Abstract-- Triacylglycerol lipase was produced from indigenous Aspergillus niger and Bacillus sp. using submerged fermentation. The production process was optimized and lipase was then purified and immobilized. The optimization process improved enzyme production by many folds. Maximum lipase activity was obtained; with microorganisms cultured in medium containing 1% olive oil and 1% soybean meal as substrate, 72hrs incubation at 30°C, pH 6.0 and medium supplementation with 2% glucose, 1% peptone for A. niger, 72hrs incubation at 37°C, pH 8.0 and medium supplementation with 1% glucose, 1.5% peptone for Bacillus sp. The enzymes were purified using ammonium sulphate precipitation and ion exchange chromatography to achieve 13-14 fold purity with a yield ranging from 53-57%. Lewatite and Amberlite were found to supports of choice for the immoblization of bacterial and fungal lipases, respectively. The immobilized lipase is a promising tool for industrial applications in terms of its retention of enzymatic activity and reusability.
Keywords-- Lipolytic; Aspergillus; Bacillus; Fermentation; Enzyme immobilization.
I. INTRODUCTION
Lipases (E.C 3.1.1.3, triacyl glycerol acylhydrolase) are hydrolytic enzymes that catalyze the hydrolysis of esters (Scheib et al., 1999). On hydrolysis, they convert triglycerides to di, mono-glycerides, fatty acid and glycerols. Lipases are versatile biocatalysts as in non-aqueous environment they catalyze various reactions like esterification, transesterification and interesterification (Abbas et al., 2002). They exhibit unique physicochemical property of catalyzing reactions at lipid-water interface and belong to the group of globular proteins having molecular weight of 20-60 KDa (Geraldine et al., 2008). Serine, histidine and aspartic acids are present at the active site. These residues coordinate to catalyze reactions. Lipases are specific to the substrates. On the basis of this specificity, they are classified into three groups which are fatty acid specific, region specific and non-specific (Imandi et al., 2010).
Lipases are abundantly present in different species of microbes, plants and animals. The energy reserved tissues of the plants have their significant amount (Sharma et al., 2001) such as Maize, castor and rape seeds. Animals can also serve as sources of lipases as they have considerable amounts of lipases in their pancreas. However, pancreatic lipases cannot be used commercially as the extract contains high concentration of trypsin, animal viruses, different hormones and other undesirable components which can bring unfavorable changes in the desired product (Vakhlu and Kour, 2006). Therefore, on industrial scale, microbes are preferred for production of lipases. Among microbes, filamentous fungi are the main producers of extracellular lipases. Different species of yeasts, fungi and bacteria are capable of producing lipases. They include Aspergillus, Mucor, Penicillium, Pseudomonas and Bacillus, etc. (Bisht et al., 2012; Huang et al., 2012)
The lipolytic microbes are present in various habitats e.g. soil contaminated with oil, factories which are involved in the processing of vegetable oils and industrial waste etc. Microbial lipases are produced by solid state and submerged fermentation. However, they are produced mainly by submerged fermentation as high enzyme productivity is achieved in this type of fermentation. The production process is affected by three main factors i.e. type of fermentation, design of the bioreactor and composition of media (Rodriguez et al., 2006). Moreover, it is also affected by parameters of fermentation. Various environmental factors also play an important role in the production of lipases which include temperature, pH, agitation and aeration (Burkert et al., 2005).
Lipase is one of the most important and versatile industrial enzyme. It is responsible for the natural generation of new flavors in different dairy products (Liu and Vaughan, 2009). Pretreatment of soybeans with lipases result in the significant reduction in cooking time thus, preventing the nutrient content of soybeans from deleterious effect of excessive cooking (Khetarpaul et al., 2004). During the processing of paper, lipases are used in combination with cellulases and ligninase for the treatment of pulp, as a result the quality and yield of paper improves enormously (Sharma et al., 2002). Lipases also find applications in the production of agrochemicals and drugs (Lotti and Alberghina, 2007). They may also be utilized to enhance degradation of fatty waste and for making polyurethane (Padmapriya et al., 2011). In the manufacturing of different types of noodles and pasta, the quality of wheat flour can be varied when treated with lipases (Ozan and Fadiloglu, 2010). Lipases may be used for biodegradation of plastics (Imandi et al., 2010).
