Papain is a plant cysteine protease of industrial importance having a two-domain structure with its catalytic cleft located at the domain interface. A structure-based rational design approach has been used to improve the thermostability of papain, without perturbing its enzymatic activity, by introducing three mutations at its interdomain region. A thermostable homologue in papain family, Ervatamin C, has been used as a template for this purpose. A single (K174R), a double (K174RV32S) and a triple (K174RV32SG36S) mutant of papain have been generated, of which the triple mutant shows maximum thermostability with the half-life (t(1/2)) extended by 94 min at 60 degrees C and 45 min at 65 degrees C compared to the wild type (WT). The temperature of maximum enzymatic activity (T(max)) and 50% maximal activity (T(50)) for the triple mutant increased by 15 and 4 degrees C, respectively. Moreover, the triple mutant exhibits a faster inactivation rate beyond T(max) which may be a desirable feature for an industrial enzyme. The values of t(1/2) and T(max) for the double mutant lie between those of the WT and the triple mutant. The single mutant however turns out to be unstable for biochemical characterization. These results have been substantiated by molecular modeling studies which also indicate highest stability for the triple mutant based on higher number of interdomain H-bonds/salt-bridges, less interdomain flexibility and lower stability free-energy compared to the WT. In silico studies also explain the unstable behavior of the single mutant.