Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene

Lipoprotein lipase, which catalyzes hydrolysis of emulsified triglycerides or water-insoluble esters, was modified with 2,4-bis(o-methoxy-polyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified lipase, in which 55% of the total amino groups in the lipase molecule, was soluble in organic solvents such as benzene, toluene, chloroform and dioxane. The modified lipase could catalyze ester synthesis reaction in benzene. When very hydrophobic substrates of lauryl alcohol and stearic acid were used, the ester synthesis reaction proceeded efficiently in the transparent benzene solution with the maximum activity of approximate 5.0 mumoles/min/mg of protein. Ester exchange and aminolysis reactions were also conducted with the modified lipase in benzene.     

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids L-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), L-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and L-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 degrees C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for L-ester-amide-PC and L-ether-amide-PC, whereas the temperature dependence is opposite for the L-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.     

Fatty-acid-modified enzymes as enantioselective catalysts in microaqueous organic media

Highly active lipase and protease complexes were prepared by non-covalent modification with stearic acid. The protein content and yield of the modified enzyme complexes depended on the enzymes' source. The increase in the transesterification activity of the modified enzymes was 15 fold for Candida rugosa lipase and porcine pancreatic lipase, with preservation of the enantioselectivity. Pseudomonas sp. lipase which showed no activity in its crude form, exhibited an activity of 38 mol/h·mg protein in the modified form. © Rapid Science Ltd. 1998     

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol⁻¹. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol⁻¹ at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h. Received: 12 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

Solid-state microstructure of poly(l-lactide) and l-lactide/meso-lactide random copolymers by atomic force microscopy (AFM)

Tapping mode atomic force microscopy was used to investigate the lamellar morphology of poly(l-lactide) and two poly(l-lactide-co-meso-lactide) random copolymers containing 3% and 6% meso-lactide. Samples were isothermally crystallized at selected temperatures, and qualitative and quantitative analyses of lamellar structure were performed using height and phase images. This is the first study of the morphology of polylactide stereocopolymers using a real-space probe, and the important effects of scanning parameters on the acquired images are described. More open spherulites with an abundance of screw dislocations between edge-on lamellar stacks were observed in samples crystallized at higher temperatures. Mean lamellar thicknesses are lower for the random copolymers compared to PLLA, particularly at lower DeltaT, in agreement with the results of our previous small-angle X-ray scattering (SAXS) experiments. Mean lamellar thicknesses derived from the current real-space examination are in good agreement with those determined previously from SAXS. Internal surfaces-from microtomed specimens-were also studied to investigate the bulk crystal morphology. Although quantitative analysis was not feasible (for reasons discussed in the text), lamellar organization similar to that seen in the surface experiments is observed at high magnifications.     

Solution studies and structural model of the extracellular domain of the human amyloid precursor protein

The amyloid precursor protein (APP) is the precursor of the beta-amyloid peptide (Abeta), which is centrally related to the genesis of Alzheimer's disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89% of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 A calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 A and 130 A, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30% of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed.     

Spin-label study of the relation between enzymatic activity and lipid-protein organization in reconstituted cytochrome c oxidase.

Lipid-depleted cytochrome c oxidase (EC 1.9.3.1) containing less than 20 microgram lipids per milligram protein was reconstituted with pure phospholipids of well-defined chemical structure and fatty acid composition without using detergents and (or) sonication. For the maximal restoration of electron transport activity, lipid-depleted cytochrome c oxidase required acidic phospholipds such as phosphatidylglycerol or phosphatidylserine or lysophospholipids such as lysophosphatidylcholine or lysophosphatidic acid, but no specific phospholipid fatty acid composition was necessary. The organization of the lipid environment of the reconstituted cytochrome c oxidase, having a well-defined lipid composition, morphology, and a high specific activity, was examined by electron spin resonance spectroscopy using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16-doxyl stearic acid) and 16-doxyl stearic acid - containing phosphatidylglycerol. The presence of boundary lipid was established in both lamellar and micellar organizations of reconstituted cytochrome c oxidase and was not necessarily related to the enzymatic activity of the complex. Our results have established that aside from structural considerations, the boundary lipid, at least in the reconstituted cytochrome c oxidase, is a necessary but not sufficient condition for the enzymatic expression of cytochrome c oxidase.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

Chitosan-cholesterol and chitosan-stearic acid interactions at the air-water interface

