Chain Triacylglycerols Research Articles

Design of high-loading and high-stability viscoelastic emulsions for polymethoxyflavones

Polymethoxyflavones (PMFs), a class of highly lipophilic phytochemicals found in citrus peels, were documented to possess various potent biofunctionalities. The efficacies of PMFs are greatly limited by their low solubility in aqueous environment and rapid metabolic activities. Moreover, the incorporation of PMFs into pharmaceutical, nutraceutical, cosmetic, and food products has been hindered by the instability of their liquid formulates and crystalline sedimentation at application and storage temperatures. In this paper, a new method of forming highly stable viscoelastic emulsions was revealed to suspend and encapsulate high loadings of PMFs (>2.5%) for prolonged storage under ambient temperature. Without utilizing any potentially toxic organic solvent during processing, oil-in-water (O/W) viscoelastic emulsions were constituted solely by GRAS status food ingredients: medium chain triacylglycerol (MCT), lecithin, and water. The viscoelastic emulsion matrices were homogeneous mixtures of PMF crystals and saturated emulsion droplets with an average particle size of approximately 400nm. Depending on their lipophilicity, the sizes of PMF crystals entrapped in the viscoelastic emulsion matrix varied considerably, with 9.3±1.3μm and 3.8±0.6μm for tangeretin and 5-demethyl tangeretin respectively. The effectiveness of viscoelastic structures to prevent the sedimentation of crystalline PMFs was confirmed by studying dissolution kinetics at 0, 15, 30, and 60 times of the emulsion dilutions. With PMF concentrations remaining constant in undiluted emulsion samples during the investigation time frame, PMF dissolution concentrations decreased to (26.3±2.8)% and (52.7±2.6)% for tangeretin and 5-demethyl tangeretin, respectively.

Growth performance, plasma Fatty acids, villous height and crypt depth of preweaning piglets fed with medium chain triacylglycerol.

A study was conducted to investigate the effects of feeding medium chain triacylglycerol (MCT) on growth performance, plasma fatty acids, villus height and crypt depth in preweaning piglets. A total of 150 new born piglets were randomly assigned into one of three treatments: i) Control (no MCT); ii) MCT with milk (MCT+milk); iii) MCT without milk (MCT+fasting). Body weight, plasma fatty acid profiles, villus height and crypt depth were measured. Final BW for the Control and MCT+fasting was lower (p<0.05) than MCT+milk. The piglets fed with MCT regardless of milk provision or fasting had greater medium chain fatty acids (MCFA) than the Control. In contrast, the Control had greater long chain fatty acid (LCFA) and unsaturated fatty acid (USFA) than the MCT piglets. The piglets fed with MCT regardless of milk provision or fasting had higher villus height for the duodenum and jejunum after 6 h of feeding. Similar observations were found in piglets fed with MCT after 6 and 8 days of treatment. This study showed that feeding MCT to the piglets before weaning improved growth performance, with a greater concentration of MCT in blood plasma as energy source and a greater height of villus in duodenum, jejunum and ileum.

Vitamin E bioaccessibility: Influence of carrier oil type on digestion and release of emulsified α-tocopherol acetate

Vitamin E is an essential micronutrient for humans and animals due to its antioxidant and non-antioxidant biological activities. The α-tocopherol acetate form is often used in foods and other products due to its high biological activity and chemical stability. In this study, we examined the influence of carrier oil type on the bioaccessibility and molecular form of emulsified vitamin E using a simulated gastrointestinal model. Oil-in-water emulsions containing α-tocopherol acetate were prepared using quillaja saponin as a natural surfactant, and either long chain triacylglycerols (LCT) or medium chain triacylglycerols (MCT) as carrier oils. The rate and extent of lipid digestion was higher for MCT- than LCT-emulsions, which was attributed to differences in the water dispersibility of the free fatty acids formed during lipolysis. Conversely, the total bioaccessibility of vitamin E after digestion was higher for LCT- than MCT-emulsions, which was attributed to the greater solubilisation capacity of mixed micelles formed from long chain fatty acids. The conversion of α-tocopherol acetate to α-tocopherol after in vitro digestion was also considerably higher for LCT- than MCT-emulsions, which may impact the subsequent absorption of vitamin E. Overall, this research has important implications for the design and fabrication of effective emulsion-based delivery systems for increasing the bioavailability of vitamin E.

