Applied Biochemistry and Biotechnology

, Volume 159, Issue 1, pp 142–154

Production and Characterization of an Alkaline Thermostable Crude Lipase from an Isolated Strain of Bacillus cereus C7

Authors

  • Sanghamitra Dutta
    • Department of Food Technology & Biochemical EngineeringJadavpur University
    • Department of Food Technology & Biochemical EngineeringJadavpur University
Article

DOI: 10.1007/s12010-009-8543-x

Cite this article as:
Dutta, S. & Ray, L. Appl Biochem Biotechnol (2009) 159: 142. doi:10.1007/s12010-009-8543-x

Abstract

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.

Keywords

LipaseBacillus cereusAlkalineThermostableProductionCharacterization

Introduction

Lipases (EC 3.1.1.3) 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 [3]. 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 [4]. 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

Microorganism

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.

Medium Composition

(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 [5]. 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 [6]. 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.

Assay Method

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 [5].

Taxonomical Studies

The selected strain was identified following Bergey’s Manual of Determinative Bacteriology [7]. 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 the strain C7 are shown in Table 1. On the basis of 16s ribosomal RNA sequence analysis (GenBank accession number AB 24464), the isolate has been identified as B. cereus. From the construction of a phylogenic tree, its nearest homolog species is found to be Bacillus thuringiensis (GenBank accession number EF 210288; Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs12010-009-8543-x/MediaObjects/12010_2009_8543_Fig1_HTML.gif
Fig. 1

Phylogenic tree (using neighbor joining method)

Table 1

Taxonomical characteristics of strain C7.

Parameters

Characteristics

Cellular characteristics

 Morphology

Straight rods, 3.24 × 1.85 μm, occurring single, pair or in short chains, nonmotile

 Staining characteristics

Gram positive, spore former, spores are terminally located

Cultural characteristics

 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

Physicochemical characteristics

 Growth at different temperatures

  45 °C

Moderate

  30 °C

Abundant

  10 °C

Scanty

 Growth at different NaCl concentration

  2%

Abundant

  5%

Scanty

  7%

None

  10%

None

 Growth at different pH

  5.4

Scanty

  6.8

Abundant

  9.6 (with 6.5% NaCl)

Moderate

Biochemical characteristics

 Ammonia from arginine

Positive

 Arginine used as a sole source

Positive

 Nitrate reduced to nitrite

Positive

 Hydrolysis of starch

Positive

 Hydrolysis of urea

Positive

 Catalase test

Positive

 Growth under anaerobic condition with/with out beef extract

Positive

 Voges–Proshaker reaction

Positive

  pH < 6

  pH > 7

 Litmus milk test

Negative

 Indole formation test

Negative

 Carbohydrate fermentation test

Acid formation

Gas production

  Glucose

Positive

Negative

  Fructose

Negative

Negative

  Sucrose

Negative

Negative

  Lactose

Negative

Negative

  Galactose

Negative

Negative

  Xylose

Negative

Negative

  Mannose

Negative

Negative

  Maltose

Positive

Negative

  Starch

Positive

Negative

  Dextrin

Negative

Negative

Inositol

Negative

Negative

Mannitol

Negative

Negative

Sorbitol

Negative

Negative

Salicin

Negative

Negative

Characterization of Crude Enzyme

Effect of Reaction Time

Enzyme substrate reaction was performed at 60 °C and pH 8 for different time period, viz., 5, 10, 15, and 20 min, other conditions remaining the same. Reaction reached its optimum value after 10 min and maximum activity was found to be 33 ± 0.567 IU/ml (Fig. 2). Then, the activity decreased with increase in reaction time.
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Fig. 2

Effect of reaction time on activity of the crude lipase from B. cereus C7

Effect of Temperature

Enzyme substrate reaction was carried out at different temperature, viz., 30 °C, 40 °C, 50 °C, 55 °C, 60 °C, 65 °C, and 70 °C, other conditions remaining the same. Lipase activity increased progressively with increase in temperature and maximum activity was found to be 32 IU/ml at 60 °C (Fig. 3). Then, the activity decreased with increase in temperature. Ghanem et al. in 2000 reported the temperature maxima of 60–65 °C for an alkalophilic thermostable lipase activity from Bacillus alkalophilus [8]. Lipase from Bacillus stearothermophilus P1 and Bacillus sp. RSJ-I also have high temperature optima [9, 10].
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Fig. 3

