Development and intracellular localization of lipase activity in rapessed (Brassica napus L.) cotyledons

In homogenates of resting rapeseeds no lipase activity (glycerolester hydrolase, EC 3.1.1.3) could be detected using a titrimetric assay procedure. Following a 30-h lag-phase after imbibition, lipase activity increased sharply, reaching its maximum at day 4 after sowing. Simultaneously triglyceride content of the cotyledons decreased sharply. At any time during the 11-day period of seedling growth examined, only an alkaline lipase activity with a pH optimum around 9 was present. White light had essentially no effect on the development of lipase activity. However, the disappearance of lipase activity from the cotyledons after fat utilization was found to depend on nitrogen nutrition of the seedlings. The activities of the glyoxysomal enzymes catalase and malate synthetase showed the usual rise and fall patterns with peak activities at day 4 after sowing, independently of the mineral nutrition of the seedlings.About 90% of the lipase activity was associated with a microsomal membrane fraction. Resolution of this fraction by sucrose density gradient centrifugation (62,000 g for 14 h) yielded three distinct membrane fractions. Maximum activities of membrane marker enzymes were recovered from the gradients at following densities: The major portion of microsomal protein and lipase activity at 1.085 kg/l; microsomal malate synthetase and phosphorylcholineglyceride transferase at 1.116 kg/l; NADH-cytochrome c reductase and phosphorylcholinecytidyl transferase at 1.133 kg/l. Evidently in rapeseed cotyledons lipase activity is associated only with a discrete microsomal membrane fraction which sediments differently from membrane fractions of the endoplasmic reticulum.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).     

Photosynthetic characteristics of chloroplasts isolated fromMesembryanthemum crystallinum L., a halophilic plant capable of Crassulacean acid metabolism

Photosynthetically highly active chloroplasts were routinely obtained by rupture of leaf protoplasts from the halophyteMesembryanthemum crystallinum which exhibited the photosynthetic characteristics of either a C₃ plant when grown with 20 mmol l^-1 NaCl in the rooting medium, or a Crassulacean-acid-metabolism (CAM) plant when grown with 400 mmol l^-1 NaCl. Photosynthesis rates of C₃ and CAM chloroplasts were 150–250 and 90–150 μmol mg^-1 chlorophyll h^-1, respectively. Because of osmotic adjustment, CAM chloroplasts required higher sorbitol concentrations (0.7–0.8 mol l^-1) in the assay medium than C₃ chloroplasts (0.3–0.4 mol l^-1) for optimum activity. Substitution of sorbitol by NaCl as the osmoticum strongly reduced photosynthesis of CAM chloroplasts. Rates of electron transport (ferricyanide reduction, uncoupled) remained unaffected over a range of sorbitol concentrations (0 to 1 mol l^-1). Sensitivity of electron transport to increasing levels of NaCl was less pronounced than the NaCl-sensitivity of CO₂ fixation by intact chloroplasts. The CAM chloroplasts showed a broad pH optimum of photosynthesis between pH 7.0 and 8.2; photosynthesis of C₃ chloroplasts dropped markedly below pH 7.6. The CAM chloroplasts maintained a higher transenvelope proton gradient than C₃ chloroplasts both in the light and dark. External pyruvate (5 mmol l^-1) inhibited photosynthesis of CAM chloroplasts, but not of C₃ chloroplasts. Inhibition was reduced by increased external concentrations of orthophosphate.     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

Production and characterization of esterase and lipase from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis>

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h⁻¹ and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l⁻¹ using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h⁻¹ (at 42.5°C, pH 7.4, and 3.6 mol l⁻¹ NaCl), 2.3 U l⁻¹ (at 50°C, pH 7.5, and 4.3 mol l⁻¹ NaCl), and 0.58 U l⁻¹ (at 50°C, pH 7.6, and 4.5 mol l⁻¹ NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l⁻¹ NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.     

Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

Proline in relation to free radical production in seedlings of Brassica juncea raised under sodium chloride stress

The production of malondialdehyde (MDA) was higher in cotyledons from NaCl-raised Brassica juncea seedlings than in control seedlings. Light accelerated the MDA-producing capacity of thylakoids isolated from both control and treated seedlings. When exposed to strong white light (920 mol photons m^-2 s^-1) the thylakoids from NaCl seedlings produced nearly 5 times more MDA than control thylakoids. In the cotyledons of NaCl seedlings, the proline level was 24-fold higher than in controls. The presence of proline during exposure of thylakoids to white light decreased MDA levels. The reduction in MDA production was higher in the thylakoids of NaCl seedlings than of controls. It is proposed that proline accumulation has an adaptive significance as it lowers the generation of free radicals and thus reduces the lipid peroxidation linked membrane deterioration under stress.     

