Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

A thermostable lipase produced by a newly isolated <Emphasis Type="Italic">Geotrichum</Emphasis>-like strain,R59

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml^–1) was obtained with urea as the nitrogen source. Growth at 30°C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60°C.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

An inducible,intracellular, alkalophilic lipase inAspergillus flavipes grown on triacylglycerols

An intracellular lipase was induced inAspergillus flavipes grown on various triacylglycerols at pH 6.0 and at 30°C, with maximum activity with sunflower oil. The lipase had an optimum pH for activity of 8.8 and retained 30% of its activity at pH 10.0. It had an optimum temperature for activity, measured over 30 min, of 45°C. It was completely inactivated at 60°C within 10 min.     

A hyper-thermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation

A hyper-thermostable, alkaline lipase from a newly-isolated, mesophilic Pseudomonas sp. was optimal at pH 11 and at 90 °C. It had a half-life of more than 13 h at 90 °C. It was activated by 30% when heated at 90 °C for 2 h. The enzyme had a greater affinity for mustard oil (K _m=40 mg ml^–1) than for olive oil (K _m=140 mg ml^–1).     

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.     

Extracellular lipase production by a sapwood-staining fungus,Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH₄)₂SO₄ gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH₄)₂SO₄and peptone as nitrogen source.The authors are with Forest Products Biotechnology, Department of Wood Science, Facully of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties

Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO₄.7H₂O, 0.05% KCl, 0.2% K₂HPO₄ and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme retained more than 90% of its activity. Enzyme activity was inhibited by Hg²⁺ and K⁺, whereas Ca²⁺ and Mn²⁺ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

Effects of oils and oil-related substrates on the synthetic activity of membrane-bound lipase from Rhizopus chinensis and optimization of the lipase fermentation media

The lipase from filamentous fungi Rhizopus chinensis, as a membrane-bound enzyme, possesses the excellent catalysis ability for esterification and transesterification reactions, and has a good potential in many industrial applications. In order to improve the synthetic activity of the lipase, the effects of oils and oil-related substrates on its production and the fermentation media optimization were investigated. Based on the results, it was suggested that oleic acid could be the important substrate for the lipase production. Among various oils and oil-related substrates, olive oil containing high content of oleic acid was the optimal one for the lipase production. Using orthogonal test and response surface methodology (RSM), the composition of fermentation media was further optimized. The optimized media for lipase synthetic activity and activity yield was composed of peptone 57.94 and 55.58 g L⁻¹, olive oil 21.94 and 22.99 g L⁻¹, maltose 12.91 and 14.34 g L⁻¹, respectively, with K₂HPO₄ 3 g L⁻¹, MgSO₄·7H₂O 5 g L⁻¹ and initial pH 6.0. Under the optimal conditions, the lipase activity and the activity yield were improved 61.5 and 93.4% comparing the results before optimization, respectively. The adequate models obtained had predicted the lipase production successfully.     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

Purification of lipase from Geotrichum candidum: conditions optimized for enzyme production using Box–Behnken design

The fungus Geotrichum candidum was selected from isolates of oil-mill waste as a potent lipase producer. Factors affecting lipase production by the fungus G. candidum in yeast-extract-peptone medium have been optimized by using a Box–Behnken design with seven variables to identify the significant correlation between effects of these variables in the production of the enzyme lipase. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9957. It was observed that the variables days (6), pH (7.0), temperature (30 °C), carbon (1.25%), nitrogen (2.0%), Tween (1.0%) and salt concentrations (0.5 mM) were the optimum conditions for maximum lipase production (87.7 LU/ml). The enzyme was purified to homogeneity with an apparent molecular mass of 32 kDa by SDS-PAGE. The optimum pH at 40 °C was 7.0 and the optimum temperature at pH 7.0 was 40 °C. The enzyme was stable within a pH range of 6.5 to 8.5 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, and EDTA. However, the presence of Ca²⁺ and Ba²⁺ ions enhanced the activity of the enzyme.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

Biochemical and molecular characterisation of a thermoactive, alkaline and detergent-stable lipase from a newly isolated Staphylococcus aureus strain

A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180 kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part of the gene encoding the mature SAL3 is cloned and sequenced. The deduced polypeptide sequence, corresponding to the mature SAL3, was very similar to the mature Staphylococcus simulans lipase sequence with two additional amino acid residues (LK) at the N-terminus of SAL3. The lipase activity is maximal at pH 9.5 and 55 °C. The specific activity of about 4200 U/mg or 3500 U/mg was measured using tributyrin or olive oil emulsion as substrate, respectively, at pH 9.5 and 55 °C.In contrast to other staphylococcal lipases previously characterised, SAL3 is found to be stable between pH 5 and 12 after 24 h incubation. The enzyme retained 50% of its activity after 60 min incubation at 60 °C. This novel lipase is able to hydrolyse its substrate in presence of various oxidizing agents as well as some surfactants and some commercial detergents, then SAL3 can be considered as a good candidate for industrial and biotechnological applications.     

Catalytic properties of mycelium-bound lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> MYA 135

A constitutive level of a mycelium-bound lipolytic activity from Aspergillus niger MYA 135 was strongly increased by 97% in medium supplemented with 2% olive oil. The constitutive lipase showed an optimal activity in the pH range of 3.0–6.5, while the mycelium-bound lipase activity produced in the presence of olive oil had two pH optima at pH 4 and 7. Interestingly, both lipolytic sources were cold-active showing high catalytic activities in the temperature range of 4–8°C. These mycelium-bound lipase activities were also very stable in reaction mixtures containing methanol and ethanol. In fact, the constitutive lipase maintained almost 100% of its activity after exposure by 1 h at 37°C in ethanol. A simple methodology to evaluate suitable transesterification activities in organic solvents was also reported.     

Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9′

Among 97 fungal strains isolated from soil collected in the arctic tundra (Spitsbergen), Penicillium chrysogenum 9 was found to be the best lipase producer. The maximum lipase activity was 68 units mL^–1 culture medium on the fifth day of incubation at pH 6.0 and 20°C. Therefore, P. chrysogenum 9 was classified as a psychrotrophic microorganism. The non-specific extracellular lipase showed a maximum activity at 30°C and pH 5.0 for natural oils or at pH 7.0 for synthetic substrates. Tributyrin was found to be the best substrate for lipase, among those tested. The K_m and V_max were calculated to be 2.33 mM and 22.1 units mL^–1, respectively, with tributyrin as substrate. The enzyme was inhibited more by EDTA than by phenylmethylsulfonyl fluoride and was reactivated by Ca²⁺. The P. chrysogenum 9 lipase was very stable in the presence of hexane and 1,4-dioxane at a concentration of 50%, whereas it was unstable in presence of xylene.     

Formation of lipolytic enzymes by Brevibacterium linens

When Brevibacterium linens ATCC 9172 was grown in shake flasks, it produced a cell-associated lipase with a specific activity of 152 to 188 U g^–1 cells depending on the composition of the growth medium. There was no growth in media containing tributyrine as the sole carbon source. The cell-associated lipase had maximum activity at pH 8.0 and 37 °C and was strongly inhibited by 3,4-dichloroisocoumarin, an inhibitor specific for serine esterases. Cell-associated activity was released from the cells by treatment with lysozyme. The kinetics of lipase formation was closely related to the amount of biomass formed during growth.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.