Characterization of Lipases from a Vegetable Oil Contaminated Soil Fungal Isolates

Lipases are enzymes that hydrolyse lipids to produce free fatty acids and glycerol. Fungi were cultured on sabouraud dextrose agar (SDA) plates and identified using microscopic techniques. Screening for lipase producers was carried out on SDA media supplemented with 3 % olive oil at ambient temperature. M. pusillus, M. canis, A. fumigatus, Yeast and T. mentagrophytes were found to produce lipases in different amounts with Yeast and M. canis being the highest producers, they were further characterised for this reason . Lipase production was carried out using submerged fermentation. Both Yeast and M. canis produced lipases maximally at 72 h. Optimum pH and temperature of activity for the lipases from Yeast were determined to be 5 and 35 o C respectively whereas, those from M. canis were 6 and 40 o C correspondingly. Yeast and M. canis lipases had preference for olive oil than vegetable and palm oils and both enzymes were activated by K + , Mg 2+ and Ca 2+ and inhibited by Fe 3+ , Hg 2+ and Co 2+ . The lipase enzyme from Yeast had V max of 0.0006 U/mL/Sec, K m of 0.4242 mM and K cat of 0.0004 S -1 while that from M. canis had corresponding V max , K m and K cat of 0.0001 U/mL/Sec, 3.2287 mM and 0.0001 S -1 .


Introduction
Lipases (E.C. 3.1.1.3) are biological catalysts that break down fats and oils releasing free fatty acids and glycerol. Additionally, they participate in esterification and transesterification reactions, lactonization, aminolysis [1], acidolysis [2], aminolysis, alcoholysis, hydrolysis [3], [4] and interesterification reactions in non-aqueous media [5]. These applications are however due to the potentials of lipases to catalyze both synthetic and hydrolytic processes. Worthy of note also, is their possession of distinctive properties such as, non-requirement of cofactors [2], wide spectrum of substrate specificities, stereoselectivity, chemoselectivity, regioselectivity, stability in organic solvents and their ability to catalyze varied reactions in both water soluble and non-water soluble systems [3]. Lipases of microbial origin differ based on the strain of the organism, composition of the growth medium, sources of nitrogen, temperature, carbon and pH [6], [7].
The all-round properties of lipases make them to have many potential applications in diverse aspects including, food processing, detergent, pharmaceutical, cosmetics, textile, agrochemical [6], paper, dairy, oleo-chemicals and leather industries [2]. They also find usefulness in the management of waste water, fine chemicals synthesis [6] and the production of other important bioactive compounds such as polyunsaturated fatty acids and carotenoids [8]. Fungi mostly produce lipases extracellularly thereby 20 Characterization of Lipases from a Vegetable Oil Contaminated Soil Fungal Isolates making their extraction easy. Fungi and other microorganisms that produce lipases are present in different habitats such as, crude and vegetable oil contaminated soils, decaying foods, oil seeds [2], fish intestine [6].
Soils particularly the ones contaminated with vegetable oil have been reported to contain lipolytic enzymes which often degrade the fats and oils in them [9]. Filamentous fungi of the many species of genera Mucor, Candida [10], Aspergillus, Rhizopus, penicillium and trichoderma have been reported to be the most prospective lipase producers [11] but despite this, many microorganisms having the ability to produce lipases which are able to breakdown fats and oils are yet to be isolated, identified and characterized [6]. [25] reported that only a very small number (about 2%) of microorganisms have been used as sources of enzyme in the whole wide world which have served mankind for the different products they are used to produce. Submerged fermentation is preferred to solid state fermentation because of the fact that growth conditions are easily controlled but the cost of substrates for enzyme production still remains a problem for any successful enzyme application presenting about 10 -30 % of the overall costs [1]. Therefore, in the present investigation, lipases production was carried out using submerged fermentation and olive oil as a substrate; production and characterization of lipases from two fungal strains were carried out with possible characteristics for desired applications.

Sample Collection
Soil samples were collected into sterile plastic bags from the dump site of Grand Cereals Limited Factory, Plateau State, Nigeria (known mainly for the production of Grand Pure Soya Oil) using a sterile spatula after which they were placed immediately in a flask packed with ice and brought to the laboratory. The samples were sealed in sterile containers and stored in the refrigerator at 4 o C until required for further usage.

