• Venkata Krishna Bayineni
  • Ravi Kumar Kadeppagari Nano Biosciences, Centre for Emerging Technologies, Jain University, Jain Global, Campus, Jakkasandra, Kanakapura Main Road, Ramanagara Dist., Karnataka, India, 562112


Objective: To improve the parent strain of Variovorax paradoxus for the production of methotrexate (MTX) degrading enzyme and to study the serum stability, release kinetics and functionality of the nano complexes of the enzyme with human serum albumin (HSA)

Methods: The activity of the enzyme was quantified by using the extinction coefficient of 8300 for the substrate, MTX. The mutant strain of V. paradoxus was isolated by exposing the cells to the UV light (302 nm) so that 50 % of the cells were killed. The enzyme was purified on DEAE-cellulose, and sephadex-G-100 columns and the purity of the enzyme was checked on 10 % SDS-polyacrylamide gel. The enzyme-HSA nano complexes were prepared by adopting desolvation-crosslinking method and their size was determined by using transmission electron microscope.

Results: MTX degrading enzymes are required to avoid the toxicity of the MTX during the treatment of cancer. The enzyme from V. paradoxus converts the MTX into non-toxic glutamate and 4-amino-N [10]-methylpteroate, and the culture utilizes the derived glutamate. Improving the production of this enzyme will be beneficial due to its therapeutic application. Different carbon and nitrogen sources didn’t improve the production of this enzyme from the parent strain of M. verrucaria. The strain improvement was carried out by using UV radiation to improve the yields of the enzyme. The mutant strain produced around 6 times higher levels of the enzyme compared to the parent suggesting its advantage for the industrial production of the enzyme. Since this enzyme is of microbial origin, it was complexed to the safe carrier, HSA and these complexes showed their size in the nano-range. The nano complexes showed longer stability compared to the native enzyme in the serum, and the enzyme was readily released from the complex suggesting the protective role of the carrier, HSA. The nano complexes showed the higher degradation of MTX in the serum compared to the native enzyme suggesting their better functionality compared to the native enzyme.

Conclusion: Usage of mutant strain will be advantageous for the industrial production of the enzyme since it produces higher levels of enzyme compared to the parent strain. Enzyme-HSA nanocomputers will be a better choice for the therapeutic applications since they show better serum stability and functionality compared to the native enzyme.

Keywords: Methotrexate, Variovorax paradoxus, Glutamate, 4-amino-N[10]-methylpteroate, Strain improvement, UV radiation, Human serum albumin, Nanocomplexes, Native enzyme


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1. Sherwood RF, Melton RG, Alwan SM, Hughes P. Purification and properties of carboxypeptidase G, from Pseudomonas sp. strain RS-16; Use of a novel triazine dye affinity method. Eur J Biochem 1985;148:447-53.
2. Levy CC, Goldman P. The enzymatic hydrolysis of methotrexate and folic acid. J Biol Chem 1967;242:2933-8.
3. Goldman P, Levy CC. Carboxypeptidase g: purification and properties. Proc Nat Acad Sci USA 1967;58:1299-306.
4. McCullough JL, Chabner BA, Bertino JR. Purification and properties of Carboxypeptidase G1. J Biol Chem 1971;246:7207-13.
5. Albrecht AM, Boldizar E, Hutchinson DJ. Carboxypeptidase is displaying differential velocity in the hydrolysis of methotrexate, 5-methyltetrahydrofolic acid and leucovorin. J Bacteriol 1978;134:506-13.
6. DeAngelis LM, Tong WP, Lin S, Fleisher M, Bertino JR. Carboxypeptidase G2 rescue after high-dose methotrexate. J Clin Oncol 1996;14:2145-9.
7. Buchen S, Ngampolo D, Melton RG, Hasan C, Zoubek A, Henze G, et al. Carboxypeptidase G2 rescue in patients with methotrexate intoxication and renal failure. Br J Cancer 2005;92:480–7.
8. Estrada LH, Chu S, Champion JA. Protein nanoparticles for intracellular delivery of therapeutic enzymes. J Pharm Sci 2014;103:1863–71.
9. Ravi-Kumar K, Venkatesh KS, Umesh-Kumar S. Evidence that cleavage of the precursor enzyme by autocatalysis caused secretion of multiple amylases by A. niger. FEBS Lett 2004;557:239–42.
10. Suresh C, Dubey AK, Srikanta S, Umesh-Kumar S, Karanth NG. Characterisation of a starch-hydrolysing enzyme of A. niger. Appl Microbiol Biotechnol 1999;51:673–5.
11. Yun YH, Jiang H, Chan R, Chen W. Sustained release of PEG-g-chitosan complexed DNA from poly (lactide-co-glycolide). J Biomater Sci Polym Ed 2005;16:1359-78.
12. Guliyeva U, Oner F, Ozsoy S, Haziroglu R. Chitosan microparticles containing plasmid DNA as potential oral gene delivery system. Eur J Pharm Biopharm 2006;62:17-25.
13. Ahn CH, Chae SY, Bae YH, Kim SW. Synthesis of biodegradable multi block co-polymers of poly (L-lysine) and poly (ethylene glycol) as a non-viral gene carrier. J Controlled Release 2004;97:567-74.
14. Omidi Y, Hollins AJ, Drayton RM, Akhtar S. Polypropylenimine dendrimer induced gene expression changes: the effect of complexation with DNA, dendrimer generation and cell type. J Drug Target 2005;13:431-43.
15. Koynova R, Mac Donald RC. Lipid transfer between cationic vesicles and lipid-DNA lipoplexes: effect of serum. Biochem Biophys Acta 2005;1714:63-70.
16. Heyes J, Palmer L, Bremner K, MacLachlan I. Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids. J Controlled Release 2005;107:276-87.
17. Ramesh R, Ito I, Saito Y, Wu Z, Mhashikar AM, Wilson DR, et al. Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery. DNA Cell Biol 2004;23:850-7.
18. Prabha S, Labhasetwar V. Nanoparticle-mediated wild-type p53 gene delivery results in sustained antiproliferative activity in breast cancer cells. Mol Pharm 2004;1:211-9.
19. Kragh-Hansen U. Structure and ligand binding properties of human serum albumin. Dan Med Bull 1990;37:57-84.
20. Kragh-Hansen U, Chuang VT, Otagiri M. Practical aspects of the ligand binding and enzymatic properties of human serum albumin. Biol Pharm Bull 2002;25:695-704.
21. Mo Y, Barnett ME, Takemoto D, Davidson H, Kompella UB. Human serum albumin nanoparticles for efficient delivery of Cu, Zn superoxide dismutase gene. Mol Vision 2007;13:746-57.
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How to Cite
Bayineni, V. K., and R. K. Kadeppagari. “PRODUCTION OF THERAPEUTIC METHOTREXATE DEGRADING ENZYME AND STUDIES ON ITS NANOCOMPLEXES WITH HUMAN SERUM ALBUMIN”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 8, no. 3, Mar. 2016, pp. 41-44,
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