Collagenase from Marine Sediment Bacillus subtilis dfs3 Using Fish Waste Substrate: Purification and Characterization

ABSTRACT This study aimed to isolate and characterize collagenolytic bacteria from marine sediment using fish waste as a substrate. Collagenase-producing bacteria were obtained by serial dilution (up to 10−6) and spread plate methods. Among 15 isolates, one strain showed the highest collagen degradation activity, indicating strong potential for fish by-product utilization. Cultural, morphological, and 16S rRNA analyses identified the isolate as Bacillus subtilis. The 16S rRNA gene was sequenced and deposited in GenBank as Bacillus subtilis dfs3 (accession JF713459.1). Collagenase activity was confirmed by clear zones of collagen hydrolysis, and the enzyme was purified using ammonium sulfate precipitation and DEAE cellulose chromatography, achieving ~11-fold purification with an activity of 7200 units/mg. The purified enzyme had a molecular mass of 45 kDa. Optimal activity occurred at pH 8.0 and 60°C, with good stability across pH 7.5–8.5 and 20–55°C. These results demonstrate a sustainable and efficient approach for collagenase production with strong industrial potential.