Immobilization of Phospholipase A<Subscript>1</Subscript> and its Application in Soybean Oil Degumming

Phospholipase A₁ (PLA₁), or Lecitase^® Ultra, was immobilized on three different supports, calcium alginate (CA), calcium alginate-chitosan (CAC), and calcium alginate-gelatin (CAG), and crosslinked with glutaraldehyde. The results indicated that PLA₁–CA retained 56.2% of the enzyme’s initial activity, whereas PLA₁–CAC and PLA₁–CAG retained 65.5 and 60.2%, respectively. Compared with free PLA₁, the optimal pH of immobilized PLA₁ shifted to the basic side by 0.5–1.0 pH units and the pH/activity profile range was considerably broadened. Similarly, the temperature-optima of PLA₁–CAC and PLA₁–CAG increased from 50 to 60 °C, and their thermal stability increased with relative activities of more than 90% that covered a wider temperature range spanning 50–65 °C. In a batch oil degumming process, the final residual phosphorus content was reduced to less than 10 mg/kg with free PLA₁, PLA₁–CAC and PLA₁–CA in less than 5, 6 and 8 h respectively while PLA₁–CAG was only able to reduce it to 15 mg/kg in 10 h. When the PLA₁–CAC was applied in a plant degumming trial, the final residual phosphorus content was reduced to 9.7 mg/kg with 99.1% recovery of soybean oil. The recoveries of immobilized PLA₁–CAC and activity of PLA₁ were 80.2 and 78.2% respectively. Therefore, it was concluded that PLA₁–CAC was the best immobilized enzyme complex for the continuous hydrolysis of phospholipids in crude vegetable oils.     

Enzymatic reduction of polyunsaturated lysophosphati‐dylcholine hydroperoxides by glutathione peroxidase‐1

Some lipid peroxides are known to be converted to their corresponding alcohols in cells containing glutathione peroxidase (GPx). In this respect, we examined the enzymatic conversion of lysophosphatidylcholine (lysoPC) hydroperoxides to hydroxyl derivatives using RBL‐2H3 cells and erythrocyte GPx‐1. First, the incubation of RBL‐2H3 cells with arachidonoyl lysoPC led to the formation of a major product, with maximal UV absorbance at 234 nm and m/z [M+H]⁺ at 560.2, corresponding to monohydroxyeicosatetraenoyl lysoPC. Similarly, linoleoyl lysoPC was also converted to its hydroxyl derivative in RBL‐2H3 cells. Separately, lysoPC hydroperoxide, generated from soybean lipoxygenase 1‐catalyzed oxygenation of linoleoyl lysoPC, arachidonoyl lysoPC or docosahexaenoyl lysoPC, was converted by GPx‐1 to the corresponding hydroxyl derivatives. When the kinetic values were determined, the K_m values (3.1–32.3 µM) of the polyunsaturated lysoPC hydroperoxides increased with decreasing number of double bonds, in contrast to a similar value of V_m among them. Moreover, the catalytic efficiency of docosahexaenoyl lysoPC hydroperoxide was much greater than that of H₂O₂ as substrate of GPx‐1. In related experiments, where phosphatidylcholine hydroperoxides were incubated with phospholipase A₂ and GPx‐1, the complete conversion of phosphatidylcholine hydroperoxides to hydroxyl derivatives was confirmed by LC/MS. Taken together, it is proposed that GPx‐1‐type enzymes may participate in the conversion of polyunsaturated lysoPC hydroperoxides to hydroxyl derivatives in cell systems.     

