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Isolation and Cloning of sprA Gene Alkaline Protease Activity from Streptomyces flavogriseus ADEM7 and Characterization of Its Antioxidant and Antitumor Activities

Doaa E. El-Hadedy1, 2,, Nesreen A. Safwat3, Abir Pertila1, 3, Rafat A. Siddiqui4,

1Microbiology Department, Egyptian Atomic Energy Authority, National Center for Radiation Research and Technology, Cairo, Egypt

2Oral Health Science Department, Temple University, Kornberg School of Dentistry, Microbiome Lab, USA / Philadelphia

3The Regional Centre for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt

4Food and Nutrition Science Laboratory, Agricultural Research Station, College of Agriculture, Virginia State University, Petersburg, VA 23806

Journal of Food and Nutrition Research. 2023, 11(5), 345-352. DOI: 10.12691/jfnr-11-5-2
Received March 22, 2023; Revised April 25, 2023; Accepted May 07, 2023

Abstract

In the food industry, alkaline protease plays an important role in the formation of value-added products. Alkaline protease also plays a significant role in waste management. In this study, to construct a stable and high-yield alkaline protease producer, we investigated a combined strategy of gamma radiation as an enhancement factor and alkaline protease PGEM-T easy construct. The present study was focused on the isolation of the sprA gene-producing alkaline protease from Streptomyces flavogriseus ADEM7 (AB723783.1). Optimum growth media, fermentation conditions, and proteolytic activity assay were evaluated. The result showed that the free cells preparation produced alkaline protease with a specific activity of 550 U/mg after being exposed to gamma radiation at 10 KGy. The sprA gene was detected using polymerase chain reaction followed by cloning in PGEM-T easy vector, transformation in E. coli JM109, and direct construct sequencing. The sprA gene encoding streptogrisin a (alkaline protease-like activity) was isolated in this study and recorded in the gene bank with accession number AB827411.1. The extracellular protease enzyme was tested for antioxidant and anticancer activities. In future work, we will extract this alkaline protease from the same strain on a larger scale using a fermenter and will characterize its antioxidant activity under in vivo conditions.

Keywords:

Streptomyces flavogriseus, alkaline protease, antioxidant and anticancer, gamma radiation
[1]  Cowan D. Industrial enzyme technology, Trends Biotechnol 1996; 4: 177-178.View Article 
 
[2]  Kumar CG, Tiwari MP, Jany KD. Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties. Process Biochem 1999; 34:441-449.View Article 
 
[3]  Kumar CG, Tiwari MP, Jany KD. Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties. Process Biochem 1999; 34:441-449.View Article 
 
[4]  M. Cotârleţ, G. E. Bahrim, Turk. J. Biochem., 2011, Optimization of cold-adapted amylases and protease production by psychrotrophic Streptomyces 4 Alga using response surface methodology. 36(2), 83-92.
 
[5]  Thumar J, Singh SP. Secretion of an alkaline protease from salt-tolerant and alkaliphilic Streptomyces clavuligerus strain MIT-1, Brazilian Journal of Microbiology 2007; 38: 766-772.View Article 
 
[6]  Vonothini G, Murugan M, Sivakumar K, Sudha S. Optimization of protease production by an actinomycete Strain, PS-18A isolated from an estuarine shrimp pond. Afr J Biotechnol 2008; 7(18): 3225-3230.
 
[7]  Vishalakshi N, Lingappa K, Amina S, Prabhakar M, Dayanand A. Production of alkaline protease from Streptomyces gulbargensis and its application in removal of blood stain. Ind J Biotechnol 2009; 8: 280-285.
 
[8]  Jayasree D, Kumari TDS, Kishor PBK, Lakshmi MV, Narasu ML. Optimization of production protocol of alkaline protease by Streptomyces pulvereceus. Inter JRI Sci Technol 1 2010; (2): 79-82.
 
