The role of plant particles,bacteria and cell-free supernatant fractions of rumen contents in the hydrolysis of trilinolein and the subsequent hydrogenation of linoleic acid

The role of different fractions of rumen contents in the hydrolysis of trilinolein and the subsequent hydrogenation of the linoleic acid has been investigated by a series ofin vitro incubations. Hydrolysis of the trilinolein to free linoleic acid occurs almost wholly in the cell-free supernatant; the liberated linoleic acid in the supernatant can be rapidly adsorbed onto food particles where it is hydrogenated to stearic acid via the C₁₈ trans-11 monoene. Some 25% of the trilinolein added as substrate was taken up by the bacteria and of this a small percentage appeared to be hydrolysed and the free linoleic acid hydrogenated to stearic acid intracellularly. No conclusive evidence was obtained to suggest that this intracellular hydrogenation proceeded by a route other than that which took place on the food particles.     

Investigation of behavior of an enzyme in a biphasic system: soybean lipoxygenase-1

Soybean lipoxygenase-1 (EC 1.13.11.12) reaction with linoleic acid as substrate was used to study the biocatalysis in a biphasic system when the reactants have surface-active properties. The poorly water-soluble substrate was initially dissolved in an apolar solvent (octane). The hydroperoxide produced was water soluble and remained in the aqueous phase (borate buffer). The bioreactor was a modified Lewis cell with a well-defined interfacial area between the two phases. Two phenomena were studied separately: the reactant transfer between the two phases and the biocatalyzed reaction in an aqueous medium. This allowed determination of the transfer and the reaction constants. Substrate transfer was found to be affected by the progress of the reaction, because linoleic acid and the hydroperoxy acid have an influence on the interfacial tension. Inactivation of the biocatalyst at the interface was observed in the bioreactor. These results indicate that it is impossible to analyze the system behavior with the method proposed in the literature, which is based on the sequential study of the substrate transfer to the aqueous phase and its biocatalysis by lipoxygenase. The interaction between transfer phenomena and reaction kinetics was studied in the biphasic system. The kinetics were different from those obtained in the aqueous medium. Catalysis and transfer influence each other reciprocally. In this compartmentalized system, cooperativity phenomena were obtained using a nonallosteric enzyme. The evolution of the system was modeled (Runge-Kutta algorithm). The curves obtained were very close to those determined experimentally.     

Lipase-mediated hydrolysis of corn DDGS oil: Kinetics of linoleic acid production

In this study, we investigated the kinetics of linoleic acid production via lipase-mediated hydrolysis of corn DDGS oil in a batch reactor with continuous mechanical agitation and developed a kinetic model that incorporated the product inhibition to study the complete hydrolysis. The model agreed very well with observed data; though situations with low enzyme dosage or low stirring rates were modeled successfully without product inhibition, actual product concentration in such situations was too low to exert any inhibitory effects. Increasing the enzyme concentration increased hydrolysis, and beyond certain enzyme concentrations, effects tended to fade away because of excessive enzyme desorption from the interface. An enzyme dosage within the range of 40–60 KLU/L of oil dispersion could be successfully applied for a substrate concentration of 25–50 g/L of DDGS oil. Increasing the agitation rates improved enzymatic hydrolysis, but a higher stirring rate of 1000 rpm moderately improved production of linoleic acid compared with a stirring rate of 750 rpm. Within the range of substrate concentrations studied, enzymatic inhibition was moderate but still evident. The high degree of hydrolysis (i.e., ∼96% of theoretical linoleic acid yield) from DDGS oil suggests this method has potential for commercial production of linoleic acid.     

