Olive-mill wastewaters: a promising substrate for microbial lipase production

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).     

Production and regulation of different lipase activities from Rhizopus chinensis in submerged fermentation by lipids

The fungal Rhizopus chinensis could produce several types of lipase, which were mainly intracellular. During the whole-cell lipase production by this strain in submerged fermentation, it was observed that two catalytic characteristics (hydrolytic and synthetic activity) of lipases were different with addition of lipids. The hydrolytic activity of the lipase was not induced by lipids efficaciously and could be detected regardless of whether substrate-related compounds were present. However, it was found that the induction of lipids for the synthetic activity lipase was significant, and that nearly no synthetic activity was detected while the medium contained no lipids. When only a little lipid (1 g/L) was added to medium, the synthetic activity increased sharply in the initial process of fermentation. Analysis of crude membrane-bound lipase by SDS-PAGE confirmed this induction. De novo biosynthesis of lipases, especially the lipase with synthetic activity occurred only when lipids existed. Cell growth and maltose repress the lipase production with synthetic activity, but have little influence on the lipase production with hydrolytic activity. Since the production process of mycelium-bound lipase with hydrolytic activity was different, it was reasonable to consider hydrolytic activity and synthetic activity for different application purposes. Whole-cell lipase obtained from fermentation process with high synthetic activity showed excellent catalytic ability in solvent free system on synthesis of ethylcaprylate and ethyloleate, the conversion could reach more than 90% in 5 h.     

Stability studies and effect of the initial oleic acid concentration on lipase production by Candida rugosa

The production of lipase by Candida rugosa in batch cultures was studied. The initial concentration of the carbon source employed, oleic acid, had an important effect on the final lipolytic activity levels. The maximum lipase/substrate yield and specific productivity obtained correspond to an initial oleic acid concentration of 2 g/l. At higher concentrations, up to 8 g/l oleic acid, specific productivity decreased. Lipase production was not observed below 1 g/l oleic acid. Lipase inactivation in culture broth due to surface forces and shear stress at the gas/liquid interface was not observed. There was no shear stress denaturation at stirring rates of 250, 500 and 750 rpm. No temperature inactivation was detected up to 50° C. Two different lipases with a similar molecular weight of 60kDa were purified from culture broth.     

Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Mass production of lipase by fed-batch culture of Pseudomonas fluorescens

Summary High concentration production of an extracellular enzyme, lipase, was achieved by a fed-batch culture of Pseudomonas fluorescens. During the cultivation, temperature, pH and dissolved oxygen concentration wwre maintained at 23°C, 6.5 and 2–5 ppm, respectively. Olive oil was used as a carbon source for microbial growth. To produce lipase effectively the specific feed rate of olive oil had to be maintained in a range of 0.04–0.06 (g oil) · (g dry cell)^-1 · h^-1. The CO₂ evolution rate was monitored to estimate the requirement of olive oil. The ratio of feed rate of olive oil to the CO₂ evolution rate was varied in the range of 20–60 g oil/mol CO₂. The higher value of the ratio accelerated microbial growth, but did not favour lipase production. Once the high cell concentration of 60 g/l had been achieved, the ratio was changed from 50 to 30 g oil/mol CO₂ to accelerate the lipase production. By this CO₂-dependent method a very high activity of lipase, 1980 units/ml, was obtained. Both the productivity and yield of lipase were prominently increased compared with a conventional batch culture.     

Production of an extracellular lipase byBeauveria bassiana

Summary The entomopathogenic fungus,Beauveria bassiana, produces an extracellular lipase when grown on a yeast extract-peptone-dextrose broth (YPD) medium. The time course of lipase production in the presence of olive oil was studied and which was shown to induce lipase. The addition of fatty acids, such as, myristic, palmitic, stearic, oleic, linoleic and arachidic acids, inhibited both growth and lipase production. Lipase production was also assessed on YPD and glucose minimal salts (GMS) medium. The addition of olive oil increased the lipase induction much more on, YPD than on the GMS. The effect of the divalent metal ions; iron, copper and magnesium, on lipase activity was studied. Whereas the iron and copper inhibited lipase activity, magnesium slightly increased lipase activity. Compounds containing a hydrolyzable ester group, such as Tweens, were found to inhibit lipase activity.     

Optimization of extracellular lipase production from the biocontrol fungus Nomuraea rileyi

Lipases are important cuticle-degrading enzymes that hydrolyze the ester bonds of waxes, fats and lipoproteins during the infection of insects by the fungus Nomuraea rileyi. Lipase production by the N. rileyi strain MJ was optimized by varying environmental and nutritional conditions in culture medium containing different vegetable oils at various concentrations with shaking at 150 rpm for 8 days at 25°C. The maximum lipase production was obtained using castor oil (30.5±0.6 U mL^?1), followed in order by coconut oil (20.8±0.4 U mL^?1), olive oil (20.8±0.4 U mL^?1) and cottonseed oil (20.6±0.4 U mL^?1). The highest lipase activity (37.7±0.4 U mL^?1) was obtained when castor oil was used at a concentration of 4% (v/v) of basal medium. When the surfactant Tween 80 was added at the fourth day rather than at the beginning of incubation, a maximum lipase activity of 44.9±3.5 U mL^?1 was obtained. The optimal temperature and pH for lipase production were 25°C and pH 8.0, respectively. This is the first report on lipase production by the biocontrol fungus N. rileyi.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Effects of different fatty acids in lipase production by Candida rugosa

Summary Oleic acid has been reported as a good inducer of lipase production by Candida rugosa. In order to know if this enzyme is induced by oleic acid itself or by a metabolite, different short chain fatty acids were tested. Butyric acid was the best carbon source to growth microorganism but it did not induce lipase production. Although caprylic and capric acid were the best inducers of lipase production, at concentrations up 1 g/l they have toxic effect in Candida rugosa growth. Thus, from the point of view of industrial production oleic acid could be considered as the best substrate tested.     

