Optimization of production of detergent-compatible and thrombolytic protease by Aspergillus flavus AT grown on agrowastes using Taguchi technique cum nanoparticles supplementation

• Valorization of groundnut shell, peel and melon seed shell to produce high titers of fungal proteases • First report of use of melon seed shell as substrate to produce enzyme • Novel Taguchi optimization cum ZnONPs increased protease production by Aspergillus flavus by 4.95 fold • The protease produced in this study was detergent compatible, removed blood stain and yielded thrombolysis of 91.3% Microbial proteases are important industrial enzymes with diverse applications. However, their production is expensive due to the cost of media, thereby necessitating the search for cheap substrates for their production. In this study, novel optimization regimen was applied to increase protease production by a local strain of Aspergillus flavus by deploying the Taguchi technique with the incorporation of ZnONPs. The isolate produced protease in both SmF and SSF by utilizing groundnut peel (GP), groundnut shell (GS) and melon seed shell (MS) with titers in the range of 24.20-45.65 U/ml for SmF and 18.15-152.90 U/g for SSF, respectively. Its protease production was further optimized in the underutilized MS, which has not been reported for protease production until now. Taguchi cum nanosupplemention in SmF increased protease production from to 37.40-185.25 U/ml as the global optimum with the inclusion of ZnONPs accounting for up to 10.74% of the production. The improved production of protease was 4.95 fold compared with the unoptimized bioprocess. The protease was effective for blood stain removing and showed enhanced proteolytic activities of 254.72-329.10 U/ml corresponding to performance of 137.50-177.65% in the presence of four detergents indicating high level of compatibility. The protease also dissolved blood clot by 91.30%, thereby making it useful as thrombolytic agent. For the first time, we report the microbial production of protease using MS-based media and enhanced its production via incorporation of nanoparticles. Thus, the detergent-compatible protease produced in this study has industrial and biomedical applications.