Purification and Characterization of a Protease Inhibitor with Anticancer Potential from Bacillus endophyticus JUPR15

Author(s): Prerana Venkatachalam* , Varalakshmi Kilingar Nadumane .

Journal Name: Current Cancer Therapy Reviews

Volume 15 , Issue 1 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Abstract:

Introduction: Protease Inhibitors (PIs) constitute a group of proteins widely distributed among all organisms and their main function includes their ability to inhibit the proteolytic activity. PIs represent an important role in the regulation of various cellular physiological and biological processes, including cell cycle, cell death, differentiation and immune response.

Material and Methods: Hence, in our search for novel anticancer compounds, we isolated microorganisms from various environmental sources and screened them for the production of protease inhibitors. Promising isolates were further checked for their protease inhibitory activity by their ability to inhibit the activity of trypsin and chymotrypsin, which were measured spectrophotometrically.

Results: The isolate identified as Bacillus endophyticus JUPR15 was found to be promising with higher inhibitory activity than the other isolates. The inhibitor was purified by cold acetone precipitation and column chromatography and further subjected to characterization studies by performing 12 % SDS-PAGE to determine the molecular weight and gelatin-PAGE assay to confirm its inhibitory activity.

Conclusion: The isolate exhibited promising anticancer activity on in-vitro Hela and HepG2 cancer cell lines, showing its application potentials.

Keywords: Bacillus endophyticus JUPR15, anticancer, protease inhibitors, Gelatin-PAGE, MTT assay, enzyme kinetics.

