Neutral Lipase Research Articles

Lipid metabolism in Rhodnius prolixus: Lessons from the genome

The kissing bug Rhodnius prolixus is both an important vector of Chagas' disease and an interesting model for investigation into the field of physiology, including lipid metabolism. The publication of this insect genome will bring a huge amount of new molecular biology data to be used in future experiments. Although this work represents a promising scenario, a preliminary analysis of the sequence data is necessary to identify and annotate the genes involved in lipid metabolism. Here, we used bioinformatics tools and gene expression analysis to explore genes from different genes families and pathways, including genes for fat breakdown, as lipases and phospholipases, and enzymes from β-oxidation, fatty acid metabolism, and acyl-CoA and glycerolipid synthesis. The R. prolixus genome encodes 31 putative lipase genes, including 21 neutral lipases and 5 acid lipases. The expression profiles of some of these genes were analyzed. We were able to identify nine phospholipase A2 genes. A variety of gene families that participate in fatty acid synthesis and modification were studied, including fatty acid synthase, elongase, desaturase and reductase. Concerning the synthesis of glycerolipids, we found a second isoform of glycerol-3-phosphate acyltransferase that was ubiquitously expressed throughout the organs. Finally, all genes involved in fatty acid β-oxidation were identified, but not a long-chain acyl-CoA dehydrogenase. These results provide fundamental data to be used in future research on insect lipid metabolism and its possible relevance to Chagas' disease transmission.

Hormone-sensitive lipase deficiency disturbs lipid composition of plasma membrane microdomains from mouse testis

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase capable of hydrolyzing acylglycerols, as well as cholesteryl and retinyl esters. In mice, HSL deficiency results in male sterility. Lipid rafts, a plasma membrane microdomain enriched in cholesterol, sphingolipids and saturated glycerophospholipids, comprise a highly dynamic clustering of proteins and lipids that play a central role in signal transduction and intercellular communication. In the present work, we examined the effect of HSL deficiency in the lipid composition of plasma membrane microdomains in mouse testis. The lack of HSL affected the density of lipid rafts, as indicated by the shifting of caveolin 1 to denser fractions in the sucrose-gradient fractionation, and altered the sterol and phospholipid composition of both lipid raft and non-raft fractions, the biochemical differences among them being less obvious in HSL−/− than in HSL+/+ mice. Compared to HSL+/+, the lipid rafts from HSL−/− mice had significantly less desmosterol and T-MAS, while the non-raft domain had increased cholesterol content. Lipid rafts from HSL−/− mice had reduced PUFA-containing phospholipid species but increased phosphatidylcholine and phosphatidylethanolamine species with monounsaturated fatty acid moieties, while the non-raft domain was enriched in phosphatidylethanolamine and phosphatidylserine species having four double bonds. The changes in both the sterols and phospholipid composition of lipid raft and non-raft microdomains may have consequences in signal transduction and contribute to the sterility observed in HSL-deficient male mice.

Incorporation of pig back fat in restructured dry cured ham to facilitate the release of unsaturated fatty acids and generation of volatile compounds

The aim of the present study was to evaluate the effects of incorporating pig back fat in restructured dry cured ham (RDH) on its free fatty acid profile (FFA) and the volatile compounds released. Fresh raw hams were skinned, boned, and cut into meat cubes (4–6 × 4–6 × 4–6 cm). The meat cubes were restructured with 0, 2, and 4% pig back fat paste into raw restructured ham. After 30 days of ripening, the FFA and volatiles in the RDH were identified using gas chromatography and gas chromatography mass spectrometry, respectively. The results showed that exogenous pig back fat incorporation from 2 to 4% gave rise to increased neutral lipase activity (p < 0.05) and to enhanced lipid degradation and unsaturated fatty acid release in the RDH sample. A total of 63 volatile compounds were isolated from RDH. The most abundant volatiles were alcohols, followed by esters and aldehydes. The chromatographic areas of the total volatile amount, short chain esters, 3‐hydroxy‐2‐butanone, 2‐nonanone, long chain hydrocarbons, and pyrazines increased with pig back fat incorporation, suggesting that exogenous pig back fat incorporation could improve the sensorial quality of restructured dry cured ham.Practical application: Hams produced from genotypes with high intramuscular fat have more intense and persistent aroma, because intramuscular triacylglycerols are a good trap for most aroma compounds and also a source of volatile (aromatic) compounds. Nowadays, mixed diet feed systems or other specific feeding strategies could improve the fat content and composition of raw ham, but led to lower nutritional and sensory quality of hams. Perhaps, the major problem for the relatively poor quality of dry cured ham may be the leanness of modern genotypes, and the pigs from these genotypes are not suitable for dry cured ham production. Thus, thinking from another perspective, through incorporating exogenous pig back fat directly into raw ham, the present work have successfully improved the sensorial properties of dry cured hams.Restructured dry cured hams (RDH) with different pig back fat levels were successfully manufactured. This work shown that large differences in the free unsaturated fatty acid and volatile compounds of RDH can be caused by the exogenous pig back fat incorporation. An increase in pig back fat level from 2 to 4% in RDH significantly enhanced the release of unsaturated fatty acid, and facilitated the generation of volatile compounds including esters, ketones, aliphatic hydrocarbons, and aromatic hydrocarbons.

