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1Arun Kumar Sharma, 1Sapna Kumari, 1Vinay Sharma*, 2Jyoti Saxena
1Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India.
2Department of Biochemical Engineering, Bipin Tripathi Kumaon Institute of Technology, Dwarahat, Uttrakhand.
Prof. Vinay Sharma
Department of Bioscience and Biotechnology,
Banasthali University, Rajasthan, India.
Background: Because of the several applications of lipases in industry, there is a necessitate to investigate their uniqueness, because lipases isolated from diverse sources may have diverse features. Objective: The aim of this work was to screen the potent lipase producing soil fungi by submerged fermentation. Materials and methods: Fungal strains were isolated from 4 different soil samples and used for production of lipase in submerged fermentation (SmF). Results: Results were assessed both in terms of lipase activity and specific activity. Among all the tested fungal strains (21), highest production of lipase (185.38 ± 2.58 U/ml/min) was obtained from isolate S3St6, which was isolated from petrol pump soil sample. Conclusion: Based on quantitative screening of 21 fungal strains in SmF, isolate S3St6 was recognized as potent producer of lipase.
Keywords: Lipases, fungi, submerged fermentation, soil sample, quantitative screening
Lipases are hydrolases category enzyme catalyze hydrolysis of long water immiscible ester molecules (triglycerides) into fatty acids and glycerol (Figure 1) (Sharma et al., 2001).
Figure 1. Lipase catalyzed reaction
Lipases are very important enzyme for industrial applications. Lipases are used in detergent industry (Saisubramanian et al., 2006), pharmaceutical industry (Hasan et al., 2006), food industry (Sharma et al., 2001), ripening of cheese (Dupuis and Boyaval, 1993), production of aroma (Ben Salah et al., 2007), synthesis of fat with more content of unsaturated fatty acids (Wang et al., 2008), production of biodiesel (Park et al., 2006) etc.
Treichel et al. (2010) reported that several investigators globally direct their activities to the screening of novel lipase producing microbes. These attempts are justified by the huge versatility of lipase applications. Because of the several applications of lipases in industry, there is a necessitate to investigate their distinctiveness, because lipases isolated from diverse sources may have diverse features.
Most of the literature reports isolation of potent lipolytic fungi from soil samples because soil is a good residence of variety of enzyme producing microbes. There are three different sources of lipase: animal, plants and microbes but fungi and bacteria secrete the majority of lipases utilized in biotechnological applications. Filamentous fungi are an attractive source of lipases since they secrete enzymes of extracellular origin (Gutarra et al., 2009).
Lipase can be industrially produced by two methods: Solid-State Fermentation (SSF) and Submerged Fermentation (SmF), but fungi are well adapted than bacteria and yeast to cultivate in SmF (Gutarra et al., 2009). SmF is broadly used in industries for enzyme production because of its advantages (process regulation) over SSF. SmF can be preferred over SSF for microbial enzyme production for increased productivity and easy Down Stream Processing (DSP) (Edwinoliver et al., 2010). In view of above background, we have screened different fungal isolates of soil by SmF in order to find out the hyperproducer isolate.
Materials and methods
Collection of soil samples
Soil samples were collected from four diverse areas (mustard crop field, wheat crop field, petrol pump of Newai Town and medicinal garden of Banasthali University).
Isolation of fungi
Fungi were obtained on PDA (potato dextrose agar) plates by serial dilution of soil samples as explained earlier by Waksman (1922) and purified by point inoculation on PDA plates.
Production of extracellular lipase
Preparation of spore suspension
Three ml of sterile distilled water was added in 6 days old slant culture of fungal isolates and it was shaken vigorously to prepare spore suspension (Sharma et al., 2015).
Transferring spore suspension in fermentation broth
One ml of spore suspension was transferred in 100 ml of production medium which was prepared in 250 ml of Erlenmeyer flask under aseptic condition of laminar air flow. The composition of production medium (g/100 ml) is as follows: bacteriological peptone, 4; olive oil, 1%; MgSO4.7H2O, 0.1; KH2PO4, 0.1; (NH4)2SO4, 0.1; sucrose, 0.5 and pH was adjusted to 7.0. Flasks were kept in an incubator at 28 ºC, 150 rpm for 6 days.
Separation of crude protein mixture from culture broth
Culture broth from the each inoculated flask was filtered via Whatmann filter paper No. 1 followed by centrifugation of filtrate at 7,000 rpm at 4 ˚C for 10 minutes. Supernatant was recovered from centrifugation tube after discarding pellet (Josephine et al., 2012). This supernatant was considered as crude protein lysate for the measurement of total protein contents and extracellular lipase activity.
Measurement of lipase activity from crude protein lysate
Preparation of standard curve of p-nitrophenol
Reference curve of p-nitrophenol was prepared within the concentration range of 2-20 µg/ml, in 0.05 M Tris HCl buffer, pH-8.0 (Verma and Sharma, 2014).
Activity of lipase was measured by spectrophotometric technique as explained previously by Winkler and Stuckmann (1979). We have used long carbon chain fatty acid triglyceride (p-nitrophenyl palmitate) as substrate for analysis of lipase activity. Isopropanaol was used as solvent for preparation of stock solution of p-nitrophenyl palmitate (pNPP; 20 mM). 75 µl of pNPP stock solution was dissolved in 2.9 ml of Tris-HCl buffer (0.05 M, pH-8.0) and pre-incubated at 70 °C for 10 minutes under shaking condition (150 rpm) in a water bath shaker followed by addition of 25 µl of crude enzyme lysate to adjust the final volume of reaction cocktail to 3 ml. Test tubes were kept for 30 minutes at 35 °C in a shaker water bath for allowing the lipase catalyzed reaction to occur. Thereafter, 1 ml of stopping reagent (chilled acetone-ethanol, 1:1 v/v) was added to cease the lipase catalyzed reaction. Yellow colour was developed due to the liberation of product (p-nitrophenol) from substrate (pNPP) by the action of lipase. Amount of p-nitrophenol was measured spectrophotometrically at 410 nm with reference to the blank/control. Blank was prepared in the same way but in the reaction mixture, crude protein lysate was added after addition of stop reagent.
