Protease-Based Enzymatic Pretreatment Enhances Biodegradability of Dairy Wastewater Purification and SEC-HPLC Molecular Evidence

Abstract The large protein clumps in dairy effluent make it harder for microbes to break down the waste, making traditional biological treatment methods less effective. This research generated an extracellular protease from Pseudomonas aeruginosa, partially purified it, and utilized it as an enzymatic pretreatment to improve substrate accessibility for biological processes. The purification method, which included ammonium sulfate precipitation and dialysis, raised the specific activity from 1.21 to 20.26 U mg −1 . This meant that the active enzyme fraction had been made stronger. Size-exclusion high-performance liquid chromatography (SEC-HPLC) was used to evaluate molecular changes in protein-rich wastewater. Following enzymatic treatment, chromatographic profiles showed a big change from early-eluting high-molecular-weight protein fractions to late-eluting low-molecular-weight peptides. This was clear proof of proteolytic cleavage. This structural transformation significantly altered on the physical and chemical properties of organic nitrogen, but it didn’t change the amount of organic matter. Biodegradability studies showed that samples that had been treated with enzymes used more oxygen in the first stage of decomposition. The high biochemical oxygen demand (BOD) and chemical oxygen demand (COD) levels did not mean that pollution was getting worse instead, they meant that microbial oxidation was happening faster because bacteria could get to soluble substrates more easily. The BOD/COD ratio showed that the speed of biodegradation was getting better. Enzymatic hydrolysis turned slowly biodegradable particulate COD into soluble parts that break down quickly. A mechanistic link exists between protein fragmentation and improved biological treatment effectiveness when enzyme purification, molecular-scale chromatographic analysis, and biodegradation performance evaluation are utilized simultaneously. The findings demonstrate that protease-based pretreatment is a viable and energy-efficient approach for mitigating hydrolysis issues in activated sludge systems. This could be a good way to deal with dairy waste that is high in protein.