Recombinant PNPLA1 catalyzes the synthesis of acylceramides and acyl acids with selective incorporation of linoleic acid

Loss-of-function mutations in patatin-like phospholipase domain-containing protein 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, and altered PNPLA1 activity is implicated in the pathogenesis of atopic dermatitis and other common skin diseases. To examine the hypothesis that PNPLA1 catalyzes the synthesis of acylceramides and acyl acids, we expressed and partially purified a soluble, truncated form of PNPLA1 in Escherichia coli, (PNPLA1_trun) along with the related protein PNPLA2 (ATGL, adipose triglyceride lipase) and coactivator CGI-58. Liposomal substrates were incubated with recombinant enzymes for 0.5–24 h and products analyzed by HPLC-UV and LC-MS. Using trilinolein or dilinolein substrates, PNPLA1_trun, like ATGL_trun, catalyzed lipolysis and acyltransferase reactions with 2–30% conversion into linoleic acid, monolinolein, and trilinolein. CGI-58 enhanced ATGL-catalyzed lipolysis as previously reported, but transacylase activity was not enhanced with ATGL or PNPLA1. In matching the proposed activity in vivo, PNPLA1 catalyzed acyl transfer from trilinolein and dilinolein donors to omega-hydroxy ceramide, omega-hydroxy glucosylceramide, and omega-hydroxy acid acceptors to form acylceramide, glucosyl-acylceramide, and acyl acid, respectively, albeit with only ∼0.05% conversion of the substrates. Notably, in experiments comparing dilinolein vs. diolein acyl donors, PNPLA1 transferred linoleate with 3:1 selectivity over oleate into acylceramide. These results support the role for PNPLA1 in the synthesis of acylceramides and acyl acids in epidermis and suggest that the enrichment of these lipids with linoleic acid could result from the substrate selectivity of PNPLA1.     

Glycine-1-14C]hippuryl-L-histidyl-L-leucine: a substrate for the radiochemical assay of angiotensin converting enzyme.

[Glycine-1-¹⁴C]hippuryl-l-histidyl-l-leucine was synthesized and evaluated as a substrate for the radiochemical assay of angiotensin converting enzyme. Hydrolysis is measured by quantitation of the liberated [glycine-1-¹⁴C]hippuric acid by liquid seintillation counting and is linear up to 30% hydrolysis. The advantages of the radiochemical assay over the spectrophotometric quantitation of the liberated hippuric acid are its increased sensitivity and lack of interference by nonionic detergents or lipids.     

Mass transfer induced interchange of the kinetic and thermodynamic control of enzymic peptide synthesis in biphasic water-organic systems

The α-chymotrypsin catalysed kinetically controlled peptide synthesis in water and in biphasic water-methyl iso-butyl ketone system was compared. Due to the substrate and product partitioning in the biphasic system an interchange of the reaction control was observed at high enzyme concentration. Under these conditions, the rate of mass transfer between the phases was the rate limiting step and the hydrolysis product concentration was found to have a transient maximum ≫ equilibrium value. In this case, most of the peptide was sythetized in a thermodynamically controlled process. In an aqueous one phase system, the peptide synthesis was kinetically controlled.     

Enzymatic production of 4-hydroxyphenylacetaldehyde by oxidation of the amino group of tyramine with a recombinant primary amine oxidase

In this study, an efficient enzymatic process for the synthesis of 4-hydroxyphenylacetaldehyde (4-HPAA) from tyramine was developed using whole cells of recombinant Escherichia coli co-expressing primary amine oxidase (PrAO) from E. coli and catalase (CAT) from Bacillus pumilus. The reaction conditions for the synthesis of 4-HPAA were systematically optimized starting from a monophasic aqueous buffer. The optimum reaction temperature, pH, and biocatalyst loading were 33 °C, 7.5, and 20 g/L wet cells, respectively. Substrate feeding strategies were employed to alleviate substrate inhibition, providing a 14.8 % increase in yield. A biphasic catalytic system was explored to avoid product inhibition and thus further improve the 4-HPAA yield. Ethyl acetate was found to be the best organic solvent, and the optimum volume ratio of the organic phase to the aqueous phase was 40 % (v/v). Under the optimized conditions on a 1 L scale, a yield of 76.5 % was obtained with a substrate concentration of 120 mM. Thus, the bioconversion was more efficient in the ethyl acetate/buffer biphasic system than in the monophasic aqueous system, and the yield of 4-HPAA was improved 1.89-fold.     

