Lipase location in Yarrowia lipolytica cells

Lipase production by Yarrowia lipolytica was growth associated and, at the beginning of cultivation, it was mainly cell-bound. Lipase release into the culture medium started when about 50% of the carbon source (olive oil or glucose) was consumed reaching its maximum concentration in the late stationary phase.     

Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica

The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was obtained from two genetically modified strains with multi-copies of the lip2 gene and further purified using gel filtration and cation exchange chromatography. Four YLLIP2 isoforms were identified and subjected to N-terminal amino-acid sequencing and mass spectrometry analysis. These isoforms differed in their glycosylation patterns and their molecular masses ranged from 36,874 to 38,481 Da, whereas the polypeptide mass was 33,385 Da. YLLIP2 substrate specificity was investigated using short (tributyrin), medium (trioctanoin) and long (olive oil) chain triglyceride substrates at various pH and bile salt concentrations, and compared with those of human gastric and pancreatic lipases. YLLIP2 was not inhibited by bile salts at micellar concentrations with any of the substrates tested, and maximum specific activities were found to be 10,760+/-115 U/mg on tributyrin, 16,920+/-480 U/mg on trioctanoin and 12,260+/-700 U/mg on olive oil at pH 6.0. YLLIP2 was found to be fairly stable and still active on long chain triglycerides (1590+/-430 U/mg) at pH 4.0, in the presence of bile salts. It is therefore a good candidate for use in enzyme replacement therapy as a means of treating pancreatic exocrine insufficiency.     

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.     

Degradation of waste waters from olive oil mills by Yarrowia lipolytica ATCC 20255 and Pseudomonas putida

Waste waters from olive oil processing may cause severe pollution in the Mediterranean area, since they have a high level of chemical oxygen demand (COD) (100–200 g/l) and contain other organic and inorganic compounds. In all olive oil producing countries, the reduction of pollution in olive oil mill waste waters at reasonable costs and using techniques suitable for most industrial applications is an unsolved problem. For this paper, the yeast Yarrowia lipolytica ATCC 20255 was grown on waste waters from an olive oil mill in a 3.5 1 fermenter under batch culture conditions. The results showed that the yeast was capable of reducing the COD value by 80% in 24 h. In this way, a useful biomass of 22.45 g/l as single cell protein (SCP) and enzyme lipase were produced. During this process, most of the organic and inorganic substances were consumed, only aromatic pollutants were still present in the fermentation effluents. Therefore, we used a phenol degrader, namely Pseudomonas putida, to reduce phenolic compounds in the fermentation effuents after removing Yarrowia lipolytica cells. P. putida was effective in reducing phenols in only 12 h.     

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.     

Oily yeasts as oleaginous cell factories

Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. More specifically, examples of oleaginous yeasts include the species: Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica. Yeast do exhibit advantages for lipid production over other microbial sources, namely, their duplication times are usually lower than 1 h, are much less affected than plants by season or climate conditions, and their cultures are more easily scaled up than those of microalgae. Additionally, some oily yeasts have been reported to accumulate oil up to 80% of their dry weight and can indeed generate different lipids from different carbon sources or from lipids present in the culture media. Thus, they can vary their lipid composition by replacing the fatty acids present in their triglycerides. Due to the diversity of microorganisms and growth conditions, oily yeasts can be useful for the production of triglycerides, surfactants, or polyunsaturated fatty acids.     

Microbial lipolysis at low temperatures.

It was found that lipase production during the growth of Pseudomonas fluorescens was not a function of the total number of bacteria. The optimal temperatures for bacterial growth and lipase production were determined as 20 and 8 degrees C, respectively. The lipolytic activity was studied in emulsions of olive oil at temperatures ranging from +8 to -30 degrees C. After an initially rapid lipolysis, the reactions retarded at different levels depending on storage temperature. Transference to a higher temperature resulted in a resumed lipolysis. Also, at low temperatures, lipolysis was studied as a function of water activity and was found to occur in dehydrated substrates.     

Media Formulation using Complex Organic Nutrients for Improved Activity,Productivity, and Yield of Candida rugosa Lipase and Esterase Enzymes

Abstract

Candida rugosa is an excellent source of multiple lipase and esterase enzymes; therefore, it is of technological importance to formulate the medium that provides high activity for each enzyme. In this work, the cultivation medium comprising complex nutrients that provided the highest activity, productivity, and yield of C. rugosa enzymes individually was formulated. Time courses of the extracellular and intracellular lipase and esterase activities of C. rugosa were represented and the role of protease in the cultivation progress was discussed. Urea, soy-peptone, yeast extract, a mixture of soy-peptone and yeast extract, cheese whey, and wheat mill bran were tested for their lipolytic and esterasic activities. Urea provided considerably higher extracellular lipase activity when compared to other nitrogen sources; however, soy-peptone provided the highest extracellular esterase activity. Hazelnut, olive, sesame, soybean, and flax seed oils affected the enzyme activities to different extents related to their fatty acid compositions. Hazelnut oil and olive oil provided the highest extracellular lipase and esterase activities, respectively, whereas sesame oil produced the highest biomass. High C₁₈ and C₁₆ ester contents of vegetable oils promoted high lipase and esterase productions, respectively. A temperature of 30°C yielded the highest extracellular and intracellular lipase and esterase activities; however, 35°C produced the highest biomass.     

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.     

Purification and Properties of a Chromobacterium Lipase with a High Molecular Weight

A lipase with a high molecular weight was purified from Chromobacterium viscosum by chromatography using the Amberlite CG–50 and Sephadex G–75. The purified lipase (Lipase A) was found to be homogeneous by disc electrophoresis.

