Wax ester formation catalysed by isoenzymes of lipolytic acyl hydrolase

Wax ester formation by esterification of a long chain fatty acid (palmitic acid) with a long chain fatty alcohol (octadecanol) was enzymically catalysed by acetone dried powder preparations of potato tubers. The enzyme responsible for wax ester formation had multiple isoenzymic forms and was identical with lipolytic acyl hydrolase, a lipid deacylating enzyme. Tubers from different varietiees of potato (Solanum tuberosum) demonstrated markedly different levels of activity and electrophoretic patterns for both wax ester formation and lipid deacylation.     

Isoenzymes of lipolytic acyl hydrolase and esterase in potato tuber

Five varieties of potato (Solanum tuberosum) were shown by gel- and free-flow-electrophoresis to exhibit multiple forms of lipolytic acyl hydrolase (LAH) and esterase enzymes. The electrophoretic patterns of LAH and esterase activities and protein differed with the variety and were characteristic for a given variety. In the variety (Golden Wonder) with the highest LAH activity (p-nitrophenylpalmitate as substrate), this was 200-fold greater than the esterase activity (p-nitrophenylacetate as substrate) and isoenzyme patterns for both enzymes were the most complex. In the variety with a very low LAH activity (Désirée), the LAH and esterase activities were similar and more simple isoenzyme patterns for these enzymes were observed.     

Lipid-degrading enzymes from potato tubers

Three varieties of potato were used to investigate the activity of lipolytic acyl hydrolase, (LAH) and lipoxygenase, (LOX), for a short period after harvest. Both enzymes displayed very low levels of activity during the first few days, followed by an increase in later storage, with the hydrolase activity of Désirée tubers remaining low. An inverse relationship was found between the total LOX activity and the percentage of activity obtained in a particulate form. Only when the total LOX content was below 0.7 units (μmol/g/min fr. wt), was it possible to obtain a highly active particulate fraction. LAH particulate activity was dependent upon both enzymes remaining low. Protoplasts were isolated by the use of cell-degrading enzymes. When the total LOX activity in the tubers was low, 50% of this activity could be obtained in intact protoplasts. Once the LOX concentration in the tubers had risen, fewer intact protoplasts were isolated. No particulate activity of either enzyme was found when these protoplasts were lysed. The two lipid-degrading enzymes were not located in the amyloplasts.     

Subcellular localization of lipoxygenase and lipolytic acyl hydrolase enzymes in plants

The subeellular localization of two lipid-degrading enzymes, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX) was studied. In potato tubers the ac     

Dibucaine,chlorpromazine, and detergents mediate membrane breakdown in potato tuber homogenates

Various strategies were evaluated for their ability to minimize the rate of breakdown of endogenous membrane lipids during cell fractionation studies with potato tubers. Buffering the homogenates at pH 7.5 to 8.0 resulted in significantly lower rates of phosphatidylcholine (PC) hydrolysis than were observed at lower pH values. Several potential inhibitors were added to homogenates to evaluate their ability to inhibit membrane lipid hydrolysis. The addition of bovine serum albumin (1% w/v) inhibited the rate of PC hydrolysis by 50%. The addition of low concentrations (25–100 μM) dibucaine (nupercaine) inhibited PC hydrolysis, but at higher concentrations (1-2 mM) it caused a 5- to 6-fold stimulation. Because dibucaine is a calmodulin antagonist, two other calmodulin antagonists (trifluoperazine and chlorpromazine) were tested and found to exhibit similar patterns of inhibition and stimulation. Similarly, the addition of low concentrations of deoxycholate also inhibited PC hydrolysis and high concentrations stimulated it. These results indicate that the hydrophobic properties of deoxycholate, dibucaine, and other calmodülin antagonists may explain their unusual effects on the rates of PC hydrolysis in potato tuber homogenates. Although the addition of exogenous calcium increased the rate of PC hydrolysis, the addition of calmodulin (bovine brain) had no effect. Other experiments revealed that the addition of 1% bovine serum albumin improved the yield and stability of mitochondrial and microsomal fractions from potato tubers. In constrast, the addition of 100 μM dibucaine caused deleterious effects.     