[1]
Alison MR. Cancer In: Enclyclopedia of Life Sciences. Nature Publishing Group 2001; 200, pp. 1-8.
[2]
Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012; 75(3): 311-35.
[3]
Koblinski E, Ahram M, Sloane BF. Unraveling the role of proteases in cancer. Clin Chim Acta 2000; 291(2): 113-35.
[4]
Lee M, Fridman R, Mobashery S. Extracellular proteases as targets for treatment of cancer metastases. Chem Soc Rev 2004; 33(7): 401-9.
[5]
Sabotic J, Kos J. Microbial and fungal protease inhibitors-current and potential applications. Appl Microbiol Biotechnol 2012; 93(4): 1351-75.
[6]
Nakahata AM, Mayer B, Ries C, et al. The effects of a plant proteinase inhibitor from Enterolobium contortisiliquum on human tumor cell lines. Biol Chem 2011; 392(4): 327-36.
[7]
Soreide K, Janssen EA, Korner H, Baak JPA. Trypsin in colorectal cancer: molecular biological mechanisms of proliferation, invasion, and metastasis. J Pathol 2006; 209(2): 147-56.
[8]
Sharony R, Yu PJ, Park J, Galloway AC, Mignatti P, Pintucci G. Protein targets of inflammatory serine proteases and cardiovascular disease. J Inflamm (Lond) 2010; 7: 45.
[9]
Safavi F, Rostami A. Role of serine proteases in inflammation: Bowman-Birk protease Inhibitor (BBI) as a potential therapy for autoimmune diseases. Exp Mol Pathol 2012; 93(3): 428-33.
[10]
Rakashanda S, Rana F, Rafiq S, Masood A, Amin S. Role of proteases in cancer: A review. Biotechnol Mol Biol Rev 2012; 7(4): 90-101.
[11]
Gora J, Latajka R. Involvement of cysteine proteases in cancer. Curr Med Chem 2015; 22(8): 944-57.
[12]
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 1998; 62(3): 597-635.
[13]
Kumar CG, Takagi H. Microbial alkaline proteases: From a bioindustrial viewpoint. Biotechnol Adv 1999; 17(7): 561-94.
[14]
Guillen JLC, Gasca TG, Labra AB. Protease inhibitors as anticancer agents. In: Ma Del Carmen Mejia Vazquez (UNAM, Samuel Navarro) Eds. New approaches in the treatment of cancer. Hauppauge, US: Nova Science Publishers 2010; pp. 91-124.
[15]
Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992; 204(2): 433-51.
[16]
Demuth HU. Recent developments in inhibiting cysteine and serine proteases. J Enzyme Inhib 1990; 3(4): 249-78.
[17]
Delston RB, Kothary MH, Shangraw KA, Tall BD. Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii. Can J Microbiol 2003; 49(8): 525-9.
[18]
Mkaouar H, Akermi N, Mariaule V, et al. Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases. Microb Cell Fact 2016; 15(1): 201.
[19]
Vergnolle N. Protease inhibition as new therapeutic strategy for GI diseases. Gut 2016; 65(7): 1215-24.
[20]
Harish BS, Uppuluri KB. Microbial serine protease inhibitors and their therapeutic applications Int J Biol Macromol 2018; 107(Pt B): 1373-87
[21]
Borg TK. It’s the matrix! ECM, proteases, and cancer. Am J Pathol 2004; 164(4): 1141-2.
[22]
Aneja KR. Cultivation techniques for isolation and enumeration of microorganisms.In: Experiments in microbiology, plant pathology and biotechnology. 4th ed. New Delhi: New Age International Publishers 2003; pp. 154-88.
[23]
Kunitz M. Crystalline soybean trypsin inhibitor: II. General properties. J Gen Physiol 1947; 30(4): 291-310.
[24]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193(1): 265-75.
[25]
Negi S, Gupta S, Banerjee R. Extraction and purification of glucoamylase and protease produced by Aspergillus awamori in a single-stage fermentation. Food Technol Biotechnol 2011; 49(3): 310-5.
[26]
Karmen C, Mayne PD, Foo AY, Parbhoo S, Rosalki SB. Measurement of biliary alkaline phosphatase by mini-column chromatography and by electrophoresis and its application to the detection of liver metastases in patients with breast cancer. J Clin Pathol 1984; 37(2): 212-7.
[27]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-5.
[28]
Felicioli R, Garzelli B, Vaccari L, Melfi D, Balestreri E. Activity staining of protein inhibitors of proteases on gelatin-containing polyacrylamide gel electrophoresis. Anal Biochem 1997; 244(1): 176-9.
[29]
Howard DR, Herr J, Hollister R. Using trypsin & soybean trypsin inhibitor to teach principles of enzyme kinetics. Am Biol Teach 2006; 68(2): 99-104.
[30]
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63.
[31]
Aoyagi T, Takeuchi T, Matsuzaki A, et al. Leupeptins, new protease inhibitors from Actinomycetes. J Antibiot (Tokyo) 1969; 22(6): 283-6.
[32]
Umezawa H, Aoyagi T, Morishima H, et al. Pepstatin, a new pepsin inhibitor produced by Actinomycetes. J Antibiot (Tokyo) 1970; 23(5): 259-62.
[33]
Taguchi S, Kikuchi H, Suzuki M, et al. Streptomyces subtilisin inhibitor-like proteins are distributed widely in Streptomycetes. Appl Environ Microbiol 1993; 59(12): 4338-41.
[34]
Karthik L, Kumar G, Keswani T, Bhattacharyya A, Chandar SS, Rao KVB. Protease inhibitors from marine actinobacteria as a potential source for antimalarial compound. PLoS One 2014; 9(3): e90972.
[35]
McKeen CD, Reilly CC, Pusey PL. Production and partial characterization of antifungal substances antagonistic to Monilinia fructicola from Bacillus subtilis. Phytopathology 1986; 76: 136-9.
[36]
Al-Awadhi FH, Salvador LA, Law BK, Paul VJ, Luesch H. Kempopeptin C, a novel marine-derived serine protease inhibitor targeting invasive breast cancer. Mar Drugs 2017; 15(9): 290.
[37]
Bijina B, Chellappan S, Basheer SM, Elyas KK, Bahkali AH, Chandrasekaran M. Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization. Process Biochem 2011; 46(12): 2291-300.
[38]
Birk Y. Protein proteinase inhibitors in legume seeds-overview. Arch Latinoam Nutr 1994; 44(4)(Suppl. 1): 26-30.
[39]
Mello GC, Marangoni S, Oliva MLV, et al. Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds. J Protein Chem 2001; 20(8): 625-32.
[40]
Bacha AB, Jemel I, Moubayed NMS, Abdelmalek IB. Purification and characterization of a newly serine protease inhibitor from Rhamnus frangula with potential for use as therapeutic drug. Biotechnology 2017; 7(2): 148.
[41]
Kidric M, Fabian H, Brzin J, Popovic T, Pain RH. Folding, stability and secondary structure of a new cysteine dimeric proteinase inhibitor. Biochem Biophys Res Commun 2002; 297(4): 962-7.
[42]
Greenwood IA, Leblanc N, Gordienko DV, Large WA. Utilization of Avizyme 1502 in corn-soybean meal diets with and without antibiotics. Eur J Phys 2002; 443: 473-82.
[43]
Jack NL, Cate NM, Rishipal RB, Hiba AB. Inhibition of matrix metalloproteinase-I activity by the soybean Bowman–Birk inhibitor. Biotechnol Lett 2004; 26(11): 901-5.
[44]
Kuzmic P, Cregar L, Millis SZ, Goldman M. Mixed-type noncompetitive inhibition of anthrax lethal factor protease by aminoglycosides. FEBS J 2006; 273(13): 3054-62.


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 15
ISSUE: 1
Year: 2019
Page: [74 - 82]
Pages: 9
DOI: 10.2174/1573394714666180321150605
Price: $58

Article Metrics

PDF: 51
HTML: 2