Chemo‐enzymatic Synthesis of Novel β‐Hydroxy‐β‐methylbutryric Acid (HMB)–Medium Chain Triacylglycerol (MCT) Complexes

Abstractβ‐Hydroxy‐β‐methylbutyrate (HMB) is reported to have ergogenic benefits such as reducing muscle wasting, promoting exercise performance and skeletal muscle hypertrophy. When used as a dietary feed, it has been found to improve colostral milk fat and the growth of piglets. Medium chain triacylglycerols (MCT) are a good energy source as they can be subjected to β‐oxidation in the liver without being stored as fats. When used as animal feed, HMB–MCT complexes are reported to stimulate growth and produce leaner meat product. A high yield two‐steps chemo‐enzymatic pathway for synthesis of HMB–MCT complexes has been developed. Ethyl HMB ester was first synthesized using a chemical catalyst. Following that, it was transesterified with MCT by using a biocatalyst. Approximately 85 wt% of HMB–MCT complexes can be achieved using this two‐step synthetic pathway. The reaction mechanism and some of the reaction parameters were also briefly elucidated.

Medium chain triglycerides dose-dependently prevent liver pathology in a rat model of non-alcoholic fatty liver disease

Metabolic syndrome is often accompanied by development of hepatic steatosis and less frequently by non-alcoholic fatty liver disease (NAFLD) leading to non-alcoholic steatohepatitis (NASH). Replacement of corn oil with medium chain triacylglycerols (MCT) in the diets of alcohol-fed rats has been shown to protect against steatosis and alcoholic liver injury. The current study was designed to determine if a similar beneficial effect of MCT occurs in a rat model of NAFLD. Groups of male rats were isocalorically overfed diets containing 10%, 35% or 70% total energy as corn oil or a 70% fat diet in which corn oil was replaced with increasing concentrations of saturated fat (18:82, beef tallow:MCT oil) from 20% to 65% for 21 days using total enteral nutrition (TEN). As dietary content of corn oil increased, hepatic steatosis and serum alanine amino transferases were elevated (P < 0.05). This was accompanied by greater expression of cytochrome P450 enzyme CYP2E1 (P < 0.05) and higher concentrations of polyunsaturated 18:2 and 20:4 fatty acids (FA) in the hepatic lipid fractions (P < 0.05). Keeping the total dietary fat at 70%, but increasing the proportion of MCT-enriched saturated fat resulted in a dose-dependent reduction in steatosis and necrosis without affecting CYP2E1 induction. There was no incorporation of C8-C10 FAs into liver lipids, but increasing the ratio of MCT to corn oil: reduced liver lipid 18:2 and 20:4 concentrations; reduced membrane susceptibility to radical attack; stimulated FA β- and ω-oxidation as a result of activation of peroxisomal proliferator activated receptor (PPAR)α, and appeared to increase mitochondrial respiration through complex III. These data suggest that replacing unsaturated fats like corn oil with MCT oil in the diet could be utilized as a potential treatment for NAFLD.

Effect of Tocopherols and Phytosterol on Color Reversion of MCT

Tocopherols and phytosterols were added to medium chain triacylglycerol (MCT) to study their effect on color reversion of vegetable oils. Four samples with 0, 6,000, 10,000 and 14,000 mg/kg phytosterols were heated at 105°C for 12 h. No significant reduction of phytosterols and obvious color change were found in the four samples. Five samples with 0, 1,000, 2,000, 5,000, 10,000 mg/kg tocopherols were also heated at 105°C for 12 h. About 2 − 5% tocopherols were absent after heating. Reduction of total tocopherol was found linearly related to color darkening. The most significant reduction is α-tocopherol. Reduction of α-tocopherol correlated more with color darkening than that of γ-tocopherol. The conclusion is phytosterol has no effect on color reversion of oils and reduction of α-tocopherol was attributed to be the most important cause of color reversion.

