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Fatty Acid and Position Specificities of an Intracellular Lipase from Geotrichum candidum
Abstract
An intracellular lipase was isolated and purified to homogeneity from mycelia of Geotrichum candidum. The lipase showed maximum activity at 35° C and pH 7.5. The enzyme preferentially hydrolyzed oleic acid glycerol ester bonds on using mono-acid triglycerides (C10:0, C18:0) as substrates. However the results indicated that the enzyme is position unspecific.
Fettsäure- und Stellungspezifität einer intracellulären Lipase von Geotrichum candidum
Eine intrazelluläre Lipase wurde aus Geotrichum candidum isoliert und bis zur Homogenität gereinigt. Das Enzym zeigte die höchste Aktivität bei 35°C und einem pH-Wert von 7.5. Die Untersuchung der Spezifität des Enzyms gegen verschiedene Fettsäureesterbindungen ergab eine ausgeprägte Spezifität gegen Ölsäureglycerinester, während keine Spezifität für Stellungsisomere nachgewiesen werden konnte.
... Literature cites that most of the values for optimum pH and temperature for lipase from Geotrichum vary between 6.0-7.0 and 20-40 °C, respectively 28 . Some studies indicate the optimum pH and/or optimum temperature is within these ranges 58,72,77,78,86,90,95,97 , however different results were obtained with modi-fications in several relevant factors for lipase production and purification, as shown in Table 2. ...
... Geotrichum lipases are frequently cited by their substrate specificity for fatty acids having at least one cis-Δ9 double bond 70,74,89,90,92,94 , but different catalytic specificities are found in different lipases from this genus 93 Table 2, and it is important to highlight that some lipases from Geotrichum showed high specificity for saturated fatty acids 41,74,76,90 . This characteristic can be related to one of the most important habitats of this genus, which is dairy products containing a significant quantity of saturated fatty acids. ...
... Geotrichum lipases are frequently cited by their substrate specificity for fatty acids having at least one cis-Δ9 double bond 70,74,89,90,92,94 , but different catalytic specificities are found in different lipases from this genus 93 Table 2, and it is important to highlight that some lipases from Geotrichum showed high specificity for saturated fatty acids 41,74,76,90 . This characteristic can be related to one of the most important habitats of this genus, which is dairy products containing a significant quantity of saturated fatty acids. ...
Lipases are enzymes produced from innumerous microorganisms, plants and animal
cells. They catalyze reactions of different lipid sources. The Geotrichum fungi are good
producers of lipases with high hydrolytic activity and specificity for unsaturated fatty
acids. A great number of studies have reported the importance of lipase from this genus
and described important fermentation parameters for the enzyme production, such as
nutrients, temperature, pH, inoculum, time of fermentation and others. Furthermore, different
strategies have been used to purify and immobilize lipases from Geotrichum and
innumerous applications are cited in different processes as polyunsaturated fatty acids
enrichment, hydrolysis and esterification of fat and oils, synthesis of aromas, biodiesel,
and many others. This review highlights fundamental aspects of the production, purification,
characterization, immobilization, and the applications of lipases produced by the
genus Geotrichum.
... Candida cylindracea Sonnet, 1988 Geotrichum candidum Tahoun, 1987;Sonnet, 1988 Chromobacterium viscasum Sugiura and Isobe, 1975 Penicillium cyclopium Iwai and Tsujisaka, 1984 Pancreatic Discriminates against Cis-4 and Cis-6 ...
... Simple agitation or stirring is not sufficient for substrates such as olive and milk triacylglycerols. Emulsification is done by homogenization or sonication of the Downloaded by [North Carolina State University] at 09:17 18 December 2013 mixture of water and triacylglycerols in the presence of additives such as polyvinyl alcohol, polyethylene glycol, gum arabic, Triton X-100, and lecithin (Yamaguchi et al., 1973;Omar et al., 1987;Tahoun, 1987;Alvarez and Stella, 1989;Phillips and Pretorius, 1991). This requires mechanical energy to increase the interfacial area and the emulsifier to stabilize the resulting emulsion. ...