Lipolytic and peroxidative enzyme expressions in cultured potato tuber cells

Potato cells (cv. Norchip) were cultured from tuber parenchymal tissue and subcultured to dissociate and habituate the despecialized cells. After several subculturings on a minimal nutrient media, this line of cells demonstrated repeatable physical growth profiles for dry weight (DW), fresh weight (FW) and protein. Two enzymes of plant lipid metabolism were investigated, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX), which respectively liberate and peroxidize fatty acids from lipid in cellular membranes. LAH, measured as p-nitrophenyl palmitate hydrolase, was present in this line of cells in easily detectable amounts (317 units g^-1 DW) albeit much lower than that found in mother tuber (9878 units g^-1 DW). The presence of LAH in this line is significant because LAH isozymes are often described as storage proteins, yet activity per gram fresh weight in these unorganized cells is reasonably constant until culture growth exits the linear phase. However, LOX, the most active free fatty acid metabolizing enzyme in potato tubers (89,800 units g^-1 DW), was not detectable in this line of callus or suspension cultured cells. The absence of LOX activity in this line of cells was verified by a number of assay approaches and was confirmed by activity staining of extracted enzymes separated in polyacrylamide gels. The absence of LOX in these cultured cells is especially important in determining the functions of this lipid peroxidation system and how it may be genetically regulated.Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named.A laboratory cooperatively operated by the Midwest Area, Agricultural Research Service, U.S. Department of Agriculture, The Minnesota Agricultural Experiment Station, the North Dakota Agrcultural Experiment Station, and the Red River Valley Potato Grower's Association.     

Role of 1-acyl-2-lyso-phosphatidylcholine acyl transferase in the biosynthesis of pulmonary phosphatidylcholine.

The majority of pulmonary dipalmitoyl phosphatidylcholine, whose surface tension lowering properties are necessary for maintenance of alveolar structural integrity and lung function, is synthesized via a reacylation mechanism. V_max and K_m values were obtained with the 1-acyl-2-lyso-PC: acyl-CoA acyl transferase system in rabbit lung microsomes and long term cultures of rabbit lung cells derived from type ll pneumonocytes enriched fractions. The data, especially for V_max, can be interpreted to indicate that this reacylation system may be an important factor in the biosynthesis of lung dipalmitoyl phosphatidylcholine.     

Membrane-degrading enzymes in the leaves of Solanum tuberosum

When homogenates of potato leaves were prepared under conditions which are typical for organelle isolation (pH 7.5 and 4°), membrane lipids underwent rapid hydrolysis (17% of phosphatidylcholine was hydrolysed in 2 hr). Leaves of 41 potato cultivars were surveyed for phospholipase activity to determine whether certain cultivars might be more suitable for the preparation of organelles. Phospholipase activities ranged from 1.04 to 11.60, μmol/min · g fr. wt and p-nitrophenyl palmitate hydrolase activity ranged from 0.0119 to 0.0502,μmol/min · g fr. wt. These phospholipase values were several hundred-fold higher than previously reported for potato leaves and nearly as high as in potato tubers. Most of the phospholipase activity in leaves was soluble and not membrane-associated as previously reported.     

Purification and properties of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase from potatoes

A rapid method for the purification of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (CQT) from potato tubers which had been stored at low temperatures is described. The method involves affinity chromatography on Blue Sepharose with biospecific desorption of CQT with its substrate, CoA. Elution of the Blue Sepharose column with a gradient of CoA leads to the resolution of CQT, a protein with MW of ca 41500, into 3 peaks of activity; the largest peak elutes first. This fraction is purified × 1440 and gives a single band of protein after PAGE which suggests a high degree of purity. The properties of the 3 fractions of CQT, with respect to substrates and to a number of inhibitors, are described. The first and last eluting CQT fractions are specific for quinate and show no activity towards shikimate. The second peak, however, shows a small activity towards shikimate but this is thought to be due to an underlying peak of a shikimate specific enzyme. The major peak of CQT activity found in potatoes stored at 0° is absent from those stored at 10° throughout the period after harvest.     

Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism

Aims: The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro‐organisms. Methods and Results: The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni‐nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35·6 kDa. Typical residues essential for lipolytic activity such as penta‐peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487 ) from Pseudomonas mendocina ymp and esterase (31%, AAY45707 ) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7·5 and 10°C. At thermal stability analysis, lipo1 was more unstable at 40°C than 10°C. Conclusions: An activity based strategy has been an effective method for fishing out a low‐temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone‐sensitive lipase family due to the enzyme’s oxyanion hole by the sequence HGGG. Significance and Impact of the Study: Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry.     

Nucleophile selectivity in the acyl transfer reaction of a designed enzyme

The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.     

Oligoarabinosides of hydroxyproline isolated from potato lectin

The lectin from potato tubers is a glycoprotein containing 50% sugars and rich in hydroxyproline and arabinose moieties. The nature of the protein-sugar linkage has been compared to that of insoluble potato cell wall protein and the arabinose was shown to exist as short oligosaccharides of 3 or 4 residues attached to hydroxyproline. In the lectin there were no large oligosaccharides attached to hydroxyproline. Lectin activity with the same specificity as that of the tuber lectin was shown to be associated with particulate membrane fractions prepared from cultured potato roots.     

Further studies on the subcellular localization of lipid-degrading enzymes

Further work on the subcellular localization of two lipid-degrading enzymes, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX) has been carried out on brassica florets, potato shoots and pea roots. In all cases, the LAH profile on sucrose and Ficoll density gradients was coincident with ‘lysosomal’ acid phosphatase activity. However, the localization of LOX activity was different for each tissue. In pea roots the activity of LOX was localized in the ‘lysosomal’ fraction, whereas with brassica florets (cauliflower and calabrese) it was present in a heavy body with a similar density to plastids and in potato shoots LOX gave only low particulate recoveries.     

Changes in the activity of hydroxycinnamyl CoA:quinate hydroxycinnamyl transferase and in the levels of chlorogenic acid in potatoes and sweet potatoes stored at various temperatures

A large increase in the activity of hydroxycinnamyl CoA:quinate hydroxycinnamyl transferase (CQT) occurred in potatoes stored at 0 and 2° and such an increase was prevented by storage at either 5 or 10°. The increase was most rapid in potatoes stored at 0° where it reached a maximum after 28 days and then declined slowly during storage for up to 6 months. Accompanying these changes in CQT were transitory increases in p-coumarate CoA ligase and PAL which occured during the first few weeks of storage at 0° and during this period there was nearly a two fold increase in the chlorogenic acid content of the tissue. The increase in chlorogenic acid did not occur at 10° when the increases in PAL, ligase and CQT were also prevented. The increase in CQT was reversed when tubers stored at 0° for 14 days were returned to 10° and this warming up period prevented further increase in CQT on return to 0°. The increase in CQT at 0° was prevented if the air in the storageatmosphere was replaced by N₂, 1 % O₂ or 10–15% CO₂. Similar increases in CQT, ligase and chlorogenic acid occurred in sweet potatoes stored at 7.5° but were prevented by storage at 15°. The role of PAL, ligase and CQT in the control of chlorogenic acid accumulation in these commodities and the significance of changes in their activities in relation to physiological changes at low temperatures are discussed.     

