The aims of this work were to establish improved conditions for lipase production by
Lipase is an enzyme that catalyzes the hydrolysis and synthesis of esters formed by the linkage of glycerol and long-chain fatty acids. Owing to properties such as catalytic activity over a wide range of temperature and pH, substrate specificity, and enantioselectivity, lipase presents important industrial application [
Lipase can be produced in submerged and solid-state cultivation by many microorganisms such as bacteria, yeast, and filamentous fungi. The utilization of solid-state cultivation (SSC) for enzyme production requires previous evaluation of important aspects, such as selection of a suitable microorganism and substrate, optimization of process parameters, and isolation and further purification of the product [
Several agroindustrial wastes have been evaluated for lipase production by microorganisms. Sugarcane bagasse was used for lipase production by
Many lipase types have been purified and biochemically characterized because their properties are very important for industrial applications. Microbial lipase usually presents high thermostability and pH stability, solvent tolerance, and high specificity for hydrolysis of long-chain unsaturated fatty acids [
Poultry fat is a low cost feedstock, which could be incorporated in delicatessen meats and has a substantial nutritional value due to its high content of unsaturated fatty acids, especially monounsaturated ones, such as oleic acid (45–50%) [
Cultures were performed in Erlenmeyer flasks (250 mL) containing 10 g of wheat bran (WB), cassava peel, barley spent grain (BSG), sugarcane bagasse, or citrus pulp (CP), which were previously washed with distilled water, dried until constant weight, and sieved (18 mesh). Nonsieved BSG and CP were incorporated into wheat bran to improve aeration of the substrates, constituting mixed substrate cultivation. Olive oil (25%, w/w) was used as initial carbon/inducer source to the substrates. Modified Vogel salts solution [
After cultivation 100 mL of distilled water was added to each flask and the mixture was incubated on a rotary shaker (250 rpm, 4°C) for 60 min. Then, the suspension was filtrated through a double layer gauze cloth and centrifuged (8000 ×g, 20 min, 4°C). The clear supernatant was used as source of crude extracellular lipase.
Lipase activity was assayed with p-nitrophenyl palmitate (p-NPP) as substrate [
Protein was determined with Coomassie blue G-250 [
The substrates were supplemented with 25% (w/w) natural triacylglycerols, palm oil, soybean oil, corn oil, canola oil, sunflower oil, linseed oil, and babassu oil, or with wastes such as poultry fat, beef tallow, lard, and cooking oil. Poultry fat used for inducing lipase production was evaluated at 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50% (w/w).
The medium was supplemented with 5% (w/w) corn steep liquor, yeast extract, soy protein, whey powder, and cotton protein. Yeast extract was evaluated at 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0% (w/w).
The effect of temperature on lipase production was verified by carrying out cultivation at 15, 20, 25, 30, 35, and 40°C.
The effect of initial moisture content of the cultures was evaluated by adding Vogel salts [
The crude extract was previously dialyzed against 0.02 M ammonium acetate buffer pH 6.9 (8 h, 3 changes, 4°C). The dialyzed extract was applied to a hydrophobic Octyl Sepharose column (HiPrep™ 16/10 Octyl Sepharose FF fast flow, GE Healthcare) previously equilibrated in the same buffer, at 2 mL/min flow rate. The column was washed with 50 mL of the same buffer and 3.0 mL fractions were collected. Elution of bound proteins was performed with 100 mL of a 0.0 to 1.0% (w/v) Triton X-100 linear gradient prepared in the same buffer. Fractions with lipase activity were pooled and sample purity was evaluated by SDS-PAGE. All purification procedures were carried out at 4°C.
The purified enzyme was previously treated with Calbiosorb™ adsorbent resin (Calbiochem®, San Diego, USA) to remove Triton X-100. The resin was equilibrated in 0.05 M ammonium acetate buffer pH 6.9 and loaded with the purified enzyme. Samples were incubated at 10°C for 45 min under slow stirring and then centrifuged (8500 ×g, 4°C, 20 min). The supernatant containing enzyme was submitted to electrophoresis. Even after treatment, residual Triton X-100 was still detected in the sample by reading absorbance at 280 nm.