Substrate specificities of lipases from corn and other seeds

Lipases from several seed species were shown to be relatively specific on triacylglycerols containing the major fatty acid components of the storage triacylglycerols in the same species. In a direct comparison using individual triacylglycerol as well as mixed triacylglycerol preparations, highest activities were observed in corn lipase on trilinolein and triolein, castor bean lipase on triricinolein, rapeseed lipase on trierucin, and elm seed lipase on tricaprin. This pattern of fatty acyl specificity was also observed on diacylglycerols, monoacylglycerols, and fatty acyl 4-methylumbelliferone, although the pattern became less distinct. The seed lipases were inactive on lecithins. Corn lipase was more active on tri- than di- or monolinolein, and released linoleic acids from both primary and secondary positions. As judged from the kinetics of hydrolysis of rac-glyceryl-2,3-stearate-1-oleate and rac-glyceryl-1,3-stearate-2-oleate, and of trilinolein and dilinoleins, corn lipase exerted some degree of preference in releasing fatty acid from the primary than the secondary position of a triacylglycerol. At the primary position, corn lipase was more active on oleyl ester than stearyl ester.     

Δ6-desaturase: improved methodology and analysis of the kinetics in a multi-enzyme system

A new method of assay for the A6-desaturation of linoleic acid was developed. This method, which uses HPLC for separation of the fatty acid substrate and product, exhibited a lower coefficient of variation (0.3%) than the reported TLC method (3.5%) [l], and avoided the step of methylation of the saponified fatty acid substrate and product. Using this new method of assay, the kinetics of the Δ6-desaturase in a multi-enzyme system were analysed. A number of factors that could have striking effects on desaturase kinetics were investigated, including the effect of (i) endogenous microsomal linoleic acid on total substrate concentration, and (ii) the pre-reaction catalysed by acyl-CoA synthetase and competing reactions catalysed by lysophospholipid acyltransferase and acyl-CoA hydrolase. Endogenous free linoleate in the hepatic microsomes was found to be 2.9 ± l.0 AM (0.5 mg microsomal protein/ml), which was comparable to added substrate concentrations (l.8 to 7.9 μM). The kinetics of the Δ6-desaturase were dissected from the kinetics of the above mentioned pre-reaction and competing reactions through a combination of experimental approaches and computer modeling. From computer modeling, a K_m and V_max of l.5 μM and 0.063 nmol/min were calculated for the Δ6-desaturase, compared to K_m and V_max of 10.7 μM and 0.08 nmol/min calculated directly from data uncorrected for endogenous substrate. It was concluded that lysophospholipid acyltransferase, acyl-CoA synthetase and endogenous linoleic acid significantly affect the kinetic measurements of hepatic microsomal Δ6-desaturase. These results have implications for kinetic analyses of all desaturases in microsomal systems.     

Kinetics of manganese lipoxygenase with a catalytic mononuclear redox center

Manganese lipoxygenase was isolated from the take-all fungus, Gaeumannomyces graminis, and the oxygenation mechanism was investigated. A kinetic isotope effect, k(H)/k(D) = 21-24, was observed with [U-(2)H]linoleic acid as a substrate. The relative biosynthesis of (11S)-hydroperoxylinoleate (11S-HPODE) and (13R)-hydroperoxylinoleate (13R-HPODE) was pH-dependent and changed by [U-(2)H]linoleic acid. Stopped-flow kinetic traces of linoleic and alpha-linolenic acids indicated catalytic lag times of approximately 45 ms, which were followed by bursts of enzyme activity for approximately 60 ms and then by steady state (k(cat) approximately 26 and approximately 47 s(-1), respectively). 11S-HPODE was isomerized by manganese lipoxygenase to 13R-HPODE and formed from linoleic acid at the same rates (k(cat) 7-9 s(-1)). Catalysis was accompanied by collisional quenching of the long wavelength fluorescence (640-685 nm) by fatty acid substrates and 13R-HPODE. Electron paramagnetic resonance (EPR) of native manganese lipoxygenase showed weak 6-fold hyperfine splitting superimposed on a broad resonance indicating two populations of Mn(II) bound to protein. The addition of linoleic acid decreased both components, and denaturation of the lipoxygenase liberated approximately 0.8 Mn(2+) atoms/lipoxygenase molecule. These observations are consistent with a mononuclear Mn(II) center in the native state, which is converted during catalysis to an EPR silent Mn(III) state. We propose that manganese lipoxygenase has kinetic and redox properties similar to iron lipoxygenases.     

