Production and Characterization of an Alkaline Thermostable Crude Lipase from an Isolated Strain of Bacillus cereus C7
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- Dutta, S. & Ray, L. Appl Biochem Biotechnol (2009) 159: 142. doi:10.1007/s12010-009-8543-x
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A bacterial strain isolated from spoiled coconut and identified as Bacillus cereus was found capable of producing alkaline thermostable extracellular lipase. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 60 °C, 10 min, and 8.0 respectively. Common surfactants except Triton X 100 and cetyltrimethylammonium bromide have no or very little inhibitory effects on enzyme activity. The enzyme was found to be stable in presence of oxidizing agents and protease enzyme. The maximum lipase production was achieved at 30–33 °C, pH 8.0 on 24 h of fermentation using 50 ml medium in a 250-ml Erlenmeyer flask. The superior carbon and nitrogen sources for lipase production were starch (2%) and ammonium sulfate (nitrogen level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and urea (nitrogen level 46.62 mg/100 ml) in combination, respectively. The maximum enzyme activity obtained was 33 ± 0.567 IU/ml.
Lipases (EC 22.214.171.124) are glycerol ester hydrolases that catalyze the hydrolysis of triacylglycerols into fatty acid, partial acylglycerols, glycerol, and under low water condition catalyze the reverse reaction [1, 2]. Although lipases are found in animals, plants, bacteria, yeast, and fungi, however, microbial lipases are commercially significant for their potential use in various industries such as food, dairy, pharmaceutical, detergents, textile, biodiesel, cosmetic industries, and in synthesis of fine chemicals, agrochemicals, and new polymeric materials . As the applications increase, the availability of lipase possessing satisfactory operating characteristics is a limiting factor. For example, lipase added in prewash soaking agents and detergent powders needs to be stable under alkaline pH and to function in the presence of surfactants . Thermal stability is another major requirement for commercial lipases because of their high activities at the elevated temperatures and stabilities in organic solvents. Therefore, a thermostable alkaline lipase is needed for industrial applications.
In the present paper, we describe the process optimization for maximum production of a bleach stable, protease stable, and thermostable alkaline lipase from an isolated strain of Bacillus cereus (GenBank accession number AB 244464) and characterization of the crude enzyme.
Materials and Methods
An alkaline thermostable lipase-producing bacterial strain was isolated from spoiled coconut and identified as B. cereus was used for the present study. It was maintained by monthly subculturing at 30 °C and stored at 4 °C.
(a) The medium for plate and slant culture was composed (g/l) of olive oil, 20; (NH4) 2SO4, 5; (NH2) 2CO, 2; MgSO4.7H20, 1; yeast extract, 0.5; and agar, 20 . The mixture was heated and emulsified and the pH was adjusted to 8 with 1 N Na2CO3. (b) The inoculum medium and fermentation medium used for lipase production contained (g/l) soluble starch, 20; peptone, 20; KH2PO4, 5; (NH4)2SO4 1; MgSO4.7H2O 1; (NH2)2CO, 1; and pH 8 (5).
Isolation of Lipase Producing Microorganism
Thirty organisms were isolated from soil and spoiled coconut using olive oil medium following plate and dilution technique . Each isolate was tested for its lipase activity.
Preparation of Crude Enzyme
Inoculum was prepared by transferring one loop of culture from slant to the inoculum medium (50/250 ml Erlenmeyer flask) and incubating the flask at 30 °C in a rotary shaker at 120 rpm for 24 h. Fermentation medium (50/250 ml Erlenmeyer flask) was inoculated with 2% (v/v) inoculum and incubated for 24 h under the same conditions. The cell-free supernatant obtained by centrifugation at 4,000 rpm for 15 min was used for determining extracellular lipase activity.
Unless otherwise stated, all experiments were run in triplicate and repeated twice. Olive oil emulsion was prepared as follows: 25 ml of olive oil and 75 ml of 2% polyvinyl alcohol solution was emulsified using a homogenizer. The reaction mixture containing 5 ml olive oil emulsion, 4 ml 0.2 M Tris buffer (pH 8.0), 110 mM CaCl2 (final concentration 10 mM), and 1 ml enzyme solution was incubated at 60 °C for 10 min. Control containing inactivated enzyme (boiled) was treated similarly. Immediately after incubation, the emulsion was destroyed by the addition of 20 ml acetone–ethanol (1:1) mixture and the liberated free fatty acid was titrated with 0.02 N sodium hydroxide. One unit of lipase was defined as the amount of enzyme, which liberated 1 μmol of fatty acid per minute. One milliliter of 0.02 N NaOH is equivalent to 100 μmol of free fatty acid .