SDS-PAGE was performed using 10% (w/v) polyacrylamide gels according to Hames [
Enzyme activity was measured at 37°C in different pH values using 0.05 M glycine-HCl buffer pH from 2.0 to 3.0 and McIlvaine buffer pH from 3.0 to 8.0. Stability to pH was carried out with the same buffers, except in pH from 8.6 to 10.0 in which 0.05 M glycine-NaOH buffer was used. Enzyme samples were (1 : 2, v/v) diluted in each buffer and incubated for 24 h at 10°C.
The optimum temperature was determined by measuring enzyme activity in temperatures from 20 to 70°C, in McIlvaine buffer pH 5.0. For thermal stability, the enzyme was incubated at 40, 45, 50, 55, and 60°C in McIlvaine buffer pH 5.0 in the absence of substrate, and the residual activity was determined in McIlvaine buffer pH 5.0 at 50°C.
The effect of organic solvents on activity and stability of crude and purified lipase was evaluated using 10% (v/v) glycerol, DMSO, propylene glycol, methanol, acetonitrile, ethanol, acetone, 1-propanol, 2-propanol, n-butanol, toluene, xylol, n-hexane, and isooctane. The effect of organic solvents on the activity was verified by adding each solvent into the enzymatic reactions. Stability experiments were carried out in sealed flasks shaken at 200 rpm, for 2 h at 30°C. Residual activities were determined in McIlvaine buffer pH 5.0 at 50°C and expressed in relation to the control without any substance.
Specificity was verified using 0.5 mM p-nitrophenyl acetate, p-nitrophenyl butyrate, p-nitrophenyl octanoate, p-nitrophenyl decanoate, p-nitrophenyl laurate, p-nitrophenyl myristate, p-nitrophenyl palmitate, and p-nitrophenyl stearate by performing enzyme assays in McIlvaine buffer pH 5.0 at 45°C and pH 5.5 at 50°C for the crude and purified lipase, respectively.
The activity of purified lipase was assayed with p-nitrophenyl palmitate from 0.0 to 1.0 mM. The Michaelis-Menten constant (
Hydrolysis of poultry fat was developed at 50°C by titration of released fatty acids. The oils (10%, w/v) were emulsified in McIlvaine buffer pH 4.0, 6.0, and 8.0, containing 0.5% (w/v) Triton X-100. The reaction was started by adding 1 mL of enzyme to 5 mL of this emulsion, and then it was maintained for 96 h at 200 rpm orbital agitation. The reaction was interrupted by adding 16 mL of an acetone : ethanol solution (1 : 1, v/v) to the mixture. The released fatty acids were titrated to pH 11 with a 0.05 M NaOH solution. From these values, the degree of hydrolysis was calculated according to [
The initial rate of reaction was calculated using the following equation:
Solid-state cultivation of
Lipase production by
Substrate | Nonsupplemented | Supplemented with olive oil | ||
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Lipase activity (U/gds) | Specific activity (U/mg prot) | Lipase activity (U/gds) | Specific activity (U/mg prot) | |
Wheat bran |
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Barley spent grain |
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Cassava peel | ND | ND |
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Sugarcane bagasse |
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Citrus pulp |
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WB + BSG (3 : 1) |
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WB + BSG (3 : 2) |
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WB + BSB (1 : 1) |
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WB + CP (3 : 1) |
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WB + CP (3 : 2) |
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WB + CP (1 : 1) |
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Cultures were carried out for 5 days, at 28°C. Substrates were supplemented with 50% Vogel salts solution without nitrogen source. Substrates with olive oil were supplemented with 25% (w/w) of olive oil. WB: wheat bran, BSG: barley spent grain, and CP: citrus pulp.
