Abstract: Microbial lipases catalyzing the hydrolysis and synthesis of esters from glycerol and fatty acids are commercially important in food, dairy, detergent and leather industries. In the present study, 15 thermo-alkalophilic bacterial strains isolated from a compost soil sample were screened for lipase production in beef extract nitrate medium containing 1% tributyrin. The strain 8C, giving maximum lipolytic activity was morphologically and biochemically identified as Bacillus sp. Lipase production from strain 8C was optimized by studying effects of incubation period, initial pH of medium, carbon and nitrogen sources, metals, substrates and thiols under static conditions. Lipase production was maximum after 7 days of incubation at 50°C and pH 9.0 and when sesame oil was used as a substrate. Supplementation of ribose or raffinose and ammonium sulphate (1%) as sole sources of carbon and nitrogen also stimulated lipase production over control. Of the different thiols tested, methionine greatly increased lipase production (175 Eu mL–1) whereas, FeCl3 (50 mM) found to be most suitable metal salt for optimal lipase production (300 Eu mL–1). Under optimized conditions, lipase production by thermo-alkalophilic Bacillus sp. 8C was increased by 6.36 fold compared to unoptimized conditions.
INTRODUCTION
Lipases (triglycerol hydrolases (EC 3.1.1.3) are the hydrolytic enzymes belonging to serine hydrolase family and catalyze conversion and synthesis of esters from glycerol and fatty acids. Lipolytic reactions are highly chemo-, regio and or enantioselective and operate at the interface of oil and water (Nguyen et al., 2010). High quality and low energy consumption of lipase catalyzed reactions make them superior over conventional steam splitting methods operating at high temperature and pressure (Treichel et al., 2010). These characteristics of lipases increased their demand in different industries including food and dairy, detergent, cosmetics, leather, paper and pulp, biodiesel and pharmaceutical (Hasan et al., 2007).
Thermophilic lipases are of special interest for industrial applications due to their stability and high activity at higher temperatures and being stable in presence of chemicals (Messias et al., 2009; Uttatree et al., 2010). Lipases being third largest selling group of enzymes with more than 1000 t of annual production and approaching to a billion dollar business. Lipases active at alkaline pH are of great importance in detergent industry where these enzymes are used for removal of fat stains and dirt (Bora and Bora, 2012). The use of alkaline lipases in detergent formulation substantially acts as a substitute for the phosphate builders in chemical detergent which are considered as major pollutant from detergent industry (Jellouli et al., 2011). The extremozymes working at high temperatures and alkaline pH decrease the need of added steps in the process, reduce the cost and increase the process efficiency (Myers et al., 2000). Lipases from different thermo-alkalophilic Bacillus sp. including Bacillus stearothermophilus (Kim et al., 2000), Bacillus licheniformis (Sangeetha et al., 2010) and Bacillus thermocatenulatus BTL-2 (Schmidt-Dannert et al., 1994) were reported earlier. The research on identification and isolation of new lipase produced in microorganisms has been intensified in recent years (Hasan et al., 2006; Shu et al., 2010; Treichel et al., 2010).
Enzyme production process is closely controlled in microorganisms and for improving its productivity. It is necessary to mitigate these controlling factors of production medium. This can be achieved by manipulating growth conditions to optimal level that generally improves the lipase productivity as evidenced in earlier studies (Gupta et al., 2007; Treichel et al., 2010; Rabbani et al., 2013). The most important parameters that critically affect the lipase production are carbon and nitrogen sources (Kumari et al., 2009; Zhao et al., 2008). However, optimization of other nutritional and growth parameters affecting lipase production will be the added advantage for improvement studies. Knowing suitable conditions required for higher lipase production will reduce the production cost, minimize production time and other critical conditions required for production process. The present research work deals with the isolation of lipase producing thermo-alkalophilic Bacillus sp. from compost soil sample and optimization of conditions required for lipase production.