We report in this work the isotherms of cholesterol and stearic acid at the air-water interface modified by different chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weight chitosans) in the aqueous subphase. The Langmuir-Blodgett films of the complexes cholesterol-chitosan and stearic acid-chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulation was performed to visualize the chitosan-lipid interactions. Strong modifications are obtained in the isotherms as a result of the chitosan interactions with cholesterol and stearic acid at the air-water interface. These modifications were dependent on the type and concentration of chitosan. Severe modifications of all phases were noticed with larger molecular areas, and the observed changes in the compressional modulus were dependent on the type of chitosan used. The complexes of chitosan-stearic acid were more flexible than the ones of chitosan-cholesterol. The AFM images demonstrated that chitosan was disaggregated by the cholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. The hydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weight chitosans produced homogeneous surfaces with cholesterol. The simulated chitosan chains interacting with cholesterol and stearic acid demonstrated the possibility of specific sites of electrostatic bonds between these molecules. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showed that the medium and high molecular weight chitosans could retain higher proportions of cholesterol compared with the other analyzed samples.     

Incorporation of bile acid of low concentration into model and biological membranes studied by 2H and 31P NMR

We have analyzed the manner of incorporation of bile acid into lipid bilayers and resultant perturbation of the bilayer structure with lower bile acid/lipid ratios relevant to the physiological conditions (approximately 1 mM) by 2H and 31P NMR methods, as an aid to understanding the possible role as an endogenous tumor promoter in colon cancer besides the primary physiological function of solubilizing lipids. On the basis of the 2H quadrupole splittings of [6,6,7,7,8-2H5]deoxycholate and [11,11,12,12-2H4]chenodeoxycholate in the presence of lamellar multibilayers of egg yolk lecithin, these bile acids were found to be incorporated in such a manner that the B-D rings lie parallel with the normal of the bilayers when the ratio of the bile acid to lipid is low (less than 0.11). When the ratio is increased, these bile acid molecules are not dispersed entirely in the bilayer but aggregate to form micelles with lipids. Further, we studied the resultant perturbation of the multibilayers of egg yolk lecithin analyzed by using the 2H quadrupole splitting of [18,18,18-2H3]stearic acid as a probe and by 31P chemical shift anisotropy. We found that the bilayer structure is retained even at the bile acid-to-lipid ratio of 0.25, although a small amount of an isotropic phase appeared such as small vesicles and micelles. The molecular ordering of fatty acyl chains was rather enhanced by the presence of 1 mM deoxycholate in erythrocyte ghosts as seen from the 2H quadrupole splitting of [16,16,16-2H3]palmitic acid, although deoxycholate caused hemolysis in this condition. The former observation can be explained by the way the lipid-protein interaction is modified by deoxycholate located in the interface between the lipids and proteins.     

Dynamical dimer structure and liquid structure of fatty acids in their binary liquid mixture: dodecanoic and 3-phenylpropionic acids system

Dimer structure and liquid structure of fatty acids in the binary liquid mixture of dodecanoic (LA) and 3-phenylpropionic acids (PPA) were studied through the measurements of DSC, self-diffusion coefficient (D), density, viscosity, 13C NMR spin-lattice relaxation time, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS). The phase diagram of LA/PPA mixture exhibited a typical eutectic pattern, which means that LA and PPA are completely immiscible in solid phase. In the liquid phase of the LA/PPA mixture, D of LA always differed from that of PPA irrespective of their compositions. This exhibited that, in the liquid phase of the binary mixture of fatty acids giving a complete eutectic in the solid phase, the fatty acid dimers are composed of the same fatty acid species irrespective of their compositions. The liquid structure of the LA/PPA mixture was clarified through the SAXS and also the SANS measurements.     

Lipase immobilization on modified zirconia nanoparticles: Studies on the effects of modifiers

Zirconia nanoparticles modified with carboxylic acid with a long alkyl chain can significantly enhance the activity of immobilized lipases used for asymmetric synthesis in organic media. In this study, various carboxylic acids with different alkyl chain lengths were grafted to zirconia. Lipase from Pseudomonas cepacia (PCL) was immobilized on the modified zirconia nanoparticles and used for the resolution of (R,S)-1-phenylethanol through acylation in isooctane. The results showed that among the straight-chained carboxylic acids that formed a closely packed layer on the support, stearic acid with a long alkyl chain gave the best activity/enantioselectivity. The initial activity obtained was about 10 times higher than that from lipase loaded onto unmodified zirconia. This improvement could be attributed to the interaction between the long hydrophobic chain of the modifier and the lipase, leading to an interfacial activation effect. The stability of the immobilized PCL was investigated, and the lipase was found to be stable for at least ten consecutive runs with a substrate concentration of 5 g/L. At a high substrate concentration of ca. 100 g/L, the activity of the lipase gradually decreased, which suggests that deactivation was induced by substrate toxicity.     