Lipase production by
Time course of lipase production by
Triacylglycerols are important inducers of lipase production and in this sense palm oil, crude babassu oil, crude linseed oil, canola oil, sunflower oil, corn oil, soybean oil, and also renewable and low cost sources such as poultry fat, lard, beef tallow, and frying oil were evaluated (Figure
Effects of different triacylglycerol sources (a) and poultry fat concentration (b) on lipase production by
Supplementation with different nitrogen sources improved lipase production by
Lipase production by
Nitrogen sources (5% w/w) | Lipase activity (U/gds) | Specific activity (U/mg prot) |
---|---|---|
Control | 31.43 ± 4.91 | 11.64 ± 1.81 |
Yeast extract | 119.91 ± 11.68 | 65.50 ± 6.38 |
Corn steep liquor | 57.96 ± 5.20 | 49.32 ± 5.35 |
Whey powder | 61.00 ± 4.20 | 93.53 ± 6.44 |
Soybean meal | 40.65 ± 4.80 | 24.12 ± 4.03 |
Soy protein | 84.60 ± 4.41 | 76.22 ± 7.62 |
Cotton seed protein | 57.95 ± 2.83 | 66.84 ± 3.72 |
Cultures were carried out on wheat bran plus barley spent grain (1 : 1, w/w), 50% moisture, and 40% (w/w) poultry fat for 5 days, at 28°C. Control was carried out in the absence of nitrogen source.
The effect of different yeast extract concentrations (Figure
Effect of yeast extract concentration on lipase production by
Cultivation in temperatures from 15 to 40°C showed the highest lipase production of 143.4 U/gds and the highest specific activity (65.8 U/mg of protein) at 30°C (Table
Effect of temperature and moisture on lipase production by
Parameter | Lipase activity (U/gds) | Specific activity (U/mg prot) |
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Temperature (°C) | ||
15 | 26.10 ± 4.42 | 13.31 ± 1.92 |
20 | 56.05 ± 6.07 | 22.97 ± 2.43 |
25 | 77.71 ± 3.36 | 28.59 ± 2.71 |
30 | 143.36 ± 9.65 | 65.76 ± 3.18 |
35 | 129.11 ± 8.15 | 43.19 ± 2.98 |
40 | 25.80 ± 6.17 | 10.05 ± 1.56 |
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Moisture (%) | ||
20 | 98.70 ± 4.87 | 78.89 ± 3.47 |
30 | 147.55 ± 8.43 | 111.32 ± 4.55 |
40 | 157.33 ± 9.25 | 136.20 ± 5.74 |
50 | 143.36 ± 7.47 | 113.32 ± 6.40 |
60 | 137.56 ± 6.34 | 74.56 ± 5.70 |
70 | 92.43 ± 5.78 | 60.80 ± 4.85 |
Cultures were carried out using wheat bran plus barley spent grain (1 : 1, w/w), 40% (w/w) poultry fat, and 3.5% (w/w) yeast extract for 5 days (above). Cultures were carried out in the same conditions and at 30°C (below).
The effect of substrate moisture on lipase production revealed optimal production with 40% (v/w) initial moisture (157.3 U/gds), in which the highest specific activity (136.2 U/mg of protein) was also observed. Cultures with 30 or 50% initial moisture resulted in intermediate lipase production of 147.5 and 143.4 U/gds, respectively.
In a previous study, the crude extract obtained in cultivation under optimal conditions was subjected to ammonium sulfate precipitation. In this step, aggregation of proteins was observed obtaining inconsistent results after four attempts of precipitation using different salt concentrations (data not shown). Then, the crude extract was used to select a resin for hydrophobic chromatography. It was observed that the lipase adsorbed in phenyl and Octyl Sepharose without ammonium sulfate; then hydrophobic chromatography was carried out using Octyl Sepharose column without previous salt equilibrium. The crude extract was subjected to dialysis against 0.02 M ammonium acetate buffer pH 6.9 and applied to hydrophobic column equilibrated in the same buffer (Figure
Purification of
Purification step | Enzyme activity (U) | Protein total (mg) | Specific activity (U/mg of protein) | Enrichment | Yield (%) |
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Crude extract | 550.02 | 193.63 | 2.84 | 1.00 | 100.00 |
Octyl Sepharose 4CL | 465.02 | 3.48 | 133.63 | 47.05 | 84.55 |
Profile of hydrophobic interaction chromatography of
SDS-PAGE of purified lipase from
The effect of pH on the activity of crude and purified lipase was determined from 2.0 to 9.0 (Figure
Optimum pH (a) and pH stability (b) and optimum temperature (c) and thermal stability (d) of crude and purified
The effect of temperature on lipase activity was evaluated from 20 to 70°C (Figure
Half-lives of crude and purified lipase from
Temperature (°C) | Half-life (h) | |
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Crude lipase | Purified lipase | |
40 | n.d. | 6.7 |
45 | n.d. | 4.2 |
50 | 23.5 | 0.9 |
55 | 1.67 | 0.3 |
60 | 0.25 | 0.3 |
n.d.: not detected after 24 hours of incubation.
