Effect of culture conditions on batch growth of Saccharomycopsis lipolytica on olive oil

Summary Batch cultures of Saccharomycopsis lipolytica were grown in minimal medium with olive oil as carbon source. Inocula of glucose-grown cells commenced growth with little lag at rates largely unaffected by variations in the stirring rate or oil concentration. However, growth rates declined when the medium pH was below 7.0. In all cultures, media pH declined with increasing cell concentration. Cell composition during exponential growth was 42% protein and 2% fat. Carbon-limited cells maintained this composition after oil exhaustion but during nitrogen- and oxygen-limited growth, protein content decreased and fat content increased although the protein decrease was only transient with oxygen limitation. Yield coefficients for triglyceride were near unity for all cultures. Free acid concentrations rose rapidly after inoculation. As fermentations progressed, free glycerol appeared and concentrations of di- and monoglycerides passed through maximal values although peak concentrations of di- and monoglycerides persisted for extended times in oxygen- and nitrogen-limited cultures respectively. The fraction of free glycerol consumed was greater in oxygen-limited than in carbon- or nitrogen-limited culture. The basic requirements for growth of yeasts on fatty wastes are discussed with reference to these observations.     

A Novel Olive Oil Degrading Thermoactinomyces Species with a High Extremely Thermostable Lipase Activity

A filamentous, Gram‐positive, spore forming aerobic bacterium was isolated from olive oil contaminated soil (Al Koura, Lebanon) on rhodamine agar plates at 60 °C. The isolate, HRK‐1 produced large quantities of an extracellular thermostable lipase which degrades olive oil. It was primarily classified as a Thermoactinomyces sp. due to the filamentous structure of its cells that bear one spore each on an un‐branched sporophore, the resistance of its spores to boiling, utilisation of sucrose as a carbon source and production of dark pigments. The isolate grew optimally at a temperature of 60 °C, a pH of 7.3 and an orbital shaking of 250 rpm. It showed an efficient olive oil degrading ability. No traces of triolein were detected after a 36‐h cultivation. A concentration of 10 % [v/v] olive oil did not inhibit its growth. Lipase production was constitutive, and did not depend on the presence of olive oil. The optimum concentration of olive oil for lipase activity was 1 % [v/v], and the activity was not enhanced at higher concentrations, but on the contrary, a decrease in enzyme activity was recorded. The lipase of HRK‐1 was preliminarily characterised in the crude cell‐free supernatant with a specific activity of 0.14 U/mg. It has an optimum activity at 60 °C and a pH of 8.0. This lipolytic enzyme showed resistance to boiling and to a wide range of metallic ions and inhibitors. The formation of this heat‐stable lipase started in the early exponential growth phase, while a maximum extracellular enzyme activity was detected at the end of the decline phase, when most of the cells appeared as spherical spores. The exceptionally high activity of lipase (2.37 U/ml) produced by HRK‐1 measured in the cell free supernatant clearly indicated the commercial importance of this isolate, especially after it showed great stability at elevated temperatures.     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Utilization of substrates during batch growth of Pseudomonas fluorescens on olive oil,lard, and mutton tallow

Summary Similar amounts of cell-associated and cell-free lipase activities were present in log phase cultures of Pseudomonas fluorescens growing on fatty substrates. The rates of triglyceride hydrolysis and fatty acid utilization were balanced as free fatty acids remained at low concentrations in the culture media at all times. The time required for growth initiation and optimal growth rates of P. fluorescens were similar on olive oil, lard and mutton tallow, provided the coalesced solid fats were mechanically dispersed during the initial stages of cultivation. Although olive oil was completely consumed at the end of cultivation, substantial amounts of tri- and diglycerides, presumably bearing saturated acyl residues, remained in lard culture and, to a lesser extent, in mutton tallow culture.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

Lipase and esterase formation by psychrophilic and mesophilic Acinetobacter species.