Molecular cloning,expression, and characterization of secretory phospholipase A<Subscript>2</Subscript> in tobacco

Phospholipase A₂ (PLA₂) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA₂ activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA₂ activity in the flower appears to have originated from both Ca²⁺-dependent and-independent PLA₂. A cDNA clone for protein with homology to animal secretory PLA₂ (sPLA₂), denoted as Nt PLA₂, was isolated from the tobacco flower. The cDNA of Nt PLA₂ encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA₂ contains 12 cysteines, a Ca²⁺ binding loop, and a catalytic domain that are commonly conserved in animal sPLA₂. The Nt PLA₂ mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA₂ was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA₂ was purified by ion exchange chromatography. It was discovered that the purified Nt PLA₂ essentially requires Ca²⁺, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA₂ was at pH 8–10 when PC was used as a substrate.     

Phase evolution during mechanochemical coreduction of V<Subscript>2</Subscript>O<Subscript>5</Subscript> and TiO<Subscript>2</Subscript> with A

The direct preparation of V-Ti solid solution alloy by coreduction of V₂O₅ and TiO₂ with Al in an attritor mill was investigated. The reduction of V₂O₅ with Al is highly exothermic, whereas reduction of TiO₂ with Al is not sufficiently exothermic for a self-sustaining reaction. A range of compositions of a mixture of V₂O₅ and TiO₂ can be so chosen as to make the overall reduction of V₂O₅ and TiO₂ with Al sufficiently exothermic for a self-sustaining reaction. Initial studies were done to identify the reaction products obtained by reducing V₂O₅ with Al. The reaction yielded the intermetallic phase (Al₃V), V, and Al₂O₃. SEM images indicated melting and solidification of the phases, leading to agglomeration. Further experiments involved mixing appropriate amounts of TiO₂ with V₂O₅ and reducing the mixture with Al. XRD data for products showed the presence of V, V₅Al₈, and Al₂O₃. X-Ray Florescence (XRF) analysis and energy dispersive analyzer (EDAX) of SEM sample images indicated the formation of V-Ti solid solution. Microstructure of the milled charges taken out prior to reaction initiation indicated morphology change in Al powder and agglomeration/segregation of reactants. As a result, the reaction of V₂O₅ with the excess Al at certain regions also promoted the formation of vanadium aluminide.     

Hydrolysis of Phosphatidylcholine-Isoprostanes (PtdCho-IP) by Peripheral Human Group IIA,V and X Secretory Phospholipases A<Subscript>2</Subscript> (sPLA<Subscript>2</Subscript>)

Biologically active F- and E/D-type-prostane ring isomers (F₂-IP and E₂/D₂-IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA₂ involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA₂, which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1–4 h (37 °C) in the absence or presence of recombinant human sPLA₂ (1–2.5 µg/ml). In the absence of exogenously added sPLA₂ the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL₃, respectively. In the presence of group V or group X sPLA₂ (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA₂ (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8–24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA₂. A co-location of sPLA₂ and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.     

Preparation of nanodisperse solid solutions based on ZrO<Subscript>2</Subscript> and HfO<Subscript>2</Subscript> from hydroperoxides

A procedure for preparing solid solutions based on zirconium and hafnium hydroperoxides is developed. This procedure is based on the coprecipitation of components in the presence of hydrogen peroxide. Amorphous nanopowders (S _sp = 80–90 m²/g, d _mean = 2–3 nm) without any indication of the agglomeration of particles are synthesized. After the decomposition of complex hydroperoxides at a temperature of 500°C, the compacted powders undergo an active sintering at a temperature of 1450°C. The particle size after heat treatment does not exceed 50 nm. As follows from the electrical and physical characteristics obtained for these materials, they can be recommended for the use as solid electrolytes and sensors of oxygen partial pressure.     