The effect of organic solvents on lipase activity is shown in Table
Effect of organic solvents on crude lipase stability produced by
Organic solvent |
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Crude enzyme | Purified enzyme | ||
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Relative activity (%) | Stability (%) | Relative activity (%) | Stability (%) | ||
Control | — | 100.0 ± 1.2 | 100.0 ± 2.0 | 100.0 ± 2.4 | 100.0 ± 3.2 |
Glycerol | −1.67 | 135.4 ± 4.2 | 99.5 ± 2.6 | 109.4 ± 5.7 | 60.2 ± 2.0 |
DMSO | −1.38 | 111.7 ± 4.0 | 99.8 ± 3.0 | 113.5 ± 2.5 | 59.5 ± 3.4 |
Propylene glycol | −0.92 | 111.5 ± 3.1 | 88.7 ± 3.5 | 97.5 ± 7.7 | 60.1 ± 3.4 |
Methanol | −0.76 | 105.1 ± 3.3 | 84.6 ± 1.9 | 103.2 ± 7.5 | 52.9 ± 6.1 |
Acetonitrile | −0.40 | 86.2 ± 1.3 | 68.1 ± 3.9 | 88.6 ± 8.2 | 58.6 ± 2.8 |
Ethanol | −0.24 | 106.2 ± 2.2 | 90.5 ± 4.8 | 106.8 ± 6.1 | 94.5 ± 2.1 |
Acetone | −0.23 | 90.5 ± 2.2 | 66.7 ± 2.4 | 85.8 ± 6.8 | 69.2 ± 1.8 |
1-Propanol | 0.07 | 97.9 ± 2.5 | 72.4 ± 1.3 | 111.5 ± 9.9 | 66.9 ± 1.8 |
2-Propanol | 0.25 | 78.3 ± 2.6 | 56.0 ± 2.4 | 83.0 ± 4.3 | 59.2 ± 1.4 |
n-Butanol | 0.80 | 92.0 ± 4.0 | 12.1 ± 2.6 | 84.1 ± 9.9 | 58.3 ± 3.5 |
Toluene | 2.50 | 91.1 ± 3.3 | 69.8 ± 1.9 | 87.2 ± 5.3 | 25.0 ± 2.6 |
Xylol | 3.15 | 65.9 ± 1.4 | 51.5 ± 1.3 | 82.0 ± 7.4 | 39.1 ± 4.2 |
n-Hexane | 3.50 | 105.9 ± 2.5 | 98.0 ± 4.7 | 98.5 ± 8.6 | 65.2 ± 3.1 |
Isooctane | 4.51 | 90.4 ± 2.4 | 95.1 ± 3.4 | 91.4 ± 7.8 | 61.8 ± 2.0 |
Assay conditions: for stability assays crude and purified lipase were incubated at 30°C at 200 rpm during 2 h and the activity was assayed with p-NPP using McIlvaine buffer pH 5.0, at 50°C.
The stability of crude lipase after 2 h incubation at 30°C was high in media containing DMSO, glycerol, n-hexane, isooctane and ethanol (more than 90%), and propylene glycol (~89%). Intermediate stability was observed with methanol, 1-propanol, acetonitrile, toluene, acetone, 2-propanol, and xylol. Butanol sharply decreased the lipase activity. The purified lipase presented high stability in ethanol (94%). Intermediate values were observed with acetone, 1-propanol, n-hexane, isooctane, glycerol, propylene glycol, DMSO, 2-propanol, acetonitrile, n-butanol, and methanol (69–53%, resp.). Lower stability was observed with xylol (39%) and toluene (25%).