Incorporation of pig back fat in restructured dry cured ham to enhance the lipase and lipoxygenase activities

The aim of the present study was to evaluate the effect of incorporating pig back fat in restructured dry cured ham (RDH) on the lipase and lipoxygenase activities. Fresh raw hams were skinned, boned, cut into meat cubes (4–6 × 4–6 × 4–6 cm). The meat cubes were restructured respectively with 0, 2, and 4% pig back fat paste into raw restructured ham. After 30 days of ripening, lipase activity in RDH was determined using a fluorescence spectrometer (λex = 350 nm, λem = 445 nm), the result was expressed as µmol of released 4‐methylumbelliferone per hour per gram protein. Lipoxygenase (LOX) activity in RDH was assayed in a 96‐Well Plate Reader M200, one unit of LOX activity was defined as an absorbance increment per minute per milligram protein at 234 nm. Results showed that exogenous pig back fat incorporation from 2 to 4% gave rise to the increased neutral lipase and LOX activities (p < 0.05), and the enhanced hydrolysis of neutral lipids and phospholipids, and production of free fatty acids, suggesting that exogenous pig back fat incorporation could improve the quality of RDH.Practical applications: Hams produced from genotypes with high intramuscular fat have more intense and persistent aroma, because intramuscular triacylglycerols are a good trap for most aroma compounds. However, traditional extensive feeding regime give rise to massive consumption of natural resource and this strategy is not always feasible because availability of natural resource is largely influenced by environment. The present work has provided a successful strategy to improve dry cured hams’ quality that incorporating exogenous pig back fat directly into raw ham from general confinement feeding, not through traditional extensive feeding, could increase the content of lipid, and enhance the neutral lipase and LOX activities in dry cured meat.Restructured dry cured hams (RDH) with different pig back fat levels were successfully manufactured. This work shown that large differences in the neutral lipase and LOX (Lipoxygenase) activities of RDH can be caused by the exogenous pig back fat incorporation. An increase in pig back fat level from 2 to 4% in RDH significantly enhanced the neutral lipase and LOX activities during ripening.

Combined effects of high-pressure and thermal treatments on lipid oxidation and enzymes in pork.

The relationship between lipid oxidation and related enzymes in pork during combined pressure-heat treatments was investigated. Minced pork was treated under a pressure range of 0.1-750 MPa and a temperature range of 30-60°C for 20 min. Thiobarbituric acid-reactive substances (TBARS) values and activities of acid lipase (AL), neutral lipase (NL), phospholipase (PL), and lipoxygenase (LOX) in pork were evaluated. NL and LOX were completely inactivated at 600 MPa-50°C and 450 MPa-60°C, and AL at 600MPa-55°C. PL had the greatest stability and was completely inactivated at 600 MPa-60°C and 750 MPa-50°C. PL activity was strongly related to lipid oxidation caused by high pressure, followed by AL and LOX. TBARS values at 600 and 750 MPa were strongly related to the inactivation rate and the ratio of PL during treatment. PL was the most important enzyme related to lipid oxidation induced by pressure.

