Solitary Colorectal cancer (CRC) is the third most type of cancer that leading causes of morbidity and mortality worldwide. Among the recent therapeutic strategies the application of probiotics for treating cancer cell is most promising one. Bifidobacterium breve is a probiotic that can be applied in treating tumor cell (for its anaerobic nature) with least adverse effect. Prodrug/enzyme therapy is one of its mechanisms of therapeutic potentiality which contains 5FC and cytosine deaminase (CD). Generally, Bifidobacterium with transgene of E coli CD is recent choice for therapy which can be avoided by increasing its own CD activity through in silico mutagenesis. In the present work, attempts were made to predict 3D structure of CD and increasing its activity using in-silico mutagenesis. The physicochemical parameters of CD of Bifidobacterium breve found through PROTPARAM tool show that it is thermally stable and hydrophilic. After prediction of its structure through SWISS-MODEL and verification through SAVES it was found to be most reliable and stable. PrankWeb predicts 21 active sites and searching for favorable and stabilizing mutations using CUPSAT for position 151 and 89 showed various values of ΔΔG in kcal/mol among which the one with highest value was selected. Mutant proteins were modeled accordingly and each of the modeled protein was docked with 5FC and the binding energy was checked. Mutants L89R and S151I had binding energy of -5.2 kcal/mol which is less than that of wild type CD (-4.6 kcal/mol) showing better interaction with 5FC. This in silico strategy can be validated in wet lab for improving the therapeutic potentiality of Bifidobacterium breve.