The regulation of pollen tube growth in Douglas-fir following high doses of ionizing radiation

The relationship between temperature and sensitivity to gibberellin A₃ (GA₃) was studied in lettuce seedlings (Lactuca sativa L. cv. Arctic). Dose/response curves for hypocotyl elongation (10^-4 mol l^-1 to 10^-8 mol l^-1) were constructed for a range of temperatures and the slope of the linear portion of the plots used as an indication of the sensitivity to GA₃. Hypocotyls were unresponsive to GA₃ below 13°C but above this temperature sensitivity increased linearly. Plots of growth rate against temperature had inflexions between 12°C and 13°C, with slopes above this point which increased with increasing GA₃ concentration. The Q₁₀ value for response increased in a similar manner. Reaction rates of NAD-dependent malate dehydrogenase and peroxidase extracted from hypocotyls varied linearly with temperature whilst nonspecific tetrazolium reduction, a membrane based activity, showed an abrupt rate change above 14°C. Pre-exposure to GA₃ had no effect on the temperature responses of soluble or particulate enzymes.Abbreviation GA₃ gibberellin A₃     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Intracellular lipase activities in heart and skeletal muscle homogenates The absence of trierucin cleavage by the heart: A possible biochemical basis for erucic acid lipidosis

Rat heart and skeletal muscle homogenstes were compared for their intracellular lipolytic activity towards a series of saturated and unsaturated triglycerides from trilaurin (C_{12: 0}) to trierucin (C_{22: 1}).It is shown that for all triglycerides esterified with fatty acids from C₁₂ to C₁₈, lipolytic activity in heart homogenates was higher than in skeletal muscle homogenates. For these triglycerides there was no relationship between the fatty acid chain length and the lipolytic activity.In both homogenates cleavage of unsaturated triglycerides was higher than cleavage of the homologous saturated triglyceride.Lipolysis of tri-Δ-11-eicosenoin (C_20:1) was similar in both homogenates but much lower than lypolysis of other triglycerides.Although cleavage of trierucin (C_22:1) was very low in skeletal muscle homogenates, it was undetectable in heart homogenates, even when enzyme concentration was increased.A mixture of triglycerides did not show preferential hydrolysis of any single triglyceride. Trierucin was the only triglyceride that did not compete for lipolytic activity and only with heart homogenates, which shows that heart lipase(s) do not cleave trierucin.The absence of lipolytic activity towards trierucin in heart homogenates could explain the selective accumulation of erucic acid-rich triglycerides in hearts of animals fed a diet with a high erucic acid content.     

Purification and properties of a F420-nonreactive,membrane-bound hydrogenase from Methanosarcina strain Gö1

The distribution of the F₄₂₀-reactive and F₄₂₀-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F₄₂₀-nonreactive enzyme. The membrane-bound F₄₂₀-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mol H₂ oxidized · min^-1 · mg protein^-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S^2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F₄₂₀-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F₄₂₀-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.Abbreviations transmembrane electrochemical gradient of H^- - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - Mb. Methanobacterium - PMSF phenylmethyl-sulfonylfluoride - Cl₃AcOH trichloroacetic acid     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Characterization of a Triacylglycerol Lipase That Liberates Arachidonic Acid from Bovine Chromaffin Cells During Secretion

Abstract: Primary cultures of chromaffin cells from bovine adrenal medullae were used as a model to study lipolytic events during stimulus-secretion coupling. It has been shown that chromaffin cells liberate arachidonic acid in addition to their main secretion product, the catecholamines. To understand more about the mechanism of arachidonic acid liberation, chromaffin cells were labeled with radioactive arachidonic acid, stimulated, and then analyzed for changes in lipid composition. After stimulation with 10^?4M acetylcholine, the radioactivity of triacylglycerols decreased to the same extent that the free arachidonic acid level rose. This finding suggests that in bovine chromaffin cells a stimulation-dependent triacylglycerol lipase (triacylglycerol hydrolase; EC 3.1.1.3) is involved in arachidonic acid liberation. Further work was performed on detection, characterization, and isolation of this enzyme. Triacylglycerol lipase activity was found in whole cell homogenates and in plasma membrane fractions isolated from adrenal medullary tissue. The plasma membrane lipase showed a pH optimum of 4.3. The apparent Michaelis constant was determined as 3.3 × 10^?4 mol/L. Ca²⁺ did not influence the enzymatic activity. To differentiate the plasma membrane triacylglycerol lipase from the previously described plasma membrane diacylglycerol lipase of chromaffin cells, the influence of RG 80267, a specific diacylglycerol lipase inhibitor, was examined. RG 80267 (50 μM) inhibited the triacylglycerol lipase by only 24%, although diacylglycerol lipase was totally inhibited with only 20 μM RG 80267. The pH optimum of homogenate lipase was broad, lying between 4 and 7. Starting from the soluble fraction of whole cell homogenates, the triacylglycerol lipase was partially purified by ultracentrifugation and size-exclusion chromatography. The molecular mass of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was found to be between 47 and 57 kDa.     