Acinetobacter O16, a psychrophilic species, produced extracellular lipase (measured by hydrolysis of olive oil, tributyrin, or beta-naphthyl laurate) when grown on a complex medium (peptone plus yeast extract). Most lipase was produced during the logarithmic phase of growth. Very little cell-bound lipase was formed. These cells also produced an esterase (measured by the hydrolysis of beta-naphthyl acetate). At first, all esterase was cell bound; significant amounts appeared in the external medium late in growth. Breaking the cells did not increase cell-bound lipase activity. After breaking of the cells, most of the cell-bound lipase and esterase activity was solubilized, even after very high speed centrifugation. No appreciable amounts of these enzymes were released by osmotic shock. Lipase formation was greatly affected by nutrient conditions. Lowering either the yeast extract of the peptone content of the normal complex medium lowered or abolished lipase formation. Esterase activity was lowered to a lesser extent. Cells growing in synthetic amino acid plus vitamin medium or in acid-hydrolyzed casein produced substantial amounts of esterase but no cell-free or cell-bound lipase. However, if sodium taurocholate was added to these media, lipase was produced. Greatest production occurred if a mixture of di- and poly-peptides was also present. Taurocholate also stimulated lipase production in the normal complex medium. Adding Tween 80 or ethanol to the normal complex medium inhibited lipase production. Sodium acetate, oleic acid, olive oil, or Tween 20 added to synthetic media did not affect lipase production. The psychrophile grew more quickly at 30 degrees C than at 15 or 20 degrees C but produced more lipase at the lower temperatures. Esterase production was about the same at 20 and 30 degrees C. A mesophilic Acinetobacter species produced the same amount of lipase and esterase at 20 and 30 degrees C. The best production of lipase by the psychrophile occurred in standing cultures.     

Hydrolysis of olive oil catalyzed by surfactant-coated<Emphasis Type="Italic">Candida rugosa</Emphasis> lipase in a hollow fiber membrane reactor

In this study, we invetigated the hydrolysis of olive oil catalyzed by a surfactant-coatedCandida rugosa lipase in a hydrophilic polyacrylonitrile hollow fiber membrane reactor and then compared the results to those using the native lipase. The organic phase was passed through the hollow inner fibers of the reactor and consisted of either the coated lipase and olive oil dissolved in isooctane or the coated lipase dissolved in pure olive oil. The aqueous phase was pumped through the outer space. After 12 h and with conditions of 30°C, 0.12 mg enzyme/mL and 0.62 M olive oil, the substrate conversion of the coated lipase reached 60%. This was twice the conversion for the same amount of native lipase that was pre-immobilized on the membrane surface. When using pure olive oil, after 12 h the substrate conversion of the coated lipase was 50%. which was 1.4 times higher than that of the native lipase.     

Phospholipid and fatty acid enrichment of Phanerochaete chrysosporium INA-12 in relation to ligninase production

Summary Ligninase activity of Phanerochaete chrysosporium INA-12 was increased when vegetable oils emulsified with sorbitan polyoxyethylene monooleate (Tween 80) were added to growth medium. Maximal enzyme yield was 22.0 nkat·ml^-1 in olive oil cultures after 4 days incubation. P. chrysosporium INA-12 was also able to utilize tall oil fatty acids for ligninase synthesis. An extracellular lipase activity was detected during the primary phase of growth in culture containing vegetable oils. On the other hand, ligninase production was 1.5-fold enhanced when olive oil cultures were supplemented with soybean asolectin as a phospholipid source. In cultures supplied with olive oil plus asolectin, P. chrysosporium INA-12 mycelium exhibited a preferential enrichment of oleic acid (C18:1), phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) as compared to lipid-free medium. PC and LPC enrichment was associated with an increased ratio of saturated versus unsaturated fatty acids of phospholipids.     

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.     

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.     

