Screening and Characterization of Lipolytic Bacteria

Screening of lipolytic bacteria. Lipase producing microbial cultures were isolated from wastewater of an oil processing plant located in Tehran, and enriched by periodic subculturing of samples in Nutrient Broth (NB) media containing 20% (v/v) and 40% (v/v) wastewater in successive. The composition of NB medium is (per liter) 5g peptone and 3g yeast extract. The pH of the medium was adjusted to 7 with 0.1M NaOH.The isolation process was performed by serial dilution of samples on tributyrin agar plates. The composition of tributyrin agar medium is (per liter) 5 g peptone, 3 g yeast extract, 10ml tributyrin and 15 g agar. Culture plates were incubated at 30°C. Colonies showing clear zones around them were picked out, purified on tributyrin agar plates and transferred to agar slants (6 (link)). Isolates having clearing zone were grown in the liquid culture and the level of lipase production was determined from the cell free culture supernatant fluid. Characterization and identification of the isolate with higher lipolytic activity was carried out both biochemically and by 16s r RNA sequencing.
Enzyme production. The composition of production medium used in this study was: (%w/v) pepton 0.2; NH₄H₂PO4 0.1; NaCl 0.25; MgSO₄ 7H₂O 0.04; CaCl₂.2H₂O 0.04; olive oil 2.0 (v/v); pH 7.0; 1–2 drops Tween 80 as emulsifier. Overnight cultures were suspended in 5ml of sterile deionised water and used as the inoculum for pre culture to obtain an initial cell density to adjust the turbidity of 0.5 McFarland standard. Submerged microbial cultures were incubated in 500 ml Erlenmeyer flasks containing 100 ml of liquid medium on a rotary shaker (150 rpm) and incubated at 30°C. After 24 hours of incubation, the culture was centrifuged at 10,000 rpm for 20 min at 4°C and the cell free culture supernatant fluid was used as the sources of extracellular enzyme.
Assay of lipase activity. Lipase activity was deter-mined spectrophotometrically at 30°C using p-nitrophenol palmitate (pNPP) as substrate. The reaction mixture was composed of 700 µl pNPP solution and 300 µl of lipase solution. The pNPP solution was prepared by adding the solution A (0.001 g pNPP in 1ml isopropanal) into solution B (0.01 g gum arabic, 0.02 g Sodium deoxycholate, 50 µl Triton X-100 and 9 ml of 50 mM Tris-HCl buffer, pH 8) with stirring until all was dissolved. Then the absorbance measured at 410 nm for the first 2 min of reaction. One unit (1U) was defined as that amount of enzyme that liberated 1µmol of pNPP per minute (ɛ:1500l/mol cm) under the test conditions (7 (link)).
Effects of culture variable on lipase production. To investigate lipase production, olive oil was replaced by other carbon sources such as glucose and tributyrin. Each of substances (1% w/v) was used as sole carbon source. The effect of nitrogen sources on the lipase production was analyzed by supplementing production medium with different nitrogen sources (0.2% w/v) like peptone, yeast extract, ammonium dihydrogen phosphate and enzyme activity was assayed. Investigation of effect of different carbon and nitrogen source on lipase activity of Pseudomonas aeruginosa KM110 was done at pH: 7.0, 30°C and 150 rpm throughout 24 h of cultivations.
Effect of pH and temperature on lipase activity and stability. The crude enzyme used for assay was the culture broth after separation of cells and particles. The enzyme was normally stored at 4°C until used. The optimal temperature for activity was determined at different temperatures (30–70°C), at pH 8.0 for 10 min. For determination of temperature stability, the reaction mixtures containing the enzyme in 50mM Tris–HCl buffer (pH 8.0) was incubated at different temperatures (37, 45, 50, 55, 65 and 70°C) for 3 h and immediately cooled. Residual enzyme activity was measured under standard enzyme test conditions. Optimal pH was determined at 30°C in buffer solutions of pH values ranging from 5 to 11 (0.05 M citrate-phosphate pH 5-7; 0.05 M Tris–HCl pH 8-9; 0; 0.05 M Glysin – NaOH pH 11). The effect of pH on enzyme stability was analyzed by the spectrophotometric assay after pre-incubation of 300 µl of enzyme solution for 1 h at 30°C, in 700 µl of the above mentioned buffer solutions (pH 5–11). Enzymatic activity was measured according to a standard protocol with pNPP as the substrate.
Effects of different ions & detergents on lipase activity. As reported from studies on other microbial lipases, a concentration as low as 1 mM of some metal ions can affect the enzyme activity. Thus, the effect of several ions (Fe2+,,Na+, Ni+, Li+, Co2+, K+, Zn2+, Hg2+, Cu2+, Mn2+, Ca2+, Mg2+) on this P. aeruginosa lipase was determined. The enzyme solution was stored for 1 h at 30°C in the presence of 1 mM of various ions (as chloride salts) prior to the colorimetric assay for remaining lipase activity. In the case of chemical detergents, activity remaining was determined after 1 h of storage of enzyme solution at 30°C in the presence of various chemical detergents (SDS, DMSO (dimethyl sulfoxide), Tween 80 and Triton X-100) at 1% concentration. Activity was measured by the spectrophotometric assay after incubation time. Remaining enzymatic activity was determined by a standard method with pNPP. Final enzyme activity was calculated relative to control activity (a parallel enzyme reaction without additions).
Taxonomic characterization of isolated bacteria. The isolate was identified via 16S rRNA sequences. Genomic DNA of Pseudomonas aeruginosa KM110 was extracted from bacterial colonies by set buffer method. The 16S rRNA gene from the genomic DNA was amplified by PCR using the following forward and reverse primers of 16S rRNA, f (5′-AGAGTTTGATCMTGGCTCAG-3′) and r (5′- TACGGYTACCTTGTTACGAC-3′).PCRwas performed in a Thermocycler (TECHNE) using a Taq polymerase (Cinnagen, Iran). The PCR program comprised initial denaturation at 96°C for 4 min, followed by 35 cycles each of 94°C for 1 min, 61°C for 30 s, 72°C for 50 s; 72°C for 4 min; and incubation at 4°C for 10 min. PCR products were purified with DNA extraction kit (Bioneer South Korea). Both strands of the PCR product were sequenced by dideoxy chain termination method. The 16S rRNA gene sequence of the KM110 was compared with those in the NCBI/EZtaxon/ Ribosomal Database Project (RPD)/ EMBL nucleotide sequence databases by using the BLAST (blastn) program http://www.ncbi.nlm.nih.gov/BLATS/), and all of the sequences were aligned using the Clustal W program (8 (link)). A phylogenetic tree and neighbor-joining phylogeny were constructed by using the MEGA soft ware package version 4.0 (9 (link)) and bootstrapping was used to estimate the reliability of the phylogenetic reconstructions (1,000 replicates).