Substrate specificity,regioselectivity and hydrolytic activity of lipases activated from Geotrichum sp.

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.     

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.     

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.     

Improvement of cell-bound lipase from Rhodotorula mucilaginosa P11I89 for use as a methanol-tolerant, whole-cell biocatalyst for production of palm-oil biodiesel

Rhodotorula mucilaginosa P11I89, isolated from oil-contaminated soil, was effectively used as the methanol-tolerant, whole-cell lipase for the synthesis of fatty acid methyl ester (FAME) via transesterification reaction in the presence of palm oil and methanol substrates at a 1:6 mole ratio. A combination of Taguchi experimental design and response surface methodology (RSM) were applied to systemically enhance transesterification activity of the whole-cell lipase or cell-bound lipase (CBL) from R. mucilaginosa P11I89 in a solvent-free system. The significant impacts of four factors including carbon sources, nitrogen sources, surfactants and pH on hydrolysis activity of extracellular and cell-bound lipases, and on the transesterification activity of CBL were evaluated using Taguchi design. Gum Arabic was the most significant component for high transesterification activity, whereas soybean oil was the most influential factor for the hydrolysis activity. Maximal CBL production of 272.72 U/L was obtained in the cultivation medium containing 2.1 % palm oil, 0.2 % NH₄NO₃ , and 0.45 % Gum Arabic, with initial pH 5.0 under shaking speed of 200 rpm at a temperature of 30?±?2 °C after 60 h incubation using Central Composite Design (CCD). Yeast cells grown under such conditions increased FAME yield from 84.0 to 92.98 % when the transesterification reaction was carried out, in comparison to those cultivated in the initial medium.     

Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity

Summary Three strains of Geotrichum candidum (ATCC 34614, NRRL Y-552 and NRRL Y-553) were examined for lipase production and activity. Variables including medium, pH, temperature, agitation rate and incubation time were examined to define the optimal culture conditions. Growth on oil in complex medium at 30°C, 300 rpm, and pH 7 produced maximal lipase activity. Fatty acid specificity of these strains and of two crude G. candidum enzyme preparations (lipase 26557 RP, Rhône Poulenc and lipase GC-4, Amano) was measured using equimolar mixtures of methyl or butyl esters of palmitic and oleic acids. The lipase from NRRL Y-553 and lipase 26557 RP displayed preferential specificity for hydrolyzing oleic acid esters, while the lipases from ATCC 34614, NRRL Y-552 and lipase GC-4 failed to discriminate between plamitic and oleic acids.     

Substrate specificity of Geotrichum candidum lipase preparations

Summary Lipases with different fatty acid specificity were produced byGeotrichum candidum depending on growth condition. The hydrolysis of olive oil was inhibited by glycerol tributyrate and was dependent on Ca-ions for running at maximal rate.     

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg²⁺, Ca²⁺, and Mn²⁺ were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.     

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.     

Inhibition of Incorporation of l-Alanine into Uridine-diphospho N-acetylmuramic Acid by Glycine

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.     

Selective lipid hydrolysis byGeotrichum candidum NRRL Y-553 lipase

Summary A crude preparation of lipases from the fungusGeotrichum candidum NRRL Y-553, which shows specificity toward cis-9 unsaturated fatty acids, was used to hydrolyze natural fats and oils, with the goal of producing industrially useful fatty acid and/or mono- and diacylglycerols products. Tallow and soy oil were hydrolyzed, producing free fatty acids high in oleic or cis-9 unsaturates, respectively. Hydrolysis of rapesced oil produced acylglycerols enriched in erucic acid.     

The effect of tweens on the lipolytic activity ofGeotrichum candidum

Geotrichum candidum produces an extracellular lipase in different media provided that the pH does not decrease too much during growth. The enzyme has a particular substrate specificity; only esters of unsaturated fatty acids which are insoluble in water, are hydrolysed. Although the lipase fails to hydrolyse tweens, the latter stimulate its production but at the same time tweens and other surface-active compounds inhibit the lipolytic action of the enzyme.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

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.     

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent K_M was 6.5 mmol l^-1, with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l^-1 NaCl and on the presence of at least 0.1 mol l^-1 NaCl in the test mixture. Desoxycholate and up to 0.1 mol l^-1 CaCl₂ also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.     