Optimum temperature and thermostability of crude lipase from B. cereus C7

Effect of pH

pH-activity profile was studied over the pH range of 7.2–9 at 60 °C, using Tris-HCl buffer and over pH range 9–10 using glycine-NaOH buffer, other conditions remaining the same (Fig. 4). The enzyme was active in the pH range 7.6–9 and maximum activity was shown at pH 8.0. There are a few reports of microbial lipases working optimally in the alkaline range of pH [1113].
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Fig. 4

Optimum pH and pH stability of the crude lipase

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

The effect of various metal ions, viz., Ca2+, Mg2+, Zn2+, Cu2+, Mn2+, Co2+, Fe3+, and Sn2+ on enzyme activity, was studied by adding the metal ions at different concentrations (viz., 0.1, 1, and 5 mM) in enzyme–substrate reaction mixture during enzyme assay. Other conditions were as usual. The results were shown in Table 2. Among the tested metal ions, Ca2+ (1 mM), Co2+ (0.1 mM), and Cu2+ (0.1 mM) significantly increased lipase activity. Mg2+ (0.1 mM) and Zn2+ (1 mM) also have stimulatory effect on lipase activity. Mn2+, Fe3+, and Sn2+ ions were found to reduce lipase activity even at 0.1 mM concentration. The negative effect of ions on the lipase is generally the result from direct inhibition of the catalytic site like many other enzymes [14]. A number of lipases produced from other microorganisms were found to be Ca dependent [1518].
Table 2

Effect of various metal ions on the activity of crude lipase.

Salt used

Relative activity (%)

Concentration of metal ions (mM)

0.1

1

5

Control

100

100

100

ZnCl2

131.03

141.5

72.41

CaCl2

138.66

204

132

MgCl2

146.66

100

100

MnCl2

30.05

12

0

CoCl2

212.56

100

38.83

CuCl2

183.03

141.77

105.94

FeCl3

75.03

34.89

0

SnCl2

53.55

26.83

0

Effect of Surfactants and Commercial Detergents

Different surfactants, viz., Triton X 100, Tween 20, Tween 80, Brij 35, Span 40, benzokonium chloride, sodium dodecyl sulfate (SDS), poly(ethylene glycol) (PEG), and cetyltrimethylammonium bromide (CTAB) at the concentration of 0.2% w/v, were added to the enzyme–substrate reaction mixture, other conditions remaining the same. Among the surfactants tested, SDS has little stimulatory effect; Tween 20, Tween 80, Brij 35, Span 40, benzokonium chloride, and PEG have a little inhibitory effect; and Triton X 100 and CTAB have inhibitory effect on lipase activity (Fig. 5).
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Fig. 5

Effect of surfactants and commercial detergents on lipase activity by B. cereus C7

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

Bile salts, viz., cholic acid, sodium deoxycholate, and sodium taurocholate at 0.25% and 0.5% (w/v) concentrations, were added separately during assay of lipase enzyme [5]. Sodium deoxycholate and sodium taurocholate increased the lipase activity while in presence of cholic acid, enzyme retained 90% of its activity (Table 3).
Table 3

Effect of bile salts on crude lipase activity.

Bile salts (0.5% w/v)

Relative activity (%)

Control

100

Cholic acid

90

Sodium taurocholate

120

Sodium deoxycholate

123

There are several reports which show that bile salts are stimulatory to the activity of some microbial lipases [19]. Watanabe et al. [20] reported inhibition of lipase activity in presence of bile salts.

Effect of Oxidizing Agents

The present lipase is highly stable (relative activity 100) toward oxidizing agent, viz., hydrogen peroxide (1% v/v) and sodium hypochlorite (1% w/v) after 1 h incubation (Table 4). Bleach stability is an important property and bleach stable enzymes are not very common. Bleach stability may be attained by site directed mutagenesis [2123] or protein engineering [24, 25]. But the present lipase is inherently stable toward oxidizing agents. Rathi et al. in 2001 [4] studied lipase stability in presence of oxidizing agents like hydrogen peroxide, sodium perborate, and sodium hypochlorite at 1% w/v or v/v. Gulati et al. [26] reported bleach stability of a novel alkaline lipase by Fusarium globulosum to 0.1 M hydrogen peroxide and sodium perborate.
Table 4

Stability of the crude lipase in presence of oxidizing agents.