Lipids in biological matter are mostly triacylglycerols (TAG). Lipolytic enzymes, primarily lipases, are indispensable for bioconversion of such lipids from one organism to another and within the organisms. In addition to their biological significance, lipases are very important in the field of food technology, nutritional and pharmaceutical sciences, chemical and detergent industries, and clinical medicine because of their ability to catalyze various reactions involving a wide range of substrates. Conventionally, lipases have been viewed as the biocatalysts for the hydrolysis of TAG (fats and oils) to free fatty acids, monoacylglycerols (MAG), diacylglycerols (DAG), and glycerol. The main advantages of lipase catalysis are selectivity, stereo-specificity, and mild reaction conditions. Despite these advantages and the fact that enzymatic splitting of fats for fatty acid production was described as early as in 1902, the lipase-catalyzed process has not replaced the commercial physicochemical process for the continuous splitting of TAG utilizing super-heated steam. The limited exploitation of lipase technology may be attributed to high enzyme cost, large reaction volume, requirement for emulsification of substrate, and risk of microbial contamination. Many of these limitations originate from the fact that lipases are employed mainly in water-rich reaction media where the solubility of the substrate TAG is very small. To circumvent this problem and to realize the full potential of lipase, researchers have explored newer approaches by manipulating the conditions under which the lipases act. Many of these novel approaches for lipase catalysis have been the outcome of the discovery that enzymes can be active in water-poor, non-polar media (Hanhan, 1952; Misiorowski and Wells, 1974; Zaks and Klibanov, 1984). Also, the finding that lipases can act in organic solvents has led lo an expansion of their applicability in a wide variety of chemical reactions. Lipase catalysis in some of the well established reaction media has previously been reviewed (Brockerhoff and Jensen, 1974; Brockman, 1984; Lilly et al., 1987; Hailing, 1990; Inada el al, 1990; Malcata et al., 1990). The present review is intended to present a compilation and comparison of novel reaction systems used for lipase catalysis. This review describes briefly the general characteristics of lipase reactions, applications of lipase in various fields, and conventional lipase technology. The lipase-mediated biochemical reactions, particularly the hydrolysis of TAG in novel reaction media is discussed in greater detail.
... When considering the types o f lipases in microorganisms, most microbial lipases are extracellular enzymes, some are intracellular and the others are membranebound. The microorganisms that produce extracellular lipase are : Pénicillium cyclopium (Lazar and Eirich, 1989), Rhizopus oligosporus (Nahas, 1988), Candida rugosa (Veeraragavan and Gibbs, 1989), and Streptomyces cinnamomeus (Sommer et al., 1997) (Marek and Bednarski, 1996), and Geotrichum candidum (Tahoun, 1987;Jacobsen et al., 1989). The examples of organisms that produce membrane-bound ...
Vegetable oils have been used as carbon sources in many fermentations to improve the antibiotic titres. However, some oil still remains at the end of the process. This study investigates how oil consumption can be increased in the production of erythromycin by Saccharopolyspora erythraea CA 340 grown on rapeseed oil. Two methods have been carried out. The first was the effect of the application of either sulphuric or phosphoric acid in the pH control. The second was the inclusion of surfactant either Disponil SMO 100 (DPN) or sodium dodecyl sulphate (SDS) in the production medium. For the effect of pH-control agent, which was studied in 2-L fermenter only, the oil consumption of the culture using phosphoric acid was higher than that using sulphuric acid while the lipase activity after 96 hr was lower. In addition, the yield of erythromycin A was also lower but not the total erythromycin. The DNA content (as a measure of biomass) was also the same. Besides that the oxygen uptake rate (OUR) and the carbon dioxide evolution rate (CER) of these cultures were also similar. However, there was a sharp drop in DOT profile after it reached 55% in the culture using sulphuric acid, which was not seen in that using phosphoric acid. For the effect of surfactant, the study was carried out in both a 2-L fermenter and shake flasks (500-ml and 2-L). In the 2-L fermenter, it appeared that Disponil at the concentration of 1.58 or 3.33 g per 29.9 g oil increased the oil uptake of this organism. The residual oil profile was 2-5 g/L lower with the earlier decrease (24 hr earlier) over a period of 168 hr. On the other hand, the DNA content was slightly higher. The OUR and CER profile also reached the maximum earlier, especially that of DPN 3.33. This surfactant also increased the rate of erythromycin synthesis before 96 hr. However, the total erythromycin concentration was lower while that of erythromycin A was similar. In contrast, SDS at 0.075% delayed the oil consumption of this culture and more residual oil remained at the end of the fermentations. It also inhibited the growth (lower contents of DNA). The OUR and CER profile in this culture was also lower than the controls. Even though the volumetric total erythromycin of this culture was lower than the controls, the yield of erythromycin per unit of consumed oil was similar while that of DPN-containing cultures was lower. There was no direct effect of DPN against the enzyme lipase when it was added in the assayed mixture and tested with either 48-hr or 96-hr culture. However, when the fermentation was carried out in 500-ml or 2-L shake flasks, none of the concentrations studied (0.1% to 1.0% (w/v)) increased the oil uptake of this organism during the 144 hr fermentation. The differences between the fixed fermenter and the shake flask fermentation are probably caused by changes in mixing and aeration. The lipase production was reduced in medium containing Disponil at concentration higher than 0.5% (w/v). The effect of the anionic surfactant, sodium dodecyl sulphate (SDS), was also investigated.