Marine invertebrate lipases: Comparative and functional genomic analysis

Lipases are key enzymes involved in lipid digestion, storage and mobilization of reserves during fasting or heightened metabolic demand. This is a highly conserved process, essential for survival. The genomes of five marine invertebrate species with distinctive digestive system were screened for the six major lipase families. The two most common families in marine invertebrates, the neutral an acid lipases, are also the main families in mammals and insects. The number of lipases varies two-fold across analyzed genomes. A high degree of orthology with mammalian lipases was observed. Interestingly, 19% of the marine invertebrate lipases have lost motifs required for catalysis. Analysis of the lid and loop regions of the neutral lipases suggests that many marine invertebrates have a functional triacylglycerol hydrolytic activity as well as some acid lipases. A revision of the expression profiles and functional activity on sequences in databases and scientific literature provided information regarding the function of these families of enzymes in marine invertebrates.

Study on lipolysis-oxidation and volatile flavour compounds of dry-cured goose with different curing salt content during production

The effect of dry-curing salt content (4% low salt (LS), 8% high salt (HS)) on lipolysis, lipid oxidation and volatile compounds in dry-cured goose was investigated in our study. The activities of acid lipase and neutral lipase increased during dry-curing, while phospholipase reached its maximum at the end of marinating. Lipoxygenase (LOX) and thiobarbituric acid reactive substances (TBARS) values increased during dry-curing and marinating then decreased during dry-ripening. Total free fatty acids (TFFA) increased at dry-curing and dry-ripening points and decreased during marinating. Total peak area of lipids derived volatile compounds (TPALDVC) and total peak area increased during entire stages. Compared to LS, HS group has higher lipolytic and LOX activities, TBARS, TFFA, unsaturated fatty acids and TPALDVC. The higher TPALDVC in HS could be attributed to higher lipid hydrolysis and oxidation during processing.

Characterization of the bovine gene LIPE and possible influence on fatty acid composition of meat

LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p<0.01), and SNP2 and SNP3 with Heneicosylic acid content (p<0.01), may be helpful to improve the quality of meat and improve health.

Perilipin 5, a lipid droplet protein adapted to mitochondrial energy utilization

We summarize recent mechanistic and physiological studies related to the role of perilipin 5 (Plin5) in regulating lipid droplet accumulation and protection to fatty acids in tissues with high lipid oxidative metabolism. Plin5 is a lipid droplet targeting protein that promotes association of lipid droplets with mitochondria and is most highly expressed in oxidative tissues, including cardiac and skeletal muscle. Recent in-vivo and in-vitro data indicate an important role for Plin5 in the regulation of cardiac lipid storage and function. Targeted overexpression of Plin5 in heart causes steatosis, mild mitochondria dysfunction, and hypertrophy in cardiac tissue, but without affecting cardiac function. In contrast, whole body ablation of Plin5 (Plin5 mice) reduces cardiac lipid droplet formation, increases cardiac fatty acid oxidation, and promotes cardiac dysfunction; cardiac defects can be prevented with antioxidative therapy. These data suggest a cytoprotective role for Plin5 to promote lipid storage but to limit fatty acid toxicity, parameters critical for tissues with high lipid oxidative metabolism. In-vivo and in-vitro data suggest that Plin5 is part of a cell-adaptive response to high lipid oxidative metabolism to protect lipid droplet storage against neutral lipases and, so, limit fatty acid accumulation. Although the specific mechanisms that underlie Plin5 lipid droplet storage protection in oxidative tissues remain to be fully elucidated, Plin5 provides a basis for the novel cytoprotective nature of lipid droplets.

Neutral Lipid Stores and Lipase PNPLA5 Contribute to Autophagosome Biogenesis

Autophagy is a fundamental cell biological process whereby eukaryotic cells form membranes in the cytoplasm to sequester diverse intracellular targets. Although significant progress has been made in understanding the origins of autophagosomal organelles, the source of lipids that support autophagic membrane formation remain an important open question. Here we show that lipid droplets as cellular stores of neutral lipids including triglycerides contribute to autophagic initiation. Lipid droplets, as previously shown, were consumed upon induction of autophagy by starvation. However, inhibition of autophagic maturation by blocking acidification or using dominant negative Atg4(C74A) that prohibits autophagosomal closure did not prevent disappearance of lipid droplets. Thus, lipid droplets continued to be utilized upon induction of autophagy, but not as autophagic substrates in a process referred to as lipophagy. We considered an alternative model whereby lipid droplets were consumed not as a part of lipophagy, but as a potential contributing source to the biogenesis of lipid precursors for nascent autophagosomes. We carried out a screen for a potential link between triglyceride mobilization and autophagy and identified a neutral lipase, PNPLA5, as being required for efficient autophagy. PNPLA5, which localized to lipid droplets, was needed for optimal initiation of autophagy. PNPLA5 was required for autophagy of diverse substrates, including degradation of autophagic adaptors, bulk proteolysis, mitochondrial quantity control, and microbial clearance. Lipid droplets contribute to autophagic capacity by enhancing it in a process dependent on PNPLA5. Thus, neutral lipid stores are mobilized during autophagy to support autophagic membrane formation.

