A newly high alkaline lipase: an ideal choice for application in detergent formulations
© Cherif et al; licensee BioMed Central Ltd. 2011
Received: 27 October 2011
Accepted: 28 November 2011
Published: 28 November 2011
Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described.
A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1) were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents.
These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations.
Lipases (EC 126.96.36.199) represent an important group of biotechnologically valuable enzymes [1–3]. They are widely distributed in nature. Although lipases have been found in many species of animals, plants, bacteria, yeast, and fungi, the enzymes from microorganisms are the most interesting because of their potential applications in various industries such as food, dairy, pharmaceutical, detergents, textile, biodiesel, and cosmetic industries and in synthesis of fine chemicals, agrochemicals, and new polymeric materials [4–6]. Detergent industries are the primary consumers of enzymes, in terms of both volume and value . The use of enzymes in detergents formulations enhances the detergents ability to remove tough stains and making the detergent environmentally safe. Nowadays, many laundry-detergent products contain cocktails of enzymes including proteases, amylases, cellulases, and lipases . As a detergent additive, the increasing usage of alkaline lipase is mainly due to its affiliation with the nonphosphate detergents. Ideally, alkaline lipases in a detergent should have high activity and stability over a broad range of temperature and pH, and should also be compatible with different components in a detergent including metal ions, surfactants and oxidants . Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments. Many staphylococci produce lipases which are released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Also, practical applications of staphylococcal enzymes may be limited due to relatively lower stabilities and catalytic activities under conditions that characterise industrial processes: high temperatures, extremes of pH values or non-aqueous solvents. In the past years, intense efforts have been focused on the engineering of enzymes with altered properties or better performance for practical applications. Therefore, screening of new microorganisms with lipolytic activities could facilate the discovery of novel lipases. Recently we isolated and optimized the production of lipase from a newly staphylococcus sp strain ESW (unpublished data). After optimization of culture conditions and medium composition, biochemical properties of crude lipase were investigated. Within this context, we report the characterisation of a thermoactive, alkaline and detergent-stable lipase (SL1) from a newly isolated staphylococcus sp strain ESW, and investigate its compatibility with various surfactants, oxidizing agents, commercial liquid and solid detergents to evaluate its potential for detergent formulation.
Tributyrin (99%, puriss) and benzamidine were from Fluk (Buchs, Switzerland); tripropionin (99%, GC) was from Jansen (Pantin, France); phosphatidylcholine, sodium deoxycholic acid (NaDC), sodium taurodeoxycholic acid (NaTDC), Tween 80, yeast extract and ethylene diamine tetraacetic acid (EDTA) were from Sigma Chemicals (St. Louis, USA); β-mercaptoethanol was from Merck (Darmshtadt, germany); all other detergents used (Ariel, Axion and Omino Bianco) were purchased locally; gum Arabic was from Mayaud Baker LTD (Dagenham, United Kingdom); pH-stat was from Metrohm (Zofingen, Switzerland).
Screening of lipolytic microorganisms
Initial screening of lipolytic microorganisms from various biotopes was carried out using a plate assay in a medium containing triacylglycerol and the fluorescent dye Rhodamine B [10, 11]. The solid medium contains 1‰ olive oil, 1% nutrient broth, 1% NaCl, 1.5 g agar and 1‰ Rhodamine B. The culture plates were incubated at 37°C, and colonies giving orange fluorescence halos around them, upon UV irradiation, were regarded as putative lipase producers . After extensive screening of lipase producers, only one bacterial colony, isolated from an hydrocarbure contaminated soil continued to give a positive signal when commercial detergent (1%) was added to the solid medium described above. The identification of this strain has been kindly determined by Dr. Abdelhafedh Dhouib (Centre de biotechnologie de Sfax, Tunisia). The biochemical properties and the morphological aspect of this microorganism showed 100% identity to Staphylococcus strain.