Cell surface and cellular debris-associated heat-stable lipolytic enzyme activities of the marine alga Nannochloropsis oceanica

Microbial surface display of lipases can be effectively employed for the development of whole-cell biocatalysts for industrial bioconversions. In the present work, we report for the first time the presence of thermostable lipolytic enzyme activities against p-nitrophenyl laurate, both on the cell surface and the cellular debris fraction of the marine microalga Nannochloropsis oceanica (strain CCMP1779). Whole cell-associated lipolytic activity (WCLA) shows a 2.5-fold stimulation after heat treatment at 100?°C for 60?min, while the activity of the respective cell debris is retained for 15?min. In contrast, heat treatment renders the soluble fraction of the disrupted cells inactive. The progress curve of cellular debris-associated lipase activity is biphasic and levels off very fast. Treatment with the surfactants SDS, Triton X-100 and CHAPS, which are known to inhibit lipase activity in various degrees, results in a loss of both cell bound and cell debris lipolytic activities (CDLA). The highest whole cell lipase catalytic efficiency was observed against p-nitrophenyl butyrate and the optimum pH for hydrolysis was determined at pH 7.0. Both unheated and heated undisrupted whole cell biocatalysts are also catalytically active against olive oil. High-salt concentrations (1M NaCl) lead to about 50% whole cell enzyme inhibition whereas the activity of heated cells increases. These findings offer novel insight into the biocatalytic properties and the biotechnological applicability of microalgal lipases from N. oceanica.     

Measurement of lipoxygenase activity in crude and partially purified potato extracts

Two assay systems, one a spectrophotometric assay at 234 nm, the other based on the oxygen electrode, were compared as methods for the routine analysis of lipoxygonase activity in crude and partially purified potato extracts. The spectrophotometric assay was unsuitable for the analysis of crude extracts and only gave meaningful results under very restricted reaction conditions. The oxygen electrode provided a reliable method for routine analysis of lipoxygenase activity.     

Purification of acyl hydrolase enzymes from the leaves of Phaseolus multiflorus

Acyl hydrolase activities have been purified from the leaves of Phaseolus multiflorus. The purification procedure involved heat treatment, DEAE-cellulose chromatography, Sephadex G-100 filtration and hexyl agarose chromatography. The elution pattern from hexyl agarose columns together with substrate competition experiments indicated the presence of two hydrolase enzymes. The first could hydrolyse oleoylglycerol and phosphatidylcholine while the second would deacylate glycosylglycerides and oleoylglycerol. Overall purification of both enzymes was ca 70-fold and the MW of the glycosylglyceride-hydrolysing enzyme was in the range 70–78000.     

Isolation and characterization of cell-wall pectic substances from potato tuber

Exhaustive treatment of potato tuber tissues by purified endo-polygalacturonase from Aspergillus japonicus solubilized 95% of the total uronides of     

Effects of dithiothreitol on dopa oxidase activity from potato tubers

A filtrate, prepared from potato tuber by grinding in an isotonic medium, has been separated into a particulate and a ‘soluble’ fraction by ultracentrifugation. Following dialysis and lyophilization, both fractions catalysed the oxidation of l-DOPA, with approximately 30% of the l-DOPA: oxygen-oxidoreductase (EC 1.14.18.1; DOPA oxidase) activity being associated with the particulate fraction. When dithiothreitol (DTT, 10^?2M was included in the grinding medium, much lower yields of DOPA oxidase were obtained and 80% appeared to be associated with the particulate fraction. DTT proved to be a powerful inhibitor of DOPA oxidase. With concentrations of DTT causing only partial inhibition, the kinetics of the inhibited rate of dopachrome formation from l-DOPA were complex. When oxygen consumption was measured inhibition was not transient. The degree of inhibition was inversely related to the DOPA oxidase activity, indicating interaction of a product of this activity with DTT. Direct determination of -SH groups in DTT using 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) showed that they were all oxidised during the initial phase of inhibition of dopachrome formation. It is concluded that the first phase of inhibition involves oxidation of DTT by an intermediate between l-DOPA and dopachrome. The second phase of inhibition also appeared to require -SH groups initially, since trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT) caused very little inhibition at all.     

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling.

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three-dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site-directed mutagenesis for the localization of the active site residues. The wild-type enzyme and mutants were expressed in Cos-1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomeric substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential "lid" domain at residues 50-74 of LCAT might be involved in the enzyme-substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three-dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish-eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.