Characterization of Neutral Lipase BT-1 Isolated from the Labial Gland of Bombus terrestris Males

BackgroundIn addition to their general role in the hydrolysis of storage lipids, bumblebee lipases can participate in the biosynthesis of fatty acids that serve as precursors of pheromones used for sexual communication.ResultsWe studied the temporal dynamics of lipolytic activity in crude extracts from the cephalic part of Bombus terrestris labial glands. Extracts from 3-day-old males displayed the highest lipolytic activity. The highest lipase gene expression level was observed in freshly emerged bumblebees, and both gene expression and lipase activity were lower in bumblebees older than 3 days. Lipase was purified from labial glands, further characterized and named as BT-1. The B. terrestris orthologue shares 88% sequence identity with B. impatiens lipase HA. The molecular weight of B. terrestris lipase BT-1 was approximately 30 kDa, the pH optimum was 8.3, and the temperature optimum was 50°C. Lipase BT-1 showed a notable preference for C8-C10 p-nitrophenyl esters, with the highest activity toward p-nitrophenyl caprylate (C8). The Michaelis constant (Km) and maximum reaction rate (Vmax) for p-nitrophenyl laurate hydrolysis were Km = 0.0011 mM and Vmax = 0.15 U/mg.ConclusionThis is the first report describing neutral lipase from the labial gland of B. terrestris. Our findings help increase understanding of its possible function in the labial gland.

Changes of Intramuscular Fat Composition, Lipid Oxidation and Lipase Activity in Biceps femoris and Semimembranosus of Xuanwei Ham During Controlled Salting Stages

Fatty acid composition of neutral lipids (NLs), phospholipids (PLs) and free fatty acids (FFAs) from intramuscular fat (IMF), lipid oxidation and lipase activity in muscle Semimembranosus (SM) and msucle Biceps femoris (BF) of dry-cured Xuanwei ham during the 90-d salting stages were analysed. The salt content increased from 0.34 to 3.52% in BF and from 0.10 to 5.42% in SM during the 90 d salting stage, respectively. PLs of IMF in both BF and SM decreased 54.70% (P<0.001) and 34.64% (P<0.05), furthermore, the saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) of PLs in both muscles were hydrolysed almost isochronously. FFAs were increased from 0.46 g 100 g−1 lipids to 2.92 g 100 g−1 lipids in BF at the end of salting, which was lower than SM (from 1.29 g 100 g−1 lipids to 9.70 g 100 g−1 lipids). The activities of acid lipase, neutral lipase and acid phospholipase all remained active in the 90 d. The thiobarbituric acid reactive substances (TBARS) was slowly increased to 1.34 mg kg−1 muscle in BF and to 2.44 mg kg−1 muscle in SM during the salting stage. In conclusion, the controlled salting process prompted the hydrolysis of PLs of IMF notably and increased the lipid oxidation of muscles within some limits.

Olive seed protein bodies store degrading enzymes involved in mobilization of oil bodies.

The major seed storage reserves in oilseeds are accumulated in protein bodies and oil bodies, and serve as an energy, carbon, and nitrogen source during germination. Here, the spatio-temporal relationships between protein bodies and several key enzymes (phospholipase A, lipase, and lipoxygenase) involved in storage lipid mobilization in cotyledon cells was analysed during in vitro seed germination. Enzyme activities were assayed in-gel and their cellular localization were determined using microscopy techniques. At seed maturity, phospholipase A and triacylglycerol lipase activities were found exclusively in protein bodies. However, after seed imbibition, these activities were shifted to the cytoplasm and the surface of the oil bodies. The activity of neutral lipases was detected by using α-naphthyl palmitate and it was associated mainly with protein bodies during the whole course of germination. This pattern of distribution was highly similar to the localization of neutral lipids, which progressively appeared in protein bodies. Lipoxygenase activity was found in both the protein bodies and on the surface of the oil bodies during the initial phase of seed germination. The association of lipoxygenase with oil bodies was temporally correlated with the appearance of phospholipase A and lipase activities on the surface of oil bodies. It is concluded that protein bodies not only serve as simple storage structures, but are also dynamic and multifunctional organelles directly involved in storage lipid mobilization during olive seed germination.