MATERIALS AND METHODS
Sample collection and isolation of lipolytic bacteria: A compost soil sample was collected from Trikut, Nanded, Maharashtra, India. One gram of air dried soil was suspended in 100 mL beef extract-nitrate broth (peptone 3%, beef extract 5% and KNO3 0.1%) supplemented with 1% tween-80 for enrichment of lipolytic bacteria. The pH of medium was adjusted to 9.0 by using 0.1 N NaOH. The flask was kept for incubation at 50°C under static conditions for 7 days. After incubation, 0.1 mL of enriched broth sample was spread on the surface of sterile beef extract-nitrate agar plates containing 1% Tween-80 in triplicates. The plates were incubated at 50°C for 7 days and observed for colonies showing zone of lipolysis. The lipolytic activity of selected isolates were further confirmed on tributyrin agar plates (peptone 3%, beef extract 5%, KNO3 0.1% and tributyrin 1%) by spot inoculation and incubating the plates under previously mentioned conditions.
Quantitative determination of enzyme activity: The selected isolates were grown in tributyrin broth (peptone 3%, beef extract 5%, KNO3 0.1% and tributyrin 1%) with pH 9.0 as mentioned before. After incubation, the culture broths were centrifuged at 10,000 rpm for 20 min at 4°C and supernatant was used as a source of crude enzyme and used for estimating lipase activity.
Lipase assay: The assay was performed by using the method described by Selvam et al. (2010), on the basis of hydrolysis of olive oil. To the reaction mixture containing 1 mL of Tris- HCl buffer of pH 9.0, 2.5 mL of deionized water and 3 mL of olive oil emulsion (10% gum arabic and 5% olive oil in deionized water), 1 mL of culture supernatant for test and 1 mL of deionized water for blank were added. The reaction mixture was mixed thoroughly by swirling and incubated at 50°C for 30 min. After incubation, enzyme substrate reaction was terminated by addition of 3 mL of 95% ethanol and mixed by swirling. The amount of fatty acid liberated due to lipase activity was estimated by titrating the contents of assay mixture against 0.05 M NaOH using thymolophthalein as pH indicator. The end point observed was from colorless to light blue.
One unit of lipase was defined as the amount of enzyme required to release 1 μmol of fatty acid per milliliter under assay conditions. The enzyme activity is expressed as Eu mL–1.
Identification of lipase producing bacterial isolate: The potential lipase producer strain 8C was selected for further studies and identified on the basis of morphological traits and biochemical pattern as per the standard criteria given in Bergy’s Manual of Systematic Bacteriology (Holt et al., 1994).
Optimization of process variables
Effect of incubation period: The 8C was inoculated in beef extract-nitrate broth of pH 9.0 containing 1% tributyrin as a substrate and incubated at 50°C for 11 days. Five milliliter culture media was removed periodically at an interval of 48 h and used for lipase assay.
Effect of initial pH of medium: Active culture of 8C was inoculated in beef extract-nitrate media containing 1% tributyrin. pH of media were adjusted to different pH (7, 8, 9, 10, 11 and 12) with the help of 0.1 N NaOH on a pH meter. The media were incubated at 50°C for 7 days. Enzyme units were calculated by performing the lipase assay.
Effect of carbon sources: To study the effect of carbon sources on lipase production by 8C, 11 different carbon sources such as xylose, fructose, dextrin, mannitol, galactose, sucrose, maltose, raffinose, ribose, lactose and gum acacia at 1% concentration were added individually to the beef extract-nitrate media. The broths were incubated at 50°C for 7 days at static condition. After incubation, broth was centrifuged and used as crude enzyme for lipase assay as mentioned earlier.
Effect of nitrogen sources: For the selection of suitable nitrogen source for lipase production by 8C different organic and inorganic nitrogen sources such as yeast extract, pyruvate, bile salts, glycerol, ammonium sulphate and arginine were screened. By supplementing those at 1% concentration in beef extract-nitrate media of pH 9.0, the active culture was inoculated and incubated at 50°C for 7 days and assayed to determine the enzyme units.
Effect of metal salts: The effect of different metal salts (NaCl, KCl, CaCl2, MgCl2, ZnCl2, NH4Cl and FeCl3 1%) on lipase production was assessed by inoculating active culture of 8C in beef extract-nitrate broth containing ribose and ammonium sulphate. The flasks were incubated at 50°C for 7 days at static condition and used for lipase assay as mentioned before.
Effect of substrates: Lipase production was accelerated by incorporation of suitable lipidic substrate. Therefore, effect of different lipidic substrates on lipase production by 8C was screened. Different substrates such as castor oil, mustard oil, eucalyptus oil, soya bean oil, coconut oil, sesame oil and clove oil at 1% concentration were supplemented individually in beef extract-nitrate media of pH 9.0. The flasks were incubated at 50°C for 7 days at static conditions. After incubation enzyme activity was determined as mentioned above.