Aliphatic Amidase from <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M8 Is Related to the Nitrilase/Cyanide Hydratase Family

A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.     

Triacylglycerol Profiles of Tilletia controversa and Tilletia tritici

The triacylglycerol (TG) profiles of teliospores of Tilletia controversa and Tilletia tritici were examined by high-performance liquid chromatography (HPLC) and gas-liquid chromatography. Boiling isopropanol was used to ensure enzyme inactivation during homogenization. The largest lipid component as determined by thin-layer chromatography was TGs. On the basis of thin-layer chromatography of crude lipid extracts, T. controversa and T. tritici do not contain a large amount of free fatty acids. TG profiles of T. controversa and T. tritici were very similar, with 18 species of TGs resolved by HPLC and gas-liquid chromatography. In both organisms, PLL (palmitic, linoleic, linoleic) was the major component, followed by LLL (trilinolein) and PLO (palmitic, linoleic, oleic). The ratio of PLO to PLL was 1:6 and 1:4 in T. tritici and T. controversa, respectively. The TGs of both organisms contain long-chain (>22 carbons) mono- and dienoic acids. Linoleic acid was the major fatty acid found in TGs from both organisms. The differences of TGs were not considered significant for differentiation purposes.     

A two-enzyme system for the transformation of unsaturated oils to 9(S)-hydroperoxy fatty acids

A two-enzyme system involving a lipase from a Pseudomonassp. and an extract of potato tubers containing lipoxygenase was used to convert triacylglycerols to 9-hydroperoxy fatty acids. Highest yields (up to 25%) were obtained with 1 to 20 g l^–1 trilinolein as substrate after 5 h under O₂ at pH 6 and 25 °C. The product structure was confirmed by ¹H NMR, MS and IR.     

Hydrolysis of bile acid conjugates by Clostridium bifermentans

Summary Two strains of Clostridium bifermentans have been investigated for their ability to hydrolyse bile acid conjugates under conditions suited to further transformation of the free acids liberated. In batch fermentation at 0.5 g/l substrate concentration, growing cells effected the near-quantitative hydrolysis of glycodeoxycholate, taurodeoxycholate and taurocholate within 48 h; glycocholate was 88% hydrolysed. At substrate concentration greater than 1.0 g/l however, taurine conjugates were less well hydrolysed. Further transformation of the liberated cholic acid to deoxycholic acid and/or 7-ketodeoxycholic acid was achieved, but quantitative conversion was not observed.     

Inhibitors of lipase activities in soybean and other oil seeds

In the cotyledon extracts of seedlings of many oil seeds, including soybean, sunflower, cucumber, and peanut, the in vitro lipase activity was too low to account for the observed in vivo lipolysis. The low in vitro lipase activity was due to the presence of lipase inhibitors in the extracts. The inhibitors from soybean were characterized based on their effects on the hydrolysis of trilinolein by corn, pancreatic, and Rhizopus lipases. The inhibitors were not dialyzable and unaltered by RNase and β-galactosidase treatment. However, they were sensitive to heating and protease digestion. The inhibitory effect of the inhibitors was expressed irrespective of the sequence of the addition of lipase, substrate, and inhibitors to the assay medium. The inhibitory effect was equally expressed when the inhibitors were added either before or after the lipase reaction had been in progress. The inhibitory effect of the inhibitors was independent of the amount of lipase present in the assay, but was dependent on the amount of substrate added. High substrate concentration eliminated totally the inhibitory effect of the inhibitors. Most of the inhibitors were recovered in the soluble fraction in subcellular fractionation. They were present in the 2-4S and not in the 7S, and 11S (storage proteins) protein fraction. There was a gradual decrease of the inhibitors in the cotyledons in the postgerminative growth. We suggest that the inhibitors are proteins which bind to the surface of the substrate micelles. The binding prevents the normal functioning of lipase which acts on the interfacial area between the aqueous medium and the micelle surface.     

