Production Optimization, Partial Purification, and Biochemical Characterization of an Extracellular Lipase from...

Lipases are industrially important biocatalysts with broad applications in biotechnology and environmental processing. In this study, an extracellular lipase-producing bacterial isolate, SAB2, recovered from soil collected in Rijal Almaa, Saudi Arabia, was identified as Proteus mirabilis using phenotypic, biochemical, and 16S rRNA gene sequence evidence. Lipase production was evaluated by a one-factor-at-a-time (OFAT) approach examining temperature, initial pH, carbon and nitrogen sources, inoculum size, and incubation period. The highest temperature-series production response was 2.660 U/mL at 20°C; the highest pH-series response was 2.833 U/mL at pH 3.0, with tar oil as the best-performing tested carbon source, tryptone as the best-performing nitrogen source, a 1 mL inoculum, and 9 days of incubation. Because factor interactions were not assessed, these conditions are interpreted as best-performing OFAT levels rather than a statistically optimized global production condition. The extracellular enzyme was partially purified by 20% ammonium sulfate precipitation followed by dialysis, increasing specific activity from 6.88 ± 0.23 to 39.7 ± 0.69 U/mg, corresponding to 5.77-fold enrichment and 41.34% recovery. SDS-PAGE showed a predominant protein band with an apparent molecular mass of approximately 25 kDa. For the partially purified preparation, the highest relative activity was observed at 25°C and pH 5 under the tested assay conditions; activity remained measurable across the tested ranges, with relatively high activity in the presence of Cu²⁺ and Fe²⁺ and lower activity with Hg²⁺ and EDTA. These findings establish P. mirabilis SAB2 as a source of an extracellular lipolytic enzyme with activity under relatively mild assay conditions. However, kinetic parameters, preincubation-based thermal and pH stability, and application-specific performance remain to be established before claims of cold adaptation, acid stability, or industrial suitability can be made.