In the present study microorganisms isolated from soil were screened for lipase production. Different culture conditions such as chemical composition of culture medium, effect of incubation time, incubation temperature, pH, and different substrates were optimized for lipase biosynthesis. The produced enzyme from lypolytic microorganisms was then purified and immobilized on resins. Further investigations may be carried out for large scale lipase production form microbes and their efficient industrial applications.
II. Materials and methods
A. Chemicals
All the chemicals were of analytical grade (Merck, Sigma, USA). Olive oil, canola oil, coconut oil, sunflower oil, pure and crude cotton seed oil, black seed oil (kalonji oil), goose berry oil (aamla oil) and soap berry oil (reetha oil) were purchased from local market.
B. Isolation and screening of lipolytic microorganisms
The fungal and bacterial strains i.e. Aspergillus niger and Bacillus sp. used in present study were isolated from soil. The microorganisms were cultured on potato dextrose agar and nutrient agar slants and incubated at 30°C and 37°C respectively for 48 hrs. The slants were maintained and stored at 4oC. The bacterial strain was identified on the basis of different morphological and biochemical tests (Holt et al., 1994). The medium proposed by Cipinyle et al. (2009) was used to detect the production of lipase from lipolytic microorganisms. The composition of the medium was (g/l) tween 80, 5.0; peptone 10.0; NaCl, 5.0; CaCl2.2H2O, 0.1 and agar 18.0, pH 7.0. Ten different culture media were screened, five each for Aspergillus niger and Bacillus sp. to compare and select the best medium for maximum cell mass and enzyme production (Table I and II).
C. Lipase production
The crude lipase production was performed in 250 ml of Erlenmeyer flasks carrying 50 ml of fermentation media. The fermentation medium used for lipase production by Aspergillus niger was composed of (g/l) soy bean meal, 10.0; olive oil, 20.0; glucose, 10.0; K2HPO4, 2.0; NaNO3, 0.5 and MgSO4.7H2O, 0.5, pH 7.0 (Iftikhar et al., 2010). The medium was inoculated and incubated at 30oC for 72 hrs at 200 rpm. On the other hand, the fermentation medium used for lipase production by Bacillus sp was composed of (g/l) yeast extract, 50.0; K2HPO4, 1.0; MgSO4.7H2O, 5.0; KCl, 5.0; FeSO4.7H2O, 1.0; olive oil, 10.0 and NaNO3, 3.0, pH 7.2 (Chakraborty and Paulraj, 2009). The medium was inoculated and incubated at 37oC for 72 hrs at 200 rpm. After 72 hrs of incubation, the fermented broth was transferred to centrifugation tubes and was centrifuged for 15 min at 6000 rpm. The supernatant was used for assay of lipase.
D. Lipase activity determination
Titrimetric
method was used for activity assay of lipase by titrating liberated fatty acids
with alkali (Aly et al., 2012). All the culture media were used for this
assay in successive treatments to select the best medium for lipase production.
The mixture of assay consisted of 1.0 ml of supernatant along with 10 ml of 10%
olive oil homogenized in 10 g of gum acacia, 5 ml of phosphate buffer (pH 7.0),
and 2.0 ml of 0.6% of CaCl2. The reaction mixture
Table I. Composition of fermentation media for lipase production by Aspergillus niger
Table II. Composition of fermentation media for lipase production by Bacillus sp.
was incubated at 30oC for 1 h in rotary shaker with 100 rpm. The enzymatic reaction was inhibited by the addition of 20 ml of acetone, ethanol mixture (1:1). The fatty acids which were released during incubation were titrated with 0.1 N NaOH.
One unit of lipase activity is defined as “The amount of enzyme which releases one micro mole (µmol) of fatty acid per minute under specified assay conditions.”
Lipase units will be determined as follows:
where 
, 
Volume of NaOH used against control flask, 
Volume of NaOH used against experimental flask, 
Normality of NaOH and 
Volume of enzyme extract.