Enrichment of very-long-chain mono-unsaturated fatty acids by lipase-catalysed hydrolysis and transesterification

Partial hydrolysis of triacylglycerols of high-erucic-acid seed oils from white mustard (Sinapis alba), oriental mustard (Brassica juncea) and honesty (Lunaria annua), catalysed by lipases from Candida cylindracea and Geotrichum candidum, leads to enrichment of erucic acid and other very-long-chain mono-unsaturated fatty acids (VLCMFA) in the acylglycerols (mono-, di- and triacylglycerol) while the C₁₈ fatty acids (oleic, linoleic and linolenic) are enriched in the fatty acid fraction. Partial hydrolysis of the high-erucic-acid triacylglycerols, catalysed by lipases from porcine pancreas, Chromobacterium viscosum, Rhizopus arrhizus and Rhizomucor miehei yields fatty acids with substantially higher levels of VLCMFA, as compared to the starting material, while the C₁₈ fatty acids are enriched in the acylglycerol fraction. Lipases from Penicillium sp. and Candida antarctica are ineffective for the fractionation of either group of fatty acids. Transesterification of the high-erucic-acid triacylglycerols with ethyl, propyl or butyl acetate or with n-butanol, catalysed by the lipase from R. miehei, leads to enrichment of VLCMFA in the alkyl (ethyl, propyl or butyl) esters, whereas the C₁₈ fatty acids are enriched in the acetylacylglycerols and acylglycerols.     

Substrate selectivity of bacterial monoacylglycerol lipase based on crystal structure

Lipases, which are conserved from bacteria to mammals, catalyze the hydrolysis of acylglycerol to free fatty acids and glycerol. Monoacylglycerol lipase (MGL) specifically catalyzes the hydrolysis of monoacylglycerol. Although there have been numerous studies of the structure of lipases, there have been few studies of MGL. Here, we report the crystal structure of authentic MGL isolated from Bacillus sp. H257 (bMGL). The crystal diffracts to 1.96 Å resolution. It belongs to space group P2₁2₁2, and the unit cell parameters are a = 99.7 Å, b = 106.1 Å and c = 43.0 Å. As in other lipases, three structural features for lipase activity are conserved in bMGL: the glycine-X-serine-X-glycine motif, catalytic triad and cap region. The structure of bMGL appears to be closed, as the cap region covers the active site entrance. The isolated bMGL hydrolyzed 2-AG, a known human MGL-specific substrate. Based on a 2-AG bound model, we discuss the substrate selectivity. The functional and structural features of bMGL provide insight how its substrate selectivity is determined and how specific inhibitors of bacterial MGL could be designed, which may be useful for development of novel antibiotics.     

Separation of colour compounds from lipase in fermentation supernatant by diafiltration

Summary Extracellular lipase was produced by growing Geotrichum candidum. A simple optical method was developed to quantify the dark-brown compounds formed during medium preparation and fermentation. The size of these molecules was in the fractionation range of Sephadex G-50. Diafiltration trials were done to screen ultrafiltration membranes for the most efficient decolonization of the cell-free lipase solution. Membranes were identified which reduced the colour by 80% with less than 5% loss of lipase activity after 4 volume exchanges in continuous diafiltration.     

Scale down and optimization of olive mill wastewaters decolorization by Geotrichum candidum

Microbiol flora acclimated in continuous pilot scale bubble column fed with OMW was analysed. The most efficient isolated fungus was identified to white-rot fungus Geotrichum candidum. Decolorization of OMW by Geotrichum candidum was investigated by using Hadamard's matrix for screening the important parameters and optimize them in order to control the biological decolorization. Agitation favours the conversion of COD removed into Geotrichum candidum biomass especially with high arthoconodia and few mycelium. Dilution of OMW and aeration enhanced the mycelium growth and rammification which that allowed polyphenols hydrolysis and then a decolorization. The initial pH of OMW is suitable for its decolorization by Geotrichum candidum growth. Ammonium sulfate concentrations tested with different OMW dilutions showed that the COD:N:S ratio of 100:5:2 is suitable for higher black colour removal. With optimized conditions Geotrichum candidum growth on OMW in laboratory scale bubble column, the OD removal reached 70% and all fractions of polyphenolic compounds of OMW were oxidized.     

Ca stimulated neutral lipase activity in castor bean lipid bodies

The membranes of lipid bodies from the endosperm of seeds of Ricinus communis have long been known to contain an acid lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3). The means by which fat hydrolysis is initiated and controlled in the endosperm of the young seedling are not yet understood, although it is generally assumed that the acid lipase is the enzyme responsible for the conversion of stored triacylglycerols to fatty acids and glycerol. However, the enzyme from seeds is not an effective catalyst at cytoplasmic pH since it has a pH optimum at 4.5 and is virtually inactive above pH 6.0. The results described in this paper show that during early growth of castor seeds the lipid bodies acquire a lipase which is active at neutral pH values. The lipase is absent from dry seeds, appears at day 3, and increases rapidly in activity until day 5. The pattern of appearance of the lipase mirrors that of other enzymes involved in the conversion of fat to sugar. The lipase is stimulated 40-fold by 30 micromolar free Ca²⁺ and the activity at pH 7.0 to 7.5 adequately accounts for the known rate of triacylglycerol hydrolysis in vivo.     

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.