Production and Optimization of a Lipase-Producing Bacteria Enterobacter hormachei Isolated from an Oil-Contaminated Soil

Abstract

Lipase enzymes possess a wide range of industrial applications. Thus, the capacity of lipase-producing bacteria to proliferate on tributyrin agar medium was used to screen for them among various sources. The lipase producing bacteria with highest zone of clearance on the screening media were preserved on the agar slants. The preserved slants were characterized by16SrRNA gene sequencing. The nucleotide sequence so obtained by the 16SrRNA gene sequence was then put through phylogenetic analysis and homology search using the NCBI’s BLAST program. The sequence of lipase producing bacteria showed maximum resemblance with Enterobacter hormaechei bacterial strain. These bacterial strains were produced by inoculating the culture in the inoculum media and allowing it to enrich over night. Subsequently, 3% of the inoculum from the inoculum media was added to the production media, which was then incubated for 48 hours in a rotary shaker. After production the media was centrifuged and supernatant was extracted and used further for optimization, Optimization of the physiochemical parameters of the bacterial strain like inoculum pH, incubation period, inoculum size was found using one factor at a time (OFAT) approach and medium parameters like different carbon source, nitrogen source, substrate, minerals, salts (11 factors) were screened using Plackett-Burman (PB) design which is a full factorial design. The Lipase activity was found by using a titrimetric method using olive oil and Arabic gum mixture as substrate mixture. The maximum lipase activity was found for inoculum pH of 5, 48 hours of incubation time, and 5% inoculum size. The results of the PB design showed the significant parameters to be glucose (carbon source), peptone (nitrogen source), KH2PO4 (salt), and NaCl (mineral). The organism of the soil sample containing bacterial strain showed maximum lipase activity of 70 U/ml and protein concentration of 4.3 μg/mL at the optimized conditions. After centrifuging the culture media that had been improved, the supernatant was collected and partially purified using dialysis and the ammonium sulfate precipitation procedure. Following precipitation, the supernatant’s activity was measured to be 74 U/mL. After being gathered, the pallet was dialyzed in a dialysis bag and added to a buffer. Both the protein content and the lipase activity were estimated. The protein concentration determined by Lowry’s technique was found to be 6.2 μg/mL, and the lipase activity was reported to be 85.22 U/mL.