Rice (Oryza sativa) lipase: Molecular cloning, functional expression and substrate specificity

Lipases are important biocatalysts showing many interesting properties with industrial applications. Previously, different isoforms of lipases, Lipase-I and Lipase-II from rice (Oryza sativa) have been purified and characterized. Lipase-II identified as the major lipase in rice bran is designated as rice bran lipase (RBL). In this study, we report the cloning and expression of the RBL in Escherichia coli and Pichia pastoris. An exploration of expression in four different E. coli expression systems analyzed: BL21(DE3)pLysS, RIL(DE3)pLysS, Rosetta(DE3)pLysS and Origami(DE3)pLysS indicated that E. coli was not a suitable host. Expression with supplement of rare codons in Rosetta (DE3)pLysS and RIL(DE3)pLysS resulted in highest expression as insoluble inclusion bodies. The hurdles of expression in E. coli were overcome by expression as a secretory protein in P. pastoris X-33. The expression of lipase in shake flasks was optimized to achieve the maximum recombinant lipase activity of 152.6U/mL. The purified recombinant lipase had a specific activity of 998U/mg toward triacetin. The pH and temperature optimum of native and recombinant enzymes were pH 7.4 and 25±2°C, respectively. Both the lipases showed higher activity toward short chain triacylglycerol and unsaturated fatty acid enriched oils. Computational modeling and molecular docking studies reveal that the catalytic efficiency of the lipase correlates with the distance of the nucleophilic Ser175-OH and the scissile ester bond. The shorter the distance, the greater is the turnover of the substrate.

The impact of single and double hydrogen bonds on crystallization and melting regimes of Ajwa and Barni lipids

The present study was conducted to assess the impact of single and double hydrogen (H2) bonds of the prominent long chain triacylglycerols (TAGs) of Ajwa and Barni lipids. The impact of double H2 bond was in situ monitored in order to mitigate the problem of fat agglomeration and crystal aggregation. HPLC data demonstrated that the contents of 1,2-dioleoyl-3-linoleoyl-sn-glycerol (OOL) of Ajwa and Barni lipids were 13.78% and 16.24%, whereas, their relative contents for 2,3-dilinoleoyl-1-oleoyl-sn-glycerol (OLL) were 14.45% and 11.05%, respectively. The impact of double H2 bonds dropped the solid fat content (SFC) of Ajwa lipid from 1% at 20°C to 0.5% at body temperature (37°C), taking Barni lipid as reference. Their impact reduced the transition temperature 2 times and gained an enthalpy (ΔH) of 4.37J/g and 2.31J/g during crystallization and melting regimes, respectively. This study revealed that the increase in the average number of double bonds within the aliphatic structure of the long chain TAGs enhanced the chemical structure of Ajwa and Barni lipids to participate as functional food ingredients.

Purification and Biochemical Characterization of Lipase from Ficus carica Latex of Tunisian East Coast Zidi Variety

AbstractLipase from Ficus carica L. (Moraceae) latex of the Zidi variety was purified 80.5‐fold with 68.5 % recovery using silica gel chromatography. The molecular weight of the enzyme was 29 kDa as determined by SDS‐PAGE. High lipolytic activity was found in the crude extract during the fruit ripening process. The activity of purified lipase (ZL) seemed to depend strongly on chain length and showed a preference to long chain triacylglycerols. Indeed, ZL specific activity was 370.3 UI/mg using olive oil as a substrate at 45 °C and pH 5.5. In contrast, activity towards short chain triacylglycerols (tributyrin) was 12‐fold lower (32 UI/mg). The enzyme was quite stable in the pH range 4–8, and thermally stable at 60 °C displaying t1/2 about 90 min using olive oil as a substrate. The values of Km app and Vm were found to be 14.3 mM and 294.1 μmol/min/mg, respectively. ZL activity was strongly reduced by Fe2+, Mg2+ and Zn2+, while significantly increased by Ca2+ and Cu2+. The enzyme was stimulated by sodium dodecyl sulfate, and Tween‐80, while Triton X‐100 and EDTA had a slight inhibitory effect. No Effect was observed in addition of PMSF and iodoacetic acid.

The insertion of four residues Isoleucines at the N-terminus of Staphylococcus simulans lipase affects its catalytic and biochemical properties

