google Scholar] [CrossRef]
- Ahmed, M.M.A.; Taha, T.M.; Abo-Dahab, N.; Hassan, F.S. Process Optimization of L-Glutaminase Production; a Tumour Inhibitor from Marine Endophytic Isolate Aspergillus sp. ALAA-2000. J. Microb. Biochem. Technol. 2016, 8, 382–389. [Google Scholar] [CrossRef] [Green Version]
- Abdelhameed, A.; Alqaysi, R.; Hilal, H.A.; Alharbi, N. Purification of L-Glutaminase from Bacillus sp. B12 and study its properties. Indian J. Forensic Med. Toxicol. 2020, 14, 1160–1166. [Google Scholar] [CrossRef]
- Saravanan, D.; Bharathi, S.; Manikkam, R.; Rama, B.; Tamil, N. Production and optimization of L-glutaminase from Vibrio sp. M9 isolated from Mahabalipuram marine sediments. World J. Pharm. Res. 2014, 3, 2117–2126. [Google Scholar]
- Reda, F.M. Kinetic properties of Streptomyces canarius L-Glutaminase and its anticancer efficiency. Braz. J. Microbiol. 2015, 46, 957–968. [Google Scholar] [CrossRef] [Green Version]
- Chasanah, E.; Tambunan, U.; Yulianti, T. Screening and characterization of L-glutaminase produced by bacteria isolated from sangihe talaud sea. Squalen Bull. Mar. Fish. Postharvest Biotechnol. 2013, 7, 115–122. [Google Scholar] [CrossRef] [Green Version]
- Ye, M.; Liu, X.; Zhao, L. Production of a Novel Salt-tolerant L-glutaminase from Bacillus amyloliquefaciens Using agro-industrial residues and its application in Chinese soy Sauce fermentation. Biotechnology 2013, 12, 25–35. [Google Scholar] [CrossRef] [Green Version]
- Karahan, M.; Karakuş, E.; Bülbül, D.; Atacı, N. Immobilization of glutaminase enzyme from Hypocria jecorina on polyacrylic acid: Preparation and biochemical characterization. Artif. Cells Nanomed. Biotechnol. 2014, 42, 262–267. [Google Scholar] [CrossRef] [PubMed]
- Imada, A.; Igarasi, S.; Nakahama, K.; Isono, M. Asparaginase and glutaminase activities of microorganism. J. Gen. Microbiol. 1973, 76, 85–99. [Google Scholar] [CrossRef] [Green Version]
- Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. [Google Scholar] [CrossRef]
- MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria, 3rd ed.; Williams and Wilkins: Baltimore, MD, USA; Philadelphia, PN, USA, 2000. [Google Scholar]
- Bergey, D.H.; Krieg, N.R.; Holt, J.G. Bergey’s Manual of Systematic Bacteriology; Williams & Wilkins: Baltimore, MD, USA, 1984. [Google Scholar]
- Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. [Google Scholar] [CrossRef]
Bacterial Isolate | Zone of Hydrolysis (mm) | Agar Diffusion Assay (mm) |
---|---|---|
Achromobacter xylosoxidans RSHG1 | 26 | 30 |
Ser # | Biochemical Test | Result |
---|---|---|
1 | Oxidase | + |
2 | Catalase | + |
3 | Mannitol Salt Agar Test | + |
4 | Simmon Citrate | + |
5 | Indole Production | + |
6 | MacConkey Agar | + |
7 | Glucose Fermentation Test | − |
8 | Arginine Hydrolase Test | + |
9 | Urease Test | + |
10 | Esculin Ferric Citrate Test | + |
11 | Gelatin Hydrolysis Test | + |
12 | β-Galactosidase Test | − |
13 | D-Glucose Assimilation Test | − |
14 | L-Arabinose Assimilation Test | − |
15 | D-Mannose Assimilation Tes | − |
16 | N-acetyl-glucosamine Assimilation Test | − |
17 | D-Maltose Assimilation Test | − |
19 | Potassium Gluconate Assimilation Test | − |
20 | Capric Acid Assimilation Test | − |
21 | Potassium Gluconate Assimilation Test | − |
22 | Capric Acid Assimilation Test | − |
23 | Adipic Acid Assimilation Test | − |
24 | Malic Acid Assimilation Test | − |
25 | Trisodium Citrate Assimilation Test | − |
26 | Phenylacetic Acid Assimilation Test | − |
Strain | Size (bp) | Sequence Homology with | Accession Number on NCBI | Identified As |
---|---|---|---|---|
RSHG1 | 759 | Achromobacter xylosoxidans | MZ868956 | Achromobacter xylosoxidans RSHG1 |
Method | Total Protein (mg) | Specific Activity (U/mg) | Purification (Fold) | Total Activity (IU) | Yield (%) |
---|---|---|---|---|---|
Crude extract | 75 | 0.52 | 1 | 23,400 | 100 |
Ammonium sulphate (70%) | 35 | 2.1 | 4.1 | 1500 | 6.4 |
Gel filtration chromatography | 1.04 | 35 | 16.6 | 874 | 3.73 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Saleem, R.; Ahmed, S. Characterization of a New L-Glutaminase Produced by Achromobacter xylosoxidans RSHG1, Isolated from an Expired Hydrolyzed L-Glutamine Sample. Catalysts 2021, 11, 1262. https://doi.org/10.3390/catal11111262
Saleem R, Ahmed S. Characterization of a New L-Glutaminase Produced by Achromobacter xylosoxidans RSHG1, Isolated from an Expired Hydrolyzed L-Glutamine Sample. Catalysts. 2021; 11(11):1262. https://doi.org/10.3390/catal11111262
Chicago/Turabian StyleSaleem, Rabia, and Safia Ahmed. 2021. "Characterization of a New L-Glutaminase Produced by Achromobacter xylosoxidans RSHG1, Isolated from an Expired Hydrolyzed L-Glutamine Sample" Catalysts 11, no. 11: 1262. https://doi.org/10.3390/catal11111262