Lipase A had an optimum pH around 7 for lipolysis of olive oil and the enzyme was stable at the range of pH 4 to 9 and below 50°C. Zn²⁺, Cu²⁺, Fe³⁺ and high concentrations of l-cysteine, iodoacetic acid and NBS had remarkable inhibitory effects. Bile salts were activator. Lipase A was more active on water insoluble esters than water soluble esters. The isoelectric point of the enzyme was pH 4.7.     

The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications

Lipases are serine hydrolases that catalyze in nature the hydrolysis of ester bonds of long chain triacylglycerol into fatty acid and glycerol. However, in favorable thermodynamic conditions, they are also able to catalyze reactions of synthesis such as esterification or amidation. The non-conventional yeast Yarrowia lipolytica possesses 16 paralogs of genes coding for lipase. However, little information on all those paralogs has been yet obtained and only three isoenzymes, namely Lip2p, Lip7p and Lip8p have been partly characterized so far. Microarray data suggest that only a few of them could be expressed and that lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to hydrophobic substrate utilization. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. Crystallographic data highlight that this latter is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation, in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies. The development of various biotechnological applications in different fields such as cheese ripening, waste treatment, drug synthesis or human therapeutics is also presented.     

Biochemical characterization of a lipase from olive fruit (Olea europaea L.)

Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies.     

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 lipids and detergents on the production and solubilization of extracellular lipase inYarrowia lipolytica

Triglycerides, oleic acid but not fatty acid-containing, nonionic detergents (Spans, Tweens) were able to stimulate the synthesis of cell-bound and soluble lipase ofYarrowia lipolytica grown in a complex medium containing citrate and urea. The optimal concentration of olive oil for induction was 0.5% (W/V). The combined effect of a high ionic strength (0.75 mol/L KCl) and of digitonin (2 mmol/L) at pH 7.6 resulted in solubilization of 80% of the cell-bound lipase and a significant activation of the enzyme. Comparison of twoY. lipolytica strains showed the effects of Mg²⁺ and Fe³⁺ concentrations in the medium on the synthesis of the enzyme to be strongly strain-dependent.     

Alkaline lipase activity from the marine protists,thraustochytrids

Two thraustochytrid protists of the genus Thraustochytrium isolated from coastal and mangrove habitats of Goa, India were studied for extracellular alkaline lipase production. Maximum lipase production was supported by a combination of peptone and yeast extract in the growth medium while strong inhibition of enzyme production was observed in presence of glucose. The inducible nature of the enzyme production was evidenced by the requirement of olive oil in the medium. Lipase production was salt-dependent and optimum production required 3.4% (w/v) crude sea salt. Ideal conditions for maximum production of lipases were therefore adopted as incubation at 30 ± 2°C for 168 h at an initial pH of 6.0 in a medium consisting of 0.5% peptone, 0.01% yeast extract, 0.5% olive oil and 3.4% crude salt. Extracellular lipase production by the two thraustochytrid isolates [designated TZ (ATCC #PRA-295) and AH-2 (ATCC #PRA-296)] was increased threefold under these optimized culture conditions. This appears to be the first report on optimization of cultivation conditions for the production of alkaline lipases by thraustochytrids.     

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.     

Role of Lipase Activators Produced by Saccharomycopsis lipolytica and Calcium Ion in Its Lipase Reaction

Lipase activator activated the reaction by Saccharomycopsis lipolytica lipase at neutral pH in the presence of calcium ions, and 5 μg of the activators were sufficient to cause the reaction to proceed at maximum activity in the presence of 2 μl of tributyrin and 0.4 units of the lipase in a total volume of 360 μl.

To define the roles of the activator and calcium ion, we studied interactions between the activator and the lipase, between the activator and a hydrophobic interface, and between the lipase and the interface. Results suggest that the interfacial adsorption of the lipase is the limiting process of lipolysis and that it is controlled by the activator and by the concentration of calcium ions.     

Chilling-induced Lipid Hydrolysis in the Oil Palm (Elaeis guineensis) Mesocarp

Oil palm fruits exposed to temperatures of 15 °C and belowshowed a significant increase in free fatty acid (FFA) contentin the mesocarp. This effect was most pronounced in fruits exposedto 5 °C when FFA levels exceeding 70% of the total oil wereobserved. The increase in FFA was accompanied by an increasein lipid-soluble phosphorus levels and a decrease in carotenecontent. Chilling did not have an effect on palm kernel oil.The results suggest the activation of a lipase in the mesocarpby low temperature stress. Key words: Lipase, oil palm, free fatty acid     

Purification and Some Properties of Two Types of Penicillium Lipase,I and II,and Conversion of Types I and II under Various Modification Conditions

The lipase produced by a strain of Penicillium crustosum Thom was fractionated into three lipase components, I～III by DEAE cellulose column chromatography, and two of them, I and II were purified and obtained in crystalline form respectively, which proved homogeneous by electrophoresis and ultracentrifugal analysis. Lipase I was an ordinary lipase with molecular weight about 29,000 hydrolyzing olive oil and tributyrin favourably in almost the same degree, while II, rather, a so-called tributyrinase with M. W. about 32,000 hydrolyzing tributyrin more efficiently than olive oil. The site of the activity on olive oil in these lipase was generally sensitive to sodium desoxycholate, ethylenediamine-tetraacetate (EDTA), and p-chloromercuribenzoate (PCMB), and lipase I was converted to a lipase II by a treatment with these reagents. Also, partial degradation of I by proteinase (‘pronase’) yielded the enzyme fragment of type II. On the other hand, treatment of the enzymes with hydrogen peroxide or sodium borohydride caused the conversion of type II into I. From the observation of UV difference spectrum during incubation with sodium desoxycholate it was indicated that the situation of tryptophane residue in enzyme molecule may have a significance in the activity of lipase I on olive oil.