Biogenesis, molecular characterization and dye degradation efficacy of alkaline protease from an estuarine associated actinobacterium Streptomyces variabilis using in-silico method

In this study, biogenesis, statistical optimization and molecular modeling of alkaline protease from an estuarine associated actinobacterium Streptomyces variabilis was carried out by Box-Behnken design. Initially, the biogenesis of alkaline protease from the selected actinobacterium was attained through submerged condition. Simultaneously, the actinobacterial mediated biogenesis of alkaline protease was statistically optimized through ‘one factor at a time approach’ using Box-Behnken design in a basal medium constitutes 2.5% w/v of NaCl concentration with pH 8.0, temperature 55°C and 2.50% of inoculum size for 94h of incubation. The analysis of variance (ANOVA) exhibited a maximum level of coefficient (R ²  = 0.9720) with more significance (P < 0.0001). In purification step, the alkaline protease expressed 21.93% of recovery with 2.93 of purification fold at the last stage using Sephadex G-100 chromatography. Followed by, the molecular mass of the enzyme was calculated as 35kDa on 10% of SDS-PAGE. The three dimensional structure of purified alkaline protease was predicted with the encoded total amino acid content 481. The maximum stability range of purified alkaline protease was denoted at pH 8, temperature 60°C and the fermentation medium constituted with 1mM of Mg ²⁺ , 3.5% of NaCl and 2.5% of casein. The kinetic parameters like K _m and V _max of purified alkaline protease showed 5.158mg/ml and 484.90 ± 2.04µg/min/mg, respectively. Further, the degradation efficacy and the interaction between the alkaline protease as well as dye molecules like acridine orange and erythrosine pink were assessed by in-silico docking method using online Swiss modeling software tool. The decolouration of dyes were evaluated through first order kinetic study with the R ² values 0.9987 & 0.9953 respectively. By keeping this view, this study could be validated that the selected actinobacterium is a potent strain for the production of alkaline protease and also used as dye decoulouring agent.