... Results demonstrated no extracellular lipolytic activity. Therefore, the high rate of hydrolysed coconut oil could be associated with intracellular or cell-bound lipolytic activities [26]. However, no lipolytic activity was detected in the cell filtrate after cell disruption using homogenization, sonication, and normal solvent extraction methods, and only cell-bound associated lipolytic activity was responsible for in situ coconut oil modification. ...
Coconut oil is a rich source of beneficial medium chain fatty acids (MCFAs) particularly lauric acid. In this study, the oil was modified into a value-added product using direct modification of substrate through fermentation (DIMOSFER) method. A coconut-based and coconut-oil-added solid-state cultivation using a Malaysian lipolytic
Geotrichum candidum
was used to convert the coconut oil into MCFAs-rich oil. Chemical characteristics of the modified coconut oils (MCOs) considering total medium chain glyceride esters were compared to those of the normal coconut oil using ELSD-RP-HPLC. Optimum amount of coconut oil hydrolysis was achieved at 29% moisture content and 10.14% oil content after 9 days of incubation, where the quantitative amounts of the modified coconut oil and MCFA were 0.330 mL/g of solid media (76.5% bioconversion) and 0.175 mL/g of solid media (53% of the MCO), respectively. MCOs demonstrated improved antibacterial activity mostly due to the presence of free lauric acid. The highest MCFAs-rich coconut oil revealed as much as 90% and 80% antibacterial activities against
Staphylococcus aureus
and
Escherichia coli
, respectively. The results of the study showed that DIMOSFER by a local lipolytic
G. candidum
can be used to produce MCFAs as natural, effective, and safe antimicrobial agent. The produced MCOs and MCFAs could be further applied in food and pharmaceutical industries.
... Several reports have discussed specificities of crude lipase preparations from G. candidum [4], but none of them have shown significant specificity for unsaturated fatty acids. This obser- * Corresponding author. ...
Substrate specificity (typoselectivity), regioselectivity and hydrolytic activity of induced lipases from three strains (4012, 4013, 4166) of Geotrichum candidum and that of Geotrichum ludwigii (48) were investigated. The lipases were induced in two types of culture media, of which the medium containing peptone as nitrogen source was proved to give better results. Olive oil was employed as inductor for the lipase activity. Activated lipases represented mostly extracelullar lipases, which penetrated through cellular membrane into medium. The activity of cell-bound lipase was also determined. Most of lipases belong to the group of specific lipases able to hydrolyse ester bonds in the positions sn-1 and sn-3 ester of triacylglycerols (1,3-selective lipases) and display specificity to saturated fatty acids. All activated lipases from Geotrichum sp., extracellular and cell-bound, were used as biocatalyst in the blackcurrant oil hydrolysis.