Surface GRASP representation of closed forms and open forms of SSL and 4Ile-SSL. The catalytic serine is colored in red. The catalytic cavity is shown in yellow. The 4Ile residues are represented in dark blue. The lid domain is colored in green. ► The insertion of four residues Isoleucines at the N-terminus of SSL. ► Expression and purification of recombinant SSL and its mutant 4Ile-SSL. ► Comparative study on the biochemical properties of r-SSL and 4Ile-SSL. ► The importance of the N-terminus region in the biochemical properties of SSL. For the sake of evaluating the effect of the hydrophobic residues insertion in the N-terminus of Staphylococcus simulans lipase (SSL), four residues of Isoleucines have been inserted at the N-terminus of this enzyme. The recombinant Staphylococcus simulans lipase (r-SSL) and its constructed mutant ( 4Ile -SSL) were expressed in Escherichia coli BL21 (DE3) and purified to homogeneity using classical chromatographic techniques. We have performed, then, a comparative study on the biochemical properties of the two enzymes. Due to the insertion of 4Ile at the N-terminus of Staphylococcus simulans lipase (SSL), some important differences in the biochemical properties between r-SSL and 4Ile -SSL have been found. We can essentially notice that, when using short chain triacylglycerols (tributyrin) as substrate, the insertion of four Isoleucines residues ( 4Ile ) was accompanied by an increase in the specific activity (3 fold) as well as in the catalytic efficiency ( k cat / K M app. ) (2 fold), as compared to the recombinant SSL. Furthermore, our results indicate that the presence of 4Ile at the N-terminus of SSL has greatly affected the pH stability of the enzyme and considerably increased its thermostability.

A thermoactive uropygial esterase from chicken: Purification, characterisation and synthesis of flavour esters

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50kDa. The purified esterase displayed its maximal activity (200U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH2-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO3 was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.

RETARDATION OF LIPID OXIDATION IN FISH OIL-ENRICHED FISH PÂTÉ- COMBINATION EFFECTS

The oxidative stability during storage of fish pâte made from cod and enriched with 5% oil was investigated. Pâtes were produced with neat fish oil, pre-emulsified fish oil, microencapsulated fish oil, inert medium chain triacylglycerol (MCT) oil or a fish/rapeseed oil mixture. Addition of fish oil decreased the oxidative stability. Fish pâte with microencapsulated fish oil or MCT oil did not oxidize, whereas oxidation was slower in fish pâte with pre-emulsified oil compared with fish pâte with neat oil. Packaging in vacuum did not decrease oxidation. Fish pâtes with emulsified oil stored at 2 or 10C were equally stable. Mixing fish oil with rapeseed oil before emulsification slightly increased the stability of the fish pâtes. Addition of antimicrobial agents, sodium benzoate and potassium sorbate increased oxidative stability. It is recommended to produce enriched fish pâte by adding pre-emulsified fish oil or microencapsulated fish oil and store at preferentially 2–10C. PRACTICAL APPLICATIONS The results from this study can directly be transferred to practical applications in the food industry. Thus, the study showed that fish oil-enriched fish pâte with an acceptable shelf life and good sensory properties can be produced if one or more of the following strategies are used: Use microencapsulated or pre-emulsified fish oil or pre-emulsified fish oil/rapeseed oil mixture as the fish oil delivery system and add antimicrobial agents to increase both microbial and oxidative stability. The fish pâte can be stored at temperatures up to 10C.

Nanoemulsion- and emulsion-based delivery systems for curcumin: Encapsulation and release properties

Curcumin has been reported to have many biological activities, but its application as a functional ingredient is currently limited because of its poor water-solubility and bioaccessibility. This study investigated the impact of different lipid-based formulations on curcumin encapsulation and bioaccessibility. Oil-in-water nanoemulsions (r<100nm), or conventional emulsions (r>100nm), were prepared with different lipids: long, medium, and short chain triacylglycerols (LCT, MCT and SCT, respectively). An in vitro model simulating small intestine digestion conditions characterised rate and extent of lipid phase digestion. A centrifugation method determined fraction of curcumin released into mixed micelles after digestion (bioaccessibility). Initial digestion rate decreased in the order SCT>MCT>LCT, while final digestion extent decreased in the order MCT>SCT>LCT. The bioaccessibility of curcumin decreased in the order MCT>LCT≫SCT and appeared to be slightly higher in conventional emulsions than in nanoemulsions.

Hydrophilic coating of mitotane-loaded lipid nanoparticles: Preliminary studies for mucosal adhesion

The aim of the present work was to load mitotane, an effective drug for adrenocortical carcinoma treatment, in solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). The SLN and NLC were successfully prepared by high shear homogenization followed by hot high pressure homogenization. Formulations were composed of cetyl palmitate as the solid lipid for SLN, whereas for NLC PEGylated stearic acid was selected as solid lipid and medium chain triacylglycerols as the liquid lipid. Tween® 80 and Span® 85 were used as surfactants for all formulations. The particle size, zeta potential, polydispersity index (PI), encapsulation efficiency (EE), and loading capacity (LC) were evaluated. The SLN showed a mean particle size of 150 nm, PI of 0.20, and surface charge −10 mV, and the EE and LC could reach up to 92.26% and 0.92%, respectively. The NLC were obtained with a mean particle size of 250 nm, PI of 0.30, zeta potential −15 mV and 84.50% EE, and 0.84% LC, respectively. Hydrophilic coating of SLN with chitosan or benzalkonium chloride was effective in changing zeta potential from negative to positive values. The results suggest that mitotane was efficiently loaded in SLN and in NLC, being potential delivery systems for improving mitotane LC and controlled drug release.

