121
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Print ISSN: 2278 - 2648 Online ISSN: 2278 - 2656
International Journal of Research in Pharmacology and Pharmacotherapeutics (Research article)
EVALUATION OF THE FUNGAL MICROORGANISMS FOR PERIPLASMIC AMIDOHYDROLASE *1
R.D.Shalima Vardhini, 2Prasad Ekke
*1
Department of Biochemistry, St. Mary’s College, Yousufguda, Hyderabad-500045, Andhra Pradesh, India. 2 Department of Biochemistry, Celesta Research Lab, Hyderabad, 500038, A.P. India.
_________________________________________________________________________ ABSTRACT The periplasmic amidohydrolase was found to have a host of medical applications, especially against Acute Lymphoblastic Leukemia (ALL). The application of this enzyme was also seen in the food processing industries by reducing the formation of acrylamide, a cancer agent. The present study evaluates a direct one step method of screening the fungal L-Asparaginase producers using the modified Czapex Dox medium.
KEY WORDS: Periplasmic Amidohydrolase, Antitumor, fungal screening method, L-Asparaginase. ______________________________________________________________________________________________
INTRODUCTION L-Asparaginase (L-Asparagine amidohydrolase E.C.3.5.1.1)1 constitutes one of the most biomedically important groups of therapeutic enzymes that catalyzes the hydrolysis of LAsparagine into Aspartic acid and Ammonia 2. The need of anti leukemia and anti lymphoma agents is far greater than any other therapeutic enzymes of which L-Asparaginase contributes one third 3. The enzyme L-Asparaginase gained importance for its remarkable properties in pharmaceutical and commercial industries. Different types of LAsparginases were screened and isolated from different microbial species and were studied for their different applications, particularly as therapeutic agents in treatment of certain types of human cancers, in particular, with the lymphoblastic leukemia 4,5. _________________________________ * Corresponding author: R.D.Shailima Vardhini,
Head, Department of Biochemistry, St. Mary’s College, Yousufguda, Hyderabad - 500045, Andhra Pradesh, India. Email address: shailima.rampogu@gmail.com
L-Asparaginase is used as an immunosuppressive agent as it was observed in some immunological responses 6. Some of the L-Asparaginase preparations showed remarkable anti tumor properties. Guinea pig serum showed to inhibit certain spontaneous and radiation induced leukemia in mice as reported by Broome 7. Kidd was the first to report this in 1953 8. L-Asparaginase is also known as Kidrolase, Elspar, Crasnitin, Leunase, and Colaspase 9 by its trade name.L- Asparagine is a nutritional requirement of both normal cells and cancer cells10. Lymphatic cells require the large amount Asparagines to keep up with their rapid, malignant growth for which they use both Asparagine from the diet as well as what they can make themselves, to a very limited extent, to satisfy their large asparagine demand. Normal cells do not require
122 R.D.Shalima Vardhini et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-1(2) 2012 [121-125]
more Asparagine to survive as they can make the required asparagine internally by the enzyme asparagine synthetases. Unlike the normal cells, the lymphatic cells die rapidly if the dietary asparagine is cut off as these cells cannot synthesize enough to meet their abundant demand as the enzyme asparagine synthetases is present at low levels 11. The drug acts by catalysis or degrades the asparagine into aspartic acid and ammonia. The aspartic acid thus formed is converted to fumaric acid in the urea cycle. This can also be converted into one of the intermediate products of the TCA cycle, the oxaloacetic acid. L-asparaginase hence is very useful in maintaining the levels of amino acids and nitrogen balance within the cells12. The important enzyme L-Asparaginase is not only used as therapeutic drug/agent but also as a food processing agent13. It is used to prevent the formation of acrylic amide, which is a carcinogen present in starchy food items. Asparagine, present in starchy food items like snacks and biscuits will convert into acrylic amides upon baking [JECFA] above 120 o C by Millard’s reaction. On adding LAsparaginase to food items before baking, asparagine is converted into another amino acid, aspartic acid and ammonia leading to the depletion of asparagines resulting in the reduced formation of acrylic amide in food items 14. L-Asparaginase isolated from two bacterial sources (E.Coli and
Erwinia Caratovara) are widely used for the treatment of acute lymphoblastic leukemia 15, 16. Many microorganisms, fungi and yeast had proven to be the best producers of L-Asparaginase. There are only a few studies on L-Asparaginase production by fungi 17. It was observed that eukaryotic microorganisms like yeast and fungi had a potential for L-Asparaginase production 18, 19. The enzyme is routinely screened using Nessler’s reagent 20. As this procedure is lengthy and time consuming, there is still a need to develop a rapid procedure. This obstacle was overcome by Gulati’s rapid plate assay method 21. It was generally observed that L-Asparaginase production was accompanied by an increase in pH of the culture filters 22. This was devised by incorporating a pH indicator, phenol red in medium containing asparagine as the sole nitrogen source. Phenol red at acidic pH is yellow and at alkaline pH turned pink around the L-Asparaginase producers. By keeping this as a standard, the aim of the present experiment is to screen the fungal microorganisms, by a fast, single, and one step method using basic nutrients (modified Czapex Dox media) and to observe the results within 18 hours. The mechanism of the enzyme23 action can be explained by the following figure.