Membrane lipid metabolism and phospholipase activity in insect Spodoptera frugiperda 9 ovarian cells

Although there is increasing use of insect ovarian Sf9 cells for the production of recombinant proteins, namely, via the baculovirus vector expression system, little is known about the lipids in the cell membrane and whether endogenous phospholipases are present for regulation of the cell membrane lipids. In this study, analysis of membrane lipids of Sf9 cells indicated the presence of phosphatidylethanolamine (PE) (diacyltype) and phosphatidylcholine as major phospholipids, followed by phosphatidylserine and phosphatidylinositol (PI), and only trace amounts of ethanolamine plasmalogen. These phospholipids contain high proportions of monoenoic fatty acids, e.g., 16∶1 and 18∶1, which comprise more than 70% of the total fatty acids although small amounts of polyunsaturated fatty acids such as 18∶2 and 20∶4 are also present. When Sf9 cells were incubated in a culture medium containing [¹⁴C]oleic acid and [¹⁴C]arachidonic acid, a large portion of the labels were incorporated into membrane phospholipids. Using [¹⁴C]arachidonoyl-phospholipids as substrates for incubation with cell homogenate and subcellular fractions, results indicate the presence of a Ca2⁺-independent phospholipase A (PLA₂) in the Sf9 cell cytosol fraction. This PLA₂ shows a high preference for hydrolysis of PE and is active at a pH range of 7–9. Unlike the brain cells which contain active phospholipase C (PLC) specific for phosphatidylinositol, only limited amount of diacylglycerol (DAG) was released from [¹⁴C]arachidonoyl-PE in the Sf9 cells. Taken together, this study demonstrates active metabolism of membrane phospholipids in Sf9 cells, most likely mediated by acyltransferases and PLA₂. Furthermore, despite the absence of PLC for PI, limited amount of DAG could be generated through hydrolysis of PE.     

Effects of preparation methods for V<Subscript>2</Subscript>O<Subscript>5</Subscript>-TiO<Subscript>2</Subscript> aerogel catalysts on the selective catalytic reduction of NO with NH<Subscript>3</Subscript>

A series of V₂O₅-TiO₂ aerogel catalysts were prepared by the sol-gel method with subsequent supercritical drying with CO₂. The main variables in the sol-gel method were the amounts of V₂O₅ and when the vanadium precursor was introduced. V₂O₅-TiO₂ xerogel and V₂O₅/TiO₂ (P-25) were also prepared for comparison. The V₂O₅-TiO₂ aerogel catalysts showed much higher surface areas and total pore volumes than V₂O₅-TiO₂ xerogel and impregnated V₂O₅/TiO₂ (P-25) catalysts. The catalysts were characterized by N₂ physisorption, X-ray diffraction (XRD), FT-Raman spectroscopy, temperature-programmed reduction with H₂ (H₂-TPR), and temperature-programmed desorption of ammonia (NH₃-TPD). The selective catalytic reduction of NOx with ammonia in the presence of excess O₂ was studied over these catalysts. Among various V₂O₅-TiO₂ catalysts, V₂O₅ supported on aerogel TiO₂ showed a wide temperature window exhibiting high NOx conversions. This superior catalytic activity is closely related to the large amounts of strong acidic sites as well as the surface vanadium species with characteristics such as easy reducibility and monomeric and polymeric vanadia surface species. This work was presented at the 7 ^th Korea-China Workshop on Clean Energy Technology held at Taiyuan, Shanxi, China, June 26–28, 2008.     