One unit of lipase activity was defined as micromole (µM) of p- nitrophenol released by hydrolytic cleavage of pNPP by one ml of lipase at 35 °C in 1 minute of reaction time.
Estimation of protein contents from crude protein lysate
Quantity of protein in culture supernatant was measured by Lowry’s technique (Lowry et al., 1951).
Preparation of standard curve of BSA (bovine serum albumin)
Reference curve of BSA was made within the concentration range of 40 to 200 µg/ml.
100 µl of crude protein lysate was transferred into a test tube followed by addition of 900 µl of distilled water to adjust the final volume to 1 ml. Thereafter, 5 ml of alkaline copper reagent was added and tubes were kept for 10 minutes at room temperature. Tubes were placed in dark for 30 minutes after addition of 0.5 ml of Folin’s reagent. Blue colour was developed at the end of incubation and quantity of this blue colour compound was measured at 660 nm with reference to blank.
All experiments (lipase assay, protein content determination) were done in triplicates and mean values as well as standard deviation of them are presented here in the respective table.
Results and Discussions
Isolation of fungi
Fungi were isolated on PDA plates. Fungi with different characteristics were appeared on PDA plates. Isolated fungi were purified by point inoculation on PDA plates. Results of purification are presented in Figure 2.
Figure 2. PDA plates showing purified colonies of fungi
Quantitative screening (Production of lipase in SmF)
Results of quantitative screening are given in Table 1-4. Table 1 presents that among all the 5 strains of medicinal garden soil, isolate S1St2 demonstrated maximum activity of lipase (64.88 ± 2.57 U/ml/min) and specific activity (3.46 ± 1.18 U/mg) at 5 days of incubation. Table 2 represents that highest production of lipase (157.23 ± 2.54 U/ml/min) was obtained from isolate S2St1 among all the tested strains of mustard field soil at 3 days of incubation. Specific activity (7.41 ± 1.68 U/mg) was also higher at day 3. Table 3 presents that isolate S3St6 exhibited highest lipase activity (185.38 ± 2.58 U/ml/min) and specific activity (21.78 ± 1.74 U/mg) at 3 days of incubation among all the 6 isolates of petrol pump soil sample. Table 4 represents that among all the 5 fungal strains of wheat field soil sample, isolate S4St5 demonstrated highest lipase activity (149.33 ± 5.01 U/ml/min) and specific activity (7.85 ± 0.77 U/mg) at 8 days of incubation.
If we compare lipase activity of all isolates then maximum production of lipase (185.38 ± 2.58 U/ml/min) was found with isolate S3St6 at 3 days of incubation, which indicate that this fungus can be used to produce large quantity of lipase in very short period of time (within 3 days of fermentation period). Isolate S4St5 was able to accumulate high quantity of lipase (149.33 ± 5.01 U/ml/min) at 8 days of incubation.
Farahbakhsh et al. (2013) studied production of lipase from Aspergillus niger by both the SmF and SSF process. Colla et al. (2015) performed isolation of fungi from diesel contaminated soil followed by quantitative screening in SmF. Niaz et al. (2014) reported isolation of fungi from decomposed food items followed by lipase production in SmF. Colla et al. (2010) reported screening of potent lipase producing soil fungi by SmF. Among the 27 fungi, highest quantity of lipase (2.81 U/ml/min) was recovered from Penicillium E-3 sp. followed by Trichoderma E-19 (2.34 U/ml/min) and Aspergillus O-8 (2.03 U/ml/min). Hosseinpour et al. (2011) carried out studies of lipase production from Aspergillus niger in SmF.
Table 1. Production of lipase in submerged culture from fungal isolates of medicinal garden soil
S1St1: soil sample 1 strain 1; S1St2: soil sample 1 strain 2.
Table 2. Production of lipase in submerged culture from fungal isolates of mustard field soil
S2St1: soil sample 2 strain 1; S2St2: soil sample 2 strain 2.
Table 3. Production of lipase in submerged culture from fungal isolates of petrol contaminated soil
S3St1: soil sample 3 strain 1; S3St2: soil sample 3 strain 2.
Table 4. Production of lipase in submerged culture from fungal isolates of wheat field soil
S4St1: soil sample 4 strain 1; S4St2: soil sample 4 strain 2.
Lipases are classified in Hydrolase class of enzymes. Lipases are produced by microbial sources at large scale especially by fungal sources using the technique of SmF. Lipase was produced from 21 soil fungal isolates in SmF in presence of olive oil and sucrose as carbon sources. Among all the isolates, maximum production of lipase (185.38 ± 2.58 U/ml/min) and specific activity (21.78 ± 1.74 U/mg) was recovered from isolate S3St6 at 72 h of incubation. So in our study S3St6 was identified as hyperproducer isolate. Further optimization studies can be carried out for enhanced secretion of lipase by hyperproducer isolate.
We are thankful to Professor Aditya Shastri, Vice-Chancellor, Banasthali University for providing research facilities in the Department.
Declaration of interest
The authors report no declarations of interest.
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