PRODUCTION OF THERAPEUTIC METHOTREXATE DEGRADING ENZYME AND STUDIES ON ITS NANOCOMPLEXES WITH HUMAN SERUM ALBUMIN
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 -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 didnâ€™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-methylpteroate, Strain improvement, UV radiation, Human serum albumin, Nanocomplexes, Native enzyme
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