Calcium-independent phospholipase A2 in isolated rabbit ventricular myocytes

We characterized phospholipase A₂ (PLA₂) activity in isolated rabbit ventricular myocytes with respect to subcellular distribution, substrate specificity, and Ca²⁺ dependency. Membrane-associated PLA₂ was found to be an order of magnitude greater than cytosolic PLA₂. Ventricular myocyte PLA₂ activity was enhanced following protease-activated receptor stimulation with thrombin and was found to be largely Ca²⁺-independent and selective for phospholipid substrates containing arachidonic acid at the sn-2 position. Immunoblot analysis using an antibody to cytosolic Ca²⁺-independent PLA₂ from Chinese hamster ovary cells recognized a membrane-associated protein with a molecular mass of approximately 80 kDa; however, differences in pH optima, response to inhibitors, and substrate selectivity of membrane-associated and cytosolic PLA₂ activity suggest the presence of multiple Ca²⁺-independent PLA₂. Pretreatment with bromoenol lactone, a specific inhibitor of Ca²⁺-independent PLA₂, significantly attenuated membrane-associated and cytosolic PLA₂ in unstimulated and thrombin-stimulated myocytes. Pretreatment with methyl arachidonyl fluorophosphonate, mepacrine, or dibucaine had no significant effect on PLA₂ activity under all conditions tested. Ventricular myocyte PLA₂ activity was significantly inhibited by ATP, GTP, and their nonhydrolyzable analogs and was regulated by protein kinase C activity. These studies demonstrate the presence of one or more unique membrane-associated Ca²⁺-independent PLA₂ in isolated ventricular myocytes that exhibit a preference for phospholipids with arachidonate at the sn-2 position and that are activated by thrombin stimulation.     

Effects of dietary corn oil and salmon oil on lipids and prostaglandin E2 in rat gastric mucosa

Three groups of male rats were fed either a corn oilenriched diet (17%, w/w), a salmon oil-enriched diet (12.5%) supplemented with corn oil (4.5%) or a low-fat diet (4.4%) for eight wk to investigate the possible relationships between dietary fatty acids and lipid composition, and prostaglandin E₂ level and phospholipase A₂ activity in the rat gastric mucosa. High-fat diets induced no important variation in total protein, phospholipid and cholesterol contents of gastric mucosa. Compared with a low-fat diet, corn oil produced a higher n−6/n−3 ratio in mucosal lipids, whereas this ratio was markedly lowered by a fish oil diet. In comparison with the low-fat diet, the production of prostaglandin E₂(PGE₂) in gastric mucosa of rats fed salmon oil was significantly, decreased by a factor of 2.8. In the corn oil group, PGE₂ production tended to decrease, but not significantly. In comparison with the low-fat diet, both specific and total gastric mucosal phospholipase A₂ activities were increased (+18 and 23%, respectively) in the salmon oil group; they were unchanged in the corn oil group. It is suggested that the decrease of gastric PGE₂ in rats fed fish oil is not provoked by a decrease in phospholipase A₂ activity but may be the result of the substitution of arachidonic acid by n−3 PUFA or activation of PGE₂ catabolism.     

Effect of CeO<Subscript>2</Subscript>-addition sequence on the performance of CeO<Subscript>2</Subscript>-modified Ni/Al<Subscript>2</Subscript>O<Subscript>3</Subscript> catalyst in autothermal reforming of iso-octane

Two types of CeO₂-modified Ni/Al₂O₃ catalysts were prepared by a consecutive impregnation method with different sequences in the impregnation of Ni and CeO₂, and their performance in autothermal reforming (ATR) of isooctane was investigated. Catalysts prepared by adding CeO₂ prior to the addition of Ni, Ni/CeO₂-Al₂O₃, produced larger amounts of hydrogen than those obtained using catalysts prepared by adding the two components in an opposite sequence, Ni-CeO₂/Al₂O₃. The results of H₂ chemisorption and temperature-programmed reduction revealed that added CeO₂ increased the dispersion of the Ni species on Al₂O₃ and suppressed the formation of NiAl₂O₄ in the catalyst such that large amounts of Ni species were present as NiO, the active species for the ATR. The elemental and thermogravimetric analyses of deactivated catalysts indicated that Ni/CeO₂-Al₂O₃, which showed a longer lifetime than Ni-CeO₂/Al₂O₃, contained lesser amounts and different types of coke on the surface.     