Enzymatic properties of polyethylene glycol-modified cholesterol esterase in organic solvents.

The solvent dependency and substrate specificity of polyethylene glycol (PEG)-modified cholesterol esterase (CEH) catalyzing cholesterol ester synthesis in organic solvents were studied. When cholesterol and linoleic acid were used as the substrates, PEG-modified CEH synthesized cholesterol linoleate only in water-immiscible organic solvents. Among some solvents capable of solubilizing all of the reaction components (PEG-modified CEH, cholesterol, and linoleic acid), chloroform was most suitable for enzymatic cholesterol linoleate synthesis, and the synthetic activity for cholesterol linoleate decreased in the order chloroform, benzene, toluene, and cyclohexane. PEG-modified CEH synthesized various cholesterol esters with significant substrate specificity. The substrate specificity for cholesterol ester synthesis in benzene was analogous to that for cholesterol ester hydrolysis in aqueous solution.     

Determination in plasma of angiotensin-converting enzyme inhibitor by inhibitor-binding assay

A method of determining a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite (RS-5139) in plasma by inhibitor-binding assay has been developed using high-performance liquid chromatography. The assay is based on the principle that the amount of inhibitor bound to the enzyme is inversely related to the amount of hippuric acid liberated on hydrolysis from the artificial substrate (hippuryl- -hystidyl- -leucine). Plasma was heated at 60°C for 15 min, to inactivate endogenous enzyme, and preincubated with rabbit-lung angiotensin-converting enzyme at 37°C for 3 min. The artificial substrate (5.75 mg/ml in pH 8.3 phosphate buffer containing sodium chloride) was added to the resulting solution, and the mixture was incubated for 30 min. The reaction was terminated by the addition of 2 M hydrochloric acid. The hippuric acid liberated on hydrolysis was extracted with ethyl acetate and determined by reversed-phase chromatography using methylparaben as an internal standard. The total concentration of the inhibitor and its metabolite were determined by this method after de-esterification by rat-plasma esterase. The standard curve was obtained by the regression analysis of log concentration against logit response. The within-day and day-to-day precision were satisfactory. The proposed method is simple, rapid and sensitive enough to determine angiotensin-converting enzyme inhibitor in plasma.     

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.     

Hydrolysis of 11-cis- and all-trans-retinyl palmitate by retinal pigment epithelium microsomes.

A partial characterization of the enzymatic hydrolysis of 11-cis- and all-trans-retinyl palmitate by bovine retinal pigment epithelium microsomes was carried out using a micro-radiometric method to quantitate liberated palmitic acid. Retinyl ester hydrolase (REH) activity was examined in the absence of detergent. Hydrolysis of 11-cis- and all-trans-retinyl palmitate was protein- and time-dependent. Optimal enzyme activity occurred at slightly alkaline pH (8-9). Apparent kinetic constants (Vmax and Km) for the 11-cis-REH were 2.1 nmol/min/mg protein and 66 microM, respectively. All-trans-REH demonstrated a lower maximum velocity of 0.3 nmol/min/mg protein and a slightly higher substrate affinity of 27 microM. Further characterization of 11-cis-retinyl palmitate hydrolysis involved monitoring formation of reaction products, 11-cis retinol and palmitic acid, which were found to be released in essentially a 1:1 stoichiometry. Addition of all-trans retinyl bromoacetate, a known inhibitor of lecithin:retinol acyltransferase reduced both 11-cis and all-trans-REH activities but to significantly different degrees (50 and 76%, respectively). Although the microsomal preparation exhibited LRAT activity, acyl transfer was not readily reversible as labeled palmitic acid was not transferred to added acyl acceptor compounds. These findings suggest that hydrolysis of 11-cis-retinyl palmitate by bovine retinal pigment epithelium microsomes may occur at a catalytic site distinct from that for the all-trans isomer and that this hydrolysis is not representative of a reverse transesterification reaction.     

Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.