Development of a food-grade organogel with high bioaccessibility and loading of curcuminoids

Curcumin, the major curcuminoid compound, is a well-known nutraceutical with many health-promoting properties. Its water insolubility, however, greatly affects its bioaccessibility and thereby bioavailability. The objective of this study was to develop a food-grade curcuminoids organogel with high bioaccessibility and high loading of curcumin. The bioaccessibility was evaluated using in vitro lipolysis experiments. It was shown that medium chain triacylglycerols (MCT) has high digestibility and thus generated high bioaccessibility of curcuminoids. High curcumin loading was achieved by the incorporation of Span 20 in the MCT. Furthermore, monostearin was used as a GRAS (generally recognised as safe) organogelator to form the food-grade organogel, in which the bioaccessibility of curcuminoids was not affected by the gel formation. This newly-developed organogel has the potential to be used for oral delivery of curcuminoids as well as incorporated in functional foods systems.

Identification and characterization of a new true lipase isolated through metagenomic approach

BackgroundMetagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant.ResultsIn preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v).ConclusionsThe combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.

Formulation of lipid core nanocapsules

Polymeric nanoparticle aqueous suspensions have been proposed as drug carriers to improve the efficacy of medicines. Considering those nanocarriers, nanocapsules are vesicular structures containing an oil core surrounded by a polymeric wall. Recently, we proposed the supramolecular model for a new kind of nanocapsule prepared with triacylglycerol, sorbitan monostearate (SM), polyester and polysorbate 80. Varying the proportions of the raw materials in the organic phase, different kinds of colloids could be obtained. So, our objective was to formulate exclusively lipid-core nanocapsules (LNC) in aqueous suspensions. In this way, the analytical approach to verify the quality of the different formulations was based on light scattering measurements (dynamic light scattering, multiple light scattering and laser diffractometry) and density gradient. The increase in the SM concentration showed a slight tendency of both sedimentation and creaming, while the increase in the oil concentration resulted in creaming. For the latter, size distribution as function of time indicated the presence of nanoemulsion simultaneously with LNC. Finally, density gradient showed an exclusive band for formulations prepared using 1:4.1:2.6 (w/w) of SM, medium chain triacylglycerol and polyester, respectively.

Erratum to: Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX1 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X33 and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X33 cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His6-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His6-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His6-ROLw) and the mutant (His6-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.

Distinction Between Esterases and Lipases: Comparative Biochemical Properties of Sequence-Related Carboxylesterases

Carboxylesterases (Carboxyl ester hydrolase) include two groups of enzymes, namely non-specific esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) which have been early differentiated on the basis of their substrate specificity. Esterases hydrolyse solutions of water-soluble short acyl chain esters and are inactive against water-insoluble long chain triacylglycerols which, in turn, are specifically hydrolyzed by lipases. Based on the comparison of the primary structures, three families of sequence-related carboxylesterases, namely the lipoprotein lipase family (L-family), the hormonesensitive lipase family (H-family) and the cholinesterase family (C-family) have been identified. Using solutions and emulsions of vinyl, glyceryl and p-nitrophenyl esters, we have reinvestigated the kinetic properties of some esterases and lipases of the H- and C-families. Results indicate that esterases and lipases, which are both active on soluble esters, can be differentiated by their value of Km. Moreover, esterase, unlike lipases, are inactive against water-insoluble esters as vinyl laurate and trioctanoylglycerol. From the the comparison of structural features of sequence-related esterases and lipases, it appears that lipases, unlike esterases, display a significant difference in the distribution of hydrophobic amino acid residues at vicinity of their active site. This observation supports the hypothesis of the existence in lipases of a particular surface domain that specifically interacts with lipid-water interfaces and contributes to the transfer a single substrate molecule from the organized lipid-water interface (supersubstrate) to the catalytic site of the enzyme.

Secretory expression and characterization of a highly Ca 2+-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca 2+. L2 lipase showed a preference for medium to long chain triacylglycerols (C 10–C 16), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.