E. Determination of dry cell mass
The cell pellets of Aspergillus niger were obtained after centrifugation at 6000 rpm. The supernatant was discarded and the pellets were washed with distilled water. After washing, they were filtered by using Whatman filter paper No. 43 and then dried for 2 hrs at 110°C. In case of Bacillus sp, the cell pellets were gained after centrifugation. These pellets were washed with distilled water and then dried at 60°C for 2 hrs.
F. Optimization of fermentation conditions
For optimizing fermentation culture conditions for lipase production, effect of different parameters were studied. The experiment conducted for lipase production was regulated to study these parameters followed by subsequent enzyme activity determination. Varying incubation time (24-120 hrs), incubation temperature (25-45°C) and pH of the medium (5.0-8.0 for Aspergillus niger and 6.0-10.0 for Bacillus sp.) were investigated. Effect of different carbon sources i.e. glucose, sucrose, fructose, starch, maltose, lactose whereas nitrogen sources i.e. peptone, yeast extract, meat extract, urea, corn steep liquor and a variety of natural oils as substrates were also investigated during the study.
G. Enzyme purification
The purification of lipase was achieved by combination of salt precipitation and ion exchange chromatography. The crude fermentation broth was centrifuged at 3000 rpm for 10 min and the supernatant obtained was used for enzyme purification. Ammonium sulphate precipitation at saturation levels from 20 to 80% was achieved by the addition of varying amounts of the salt to the broth supernatant in combination with stirring and centrifugation (3000 rpm for 10 min) at 4ºC. All the pellets obtained at different salt saturation levels were re-suspended in phosphate buffer (pH 7.3) and dialyzed against the same buffer using a dialysis tubing – visking size 9 (MWCO 12-14000 Da) and assayed for enzyme activity and protein content (Bradford et al., 1976). The pellets showing lipase activity were pooled and subjected to further purification through ion exchange chromatography.
The pooled sample was loaded onto DEAE Cellulose Fast Flow column equilibrated with 100mM potassium phospahte buffer (pH 6.5). The column was connected to a Biologic LPC system (BioRad). The bound proteins were eluted with a NaCl salt gradient (0–0.8 M) in the 100mM potassium phospahte buffer (pH 6.5). The flow rate was adjusted as 1.25 ml/min. A single peaks showing lipase activity was collected in a fraction collector and was further concentrated through freeze drying.
H. Immobilization of purified lipase
Three ionic resins namely Amberlite XAD 761, Lewatite VPOC, and
Duolite A568 were used for the immobilization of purified lipase. The resins
were first equilibrated with phosphate buffer (100 mM pH 7.0) at room
temperature for 16 hrs after which, the mixture was vacuum filtered through a
membrane filter (0.2µm). The resins collected on the filter were then incubated
with the lipase dissolved in phosphate buffer at 4°C for 2 hrs. The vacuum
filtered immobilizates were used for further studies. Immobilized enzyme was
re-used for upto 10 times to determine the re-usability of the immobilizate.
The immobilized enzyme was washed after every assay with phos phate buffer and
was re-used in a fresh assay. The Bradford method (Bradford et al.,
1976) was used for protein quantification. The enzyme activity of the first
assay was
Figure 1. Effect of different culture media on lipase production from Aspergillus niger*. Y- error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
*Incubation temperature 30oC, incubation time 72 hrs, pH 5.5
considered as 100% relative activity.
I. Statistical Analysis
All the experiments were carried out in triplicates and the results are presented in the form of mean values. The results were subjected to statistical analysis using software CoStat 3.03 CoHort Software, Berkeley, CA 94701. Duncan’s multiple range test was applied under one way ANOVA. Significance has been presented in the form of probability (p < 0.05) values.
III. Results and discussion
A. Screening of lipolytic microbes
The isolation of lipase producing bacterial and fungal strains from different habitats was carried out on Tween 80 agar plates using serial dilutions. The microbial colonies which exhibited white colored zone around their colonies were isolated. 20 bacterial and 20 fungal strains were selected. The selected strains were grown in the fermentation media and screened for lipase production through submerged fermentation. The fungal and bacterial strain, IIB-H and IIB-F showed highest activities of lipase i,e. 5.46±0.06 U/ml and 4.0±0.027 U/ml, respectively (IIB: Institute of Industrial Biotechnology, H and F: sequence of isolation).
