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Chemical surfactants find a wide range of applications in leather manufacturing practice and they are present in the wastewater at a concentration of 10–200 mg/L. The wastewater after primary, secondary biological, and tertiary treatments contains chemical surfactants at a considerable concentration (60 mg/L). In the present investigation, lipolytic micro-organisms that are capable of utilizing wetting agents (vegetable fatliquor) as the substrate were used to produce lipase. The various conditions such as time, pH, temperature, and concentration of vegetable fatliquor were optimized for the production of lipase. The lipase of an activity 345 U/mL with two different molecular weights 62 and 80 kDa was produced from Lysinibacillus sp. The predominant amino acid present in the lipase was found to be glutamic acid. The lipase was characterized using FT-IR, circular dichroism, and XRD spectroscopy. The purified lipase could be used for destabilization of tannery vegetable fatliquor (TVFL) present in t...