The interactions between 14 isolates of Geotrichum candidum and 14 isolates of Brevibacterium linens were analyzed to understand their behavior in cheese making. Mixed cultures were grown in a peptone broth containing various NaCl concentrations and various pH. The pH and the concentration of NaCl affected the interactions. According to the pH, in many strain pairs, B. linens strongly stimulated the growth of G. candidum, and this interaction varied depending on the pH. Inhibition occurred only at pH 5.0. In fewer pairs, G. candidum moderately stimulated the growth of B. linens, and the optimal pH for this stimulation and the inhibition were 7.0 and 5.0, respectively. The number of pairs in which the growth of G. candidum was increased by the presence of B. linens was highest with 1.5% added NaCl. With increasing NaCl concentration, stimulation of the growth of B. linens by G. candidum decreased because of the sensitivity of G. candidum and resistance of B. linens to NaCl. Brevibacterium linens isolates were similarly grown in the presence of filtrates of stationary phase cultures of G. candidum. The filtrates exhibited effects analogous to, but larger than, those of the strains from which the filtrates were prepared.
Geotrichum candidum ATCC34614 produces a major (I) and three minor (II, HI and TV) forms of lipase with similar molecular masses, but different
positional and fatty acid specificities. The major and one of the minor (II) forms were confirmed to cleave both the inside
and outside ester bonds of triolein indiscriminately. The non-positional specificity was also retained on l,3-dipalmitoyl-2-oleoyl-glycerol
(POP) hydrolysis. In contrast, the remaining two forms (III and IV) showed unusual positional specificity; they cleaved the
inside (2-position) ester bond of triolein at nearly twice the rate of cleavage at the l(3)-position. The preference for the
inside ester bond was increased upon POP hydrolysis.
A new component of Geotrichum candidum lipase with a unique positional specificity was isolated from culture broth together with a known major component. The purification included DEAE-Sephadex A-50 ion exchange chromatography. Sephadex G-100 gel filtration, and Butyl Toyopearl 650S hydrophobic interaction chromatography. The newly isolated component, though only a minor one, cleaved the 2-positioned ester bond nearly twice as fast as the 1(3)-positioned ester bond of a triglyceride molecule. In contrast, the major component hydrolysed all the ester bonds indiscriminately, which is consistent with the widely accepted positional specificity of the lipase from G. candidum.
A new form of Geotrichum candidum lipase with a unique positional specificity was found to exist in the culture broth as a minor component together with the well-documented major form. Unlike the major form, which cleaves both the inside and outside ester bonds of triglyceride indiscriminately, the newly isolated form showed some preference for the inside (2-position) ester bond. The new enzyme was also characterized by its own fatty acid specificity, i.e., an outstandingly high activity towards triolein and methyl oleate among the simple triglycerides and fatty acid methyl esters tested. Moreover, the enzyme possessed a specific activity three times as high as the major form. Notable difference in circular dichroism spectra were observed between the two forms, indicating distinct conformational differences. Edman degradation revealed that the N-terminal sequence of the new form differed from that of the major form, thus demonstrating the existence of a novel lipase gene on the chromosome.
The wide genotypic and phenotypic diversity of Geotrichum candidum strains does not facilitate its classification as yeast or a yeast-like fungus that is still a matter of debate. Whatever its classification, G. candidum possesses many different metabolic pathways that are of particular interest to the dairy industry. G. candidum is of importance in the maturation of cheese, and much is known about its direct contribution to cheese ripening and flavour formation. Its diverse metabolic potential means that G. candidum can play an important role in the ripening of many soft and semi-hard cheeses and make a positive contribution to the development of taste and aroma. It may also influence the growth of other microorganisms, both valuable and detrimental. The significance of the presence of G. candidum in cheese depends on the particular type of production and on the presence of biotypes featuring specific types of metabolism. However, in situ metabolic pathways involved in cheese ripening and their regulations are mainly unknown. The information available provides a good understanding of the potential of G. candidum strains that are used in cheese manufacture, and permits a better choice of strain depending on the characteristics required. The biochemical activities of G. candidum and its application in the dairy industry are presented in this review.
Addition of lard or sodium oleate to the medium used for lipase production by Pseudomonas fragi resulted in a decreased accumulation of lipase in the culture supernatant fluid without affecting cell growth. The production and activity of lipase was inhibited by lard, sodium oleate, and the salts of other unsaturated fatty acids. Some divalent cations, Tweens, lecithin, and bovine serum prevented oleate inhibition, but did not reverse it. Similar inhibitory actions were observed with Geotrichum candidum lipase, but not with a staphylococcal lipase or pancreatic lipase. A protective effect by protein in crude enzyme preparations is indicated. The ability of oleate to lower surface tension does not appear to be related to its ability to inhibit lipase.