The selected bacterial and fungal strains were identified on the basis of different morphological and biochemical characteristics (Holt et al., 1994. They were further identified by 16S rRNA sequencing in another study. The selected bacterial strain was identified as Bacillus pumilus while the fungal strain was identified as Aspergillus niger.
B. Screening of culture media
For lipase production, ten different culture media were screened. In case of fungi, the culture medium M3 (g/l: Peptone, 10.0; Yeast extract, 1.0; MgSO4.7H2O, 0.25; KH2PO4, 1.0; NaNO3, 0.25; Olive oil, 5.0, pH 5.5) exhibited maximum units of lipase (5.3±0.12 U/ml). Moreover, highest cell mass from Aspergillus niger (55.1 mg/ml) was also obtained in the presence of M3 culture medium. The remaining culture media showed less lipase production (Fig. 1). The decreasing order of lipase yield in different media was M4>M1>M2>M5.
Figure 2. Effect of culture media on lipase production from Bacillus pumilus*. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
*Incubation temperature 37ºC, incubation time 72 hrs, pH 7.0
Similarly, in case of Bacillus sp, the culture medium M1 (g/l Peptone, 4.0; Glucose, 5.0; Yeast extract, 3.0; K2HPO4, 2.0; Olive oil, 10.0; NH4Cl, 8.0; FeSO4.7H2O, 0.06; MgSO4.7H2O, 0.4; NaCl, 1; KCl, 0.1, pH 7.0) gave the highest production of lipase (4.6±0.02 U/ml) and maximum cell mass of Bacillus sp (5.8 mg/ml). As compared to culture medium M1, the rest of the media showed decreased production of lipase (Fig. 2). The decreasing order of lipase yield in different media was M2>M5>M4>M3.
Due to high production of lipase from Bacillus pumilus and Aspergillus niger, the culture media M1 and M3 were selected for further analysis. The maximum production of lipase may occur due to the presence of peptone and glucose in the culture media which are readily metabolizable nitrogen and carbon sources, respectively. Moreover, these media (M1 and M3) contain variety of nutrients that favor microbial growth and enzyme production. KCl and NaCl are the mineral salts that are capable of enhancing the growth of microorganisms. K2HPO4 and KH2PO4 act as the most effective source of potassium and phosphorus. The presence of NaNO3 increases the fungal growth in the media (Chahinian et al., 2000). The enhanced fungal growth may be due to the specific requirement of sodium or nitrate ions or partial replacement of other limiting component in medium (Jones and Jennings, 1965). On the other hand, other culture media did not support significant amount of lipase production. As compared to simple culture media, the production of lipase is increased in a complex culture media. These media either lack these nutrients or the nutrients are not present in significant amount that increased the production of lipase.
C. Effect of incubation time on lipase production
The effect of varying incubation
times (24-120 hrs) on the production of lipase from both the Aspergillus
niger and Bacillus sp. was studied. Aspergillus niger gave
maximum activity of lipase (6.2±0.31 U/ml) after 72 hrs of incubation.
Similarly, Bacillus sp. also showed highest activity of lipase (5.1±0.09
U/ml) after 72 hrs of incubation. Initially, the lipase production was low and
gradually began to increase and reached maximum at 72 hrs. However, further
increase in the incubation time lowered
Figure 3. Effect of incubation time on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
the production of lipase (Fig. 3). Maximum cell mass of 57.2 mg/ml was obtained from Aspergillus niger and Bacillus sp gave 5.6 mg/ml of cell mass after 72 hrs of incubation.