Comparison of differently modified Pseudomonascepacia lipases in enantioselective preparation of a chiral alcohol for agrochemical use

Three methods for enzyme modification/immobilization were compared to enhance the catalytic performance of a commercially available lipase, Lipase PS from Pseudomonascepacia, in highly enantioselective transesterification of an agrochemically useful sec-alcohol, (R,?S)-HMPC [=(R,?S)-4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopenten-1-one], with vinyl acetate as both acyl donor and reaction medium. The stearic acid-coated lipase showed the highest catalytic activity, with a specific activity improved by 54 times over the native lipase. The microcrystal salt-supported lipase and celite-adsorbed lipase also displayed much better performance as compared with the native lipase. All the three modified lipase preparations showed a similar thermal stability to that of the native enzyme. The enantioselectivity (E-value) was also quite satisfactory in all the cases (E>100 at 30°C), though a trend of slight decline was also observed with the temperature increase in the range of 25–60°C. The optimum aqueous pH, from which the modified lipases were prepared, was 6.0–7.0. A low water activity (a_w) of ca. 0.1 was favorable for all the three modified lipases. The stearic acid-coated lipase displayed prominent advantages in catalyzing the transesterification reaction at a very high (R,?S)-HMPC concentration up to 1.0?M.     

CD and small-angle x-ray scattering of silk fibroin in solution

We investigated the structure of silk fibroin dissolved in water and in water-organic solvent mixtures by CD and small-angle x-ray scattering (SAXS). CD spectra indicated a disordered secondary structure in water and a beta-sheet conformation in aqueous organic solvents, such as methanol, dioxane, and trifluoroethanol (in trifluoroethanol a transient form evolving toward beta-sheet conformation was seen just after dissolution). The SAXS technique indicated the presence of fibroin particles of lamellar shape. The molecular weight was 188,000 daltons in water and 302,000 daltons in aqueous methanol.     

X-ray diffraction study on ordered, disordered and reconstituted intercellular lipid lamellar structure in stratum corneum

From small angle X-ray diffraction for the stratum corneum of hairless mouse, it was obtained that in the normal stratum corneum, the 1st, 2nd and 3rd order diffraction peaks for the intercellular lipid lamellar structure appear at 13.8, 6.87 and 4.59 nm, respectively and also a broad hump for the 4th order reflection appears as observed by the previous researchers. In the damaged stratum corneum prepared by the treatment of sodium dodecyl sulfate, these small-angle diffraction peaks disappear and only the broad maxima remain around the 1st, 2nd and 3rd order diffraction peaks. These facts indicate that in the normal stratum the lamellar structure is ordered and in the damaged stratum corneum the lamellar structure is disordered. Furthermore, in the reconstituted lamellar structure obtained by immersing into the dilute suspension of the mixture of ceramide 3, cholesterol and stearic acid, the 1st, 2nd and 3rd order diffraction peaks reappear at 13.3, 6.67 and 4.44 nm, respectively. This fact indicates that the reorganization of the ordered lamellar structure takes place by adding the mixture to the damaged stratum corneum.     

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed tributyrin hydrolysis. Effect of trypsin treatment

Hepatic triacylglycerol lipase (EC 3.1.1.3) hydrolyzes water-insoluble fatty acid esters, e.g., trioleoylglycerol (lipase activity) and water-soluble fatty acid esters, e.g., tributyrin (esterase activity). Esterase activity of hepatic triacylglycerol lipase is enhanced by triolein emulsion and phospholipid vesicles [1]. The catalytic mechanism and structure of human hepatic triacylglycerol lipase isolated from human post-heparin plasma and the effect of trypsin treatment on the lipase and esterase activities of the enzyme were examined. Treatment of hepatic triacylglycerol lipase with trypsin resulted in loss of its lipase activity, but had no effect on its esterase activity. Chromatography of hepatic triacylglycerol lipase on Bio-Gel A5m showed that hepatic triacylglycerol lipase binds to dipalmitoylphosphatidylcholine vesicles. However, on chromatography of the trypsin-treated enzyme after incubation with dipalmitoylphosphatidylcholine vesicles, a part of hepatic triacylglycerol lipase that retained esterase activity was eluted separately from the dipalmitoylphosphatidylcholine vesicles. Addition of vesicles of dipalmitoylphosphatidylcholine to the trypsin-treated enzyme did not enhance its esterase activity. These results are consistent with the hypothesis that hepatic triacylglycerol lipase has a catalytic site that hydrolyzes tributyrin and a lipid interface recognition site, and that these sites are different: trypsin modified the lipid interface recognition site of the hepatic triacylglycerol lipase but not the catalytic site.     