MECHANISM OF THE ENZYME ACTION
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123 R.D.Shalima Vardhini et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-1(2) 2012 [121-125]
MATERIALS AND METHODS Five fungi (F1, F2, F3, F4, F5) used for the study were isolated from soil samples. The dilution platemethod was employed for the isolation of fungal strains 24. The media used is the modified Czapex Dox media which consist of minimum salts and the pH is adjusted to 6.8. 50ml of each of the medium was taken in three different petri plates. The dye was prepared by using 2% of the phenol red dye stock in ethanol with the pH was adjusted to 7.0 with standard 1 mol-1 NaOH. The stock ranged from 0.05ml to 0. 5ml. Different
concentration of the dye i.e. 0.001%, 0.01% and 0.1% were added to 50ml of the media taken in 3 different petri dishes. One control plate devoid of the phenol red dye was also maintained to evaluate the simple screening method to identify the fungi capable of producing L-Asparaginase. After standardization, the plates were incubated at 37o C overnight.
Table: 1 Media Composition S.No
Chemicals
Concentration g/lit
1
Glycerol
6
2
L-Asparagine
4
3
KCl
1
4
MgSo4
2
5
KH2Po4
3
6
Agar
20
Results After overnight incubation the plates were analyzed for pink zone formation against the
Fig-1: Control
control. The five fungal organisms showed different dimensions of pink zone corresponding to different concentration of the dye.
Fig-2: Pink Zone www.ijrpp.com
124 R.D.Shalima Vardhini et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-1(2) 2012 [121-125]
Table- 2: Diameter of pink zone. S.No
Phenol Red Concentration Diameter of Pink Zone in Cm F1
F2
0.86
0.68 0.90
0.3
0.45
2.
0.01%
1.03
0.83 0.96
0.69
0.80
3.
0.1%
1.62
0.98 1.09
0.92
0.98
7.
8. 9. 10.
Acknowledgements I thank the Management and the Principle St. Mary’s College, Yousufguda, Hyderabad for their continuous support and encouragement. I also would like to thank Mr. K. Onesmies Elbert and Celesta Research Lab, Hyderabad.
References
3.
4.
5.
6.
F5
0.001%
In the present study, we could successfully evaluate a method for screening the fungal organisms that produce L-Asparaginase in much less time and using minimum nutrients. This process hence would serve as a direct screening method for fungal L-Asparaginase producers.
2.
F4
1.
Discussions
1.
F3
Siddalingeshwar et al. J. advanced scientific Research, 2010, 1(1); 55-66. Jha et al., IJPSR, 2012; Vol. 3(9): 30763090 Warangkar, S.C et al C.N Journal of Sell and Tissue Research Vol.9 (3) 1963-1968 (2009). Freedman M.J.Agrie Food chem., 2003 Chemistry, biochemistry, and safety of acryl amide. Kristina Kuburava Molecular Nutrition and Food Research Volume53, Issue 12 1532-1589, December 2009. Ali, S.S et al (1994), Fungal Asparginases with potential antitumor activity, Ind.J.Mic roluol. 34,73-76.
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11.
12. 13.
14.
15.
16.
17.
Broome, J.D (1961) Evidence that the LL-Asparaginase activity of guinea pig serum is responsible for its antilymphoma effects. Nature 191, 1114-1115. Kidd, J.G., J Exp.Med.98,565 (1953) Geckil, H, et al., Appl. Microbial. Biotechnol.63 (2005) 691. Mc Credie KB, et al L-Asparaginase for treatment of cancer-Cancer J.Clin 1973; 23:220-227. Nakamura Ct, et al Pancreatitis and perotitis following therapy with L-LAsparaginase.Int.pediatrics 1999; 14: 2527. Mi-Kyung Yun, et al J.Health. J.Mol Biol. 2007 June8; 369(3):794-811. Freedman M.J.Agrie Food chem., 2003 Chemistry, biochemistry, and safety acryl amide. Kristina kukkrova Molecular Nutrition and food Research [Volume 53 issue 12] pages 1532-1539, December 2009. Joseph Roberts, et al. The Anti tumor activity of E.coli- Asparaginase. [Cancer Research 26, 2213 -2217. October] Keating M.J.Holmes, et al (1993) LAsparaginase and PEG L-Asparaginase past, present and future. Leuk.Lympmoma.10, 153-157. Lapmark K, et al. L-Asparaginase production by Bipolarize species. BR 438 isolated from brown rice in Thailand, Chinag Mai J.Sci 2010; 37:160-164.
125 R.D.Shalima Vardhini et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-1(2) 2012 [121-125]
18. Wade HE, et al .J.Gen.Microbial, 1971; 69:299-312. 19. Pinheiro, et al. Biomaterial and Diagnostic BD06, 2001;245-244. 20. Imade. A et al (1973) L-Asparaginase and Glutaminase activities of Mo.Journal of general Microbiology 76, 85-99. 21. R.Gulati et al, A rapid plate assay for screening L-Asparaginase producing microorganism, Lettero in Applied Microbiology 1997, 24, 23-26. 22. De Jong. (1972) L-Asparaginase production by Streptomyces griseus, Applied Microbiology 23, 1163-1164. 23. Hill J.Roberts et al; L-Asparaginase therapy of leukemia and other malignant neoplanm.1967 JAMA; 202:882. 24. Palaniswamy M ,et al. Isolation, identification and screening of potential xylanolytic enzyme for litter degrading fungi, Afri J Biotech 2008; 7: 1978-1982.
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