Lipase in lipid bodies of cotyledons of rape and mustard seedlings

Lipolytic activity was absent in the crude cotyledon extract of ungerminated rapeseed (Brassica napm L. var. Dwarf Essex), and increased to a peak at day 4 in seedling growth, concomitant with the decrease in total lipids. About 50% of the lipase activity was recovered in the lipid bodies isolated from the cotyledon extract by flotation centrifugation. Isolated lipid bodies underwent autolysis of internal triacylglycerols resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies had been extracted with diethyl ether, the lipase was recovered in the remaining membrane fraction. The lipase had a maximal activity at pH 6.5 on trierucin, trilinolein, or endogenous triacylglycerols, and at pH 8.0 on N-methylindoxylmyristate. The lipase was most active on trierucin and trilinolein, and hydrolyzed the related di- and monoacylglycerols at lower rates. There was little enhancement of the lipase activity in the presence of NaCl, CaCl₂, or detergents, and detergents in general reduced the activity. The hydrolysis of trierucin was linear until about 50% of the trierucin had been converted to erucic acid, and there was little accumulation of dierucin and monoerucin. Lipase extracted from lipid bodies isolated from germinated rapeseed of the variety Tower, which contains little or no erucic acids in the storage triacylglycerols, also had the highest activities on trierucin and trilinolein. A comparative study on mustard seed (Brassica juncea) revealed that the mustard lipase possessed characteristics very similar to those of the rapeseed lipase.     

Changes in respiration of mitochondria isolated from cotyledons of ethylene-treated pea seedlings

Treatment of intact, germinating pea (Pisum sativum L. cv. Homesteader) seedlings with ethylene enhanced the cyanide-resistant respiration of mitochondria isolated from the cotyledons. The level of enhancement depended on the concentration of ethylene. Thus, exposure to 0.9 l·l^-1 of ethylene in air for days 4–6 of germination had little effect on cyanide-resistant respiration, while exposure to 130 l·l^-1 increased it from 10 to 50 nmol O₂·min^-1·(mg protein)^-1. The length of exposure to ethylene also affected the degree of enhancement. According to some literature data, lipoxygenase (EC 1.13.11.12) activity can be mistaken for cyanide-resistant respiration, but in our preparations of purified pea mitochondria ethylene had no effect on lipoxygenase activity, nor did the gas disrupt the outer mitochondrial membrane. Bahr and Bonner plots of respiration in the presence of salicylhydroxamic acid (SHAM) indicated that ethylene did not affect respiration proceeding via the cytochrome pathway. Thus, increases in total respiration in mitochondria from cotyledons of ethylene-treated pea seedlings reflect increases in cyanide-resistant respiration.Abbreviations Cyt c cytochrome c - SHAM salicylhydroxamic acid     

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca²⁺-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using ??-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T_1/2 was 1 h at 100°C. The K _m of the partial purified enzyme was 1 mM and the V _max was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca²⁺ and Hg²⁺ stimulated lipase activity, whereas Zn²⁺, Co²⁺, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.     

Modulation of lipolytic activity in isolated canine cardiac sarcolemma by isoproterenol and propranolol.

Cardiac sarcolemmal preparations isolated from dog were tested for membrane-associated phospholipase A and lipoprotein lipase activities. The sarcolemma hydrolyzed 1-acyl 2¹⁴C-linoleoyl 3-glycero-phosphorylethanolamine at pH 7.0 to form predominantly ¹⁴C-lyso PE with 5 mM EDTA and ¹⁴C-free fatty acid with 5 mM Ca²⁺ suggesting the presence of both phospholipases A₁ and A₂ and/or lysophospholipase activities in these preparations. Sarcolemmal PLA activity was stimulated 300% by 10^?5 to 10^?6 M d1-isoproterenol; this stimulation was blocked by 10^?4 M d1-propranolol. Lipoprotein lipase activity associated with the sarcolemmal fraction was enhanced 10-fold by 10^?5 M d1-isoproterenol; stimulation was blocked by d1-propranolol. Thus, the activities of membrane-bound lipolytic enzymes appear to be modulated by β-adrenergic agents in canine cardiac sarcolemma and could affect lipid dependent enzymes and/or membrane permeability.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.     

Sophorolipid Production with High Yields on Whey Concentrate and Rapeseed Oil without Consumption of Lactose

Sophorolipids were produced by single-step batch cultivation of Candida bombicola ATCC 22214 on deproteinized whey concentrate and repeated feed of rapeseed oil. A mild sterilization method for whey was developed. High yields of 280 g dry sophorolipids l^–1 were obtained from deproteinized whey concentrate containing 100 g lactose l^–1 and 300 g rapeseed oil l^–1. Surprisingly, the whey lactose was not consumed by the organism. Growth only on the oil was assumed and a high lipase activity of 24 U per g cell dry weight resulted.     

High cell density cultivation of Brevibacterium linens and formation of proteinases and lipase

Brevibacterium linens forms hydrolytic enzymes which can be used to accelerate the ripening of cheese without causing bitterness. B. linens ATCC 9172 was grown to a high cell density (50 g dry wt l^–1 after 60 h) in a mineral medium containing lactic acid, soy-peptone and ammonium sulphate by applying a continuous feed of nutrients. The maximal activities of l-leucine aminopeptidase and cell-associated proteinase were 286 U l^–1 and 202 U l^–1, respectively. The cell-associated lipolytic activity exhibited a strong and sudden increase at 46 h, resulting in a maximum of 9.5 U g^–1 dry wt; thus the volumetric productivity of proteolytic and lipolytic activity was 4220 U l^–1 h^–1 and 7.3 U l^–1 h^–1, respectively.