[9]  James PDA, Iqbal M, Edwards C, Miller PGG. Optimization of Production Protocol of Alkaline Protease by Streptomyces pulvereceus. Curr Microbial 1991; 22 377-382.View Article 
 
[10]  Tsuchida OY, Yamagata T, Ishizuka T, Arai J, Yamada M, Takeuchi, et al. An alkaline protease of an alkalophilic Bacillus Sp., Curr Microbiol 1986; 14:7-12. OY, Yamagata T, Ishizuka T, Arai J, Yamada M, Takeuchi, et al. An alkaline protease of an alkalophilic Bacillus Sp., Curr Microbiol 1986; 14: 7-12.View Article 
 
[11]  D.E.El-Hadedy, Eman W. El-Gammal, and Mortaza M. Saad Alkaline Protease Production with Immobilized Cells of Streptomyces flavogriseus (NRC) on Various Radiated Matrices by Entrapment Technique. European Journal of Biotechnology and Bioscience 2014; 2 (3): 5-16.
 
[12]  Lowry, O.H., et al. (1951) Protein measurement with folin phenol reagent. The Journal of Biological Chemistry, 193, 265-275.View Article  PubMed 
 
[13]  Engelke, G., Gutowski-Eckel, Z., Hammelmann, M., Entian, K.-D., 1992. Biosynthesis of the lantibiotic nisin; genomic organization and membrane localization of the nisB protein. Appl. Environ. Microbiol. 58, 3730-3743.View Article  PubMed 
 
[14]  Horn, N., Swindell, S., Dodd, H., Gasson, M., (1991). Nisin biosynthesis genes are encoded by a novel conjugative transposon. Mol. Gen. Genet. 228, 129-135.View Article  PubMed 
 
[15]  Sambrook, J.; Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, Vol 1, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
 
[16]  Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods; 65: 55-63.View Article  PubMed 
 
[17]  A. S. Abd-El-Aziz, E. G. El-Ghezlani, M. M. Elaasser, T. H. Afifi, R. M. Okasha (2020). First example of cationic cyclopentadienyl iron based chromene complexes and polymers: synthesis, characterization, and biological applications. Journal of Inorganic and Organometallic Polymers and Materials 30: 131-146.View Article 
 
[18]  Al Zahrani, N.A.; El-Shishtawy, R.M.; Elaasser, M.M. and Asiri, A.M. (2020): Synthesis of Novel Chalcone-Based Phenothiazine Derivatives as Antioxidant and Anticancer Agents. Molecules 2020, 25, 4566.View Article  PubMed 
 
[19]  James, M.N.G., A.R. Sielecki, G.D. Brayer, L.T.J. Delbaere, and C.-. Bauer.1980. Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 A resolution. J.Mol.Biol. 144: 43-88.View Article  PubMed 
 
[20]  Fujinaga, M., L.T.J. Delbaere, G.D. Brayer, and M.N.G. James. 1985. Refined structure of ax-lytic protease at 1.7 A resolution; analysis of hydrogen bonding and solvent structure. J.Mol.Biol. 183: 479-502.View Article  PubMed 
 
[21]  Jurasek, L., P.Johnson, R.W.Olafson,and .B.Smillie.1971.An improved fractionation system for pronase on CM- Sephadex. Can.J. Biochem. 49: 1195-1201.View Article  PubMed 
 
[22]  Vijayasurya, Gayathri Devi M.G., Tintu S.P. and Manjusha W.A. (2014). In vitro cytotoxicity and antimicrobial screening of protease enzyme isolated from marine bacteria. Ind. J.Sci. Res. and Tech. 2014 2(5): 18-22.
 
[23]  Nicholas James Gonzalez and Linda Lee Isaacs (1999). Evaluation of Pancreatic Proteolytic Enzyme Treatment of Adenocarcinoma of the Pancreas, With Nutrition and Detoxification Support. NUTRITION AND CANCER, 33(2), 117-124.View Article  PubMed 
 
[24]  Abidi, F. Aissaoui, N. Lazar, S. and Marzouki, M.N. 2014. Purification and biochemical characterization of a novel alkaline protease from Aspergillus niger. Use in Antioxidant peptides production. J. Mater. Environ. Sci. 5 (5) (2014) 1490-1499.