Effect of thiols: To determine the effect of thiols on lipase production, different thiol containing compounds (methionine, dithiothreitol, β-mercaptoethanol, cysteine, sodium sulphite and thiourea) were added individually to beef extract-nitrate medium at 50 mM concentration. After 7 days of incubation at 50°C under static conditions, the enzyme activity was determined as stated earlier.
RESULTS AND DISCUSSION
Isolation and identification of lipolytic bacteria: In the present study, 15 bacterial isolates obtained from compost soil were tested for lipase production potential. Out of the 15 isolates, 5 (1C, 2C, 3C, 8C and 15C) showed good lipolytic activity on tributyrin agar and broth (Table 1). The isolate 8C showing higher activities (28 mm, 55 Eu mL–1), was selected as a potent lipase producer strain for further studies.
The isolate 8C was characterized morphologically and biochemically as per the criteria given in Bergy’s Manual of Systematic Bacteriology. The isolate was gram positive, non-motile, catalase positive rod, with ability to ferment lactose, mannose, maltose, fructose and ribose while not utilizing salicin, mannitol and arabinose. The isolate was positive for vogues-proskeur test, hydrolyzed starch and showed citrate utilization capacity. Based on these characteristics the isolate 8C was identified as Bacillus sp. and used further for optimization studies. Thermophilic Bacillus sp. have previously shown the lipase production at 50-60°C (Kim et al., 2000; Bora and Bora, 2012). However, the reports on lipase production by Bacillus sp. under thermo-alkalophilic conditions are scanty.
Optimization of process variables
Effect of incubation period: Lipase production by Bacillus sp. started after 72 h at 50°C and increased thereafter with the increase in incubation time. The maximum enzyme activity (125 Eu mL–1) was obtained by 7 days of incubation and then decreased gradually from 8th day to 11th day of incubation (Fig. 1). The high incubation time required for enzyme production might be due to the growth of microorganism at high temperature (50°C) under alkaline (pH-9.0) and static conditions. Under these conditions the organism may have high lag period before its exponential growth. The gradual decrease in enzyme production after 7 days might be due to the accumulation of toxic end products of metabolism or exhaustion of available nutrients in medium.
| Table 1: | Qualitative and quantitative screening of isolates showing lipolytic activity |
| Fig. 1: | Effect of incubation period on lipase production by Bacillus sp. 8C. Each value represents Mean±percentage error |
Kumar et al. (2012) have found that Bacillus sp gave the maximum yield of (1.2 Eu mL–1) lipase after 72 h of incubation whereas Sharma et al. (2002) showed the lipase production was highest after 12 h of incubation.
Effect of initial pH of medium: Changes in the initial pH of production medium may induce the production of new metabolites that affect the biosynthesis of desired products. Hence, we examined the effect of pH on lipase production by Bacillus sp. and the results are summarized in Fig. 2. pH 9.0 was found to be the optimum for enzyme production (125 Eu mL–1). Most of the published literature responds pH 8.0 as optimum pH for lipase production in thermophilic Bacillus sp. (Hasan and Hameed, 2001; Kim et al., 1994; Duza and Mastan, 2014). Increased cultivation of the organism at an unfavorable pH, reduce the growth rate of organism and also has marked effects on the level of enzyme production (Bhosale et al., 2011) as evidenced by drastically reduced enzyme production at all other pH.
Effect of carbon sources: Lipase production by thermo-alkalophilic Bacillus sp. was determined by measuring the enzyme activity when the strain was growing on different carbon sources. The highest lipase production was obtained with 1% ribose and raffinose (165 Eu mL–1) followed by gum acacia (140 Eu mL–1) and lactose (115 Eu mL–1). Dextrin, sucrose, maltose and most of the carbon sources except xylose could stimulate lipase activity as they supported the growth of thermo-alkalophilic Bacillus sp. (Fig. 3). These results are in agreement with those reported by Sztajer and Maliszewska (1989).
| Fig. 2: | Effect of pH on lipase production by Bacillus sp. 8C. Each value represents Mean±percentage error |
| Fig. 3: | Effect of different carbon sources on lipase production by Bacillus sp. 8C. Each value represents Mean±percentage error |
Ability of an organism to drive a metabolic reaction and grow in presence of specific carbon source is dependent on typical enzymatic machinery present in the cell. In the present study, Bacillus grew well in presence of ribose and raffinose. Gum acacia, lactose and mannitol also supported for maximum enzyme production. However, lowest enzyme units recorded in the presence of xylose indicating that xylose is not suitable for lipase production.