Lipases from Aspergillus niger and Rhizopus delemar hydrolyzed triolein and produced l,2 (2,3)-diolein and 2-monoolein. These two lipases appears to have strong specificity towards the outer chains of the triglyceride. Comparing the proportions of fatty acids in position 1 (3) of cocoa butter with proportions of fatty acids liberated after limited hydrolysis of cocoa butter, it becomes clear that these two lipases do not hydrolyze the ester bond in position 2 of the triglyceride.On the other hand, lipases from Geotrichum candidum Link and Penicillium cyclopium Westring attacked the fatty acid chains regardless of their positions. Geotrichum candidum lipase liberated oleic acid and palmitic acid in preference to stearic acid from cocoa butter.
Die Spezifität der Lipase von Geotrichum candidum auf verschiedene glyceridgebundene Fettsäuren wird durch Lipolyse synthetischer Triglyceride mit nachfolgender Analyse der Reaktionsprodukte untersucht. Dabei werden folgende Resultate erzielt: Gesättigte Fettsäuren werden in geringem Maße aus Glyceridsubstraten abgespalten. Unter den gesättigten Fettsäuren wird dabei eine gewisse Bevorzugung von Laurin‐, Myristin‐ und Palmitinsäre festgestellt.
Das Enzym besitzt eine ausgeprägte Spezifität auf cis‐9‐ungesättigte Fettsäuren, wobei eine gewisse Abstufung von Ölsäure über Palmitolein‐ und Linolsäure zur Linolensäure zu verzeichnen ist. Ein hoher Anteil von cis‐9‐ungesättigten Fettsäuren an der Fettsäurezusammensetzung der Glyceridsubstrate wirkt sich positiv auf die Spezifität der Lipase von Geotrichum candidum aus. Das Enzym zeigt eine geringe Bevorzugung von Ölsäure, die in den primären Glyceridpositionen verestert ist.
Stellungsisomere von cis‐9‐ungesättigten Fettsäuren werden nicht oder in geringem Maße aus Glyceridsubstraten abgespalten.
Elaidinsäure wird bei der Lipolyse von Glyceridsubstraten in geringerer Menge als die cis‐isomere Ölsäure in Freiheit gesetzt.
Die ausgeprägte Spezifität der Lipase von Geotrichum candidum auf cis‐9‐ungesättigte Fettsäuren wird mit der Struktur des aktiven Enzymzentrums begründet. Neben einem optimalen Abstand zwischen Bindungs‐ und Wirkungszentrum des Enzyms von ca. 12 Å werden die Affinität des Bindungszentrums auf cis‐Doppelbindungen sowie die sterische Anordnung der restlichen Eiweißkette des Enzyms für die bisher einzigartige Spezifität verantwortlich gemacht.
Es Wird über die industrielle Herstellung hochkonzentrierter Monoglyzeride durch Umesterung von Fetten und Konzentrierung durch Kurzwegdestillation aus der Sicht eines Anlagenbauers berichtet. Neben der Verfahrensbeschreibung wird die apparative Ausführung von Produktionsanlagen vorgestellt. Auf kritische Bedingungen, soweit sie Qualität und Ausbeute wesentlich beeinflussen, wird detailliert eingegangen. Der Rohstoffeinsatz ist aus einem Mengenbilanzschema ersichtlich, aus dem sich zusammen mit den genannten Energieverbrauchszahlen eine gute Abschätzung Wirtschaftlichkeit des Verfahrens ergibt.Procedure of Monoglyceride Concentrate ProductionThe industrial production of high concentrated monoglycerides by interesterification of fats and concentration by molecular distillation is presented from the view of a plant constructor. Besides the description of procedure the technical design of production plants is demonstrated. Critical conditions are discussed in detail as far as they have an important influence on quality and yield. The raw material input is evident from a quantitative balance from which together with the mentioned figures of energy consumption follows a good estimation of efficiency of the procedure.