Substrate hydrolysis reactions were performed for purified lipase with p-NPP (0.0 to 1.0 mM) to determine
Hydrolytic activity of crude and purified lipase was evaluated on p-nitrophenyl ester substrates (Figure
Activity of crude and purified
The hydrolysis of poultry fat and initial hydrolysis rate in different pH values are shown in Figure
Parameters of poultry fat hydrolysis using crude
pH |
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Crude enzyme | ||
4.0 | 33.17 | 0.905 |
6.0 | 18.75 | 0.812 |
8.0 | 3.60 | 0.838 |
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Purified enzyme | ||
4.0 | 7.20 | 0.720 |
6.0 | 6.50 | 0.733 |
8.0 | 1.91 | 0.754 |
Poultry fat hydrolysis profiles by crude (a) and purified (b)
Solid-state cultivation for lipase production using yeast cells is a nonconventional practice since the access to nutrients is a limiting factor for this group of microorganisms due to the absence of well-developed hyphae and enzymes production capable of releasing soluble monomers or dimers for microbial nutrition and growth. Substrates such as wheat bran, barley spent grain, cassava peel, sugarcane bagasse, and citrus pulp used for
Mixed substrates formulation of wheat bran plus barley spent grain or wheat bran plus citrus pulp supplemented with olive oil increased the enzyme production by 322 and 174%, respectively, in relation to individual wheat bran, which may be related to an increase in spatial distribution or/and larger surface contact between yeast and substrate during microbial colonization. Benjamin and Pandey [
Supplementation of substrates with renewable triacylglycerol such as 25% poultry fat instead of olive oil increased the lipase production to 132%, and at 40% it was increased to 171.2%. This is an important characteristic since olive oil is the most usual inducer in many submerged processes due the high level of oleic acid in its fatty acids composition [
Although wheat bran and barley spent grain can provide high quantity of protein, the supplementation with organic nitrogen sources may be better accessed resulting in higher enzyme production. Yeast extract supplementation increased lipase production to 381.5% and when it was employed at 3.5% (w/w) enzyme production increased to 456.9%. Nitrogen sources play an important role in the synthesis of lipase; organic nitrogen sources supply cells with growth factors and amino acids, which are required for cell metabolism and enzyme synthesis [
Temperature and moisture are environmental factors that affect microbial growth and enzyme production. Optimum temperature for lipase production by
The lipase produced by solid-state fermentation under previous established conditions was purified using hydrophobic interaction chromatography. The procedure was simple involving only one chromatographic step, which did not require ammonium sulfate. Similarly, Bastida et al. [
The purified lipase presented molecular mass of 18.5 kDa,
Biochemical characterization of crude and purified lipase is important for further industrial applications in hydrolysis or synthesis reactions. Crude lipase from
The crude lipase was clearly more thermally stable than the purified lipase. Half-lives of the purified lipase were 26.1- and 5.6-fold decreased at 50 and 55°C, respectively. This finding is also observed for the crude and partially purified pectinolytic enzymes from
Lipase stability in organic solvents is an essential prerequisite for its application in organic synthesis, since synthetic reactions with enzymes are often performed in organic media to displace the thermodynamic equilibrium towards synthesis [
Substrate specificity is important for many industrial applications in food industry and biodiesel production. The crude and purified lipase from
Hydrolysis of triacylglycerol is an important industrial operation; the products, fatty acids and glycerol, are basic raw materials with wide range of applications. The fatty acids are used as feedstock for the production of oleochemicals such as fatty alcohols, fatty amines, and fatty esters. In this study, poultry fat was subjected to hydrolysis using crude and purified lipase produced by
The alkaline hydrolysis is the most important current route used for triacylglycerol hydrolysis and it requires acidification of the formed soaps to obtain fatty acids [
This study demonstrated that the lipase from
The authors declare that they have no competing interests.
The authors would like to thank the National Council of Technological and Scientific Development (CNPq) for the scholarship awarded to the first author and financial support (Process no. 455754/2014-4), CAPES for the scholarship awarded to K. B. Dias, and UNESP for payment of this article publication.