The selected strain was identified following Bergey’s Manual of Determinative Bacteriology . DNA base composition was determined by Bangalore Genei.
Results and Discussion
Screening of Lipase Producing Organism
Of the 30 isolates, only ten samples were found to be capable of producing lipase. The isolate C7 was selected as the potent strain and used in further studies (results not shown).
Taxonomical Studies of the Isolate C7
Taxonomical characteristics of strain C7.
Straight rods, 3.24 × 1.85 μm, occurring single, pair or in short chains, nonmotile
Gram positive, spore former, spores are terminally located
Nutrient broth (stationary condition) 48 h
Moderate growth, flocculent sedimentation, ring formation, no pellicle formation
Nutrient broth (shaking condition) 48 h
Abundant growth, turbidity, ring formation, no pellicle formation, off white color
Nutrient agar colonies
Irregular shaped (1 mm diameter), opaque, smooth, flat, off white with entire edge, dry
Growth at different temperatures
Growth at different NaCl concentration
Growth at different pH
9.6 (with 6.5% NaCl)
Ammonia from arginine
Arginine used as a sole source
Nitrate reduced to nitrite
Hydrolysis of starch
Hydrolysis of urea
Growth under anaerobic condition with/with out beef extract
pH < 6
pH > 7
Litmus milk test
Indole formation test
Carbohydrate fermentation test
Characterization of Crude Enzyme
Effect of Reaction Time
Effect of Temperature
Effect of pH
pH Stability and Thermal Stability
The alkaline pH stability of lipase was determined by incubating them at pH values 7.2 to 9 for 1 h and then the preservation stability of lipase was determined by standard assay procedure. Figure 4 shows that the enzyme is stable in this pH range, so it can be said that crude lipase is active at alkaline pH.
To determine the thermal stability of the crude lipase, the enzyme was incubated at different temperature (4–70 °C) for 1 h at pH 8.0 using 0.2 M Tris-HCl buffer. The residual activities were evaluated according to the standard assay procedure. As shown in Fig. 3, the enzyme is thermostable up to 60 °C.
Effect of Metal Ions
Effect of various metal ions on the activity of crude lipase.
Relative activity (%)
Concentration of metal ions (mM)
Effect of Surfactants and Commercial Detergents
Commercial detergents, viz., Ariel, Tide, Rin, Surf excel blue, Surf excel quick wash, Sunlight, and Ezee from local market, were also used at 0.2% concentration in enzyme–substrate reaction mixture and enzyme assay was carried out as usual. It has been observed that Ariel enhanced the enzyme activity, whereas the enzyme retained 60% or more activity in presence of commercial detergents like Rin, Surf excel blue, Surf excel quick wash, Sunlight, and Tide. Only Ezee showed significant inhibitory effect on lipase activity.
Effect of Bile Salts
Effect of bile salts on crude lipase activity.
Bile salts (0.5% w/v)
Relative activity (%)
There are several reports which show that bile salts are stimulatory to the activity of some microbial lipases . Watanabe et al.  reported inhibition of lipase activity in presence of bile salts.
Effect of Oxidizing Agents
Stability of the crude lipase in presence of oxidizing agents.
Oxidizing agents (1% v/v or w/v)
Relative activity (%)
Effect of Protease
Stability of the crude lipase in presence of protease.
Protease (0.5 mg of 1240 U/mg)
Relative activity (%)
Effect of Environmental Conditions on Production of Lipase
Nutritional Factors Affecting Lipase Production
Effect of inorganic nitrogen sources on production of lipase.
Lipase activity (IU/ml) in presence of
Peptone 2% (N2 level 297 mg/100 ml) + urea 0.1% (N2 level 46.62 mg/100 ml)
Peptone 2% (N2 level 97 mg/100 ml)
Diammonium hydrogen phosphate
Ammonium dihydrogen phosphate
Authors gratefully thank the Council of Scientific and Industrial Research (CSIR) for providing research fellowship and funding.