Isolation and Identification of Lipolytic Fungal Species from Vegetable Oil Contaminated Soil
A six-fold serial dilution of the soil samples was made in sterile distilled water followed by the addition of 1 mL inoculum of 10 4 dilution on sabouraud dextrose agar (SDA) media. The fungal inoculated plates were incubated at 25 °C for 5 days. Colonies which appeared were subcultured in order to have a pure culture of each isolate and stored in SDA slants at 4 °C. Identification of the isolates was carried out by observing their growth features (such as pigmentation, texture and form) and microscopically based on the growth from their aerial hyphae and structures like conidia, spores, hyphae and sporangiophore or conidiophore.

Preliminary Screening for Lipase Producers
The fungal isolates were screened by their ability to produce lipase following incubation on SDA media supplemented separately with 3 % (v/v) olive oil and analyzing the formation of clear zones around colonies according to the method described by [7]. The abilities of the isolates to produce lipase were at that moment compared by measuring the zone of clearance. Isolates having lipolytic halo radius (R) / colony radius (r) ratio of greater than 5 mm were selected for further studies.

Production of Lipase from Selected Fungal Strains of Vegetable Oil Contaminated Soil
Lipase production was carried out in broth containing (g/500 mL): MgSO 4 . 7H 2 O (0.1), KH 2 PO 4 (0.5), K 2 HPO 4 (1.5), Na 2 SO 4 (1.0), yeast extract (2.5), peptone (2.5), glucose (1.0) and olive oil (2% v/v), pH 7.0. Productions were carried out in 50 mL production media with inoculation of a 6 mm of the fungal spores inside a 250 mL Erlenmeyer flasks under shaking speed of 150 rpm at room temperature for successively 7 days. The broth was harvested at 24 h interval and centrifuged at 4000 rpm for 40 min at 10 o C to remove cells. Total protein as well as lipase activity were determined from the supernatant.

Assay for Lipase Activity
Lipase activity was assayed spectrophotometrically at 450 nm according to the method of [12] with slight modification. To 800 µL of substrate solution (containing 3% v/v Tween 80 in 20 mM Tris-HCl with pH of 8.5 and 80 mM CaCl 2 ), 200 µL of the enzyme was added. The hydrolysis rate of Tween 80 was monitored directly by measuring the change in absorbance over a period 5 min.

Total Protein Determination
Protein concentration was measured spectrophotometrically at 590 nm as described by [13]. 1 mL of the sample was added to 5 mL of Bradford reagent, the mixture was allowed to stand for 5 min at room temperature after which absorbance was read. Total protein was determined by extrapolating from the egg albumin (EA) standard curve which was constructed using a solution containing 1 mg/mL EA.

Effect of pH
The effect of pH on the activity of lipases was studied by incubating 200 µL of the enzymes with 1M Tris-HCl buffer over a pH range of 4 to 10 and assayed for lipase activity. A plot of lipase activity against pH was used to determine the optimum pH for the lipases.

Effect of Temperature
The effect of temperature on the activity of lipases was studied by assaying from lipase activity followed by incubation at temperature range of 20 to 55 °C. A plot of lipase activity against temperature was used to determine the optimum temperature of activity for the lipases.

Effect of Different Substrates on Lipase Activity
The effect of different substrates on lipase activities was studied; 1% Tween 80 was substituted with olive oil, palm oil and groundnut oil and assayed as described above.

Effect of Different Metal Ions on Lipase Activity
The effect of different metal ions on lipase activities was studied by using K + , Co 2+ , Mg 2+ , Hg 2+ , Cu 2+ , Ca 2+ and Fe 3+ . 0.2 µL of 1mM of each metal ion already prepared in 1M Tris-HCl buffer was added to the substrate mixture and treated as described in assay for lipase activity above.

Determination of K m , V max and K cat
The kinetic constants K m and V max for lipases were determined from the Lineweaver -Burk plot following a change in substrate concentration over the range of 0.02 -1.80 v/v of Tween 80 and assayed. K cat of the lipases were as well determined.

Thermal Inactivation Studies
The thermal inactivation studies was carried out in a water bath over temperature range of 20 -90 °C using 1M Tris-HCl buffer for 30 min. Thereafter, the test tubes were allowed to cool to room temperature and assayed for lipase activity. Arrhenius plot was used to determine the energy of deactivation for the enzymes as thermal inactivation usually follows the first order kinetics [14]. The residual activity of the enzymes was also calculated in order to determine the half-life of the enzymes.

Statistical Analysis
Experiments were performed in triplicates and the means of results determined were calculated using Statistical Package for Social Science (SPSS) version 23.