Extracellular lipid metabolism influences the survival of ovarian cancer cells

Lysophosphatidic acid (LPA) is an extracellular lipid mediator consisting of a fatty acid and a phosphate group linked to the glycerol backbone. Here, we show that 1-oleoyl- and 1-palmitoyl-LPA, but not 1-stearoyl- or alkyl-LPA, enhance HNOA ovarian cancer cell survival. Other lysophospholipids with oleic or lauric acid, but not stearic acid, also induce the survival effects. HNOA cells have the lipase activities that cleave LPA to generate fatty acid. Oleic acid stimulates HNOA cell survival via increased glucose utilization. Our findings suggest that extracellular lysolipid metabolism might play an important role in HNOA cell growth.     

Time-resolved X-ray diffraction study on poly[(R)-3-hydroxybutyrate] films during two-step-drawing: generation mechanism of planar zigzag structure

Uniaxially oriented films with high tensile strength were processed from ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] (P(3HB)) by a method combining hot-drawing near the melting point of the polymer and two-step-drawing at room temperature. In a two-step-drawn and subsequently annealed film, P(3HB) molecular chains fall into two states: 2/1 helix (alpha-form) and planar zigzag (beta-form) conformations. The mechanism for generating the beta-form during two-step-drawing was investigated by time-resolved synchrotron wide- and small-angle X-ray scattering measurements (WAXD and SAXS), together with the measurement of stress-strain curves. It was found that the improvement of mechanical properties is due to not only the orientation of molecular chains but also the generation of the beta-form during the drawing. The crystal and molecular structures of the alpha-form remained unchanged until the yield point of the stress-strain curve. At the yield point, the long period obtained from SAXS doubled and a new reflection indicative of the beta-form was observed on the equatorial line in WAXD. The intensity of the reflection from the beta-form increased with an increase in the two-step-drawing ratio at room temperature. The SAXS pattern changed from a two-point reflection along the meridian to a cross pattern with streaking on the equatorial line, demonstrating the close alignment of shish-kebab structures. The reflection intensity, crystal orientation and crystal size of the alpha-form decreased during two-step-drawing. Based on these results, the beta-form is mainly introduced from the orientation of free molecular chains in the amorphous regions between alpha-form lamellar crystals, but the structural transformation of molecular chains also occurs from the alpha-form to the beta-form at the deformed lamellar crystals.     

硬脂酸和月桂酸在Novozym 435催化下对芦丁酯化及分子筛对酯化率的影响

为了增加芦丁的脂溶性从而使其具有更优秀的抗氧化活性,以硬脂酸和月桂酸为酰基供体,在脂肪酶Novozym 435催化下对芦丁选择性酯化.经色谱柱提纯,得到两种带不同长度烃基的芦丁脂肪酸酯.用红外光谱和核磁共振波谱对芦丁硬脂酸进行了结构鉴定,表明该类酯化物的酯化反应位为鼠李糖的C4′″位羟基.以高效液相色谱监测酯化反应进程,分子筛添加时间对酯化率的研究结果显示,分子筛对酯化率和反应速率有提高的作用.分子筛添加时间对酯化率有影响.对于硬脂酸为酰基供体的情况,反应24h后添加分子筛的酯化反应可以得到最大的酯化转化率46%.以月桂酸为酰基供体的酯化反应,反应11 h后添加分子筛可以得到最大的酯化转化率64.5%.     

Fusion of lamellar body with plasma membrane is driven by the dual action of annexin II tetramer and arachidonic acid

Annexin II has been implicated in membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells. Most previous studies were based on the fusion assays by using model membranes. In the present study, we investigated annexin II-mediated membrane fusion by using isolated lamellar bodies and plasma membrane as determined by the relief of octadecyl rhodamine B (R18) self-quenching. Immunodepletion of annexin II from type II cell cytosol reduced its fusion activity. Purified annexin II tetramer (AIIt) induced the fusion of lamellar bodies with the plasma membrane in a dose-dependent manner. This fusion is Ca2+-dependent and is highly specific to AIIt because other annexins (I and II monomer, III, IV, V, and VI) were unable to induce the fusion. Modification of the different functional residues of AIIt by N-ethylmaleimide, nitric oxide, or peroxynitrite abolished AIIt-mediated fusion. Arachidonic acid enhanced AIIt-mediated fusion and reduced its Ca2+ requirement to an intracellularly achievable level. This effect is due to membrane-bound arachidonic acid, not free arachidonic acid. Other fatty acids including linolenic acid, palmitoleic acid, myristoleic acid, stearic acid, palmitic acid, and myristic acid had little effect. AIIt-mediated fusion was suppressed by the removal of arachidonic acid from lamellar body and plasma membrane using bovine serum albumin. The addition of arachidonic acid back to the arachidonic acid-depleted membranes restored its fusion activity. Our results suggest that the fusion between lamellar bodies with the plasma membrane is driven by the synergistic action of AIIt and arachidonic acid.