The production of lipase was decreased due to the exhaustion of essential nutrients present in the fermentation medium and accumulation of different by products, thus resulting in decreased yield of desired product. Nadia et al. (2010) obtained maximum activity of lipase after 72 hrs of incubation from Mucor racemosus. Mukesh et al. (2012) obtained highest lipase activity from Bacillus sp. after 72 hrs of incubation. Similarly, Tembhurkar et al. (2012) also reported that maximum lipase production was achieved from Pseudomonas sp. after 72 hrs of incubation. Thus, 72 hrs of incubation was optimized for lipase production from Aspergillus niger and Bacillus pumilus.
D. Screening of different substrates
The effect of variety of natural oils such as olive oil, canola oil, coconut oil, sunflower oil, pure and crude cotton seed oil, black seed oil (kalonji oil), goose berry oil (aamla oil) and soap berry oil (reetha oil) on the lipase production was analyzed. Both Aspergillus niger and Bacillus sp. gave maximum activity of lipase in the presence of olive oil (7.3±0.09 U/ml and 5.3±0.03 U/ml, respectively). However, Aspergillus niger also produced significant amount of lipase (6±0.21 U/ml) in the presence of crude cottonseed oil whereas moderate production of li pase (4.6±0.19 U/ml) by Bacillus sp. was achieved in the presence of coconut oil. Other substrates did not support the high yield of lipase (Fig. 4). Highest cell mass of 62.6 mg/ml from Aspergillus niger and 3.8 mg/ml from Bacillus was obtained in the presence of olive oil.
The effect of different
concentrations of olive oil on lipase production was also evaluated. Maximum
activity of lipase (7.3±0.07 U/ml) was obtained from Aspergillus niger
in the presence of 1% olive oil. Bacillus sp. also
Figure 4. Effect of different substrates (oils) on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
Figure 5. Effect of different concentrations of olive oil on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
gave the highest activity of lipase (5.3±0.21 U/ml) in the presence of 1% of olive oil. Further increase in the amount of olive oil caused a considerable decline in the activity of lipase (Fig. 5). The culture media containing 1% of olive oil as substrate influenced the growth of both A. niger and Bacillus sp. i.e. 65.9 mg/ml of cell mass from Aspergillus niger and 4.6 mg/ml from Bacillus sp.
Olive oil has a significant effect on lipase production. In the presence of olive oil, the synthesis of lipase is approximately increased to 13 folds. It has an ability to evoke the secretion of lipase from the microbial cells. However, it does not have a prominent impact on the microbial growth. Prasad and Manjunath (2012) gained maximum activity of lipase from different species of Bacillus, Pseudomonas and Staphlococcus while utilizing olive oil as substrate.
Figure 6. Effect of different carbon sources on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
The lipase production decreases with an increase in the concentration of olive oil. Excess amount of olive oil in the culture medium may have an inhibitory effect on the production of lipase. As, high concentration of olive oil causes less transfer of oxygen in the culture medium, thus the microbial growth and their metabolism is affected (Gaspar et al., 1999). Nadia et al. (2010) also obtained maximum lipase activity from Mucor racemosus in the presence of 1% olive oil. Similarly, Sirisha et al. (2010) gained highest lipase activity from Staphylococcus in the presence of 1% olive oil and Tembhurkar et al. (2012) reported that Pseudomonas sp. also gave maximum lipase activity in the presence of 1% olive oil.
E. Effect of different carbon sources
The effect of various carbon sources i.e. glucose, sucrose, fructose, starch, maltose and lactose on the production of lipase from both Aspergillus niger and Bacillus sp was investigated. Highest yield of lipase from Aspergillus niger and Bacillus sp was achieved in the presence of glucose (6.67±0.13 U/ml and 5.3±0.09 U/ml, respectively). Rest of the carbon sources did not play a significant role in lipase synthesis (Fig. 6). Maximum cell mass of 70.1 mg/ml from Aspergillus niger and 7.3 mg/ml of cell mass from Bacillus sp was obtained in the presence of glucose.
The influence of varying concentrations of glucose on the production of lipase was also studied. Aspergillus niger gave maximum activity of lipase (7.3±0.09 U/ml) and highest cell mass (78.2 mg/ml) in the presence of 2% glucose whereas Bacillus sp gave maximum activity of lipase (5±0.26 U/ml) and cell mass (6.5 mg/ml), in the presence of 1% glucose. Further increase or decrease in glucose concentration caused a decrease in lipase production (Fig. 7).