Results and Discussion
Fungi are good sources of enzymes and reports from previous literatures have it that soils contaminated with oil often retain microbial diversity [15]. Out of the 9 fungi isolated from the vegetable oil contaminated soil following incubation on SDA, 5 isolates exhibited different magnitudes of zones of clearance when 3 % olive was used as a substrate (Table 2). This indicates the occurrence of degradation reaction of olive oil by extracellular enzymes, inducing the production of the lipase enzyme. From the 5 lipase producers, Yeast and Microsporum canis being the best with zones of hydrolysis of 5.63 and 5.16 mm respectively were selected for lipase production and characterization. Various microorganisms such as bacteria, fungi and yeast are recognised as potential producers of enzymes which employ these enzymes in nutrients degradation process [6]. Plate   Quantitative lipase production was carried out using submerged fermentation for a period of 7 days during which lipase activity and total protein were determined. Both Yeast and Microsporum canis produced lipases maximally at 72 h (Figures 1 and 2). A number of other studies also reported lipase production by submerged fermentation. [16] reported maximum lipase production from Candida rugosa at 50 h using olive oil as a substrate and [17] on the other hand reported maximum lipase production using olive mill wastewater from Candida cylindracea NRRLY-175 at 175 h. The time of incubation for enzyme production is usually guided by the characteristics of the culture medium and is dependent on growth rate [18].
Lipase from Yeast had an optimum pH of 5 while that from M. camis was determined to be 6 ( Figure 3). Various studies with lipases reported acidic pH values which correlate with the acidic pH values of the present study. [19] reported an optimum pH of 6.5 from A. niger and [8] reported a pH value of 5.2 for the Rh. oryzae enzyme studied. Both lipases found in the present study may be useful in the leather industry because the use of acidic active lipases has been reported in the treatment of animal skins which have been kept in pickled form [8].   [19] reported 30-35 o C as the optimum temperature for an olive oil-induced lipase from Aspergillus niger MYA 135 while [14] reported that lipase from Cellulomonas flavigena UNP3 was optimally active at 30 o C. The optimum temperature obtained for lipase from Yeast corresponds to the highest temperature as reported by [19] and that of lipase from M. canis tallies with the optimum temperature of 40 o C as reported by [20] and [21] from Aneurinibacillus aneurinilyticus strain LP-II and A. niger respectively. Both lipases from Yeast and M. canis had moderate temperature values making them possibly useful in bioprocesses operating at moderate temperatures.
Palm, groundnut and olive oils were substituted with tween 80 in order to determine their effects on lipase activities (Table 2). Generally, activities were found to be highest when olive oil was used as a substrate in this study. Palm and vegetable oils had the same activities for the lipase enzyme from Yeast while the lipase from M. canis had preference for palm oil to vegetable oil. The findings of the present study conform to those of other studies [22], [23], [24] suggesting olive oil as potent substrate for lipases.  The effect of metal ions on the activities of lipases from Yeast and M. Canis were investigated and the results are shown in Figure 5. While Cu 2+ had no effect on lipase activity from Yeast, it inhibited the activity of lipase from M. canis. K + , Mg 2+ , and Ca 2+ increased the activity of lipases from both Yeast and M. canis. [14] reported an activation of lipase activity by Mg 2+ and Ca 2+ from different studies which is in tandem to the outcomes of the present study. On the other hand, Fe 3+ , Hg 2+ and Co 2+ showed inhibitory effects for the lipases studied. Figures 6 and 7 [8] found an estimated value of 1.13 and 1.08 mM for purified lipases corresponding to Rhizomucor miechei and Rhizopus oryzae using p-nitrophenyl palmitate (pNPP) as substrate. It can therefore be concluded that lipases from Yeast has higher affinity for the substrate than the already mentioned suggesting it to be a potential biotechnological machinery.   (Figures 8 and 9). The energy of deactivation determined in the present study were lower than the value of 64.32 KJ/mol of lipase from Cellulomonas flavigena UNP3 earlier reported by [14]. The calculated half-life for lipase from Yeast was 0.23 hr whereas that from M. canis was 0.58 hr. 26 Characterization of Lipases from a Vegetable Oil Contaminated Soil Fungal Isolates

Conclusions
Yeast and M. canis fungal strains isolated from a vegetable oil contaminated soil were found to be good lipase producers. The lipases studied were optimally active at acidic pH and at moderate temperature with immense affinities for different substrates. The findings of the present study suggest lipases from these sources could find usefulness in different bioprocesses.