Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4.

Abstract

The oceanic actinobacteria have strong potential to secrete novel enzymes with unique properties useful for biotechnological applications. The Nocardiopsis dassonvillei strain VCS-4, associated with seaweed Caulerpa scalpeliformis, was a halo-alkaline protease producer. Further investigation focuses on medium optimization and the use of agro-industrial waste for economically feasible, high-yield protease production. A total of 12 experimental runs were designed using Minitab-20 software and Placket-Burman design. Among the 7 physicochemical parameters analyzed, incubation time and gelatin were detected as significant factors responsible for higher protease production. Incubation time and gelatin were further analyzed using OVATs. Optimal protease production was achieved with 2% gelatin, 0.1% yeast extract, 0.1% bacteriological peptone, 7% NaCl, pH 8, 5% inoculum, and a 7-day incubation period, resulting in a maximum protease activity (Pmax) of 363.97 U/mL, generation time of 11.9h, specific growth rate of 0.161g/mL/h, and protease productivity (Qp) of 61.65 U/mL/h. Moreover, utilizing groundnut cake as an agro-industrial waste led to enhanced production parameters: Pmax of 408.42 U/mL, generation time of 9.74h, specific growth rate of 0.361g/mL/h, and Qp of 68.07 U/mL/h. The immobilization of crude protease was achieved using Seralite SRC 120 as a support matrix resulting in 470.38 U/g immobilization, 88.20% immobilization yield, and 28.90% recovery activity. Characterization of both crude and immobilized proteases revealed optimal activity at pH 10 and 70°C. Immobilization enhanced the shelf-life, reusability, and stability of VCS-4 protease under extreme conditions.