Media and culture conditions
Staphylococcus sp. was incubated overnight at 37°C and 200 rpm in 1-liter-shaking flasks with 100 mL of Luria-Bertani broth medium composed of (g/L): peptone, 10.0; yeast extract, 5.0; NaCl, 5.0; 1% olive oil; pH 7.0. (medium A). Overnight Staphylococcus sp. cultures used as inocula were cultivated in 1-liter shaking flasks with 100 ml of the medium A supplemented with 1% olive oil (medium B). The culture was incubated aerobically during 36 h on a rotary shaker set at 160 rpm and at a temperature of 37°C. The cultures were centrifuged at 12 000 rpm for 15 min at 4°C, and the cell-free supernatants were used for estimation of lipase activity. Growth was followed by measuring the cultures optical density (OD) at 600 nm.
Lipase activity determination
The lipase activity was measured titrimetrically at pH 12 and 60°C with a pH-stat under standard conditions using tributyrin (0.25 mL) in 30 ml of 2.5 mM Tris-HCl pH 12, 2 mM CaCl2, 1 mM NaDC or olive oil emulsion (10 mL in 20 mL of 9‰ NaCl pH 12, 2 mM CaCl2, 2 mM NaDC) as substrate. Lipase activity was also measured at pH 7 and 37°C using TC3 as substrate (0.25 mL TC3) in 30 mL of 2.5 mM phosphate buffer pH 7, 2 mM CaCl2. The olive oil emulsion was obtained by mixing (3 × 30 s in a Waring blender) 10 mL of olive oil in 90 ml of 10% GA. When measuring SL1 lipase activity in the absence of CaCl2, EDTA or EGTA was added to the lipolytic system. Lipolytic activity was expressed as units. One unit corresponds to 1 μmol of fatty acid released per minute.
Determination of substrate specificities
Activity of the crude lipase towards different triacylglycerols was determined by pH-stat assay under optimal conditions (pH 12.0 and 60°C). The triacylglycerols triacetin (TC2), tripiopionin (TC3), tributyrin (TC4), trioctanoin (TC8), and triolein (C18) at a final concentration of 10 mM. The triolein was emulsified immediately before use in 10% gum Arabic solution as described previously .
Effect of pH and temperature on SL1 activity and stability
SL1 activity was tested in various buffers at different pH (5-13) at 60°C. The pH stability of the lipase was determined by incubating the enzyme at different pH (3-12) for 24 h at room temperature. The residual activity was determined, after centrifugation, under standard assay method .
The optimum temperature for the SL1 activity was determined by carrying out the enzyme assay at different temperatures (25-65°C) at pH 12. The effect of temperature on lipase stability was determined by incubating the enzyme solution at different temperatures (30-60°C) for 60 min. The residual activity was determined, after centrifugation, under standard assay method.
Effects of metal ions on enzyme activity
The effect of various metal ions on lipase activity was investigated by adding divalent metal ions (Ca2+, Mn2+, Zn2+, Cu2+, Ba2+, Mg2+) to the reaction mixture. The activity of the crude enzyme without metallic ions was considered as 100%.
Effect of surfactants and detergents on enzyme stability
The suitability of Staphylococcus sp crude enzyme as a detergent additive was determined by testing its stability in the presence of some surfactants such as SDS (sodium dodecyl sulphate), Triton X-100, Tween 20, and oxidizing agents such as hydrogen peroxide (H2O2) and sodium perborate (NaBO3). Crude enzyme containing alkaline lipase, at 15 U/mL was incubated with different additives for 1 h at 40°C and then the residual enzyme activities were determined under standard assay conditions. The activity of the crude enzyme, incubated under similar conditions without any additive was taken as 100%. The compatibility of the ESW enzymatic preparation with commercial solid and liquid laundry detergents was also studied. The solid detergents tested were Dixan (Henkel, Spain), Nadhif (Henkel-Alki, Tunisia), Ariel (Procter and Gamble, Switzerland) and Axion (Colgate-Palmolive, France). The liquid detergents tested were Dixan (Henkel, Spain), Nadhif (Henkel-Alki, Tunisia) and Lav+ (Best LAV, Tunisia). Solid detergents were diluted in tap water to give a final concentration of 5 mg/l and liquid detergents were diluted 100-fold to simulate washing conditions. The endogenous enzymes contained in these detergents were inactivated by heating the diluted detergents for 30 min at 80°C prior to the addition of the ESW crude enzyme. Crude enzyme containing alkaline lipase, at 15 U/mL, was added to solid detergents diluted in tap water and incubated in various detergent solutions for 1 h at different temperatures, and then the residual enzyme activity was determined under standard assay conditions. To allow further comparison, the effect of surfactants, commercial detergents and oxidizing agents on a commercial lipase stability (Lipolase®, marketed by Novo Nordisk, Denmark), was also studied under the same experimental conditions. The enzyme activity of the control sample (without any detergent), incubated under the same conditions, was taken as 100%.
