Fifty Soil samples were collected from Khartoum University farm in Shambat (Rhizosphere depth 10 cm of guar, Abusabein and sun flower fields), the soil was alkaline; pH 8.5 and was salty clay. In addition 15 samples were collected from effluent oil at the industrial area Omdurman. Each sample was placed into sterilized plastic package and transferred to laboratory and they kept at room temperature.

Isolation of Bacillus was carried out according to [9]. 10 g from each sample were taken and dissolved in 90 ml distilled water. Soil suspension was heated at 80˚C for 15 minutes. The five dilutions of soil and effluent oil were made (10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵). And 6 plates of nutrient agar for each sample were inoculated (by the spread plate method) with 0.1 ml of heated dilutions (10⁻³, 10⁻⁴, 10⁻⁵) and incubated aerobically in 37˚C for 24 hours. Then colonies were purified and prepared for the further work.

Identification of pure culture of each Bacillus isolates was carried out according to [10] [11].

DNA extraction from Bacillus isolates was carried out using a CTAB based protocol described by [12]. Classification of the isolates at the molecular level was carried out using RAPD-PCR technique according to [13]. By three Primer, (1) OPL3 (5-CCA GCA GCT T-3), Primer (2) OPL8 (5-AGC AGG TGG A-3), and Primer (3) OPQ1 (5-GGG ACG ATG G-3).

Typical Bacillus isolates was screened for producing lipase enzymes. Using chromogenic plate’s assay, which was prepared as followed by [14]. using phenol red (0.1%) along with 1% lipidic substrate olive oil, 10 mM CaCl₂ and 2% agar pH is adjusted to 7.3 - 7.4 using 0.1 N NaOH. Then, the plates were incubated with young culture 24 h from the different isolates then incubation for 7 days at 37˚C. After that the plates should be examined for change in color from pink to yellow.

The enzyme was produced from the purified isolates that showed a positive reaction on olive oil/phenol red plates test. The tested strains were cultured at 37˚C in 100 ml Erlenmeyer flasks containing 50 ml of, the broth of production media contained peptone 0.2%, NH₄H₂PO₄ 0.1%, NaCl 0.25%, MgSO₄∙7H₂O 0.04%, CaCl₂∙2H₂O 0.04%, Olive oil 2 ml and Tween-20 2 to 3 drops. One loop full of bacterial culture was inoculated in each flask and incubated at 37˚C. The pH was adjusted at pH 7. Lipase was extracted according to [15], from the production medium after incubation period (24, 48, 72 and 96 h) by centrifugation at 6000 rpm for 10 min.

Lipase activity was assayed according to [16], with slightly modification, using p-nitro phenyl palmitate (p-NPP) as substrate. Enzyme solution (50 µl) was added to 950 µl of the substrate solution consisting of one part solution A (3.0 mM pNPP in 2-propanol) and nine parts solution B (100 mM potassium phosphate buffer pH 7.0, 0.4% Triton X-100 and 0.1% gum arabic), which was freshly prepared before use. The reaction mixture was incubated at 37˚C for 20 min and absorbance was read at 410 nm. Lipase activity and specific activities was calculated as the following formula:

Lipaseactivity ( U / ml ) = A ∗ B C ∗ D ∗ E (1)

where,

A—µmol of p-Nitro phenol released;

B—Total volume;

C—Volume used in spectrophotometric determination;

D—Volume of enzyme used in assay;

E—Time of incubation.

Michaelis-Menten constant for lipase substrate pNPP was determined according to [18]. With some modification pNPP instead pNPL (p-nitropheny laurate) and concentration by incubating with concentrations of substrate ranging from 0.05 to 5.0 mmol/l with lipase from three have chosen isolates. The value of constant was calculated according to the Hanes-Woolf plot.

AII isolate subjected to chromogenic plates assay; isolates were showed a positive result to the test by convert the color of medium from red to yellow indicate that lipase was produced by different Bacillus strains. The free fatty acids liberated by the lipolysis organisms lower the pH resulting in the change of color indicating the formation of lipase [19]. Therefore the isolates were found to be a lipase producer, capable of utilizing the substrate (olive oil) to produce yellow color zone. This result agreed with [20] [21], they proved that the use of phenol red in lipase assay was highly reproducible with sensitivity level as low as 0.5 p-nitrophenyl palmitate (p-NPP) enzyme units within 15 min. Therefore, by using olive oil as the lipidic substrates in phenol red agar, the presence of lipolysis activity could be indicated by the yellow coloration. This assay is based on the principle where free fatty acids were released from the bacterial lipolysis reaction. Lipase from Bacillus isolates was produced in liquid production medium. As a more accurate, quantitative method to support chromogenic plates assay for use in screening for Bacillus producing lipase. The lipase activities of isolates ranged between 3.4 U/ml (isolate O3) to 63.4 U/ml isolate G14 (Table 1). Among the tested isolates, three isolates B10, O1 and G14 showed high potential in Lipase production and had an average lipase activity of 28.3, 41.2 and 63.4 Unit/ml, respectively.

The lipase kinetics from Bacillus isolates O1, B10 and G14 were measured at the pH and temperature optima of the respective lipase in 0.01 M potassium phosphate buffer and in different pNPP concentration as substrate (0.05, 0.5, 1.0, 2.5 and 5 mM). The V_max and K_m values of lipase from isolates G14, OI and B10 were 17.6, 135 and 24.4 µmole/min and 1.3, 1.6 and 0.681 mM, respectively. [28] found that K_m and V_max values of the lipase purified from Bacillus Stearothermophilus HU1 of 0.235 mM and 161.2 mmol/min/mL, respectively, as the reported by [29] K_m and V_max values, were 105.26 mmol and 0.116 mmol∙min⁻¹∙g⁻¹, respectively from Bacillus sp. ITP-001 using olive oil as the substrate.