S ummary
The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
Extracellular microbial lipases can be used as catalysts for the interesterification of oils and fats. Use of specific lipases
gives products which are unobtainable by chemical interesterification methods. Some of these products have properties of value
to the oils and fats industry. The catalysts for enzymatic interesterification are prepared by coating inorganic support materials
with the lipases. For batch interesterification reactions, the catalyst particles are activated by addition of a small amount
of water and then stirred with a reactant mixture dissolved in petroleum ether. At the end of the reaction period, the catalyst
particles are removed by filtration, and the interesterified triglycerides isolated by conventional fat fractionation techniques.
The catalyst can be used in subsequent batch reactions. As an alternative to the batch reaction system, continuous enzymatic
interesterification processes can be operated by pumping water containg feedstock through a packed bed of activated catalyst.
Pancreatic lipase hydrolyzed fatty acids in equimolar quantities from thesn-1- and 3-positions of three synthetic enantiomeric triglycerides, two of which could make a racemic pair. The monoglycerides
from digestions of five enantiomeric triglycerides were at least 99% representative of the 2-position. The data confirm that
pancreatic lipase did not distinguish between thesn-1- and 3-positions and that with these triglycerides pancreatic lipolysis can be used to help establish structure.
The lipase (EC 3.1.1.3) from the microorganism Candida cylindracea is very active on long chain triglycerides at pH 8.0 and 37°. Under these conditions the three chains of the glycerol moiety are hydrolyzed. Free glycerol appears in the same time as the other reaction products, however, monoglycerides are present only in very minute amounts, all along the course of lipolysis. It can thus be supposed that the enzyme is able to hydrolyse all the ester bonds of the glycerol.A comparison between the proportions of the fatty acid chains in intact olive oil and cocoa butter and the proportions of fatty acids liberated after a limited hydrolysis of these lipids shows that the lipase of Candida liberates all types of acyl chains, regardless of their position in the glycerol. However, palmitic and oleic chains are liberated before stearic acid when they are present together in a given glyceride.When synthetic glycerides, containing only palmitic and oleic acid chains, are submitted to a partial lipolysis by Candida lipase, the composition of the liberated fatty acids and the composition of the acyl chains in the intact triglycerides are similar. However, oleic acid is liberated before palmitic acid, regardless of their respective position, as would appear from the composition of diglycerides and monoglycerides.Since 1,3-dihexadecyl-ether-2-oleoyl-glycerol is readily hydrolyzed it can be concluded that the Candida lipase is able to attack secondary ester groups of glycerol without the help of an isomerase.
Addition of lard or sodium oleate to the medium used for lipase production by Pseudomonas fragi resulted in a decreased accumulation of lipase in the culture supernatant fluid without affecting cell growth. The production and activity of lipase was inhibited by lard, sodium oleate, and the salts of other unsaturated fatty acids. Some divalent cations, Tweens, lecithin, and bovine serum prevented oleate inhibition, but did not reverse it. Similar inhibitory actions were observed with Geotrichum candidum lipase, but not with a staphylococcal lipase or pancreatic lipase. A protective effect by protein in crude enzyme preparations is indicated. The ability of oleate to lower surface tension does not appear to be related to its ability to inhibit lipase.
A procedure is described for determining the stereospecific structure of triacid triglycerides containing oleic acid. The method utilizes the unique specificity of the lipase system fromGeotricum candidum for hydrolyzing fatty acids which containcis-9-unsaturation.
The procedure involves a pancreatic lipase hydrolysis of 10–20 mg of substrate to determine the fatty acids in the β-position and the incubation of another 50 mg of triglyceride withG. candidum lipase to obtain diglyceride for further treatment. The α,α- and α,β-diglycerides are collected separately, converted to phenyl phosphatides and treated with phospholipase A. The analysis of the monoglycerides, produced with pancreatic lipase and the analysis of the β-lysophosphatides allows the determination of the ratios of the original 2 position oleic acidsn-triglycerides while the analysis of the α-lysophosphatides aids in the determination of the originalsn-triglycerides which contained oleic acid in the 3 position. The 1 position oleic acidsn-triglycerides are calculated by difference.
Racemic and enantiomeric triglycerides containing palmitic, stearic and oleic acids were synthesized and used to establish the limits of the new method. The good agreement between the actual and observed values for a mixture of isomers indicates that the procedure will be useful in the analysis of triacid triglycerides which contain oleic acid. The application of the procedure to triacid triglycerides which contain other unsaturated fatty acids is discussed.
Ono United States Patent
- T Tanakaande