Novel Hydrogen Sulfide (H2S)-Releasing BW-HS-101 and Its Non-H2S Releasing Derivative in Modulation of Microscopic and Molecular Parameters of Gastric Mucosal Barrier

Hydrogen sulfide (H₂S) is an endogenously produced molecule with anti-inflammatory and cytoprotective properties. We aimed to investigate for the first time if a novel, esterase-sensitive H₂S-prodrug, BW-HS-101 with the ability to release H₂S in a controllable manner, prevents gastric mucosa against acetylsalicylic acid-induced gastropathy on microscopic and molecular levels. Wistar rats were pretreated intragastrically with vehicle, BW-HS-101 (0.5–50 μmol/kg) or its analogue without the ability to release H₂S, BW-iHS-101 prior to ASA administration (125 mg/kg, intragastrically). BW-HS-101 was administered alone or in combination with nitroarginine (L-NNA, 20 mg/kg, intraperitoneally) or zinc protoporphyrin IX (10 mg/kg, intraperitoneally). Gastroprotective effects of BW-HS-101 were additionally evaluated against necrotic damage induced by intragastrical administration of 75% ethanol. Gastric mucosal damage was assessed microscopically, and gastric blood flow was determined by laser flowmetry. Gastric mucosal DNA oxidation and PGE₂ concentration were assessed by ELISA. Serum and/or gastric protein concentrations of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, VEGF, GM-CSF, IFN-γ, TNF-α, and EGF were determined by a microbeads/fluorescent-based multiplex assay. Changes in gastric mucosal iNOS, HMOX-1, SOCS3, IL1-R1, IL1-R2, TNF-R2, COX-1, and COX-2 mRNA were assessed by real-time PCR. BW-HS-101 or BW-iHS-101 applied at a dose of 50 μmol/kg protected gastric mucosa against ASA-induced gastric damage and prevented a decrease in the gastric blood flow level. H₂S prodrug decreased DNA oxidation, systemic and gastric mucosal inflammation with accompanied upregulation of SOCS3, and EGF and HMOX-1 expression. Pharmacological inhibition of nitric oxide (NO) synthase but not carbon monoxide (CO)/heme oxygenase (HMOX) activity by L-NNA or ZnPP, respectively, reversed the gastroprotective effect of BW-HS-101. BW-HS-101 also protected against ethanol-induced gastric injury formation. We conclude that BW-HS-101, due to its ability to release H₂S in a controllable manner, prevents gastric mucosa against drugs-induced gastropathy, inflammation and DNA oxidation, and upregulate gastric microcirculation. Gastroprotective effects of this H₂S prodrug involves endogenous NO but not CO activity and could be mediated by cytoprotective and anti-inflammatory SOCS3 and EGF pathways.     

Phase cooperation of V<Subscript>2</Subscript>O<Subscript>5</Subscript> and Bi<Subscript>2</Subscript>O<Subscript>3</Subscript> in the selective oxidation of H<Subscript>2</Subscript>S containing ammonia and water

The selective oxidation of hydrogen sulfide containing excess water and ammonia was studied over vanadium oxide-based catalysts. The investigation was focused on the role of V₂O₅, and phase cooperation between V₂O₅ and Bi₂O₃ in this reaction. The conversion of H₂S continued to decrease since V₂O₅ was gradually reduced by treatment with H₂S. The activity of V₂O₅ was recovered by contact with oxygen. A strong synergistic phenomenon in catalytic activity was observed for the mechanically mixed catalysts of V₂O₅ and Bi₂O₃. Temperature-programmed reduction (TPR) and oxidation (TPO) and two bed reaction tests were performed to explain this synergistic effect by the reoxidation ability of Bi₂O₃. This paper is dedicated to Professor Wha Young Lee on the occasion of his retirement from Seoul National University.     

Characterization of Secretory Phospholipase A<Subscript>2</Subscript> with Phospholipase A<Subscript>1</Subscript> Activity in Tobacco, <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> (L.)

A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.     