Microbes exhibit high rate
of activity when they are grown in a medium containing glucose. Glucose is a
highly oxidizable carbon source and is readily available due to its solubility
in water. As compared to other carbon sources, it has a marked stimulating
impact on lipase synthesis moreover; it also increases the biomass content of
the microbes (Fadiloglu and Erkmen, 2002). Fadiloglu
Figure 7. Effect of different glucose concentrations on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
Figure 8. Effect of different nitrogen sources on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
and Erkmen (2002) obtained maximum production of lipase from Candida rugosa in the presence of glucose. Heravi et al. (2008) obtained maximum production of lipase in the presence of glucose from Bacillus sp.
The amount of glucose in the culture medium plays a significant role in the production of lipase. The optimum concentration of glucose stimulates the high production of lipase (Hosseinpour et al., 2011). In the presence of low glucose concentration, initially growth of the cells takes place and after attaining appropriate growth, the rapid synthesis of lipase starts as glucose stimulates the microbial cells to release lipase. Very high concentration of glucose supports the growth of microbes but it has an inhibitory effect on lipase synthesis (Fadiloglu and Erkmen, 2002). Shahidan et al. (2011) obtained maximum activity of lipase from Pichia pastoris in the presence of 2% glucose while Qamsari et al. (2011) obtained maximum lipase production from Pseudomonas aeruginosa in the presence of 1% glucose.
The culture medium supplemented with peptone has a significant effect on the production of lipase. Due to its presence, high microbial growth rate and increased lipase production is attained. As compared to other organic nitrogen sources, peptone contains certain amino acids and co-factors that are required during the synthesis of lipase (Freire et al., 1997). For the utilization of peptone, microbes secrete protease in order to achieve the degradation of the particular nitrogen source. In the presence of peptone, microbes synthesized less amount of protease thus, more production and less degradation of lipase was achieved (Salleh et al., 2006). Fadiloglu and Erkmen (2002) obtained maximum activity of lipase from Candida rugosa in the presence of peptone. Burkert et al. (2005) achieved highest lipase activity in the presence of peptone from Geotrichum candidum while Wolski et al. (2009) also reported that maximum activity of lipase was gained from Penicillium sp. when peptone was used as nitrogen source. Mukesh et al. (2012) achieved maximum activity of lipase from Bacillus sp in the presence of peptone. Prasad and Manjunath (2012) reported that in the presence of peptone, different species of Bacillus, Serratia and Staphlococcus gave highest lipase activity. Thus, peptone was optimized for the maximum production of lipase from Aspergillus niger and Bacillus sp.
The rate of proteolysis of the lipase is greatly enhanced in the presence of high concentration of peptone due to more production of protease enzyme. Nadia et al. (2010) obtained maximum production of lipase by Mucor racemosus in the presence of 1% peptone while Olusesan et al. (2011) achieved highest lipase activity from Bacillus subtilis in the presence of 1.5% peptone.
F. Effect of initial pH of culture medium on lipase production
The effect of pH of the medium (5.0-8.0 for Aspergillus niger and 6.0-10.0 for Bacillus sp.) on the production of lipase was investigated. In case of Aspergillus niger, maximum activity of lipase (6.6±0.31 U/ml) was obtained when the pH of the culture medium was adjusted at 6.0. Similarly, Bacillus sp. expressed highest lipase activity (4.67±0.15 U/ml) when the pH of the culture medium was adjusted at 8.0. Gradual decrease of lipase activity was observed when the pH was either higher or lower than 6.0 and 8.0 for Aspergillus niger and Bacillus pumilus, respectively (Fig. 9). When the pH of medium was maintained at 6.0, maximum cell mass of 91.2 mg/ml was obtained from Aspergillus niger whereas at pH 8.0, highest cell mass of 5.8 mg/ml from Bacillus sp was observed.