Determination of protein concentration
Protein concentration was determined as described previously by Bradford  using bovine serum albumin (BSA) as the standard.
All results are expressed as the mean ± standard deviation (± SD). The experiment was conducted at least 3 times, and each treatment had 3 replicates. Thus, for most data points, the n = 3. The SAS System for Windows, V8 (SAS Institute, Gary, NC) was used for statistical evaluations. Means ± S.D. were calculated for normalizing the control as 100%. Differences among treatment and control groups were tested by one-way analysis of variance (ANOVA), followed by pair-wise comparisons between groups using Tukey's test. Differences at p < 0.05 were considered significant.
Results and Discussion
Production of lipase
The medium B (100 mL) was incubated with different amounts of inoculum from the overnight Staphylococcus sp. culture. The maximum lipase production (15 U/mL of culture medium) was obtained after 30-h incubation, with an initial absorbance (OD) measured at 600 nm of 0.2 and an inoculum size of 3 × 108 cells/L. Our results show that the time course of lipase production followed at 37°C with cell growth. The lipase activity was observed to start soon after incubation and reached the maximum (30 U/mL) at the end of the exponential phase corresponding to 30 h of cultivation (data not shown). Lipases are generally produced using carbon source such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source . In fact, the production of SL1 is induced by the presence of long chain triacylglycerols (like olive oil).
Interfacial activation of SL1
Effects of pH and temperature on SL1 activity and stability
Effects of calcium and other metal ions on SL1 activity
Effect of some metal ions (2 mM) on alkaline lipase activity of Staphylococcus sp ESW.
Relative lipase activity (%)
120 ± 1.5
108 ± 1.0
110 ± 1.7
100 ± 1.5
100 ± 1.0
100 ± 1.2
100 ± 1.0
Effect of surfactants and oxidizing agents on SL1 stability
Stability of alkaline lipase of Staphylococcus sp ESW in the presence of various detergent components.
Residual activity (%)
100 ± 1.0
100 ± 1.0
100 ± 1.5
80 ± 1.0
100 ± 1.7
60 ± 1.0
92 ± 1.2
40 ± 1.2
90 ± 1.0
80 ± 1.0
85 ± 1.5
70 ± 1.5
80 ± 1.9
65 ± 1.0
80 ± 1.1
60 ± 1.0
80 ± 1.5
60 ± 1.9
Stability of SL1 with commercial solid and liquid detergents
This work describes the characterization of the crude enzymatic preparation containing a lipase produced by a novel Staphylococcus sp strain. The lipase preparation shows a high activity and stability in high alkaline pH and high temperatures. It showed stability not only towards the non-ionic surfactants like Triton X-100 and Tween 20, but also towards the strong anionic surfactant, SDS and oxidizing agents. Furthermore, the crude enzyme exhibited a high stability in the presence of various commercial liquid and solid laundry detergents. Considering its promising properties, one can say that SL1 can be considered as a potential candidate to be used as in biotechnology and essentially for application in the detergent industry.
This work is part of a post-doctoral thesis by Slim Cherif. This work received financial support from "Ministère de l'enseignement supérieur et de la recherche et de la technologie" granted to the « Laboratoire des Bioprocédés Environnementaux, pôle d'excellence régional (PER, AUF), Centre de Biotechnologie de Sfax, Tunisie.
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