Oxidizing agents (1% v/v or w/v)

Relative activity (%)

Control

100

Hydrogen peroxide

100

Sodium hypochlorite

100

Effect of Protease

The B. cereus lipase was found to be stable in presence of protease (trypsin 0.5 mg of 1240 IU/mg; Table 5). So, it has an added advantage when used in combination with proteases in detergent formulations. Protease and lipase, both enzymes, are used in detergent formulation, so the lipase used as additive in the detergent must be resistant to protease. Rathi et al. [4] in 2001 reported that Burkholderia cepacia lipase was stable to different alkaline proteases (0.5 mg) from different microbial sources. Gulati et al. [26] reported that F. globulosum lipase exhibited excellent stability in presence of Aspergillus saitoi protease (88% residual activity) and Bacillus licheniformis protease (100% residual activity) even after 1 h incubation.
Table 5

Stability of the crude lipase in presence of protease.

Protease (0.5 mg of 1240 U/mg)

Relative activity (%)

Control

100

Trypsin

100

Effect of Environmental Conditions on Production of Lipase

Lipase production was carried out using different volumes of medium, viz., 40, 50, 60, and 70 ml in 250 ml Erlenmeyer flask, other conditions remaining the same. Samples were collected at 24-h intervals up to 72 h and tested for enzyme activity. Volume of medium and fermentation time had a significant effect on maximum production of lipase (Fig. 6). Maximum lipase production was observed at 24 h when 50 ml medium was used. Effect of initial pH of the culture medium on lipase production was studied using a wide range of pH 7–10 and maximum production was found at pH 8.0 (Fig. 7).
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Fig. 6

Effect of fermentation time and medium volume on production of lipase by B. cereus C7

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Fig. 7

Effect of initial pH on lipase production by B. cereus C7

Lipase production was carried out at different temperatures, viz., 25 °C, 30 °C, 33 °C, 35 °C, and 38 °C. Maximum lipase activity 35 ± 0.826 IU/ml (Fig. 8) was achieved by growing the selected strain at 30–33 °C and 24 h of fermentation. Effects of age of inoculum (range used 20–48 h) and volume of inoculum (range used 0.5–3%) on lipase production were also studied; most suitable conditions were found to be 24 h and 2%, respectively (results not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs12010-009-8543-x/MediaObjects/12010_2009_8543_Fig8_HTML.gif
Fig. 8

Effect of temperature on lipase production by B. cereus C7

Nutritional Factors Affecting Lipase Production

Carbohydrates, a variety of oil and some salts, were used as carbon source for lipase production, other ingredients of the medium and production parameters remaining the same. The results were shown in the Fig. 9. From the experiment, starch was found to be the superior carbon source (enzyme activity 33.5 ± 0.987 IU/ml). Moderate to good amount of lipase activity was obtained with sodium acetate, sodium citrate, fructose, glucose, maltose, xylose, mannitol, sorbitol, and arabinose. Among the different types of oil tested, except rice bran oil and soybean oil, moderate to good amount of lipase activity was observed with other oils. Then, different levels of starch were used for enzyme production and maximum enzyme production was observed at 2% starch.
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Fig. 9

Lipase production by B. cereus C7 using various carbon sources (2% w/v)

Simple nitrogen sources like ammonium nitrate, ammonium chloride, ammonium sulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and sodium nitrate (N2 level 21.2 mg/100 ml) were used along with peptone (N2 level 297 mg/100 ml) (a) in presence of urea (N2 level 46.62 mg/100 ml) and (b) in absence of urea (Table 6). Ammonium sulfate (nitrogen level 21.2 mg/100 ml) along with peptone (N2 level 297 mg/100 ml) and urea (N2 level 46.62 mg/100 ml) having total nitrogen level 364.82 mg/100 ml gave the maximum production of lipase. According to Ferie et al., peptone contains certain cofactors and amino acids, which match the physiological requirements for lipase biosynthesis [27]. Lima et al. found that lipase production in Penicillium aurantiogriseum was stimulated using ammonium sulfate [28]. Urea was found to increase lipase production from a bacterial isolate SJ-15 [29] and a fungus Rhodotolura glutinis [30].
Table 6

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)

Ammonium nitrate

30.6

28.98

Ammonium chloride

27.8

21

Ammonium sulfate

31.95

30.07

Diammonium hydrogen phosphate

23.24

21.25

Ammonium dihydrogen phosphate

19.5

17

Sodium nitrate

24.07

22.1

Acknowledgments

Authors gratefully thank the Council of Scientific and Industrial Research (CSIR) for providing research fellowship and funding.

Copyright information

© Humana Press 2009