Effect of nitrogen sources: The addition effect of different nitrogen sources on enzyme production is shown in Fig. 4. The maximum production of lipase was observed with 1% ammonium sulphate (140 Eu mL–1) compared to glycerol (35 Eu mL–1) and pyruvate (50 Eu mL–1). Lipase production was also noticeable in presence of peptone, arginine and bile salts. Ammonium sulphate is highly soluble in water. Hence, readily available nitrogen source for microbial growth. Further, its high ionic strength releases ammonium ions in aqueous media that can be easily incorporated as part of different amino acids and hence plays important role in protein synthesis process.
Effect of metal salts: The effect of different metal salts added to the production medium on lipase production is presented in Fig. 5. Salts of ferric ions (FeCl3) highly stimulated (300 Eu mL–1) the lipase production by Bacillus sp. whereas, enzyme production was considerably high when ZnCl2 and MgCl2 were supplemented in the medium. Presence of KCl did not affected enzyme production and the production increase was marginal in presence of CaCl2, NaCl and MnCl2.
| Fig. 4: | Effect of different nitrogen sources on lipase production by Bacillus sp. 8C. Each value represents Mean±percentage error |
| Fig. 5: | Effect of metal salts on lipase production by Bacillus sp. 8C. Each value represents Mean±percentage error |
Effect of different substrates: Supplementation of different lipidic substrate in production medium greatly affected the lipase production capacity of Bacillus sp. The enzyme production was unchanged when tributyrin in production media was replaced with castor oil, eucalyptus oil, soya bean oil and coconut oil as indicated in Fig. 6. However, production was highly stimulated when sesame oil was used as a substrate (165 Eu mL–1) and was increased considerably in presence of mustard oil (110 Eu mL–1) and clove oil (77 Eu mL–1). Lipase is an extra-cellular inducible enzyme whose production is only detectable in presence of suitable substrate. Further, the growth and enzyme production capacity of an organism while growing on different substrate depend on its substrate utilization efficiency that ultimately depends on the presence of specific metabolic route required for metabolism of substrate.
Effect of thiols: In present study, we also examined the effects of different thiol compounds on lipase production by Bacillus sp. Interestingly, supplementation of different thiols in production medium either did not affected or stimulated the lipase production (Fig. 7). The effect was not detectable in the presence of dithiothreitol, β-mercaptoethanol and thiourea. However, addition of sodium sulphite, cysteine, sodium thiosulphate and methionine gradually increased the lipase production.
| Fig. 6: | Effect of different substrates on lipase production by Bacillus sp. 8C. Each value represents Mean±percentage error |
| Fig. 7: | Effect of different thiols on lipase production by Bacillus sp. 8C. Each value represents Mean±percentage error |
Methionine and cysteine are sulphur containing amino acids that play an important role in stabilizing tertiary structure of protein molecules as well as they act as good nitrogen source for microbial growth. Enhanced lipase production in presence of methionine and cysteine indicated good growth of Bacillus sp. that might have supported higher production of enzyme.
The enzyme activity determined in tributyrin beef extract-nitrate broth before optimization was 55 Eu mL–1 and the production found in optimized medium (peptone 3%, beef extract 5% , KNO3 0.1%, ribose 1%, ammonium sulphate 1%, sesame oil 1%, FeCl3.7H2O 1% and methionine 50 mM) was 350 Eu mL–1 indicating 6.36 fold increase in lipase production by thermo-alkalophilic Bacillus sp. 8C.
CONCLUSION
In the present study, a thermo-alkalophilic lipase producer strain of Bacillus sp. 8C has been optimized for improved lipase production. The parameters and conditions used for lipase optimization studies are simple and easy to manage. This study first time demonstrated the role of raffinose and ribose sugars and thiols like methionine in improvement of thermo-alkalophilic Bacillus produced lipase yield.
ACKNOWLEDGMENT
The authors wish to acknowledge School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India for providing support and necessary facilities to complete this research work.