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.     

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.     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

Physiology of lipase formation inPenicillium candidum

Penicillium candidum grew and produced lipase in a culture medium supplemented with 0.2% olive oil. Significant enzyme production required the presence of olive, oil and was prevented by cycloheximide. Polyacrylamide gel electrophoresis of filtrates from olive oil fermentations gave a single band of lipase activity (MW 80 KDa). Among the olive oil components only oleate allowed significant lipase production. Other carboxylic and saturated fatty acids containing similar or lower numbers of carbon atoms, did not cause derepression of lipase formation.     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.     

Production and properties of an alkaline,thermophilic lipase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> NS2W

Eighteen bacterial strains were isolated from soil samples and screened for alkaline, thermophilic lipase production. Pseudomonas fluorescens NS2W was selected and its production of lipase was optimized in shake flasks using a statistical experimental design. Cell growth and lipase production were studied in shake flasks and in a 1-l fermenter in the optimized medium. Maximum lipase yields were 69.7 and 68.7 U ml⁻¹, respectively. The optimized medium resulted in about a five-fold increase in the enzyme production, compared to that obtained in the basal medium. The lipase had an optimal activity at pH 9.0 and was stable over a wide pH range of 3–11 with more than 70% activity retention. The lipase had an optimal activity at 55°C and was stable up to 60°C with more than 70% activity retention for at least 2 h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 344–348 DOI: 10.1038/sj/jim/7000254 Received 06 September 2001/ Accepted in revised form 15 March 2002     

华根霉合成活性脂肪酶液态发酵菌体形态调控的研究

华根霉(CCTCC M201021)膜结合脂肪酶在非水相中具有突出的催化酯合成的能力,在生物香料、生物柴油生产等工业应用中具有良好的前景.与许多丝状真茵形态影响其初级及次级代谢产物的生产相类似,华根霉在液态发酵中也会形成不同的茵体形态,显著影响合成活性膜结合脂肪酶的发酵水平.本研究以华根霉菌体形态及脂肪酶合成活性为指标...     

Carbon source impact on Yarrowia lipolytica KKP 379 lipase production

The production of lipases by microorganisms is strongly influenced by the culture conditions. The optimum culture conditions for enzyme production are strain- and species-dependent. The aim of this study was to evaluate the impact of the carbon source used in the culture medium on the profile of lipases produced by Yarrowia lipolytica KKP 379. We observed a different pattern of extracellular and cell-bound lipase production, which was the highest in the early exponential phase. The extracellular lipase activity increased in the late exponential phase due to the lower accumulation of lipase molecules in cell walls. The best carbon source for extracellular lipase production by Y. lipolytica KKP 379 was olive oil. Glucose, dodecane and olive oil had a positive effect on biomass yield. Dodecane and/or glycerol utilization in microbiological lipase production was possible, but this process could not proceed without the addition of some activators such as olive oil in the cultivation medium.     

Fatty acid preference of mycelium-bound lipase from a locally isolated strain of <Emphasis Type="Italic">Geotrichum candidum</Emphasis>

Mycelium-bound lipase (MBL) was prepared using a strain of Geotrichum candidum isolated from local soil. At the time of maximum lipase activity (54 h), the mycelia to which the lipase was bound were harvested by filtration and centrifugation. Dry MBL was prepared by lyophilizing the mycelia obtained. The yield of MBL was 3.66 g/l with a protein content of 44.11 mg/g. The lipase activity and specific lipase activity were 22.59 and 510 U/g protein, respectively. The moisture content of the MBL was 3.85%. The activity of free (extracellular) lipase in the culture supernatant (after removal of mycelia) was less than 0.2 U/ml. The MBL showed selectivity for oleic acid over palmitic acid during hydrolysis of palm olein, indicating that the lipase from G. candidum displayed high substrate selectivity for unsaturated fatty acid containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.     

Significant improvement of Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase production using response surface methodology

The medium optimization for the production of the Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase was carried out in shake flask cultures using safflower high oleic oil. In the first step of optimization, a two level fractional factorial design allowed the identification of the concentration of nutrient broth and temperature as the main variables significantly affecting lipase production (P<0.05). In a second step, a D-optimal design was applied to determine the variables optimal values, defined as those yielding maximal lipase production in shaken flasks, thus demonstrating that the optimal concentration of nutrient broth was 3.8 g/l and the optimal culture temperature was 39.5°C. The model was experimentally validated, yielding a lipase production of 2283.70 ± 118.36 U/mL which represents a 6.7-fold increase in comparison to the non-optimized medium.     

Production of a novel extracellular acidic lipase from Pseudomonas gessardii using slaughterhouse waste as a substrate

An isolate exhibiting high extracellular lipolytic activity was identified as Pseudomonas gessardii by 16S rDNA gene sequence analysis. The slaughterhouse waste, goat tallow, was used as a lipid substrate for the production of acidic lipase by P. gessardii. The maximum lipase activity of 156 U/ml was observed at an acidic pH of 3.5 and at 0.31 g substrate concentration. The purification steps resulted in the isolation of acidic lipase with a specific activity of 1,473 U/mg and a molecular weight of 94 kDa. One interesting feature of this purified lipase is its stability at highly acidic pH ranging from 2.0 to 5.5 with a high molecular weight. The amino acid composition was determined using HPLC. This acidic lipase has potential applications in the medicinal field as a substitute for pancreatic lipases for enzyme therapy, oleochemical and in biotechnological industries.