The decrease in lipase production occurs as acidic pH causes an inhibitory effect on the enzyme activity. A change in pH affects the essential amino acids which are present in the active site of the enzyme. Kasana et al. (2008) obtained maximum activity of lipase at pH 8.0 with Acinetobacter whereas, Tembhurkar et al. (2004) obtained highest lipase activity from Pseudomonas sp. at pH 8.0. Moreover, Mukesh et al. (2012) also obtained highest lipase activity at pH 8.0 from Bacillus sp.
Figure 9. Effect of initial pH of culture medium on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
Figure 10. Effect of incubation temperature on lipase production. Y-error bars indicate the standard deviation of means among three parallel replicates. The alphabets differ significantly at p≤0.05 level.
Generally, fungi have an ability of altering the pH of the medium in which they grow. This change occurs due to the uptake of various anions and cations existing in the culture medium. Fungi are acidophilic in nature and vary the pH of the medium within acidic range of pH scale (Nithiyaaet al., 2012). Ali et al. (2010) obtained maximum activity of lipase at pH 6.0 from Candida lipolytica. Ulker et al. (2011) also obtained maximum lipase activity at pH 6.0 from Trichoderma harzianum which is in accordance with our studies.
G. Effect of incubation temperature on lipase production
The effect of incubation temperature (25-45°C) on the production of lipase from both the Aspergillus niger and Bacillus sp. was investigated. Maximum activity of lipase (6.67±0.29 U/ml) was achieved at 30°C from Aspergillus niger whereas Bacillus sp. gave highest lipase activity (5.3±0.13 U/ml) at 37°C. Decrease in lipase activity was observed with the increase in incubation temperature (Fig. 10). At the optimized temperature of 30°C, maximum cell mass of 68.2 mg/ml from Aspergillus niger was obtained. In case of Bacillus sp. 5.8 mg/ml of cell mass was obtained at 37°C.
At high temperature, significant decrease in the yield of lipase was observed because high temperature has an inhibitory effect on the lipase production and microbial growth. With an increased temperature, the rate of reaction is enhanced due to increased kinetic energy of the molecules. As the temperature exceeds the optimum level, the breakdown of hydrophobic and hydrogen bonds takes place, thus disrupting the structure of the enzyme. Similarly, at low temperature, within the cell and in the culture medium, the transportation of nutrients is hindered, thus less yield of product is gained (Rajoka and Malik, 1997). Hosseinpour et al. (2011) reported that maximum activity of lipase was obtained from Aspergillus niger at 30°C. In case of bacteria, Sirisha et al. (2010) obtained maximum lipase activity at 37°C from Pseudomonas aeruginosa and Staphylococcus, respectively.
H. Purification and immobilization of lipase
Ammonium sulphate precipitation at saturation levels from 20-80% showed that the maximum fungal enzyme activity was found in the 60% fraction (5 U/ml) while maximum bacterial lipase activity was found in the 80% fraction (4.8 U/ml) with specific activities of 2.78 U/mg and 3.7 U/mg, respectively (Table III). The ammonium sulphate precipitated fractions showing lipase activity were further purified using DEAE Cellulose ion exchange chromatography. The fungal lipase after chromatographic purification showed 3.9 U/ml while the bacterial lipase showed 3.0 U/ml of enzyme activity with specific activities of 10.8 U/mg and 10.34 U/mg, respectively. The final 13 and 14 fold purity with a yield of 57 and 53% was achieved after ion exchange chromatography (Table III).
Three different supports i.e., Amberlite XAD 761, Lewatite VPOC, and Duolite A568 were used for the immobilization of purified fungal and bacterial lipases and the activities of the immobilized enzymes were determined (Table IV). The enzymes have different tendencies to bind to these supports through ionic interactions depending on their physical and chemical properties.
The bacterial enzyme immobilized on Amberlite XAD 761 showed maximum activity while the fungal lipase immobilized on Lewatite VPOC showed maximum enzyme activity. Therefore, Amberlite XAD 761 and Lewatite VPOC were found to be best supports for immobilization of fungal and bacterial lipases, respectively. Wang et al. (2011) hace shown that 96.99% of immobilized lipase activity on NKA-9 resin can be achieved using 0.5% genipin and 86.18% on S-8 with a 0.25% genipin.