Ni-Co bimetallic catalyst for CH<Subscript>4</Subscript> reforming with CO<Subscript>2</Subscript>

A co-precipitation method was employed to prepare Ni/Al₂O₃-ZrO₂, Co/Al₂ O₃-ZrO₂ and Ni-Co/Al₂O₃-ZrO₂ catalysts. Their properties were characterized by N₂ adsorption (BET), thermogravimetric analysis (TGA), temperature-programmed reduction (TPR), temperature-programmed desorption (CO₂-TPD), and temperature-programmed surface reaction (CH₄-TPSR and CO₂-TPSR). Ni-Co/Al₂O₃-ZrO₂ bimetallic catalyst has good performance in the reduction of active components Ni, Co and CO₂ adsorption. Compared with mono-metallic catalyst, bimetallic catalyst could provide more active sites and CO₂ adsorption sites (C + CO₂ = 2CO) for the methane-reforming reaction, and a more appropriate force formed between active components and composite support (SMSI) for the catalytic reaction. According to the CH₄-CO₂-TPSR, there were 80.9% and 81.5% higher CH₄ and CO₂ conversion over Ni-Co/Al₂O₃-ZrO₂ catalyst, and its better resistance to carbon deposition, less than 0.5% of coke after 4 h reaction, was found by TGA. The high activity and excellent anti-coking of the Ni-Co/Al₂O₃-ZrO₂ catalyst were closely related to the synergy between Ni and Co active metal, the strong metal-support interaction and the use of composite support.     

Effects of La<Subscript>2</Subscript>O<Subscript>3</Subscript> on ZrO<Subscript>2</Subscript> supported Ni catalysts for autothermal reforming of CH<Subscript>4</Subscript>

The effect of La₂O₃ content in Ni-La-Zr catalyst was investigated for the autothermal reforming (ATR) of CH₄. The catalysts were prepared by the coprecipitation method and had a mesoporous structure. Temperature programmed reduction (TPR) and X-ray photoelectron spectroscopy (XPS) indicated that a strong interaction developed between Ni species and the support with the addition of La₂O₃. Thermogravimetric analysis (TGA) and H₂-pulse chemisorption showed that the addition of La₂O₃ led to well dispersed NiO molecules on the support. Ni-La-Zr catalysts gave much higher CH₄ conversion than Ni-Zr catalyst. The Ni-La-Zr containing 3.2 wt% La₂O₃ showed the highest activity. The optimum conditions for maximal CH₄ conversion and H₂ yield were H₂O/CH₄=1.00, O₂/CH₄=0.75. Under these conditions, CH₄ conversion of 83% was achieved at 700 °C. In excess O₂ (O₂/CH₄>0.88), the catalytic activity was decreased due to sintering of the catalyst.     

Synthesis and study of novel catalysts based on hollandite K<Subscript>2</Subscript>Ga<Subscript>2</Subscript>Ti<Subscript>6</Subscript>O<Subscript>16</Subscript>

The results of experimental studies of synthesis of the hollandite phase K₂Ga₂Ti₆O₁₆ using initial mixtures of different dispersion compositions obtained by two methods, i.e., mechanical dispersion (MD) (followed by solid-phase sintering) and sol-gel method (Pechini method, MP), are reported. The catalytic properties of the obtained materials in the reactions of carbon monoxide (CO) and hydrogen (H₂) oxidation have been determined. It has been demonstrated that an increase in the catalytic activity in the CO oxidation reaction is observed on the samples obtained using the sol-gel method, in which the hollandite phase content is higher and crystallization is more complete. The samples obtained using the MD method are characterized by a low porosity and activity in comparison with those produced by the Pechini method.     