Effective production of Pseudomonas fluorescens lipase by semi-batch culture with turbidity-dependent automatic feeding of both olive oil and iron ion

Summary An automatic feeding system to supply olive oil in semi-batch culture was established by monitoring cell concentration with a laser turbidimeter combined with a microcomputer and a pulse motor. In this automatic feeding system, specific olive oil supply rate (g olive oil) · (g dry cell)^-1 · h^-1, q ₀, was changed in an appropriate range. Attempts were made to produce lipase by a turbidity-dependent automatic fed-batch culture of Pseudomonas fluorescens. It was found from the semi-batch cultures with turbidity-dependent feeding of olive oil and with varied initial Fe ion concentrations that excess Fe ion was inhibitory to formation of the lipase. Turbidity-dependent automatic simultaneous feeding of olive oil and Fe ion was performed to obtain semi-deficiencies of both the oily substrate in the culture liquid and Fe content of the cells. Using this semi-batch culture, high lipase activity, 5600 units/ml, was attained at an optimal value of q ₀.     

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.     

Lipase Induction in Mucor hiemalis

The influence on lipase induction in Mucor hiemalis of different types of triglycerides containing mainly oleic acid (olive oil), erucic acid (mustard oil), or saturated fatty acids of 8 to 16 carbons (coconut oil) was studied. The fungus was grown in shake flasks in a fermentation medium containing peptone, minerals, and glucose or one of the oils as the carbon source. Maximum lipase was produced when the initial pH of the fermentation medium was kept at 4.0. Addition of Ca²⁺ to the medium did not increase lipase production. The optimum pH for activity of both the mycelial and extracellular lipases was found to be 7.0. The fungus produced a significant amount of lipase in the presence of glucose, but the lipase activity increased markedly when olive oil was added to the medium at the beginning of the fermentation. Addition of olive oil at a later stage did not induce as much enzyme. Studies with washed mycelia showed that a greater amount of lipase was released when olive oil was present than when glucose was present. Among the various types of triglycerides used as the carbon source, olive oil was found to be most effective in inducing the lipase. Olive oil and mustard oil fatty acids inhibited the lipase more than those of coconut oil. The lipase induced by a particular type of triglyceride did not seem to be specific for the same triglyceride, nor was it inhibited specifically by it. Irrespective of the triglyceride used in the fermentation medium, the lipase produced was most active against coconut oil triglyceride, and this specificity, as shown by lipase activities in an n-heptane system, was not found to be due to a better emulsification of this oil. The lipase of M. hiemalis can be considered to be both constitutive and inducible.     

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.     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。     

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.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Effect of solvents on hydrolysis of olive oil by immobilized lipase in reverse phase system

Summary Lipase fromCandida rugosa was immobilized by adsorption on three supports which could contain water available for the hydrolysis of olive oil in a reverse phase system. To select the most suitable solvent for this system, the effect of organic solvents on the stability and catalytic activity of immobilized lipase for the hydrolysis reaction has been examined. The results revealed that isooctane was superior to any other solvents tested in this study for enzymatic fat splitting in a reverse phase system. Also the effect of the solvent polarity on the hydrolysis of olive oil has been examined in detail using various organic solvents mixed with an equivolume of isooctane. It was found that the hydrolysis of olive oil by immobilized lipase was markedly affected by the polarity of reaction solvents.     

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.     

Studies on Mucor Lipases

Mucor lipolyticus Aac-0102, a new species of Mucor, accumulated lipase in culture fluid when grown in a medium composed of soluble starch, soy bean meal, (NH₄)₂SO₄, and K₂HPO₄. This strain was the most lipolytic of the genus Mucor surveyed.

The culture fluid of this strain hydrolyzed various kinds of fatty acid esters, such as glycerides, Tweens or Spans and optimum activity for the hydrolysis of olive oil occurred at pH 8.0. This pH optimum was common to the lipases of the type cultures Mucor tested. The lipase of Mucor species may be different from that of Rhizopus species or other molds, since their pH optima are not the same.