Phospholipases A<Subscript>1</Subscript> from <Emphasis Type="Italic">Armillaria ostoyae</Emphasis> Provide Insight into the Substrate Recognition of α/β-Hydrolase Fold Enzymes

Four enzymes with phospholipase A₁ (PLA₁) activity were purified from the fruiting bodies of the basidiomycete Armillaria ostoyae. The enzymes (PLA₁-1, -2, -3 and -4) showed similar isoelectric points (4.3, 3.9, 4.0 and 4.0) and apparent molecular masses in the range of 35–47 kDa. Mass spectrometric analyses of proteolytic fragments revealed sequences homologous to α/β-hydrolase fold enzymes. The enzymes share one conserved region with fungal phospholipases B and the active site sequence with bacterial esterases and PLA₁s. PLA₁-1 cleaves phospholipids and lysophospholipids with an optimum activity at pH 5.3. In contrast, PLA₁-2, -3 and -4 are characterized by broad pH optima in the slightly acidic to neutral range and are additionally capable of hydrolyzing mono- and diglycerides as well as fatty acid methyl esters. All enzymes favor glycerol-based lipids with a single medium-sized fatty acid moiety in the sn-1 position but show reduced activity towards the corresponding 1,2-diacyl derivatives with bulky long-chain or inflexible saturated fatty acid moieties in the sn-2 position. The enzymes prefer zwitterionic phospholipid substrates and are unable to hydrolyze triglycerides. From the selectivity of these broad-spectrum α/β-hydrolase fold enzymes towards the different classes of their substrates a regiospecific steric hindrance and a head group recognition are concluded.     

Mechanism of Phospholipid Hydrolysis for Oyster <Emphasis Type="Italic">Crassostrea plicatula</Emphasis> Phospholipids During Storage Using Shotgun Lipidomics

A fast and efficient shotgun lipidomics strategy was applied to analyze phospholipids (PL) in the oyster Crassostrea plicatula, including 29 species of phosphatidylcholine (PtdCho), 23 species of phosphatidylethanolamine (PtdEtn), 11 species of phosphatidylserine (PtdSer), 6 species of phosphatidylinositol (PtdIns), and 17 species of lysophospholipids (Lyso-PL). During storage at 4 °C for 7 days, the PL content decreased by 68.08%, but a significant increase in the FFA content was observed (from 63.11 to 318.72 μg/g). PtdCho and PtdIns decreased relatively by 64.97 and 67.49%, and PtdSer decreased most markedly by 74.15%. However, the PtdEtn content increased slightly during the early stages of storage but subsequently began to decrease. Moreover, PL with eicosapentaenoic acid (EPA-PL) and docosahexaenoic acid (DHA-PL) decreased by 51.77 and 50.61%, whereas plasmalogens were relatively stable showing only a 25.46% decrease. In particular, through enzyme activity analysis of lipase, phospholipase A₁ (PLA₁), phospholipase A₂ (PLA₂), phospholipase C (PLC), and phospholipase D (PLD), it was observed that the activities of all these enzymes increased at the early stage at 4 °C, but their activities were at lower levels when the oysters were stored at ?20 °C. During the storage period at 4 °C, correlation analysis suggests that the degradation of PtdCho was mostly correlated to PLA₂ (p < 0.05), whereas PtdEtn and PtdSer were more markedly correlated to lipase and PLD, respectively. The above result indicates that the hydrolysis mechanism of PL during seafood storage was correlated to the lipid hydrolytic enzyme activities under different storage temperatures.     

Effect of TiO<Subscript>2</Subscript> loading on the activity of V/TiO<Subscript>2</Subscript>-Al<Subscript>2</Subscript>O<Subscript>3</Subscript> in the catalytic oxidehydrogenation of ethylbenzene with carbon dioxide

TiO₂-Al₂O₃ mixed oxides with different compositions ranging from 40wt-% to 95wt-% of TiO₂ were prepared by sol-gel method and impregnated with different amounts of VO _x . Supports and catalysts were characterized by X-ray diffraction (XRD), physisorption, temperature preprogrammed reduction (H₂-TPR), and ammonia temperature programmed desorption (NH₃-TPD). TiO₂ content in the support had obvious effect on the crystal structure, texture characteristic, acid property, and catalytic activity in dehydrogenation of ethylbenzene (EB) with carbon dioxide. The highest catalytic activity was acquired when the TiO₂ content was 50 wt-%.