Lysosomal acid lipase and lipophagy are constitutive negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells

Lipolytic breakdown of endogenous lipid pools in pancreatic beta cells contributes to glucose-stimulated insulin secretion (GSIS) and is thought to be mediated by acute activation of neutral lipases in the amplification pathway. Recently it has been shown in other cell types that endogenous lipid can be metabolised by autophagy, and this lipophagy is catalysed by lysosomal acid lipase (LAL). This study aimed to elucidate a role for LAL and lipophagy in pancreatic beta cells. We employed pharmacological and/or genetic inhibition of autophagy and LAL in MIN6 cells and primary islets. Insulin secretion following inhibition was measured using RIA. Lipid accumulation was assessed by MS and confocal microscopy (to visualise lipid droplets) and autophagic flux was analysed by western blot. Insulin secretion was increased following chronic (≥ 8 h) inhibition of LAL. This was more pronounced with glucose than with non-nutrient stimuli and was accompanied by augmentation of neutral lipid species. Similarly, following inhibition of autophagy in MIN6 cells, the number of lipid droplets was increased and GSIS was potentiated. Inhibition of LAL or autophagy in primary islets also increased insulin secretion. This augmentation of GSIS following LAL or autophagy inhibition was dependent on the acute activation of neutral lipases. Our data suggest that lysosomal lipid degradation, using LAL and potentially lipophagy, contributes to neutral lipid turnover in beta cells. It also serves as a constitutive negative regulator of GSIS by depletion of substrate for the non-lysosomal neutral lipases that are activated acutely by glucose.

Effects of increasing the dietary lipid levels on the growth performance, body composition and digestive enzyme activities of the teleost pejerrey (Odontesthes bonariensis)

The present study aimed at determining the growth performance, body composition and digestive enzyme activities of pejerrey (Odontesthes bonariensis) juveniles fed with graded levels of dietary lipids: 6% (L6), 10% (L10) and 25% (L25). Following a 14-week growth trial, weight increase decreased in group L6. The protein productive value increased in groups L10 and L25, whereas group L10 showed the highest lipid productive value. Hepatosomatic and mesenteric fat indexes decreased in group L6. Body protein content decreased with the increase in dietary lipids (L6>L10>L25); whereas body lipid content, and nitrogen and energy retentions were the lowest in group L6. The muscle fatty acid composition was differentially regulated by the diets except EPA, DHA and n-3 PUFA contents. The total n-6 PUFA decreased in group L6. The n-3 PUFA tended to accumulate in the muscle of the fish; group L10 exhibiting the lowest accumulation rate. The total activity of neutral lipase was stimulated in group L25 whereas the specific activity of pancreatic lipase decreased in this same group. While group L6 showed the lowest total activity of alkaline proteases, group L10 showed the highest total activity of trypsin. A decrease in the specific activity of amylase in group L25 followed the decrease in dietary starch. In summary, 25% of dietary lipids did not have additional stimulatory effects on the digestive proteases activity compared to 10%, but negatively affected pancreatic lipase and trypsin activities. Moreover, the decrease of the latter in group L25 suggests that approximately 10% of dietary lipids might support the growth of pejerrey juveniles reared in captivity.

Screening of thermophilic neutral lipase-producing Pseudomonas sp. ZBC1 and improving its enzymatic activity

From oil-contaminated soil, three lipase-producing microorganisms were selected as good lipase producers using rhodamine B-olive oil plate agar and they were identified as from Pseudomonas, Burkholderia and Klebsiella genera by morphology, biochemical characterization and 16S rRNA gene sequencing. Among the three strains, Pseudomonas sp. ZBC1 was found to produce thermophilic neutral lipase which exhibited maximum activity at 80oC and pH 7.0. Plackett-Burman design (PB design) and response surface methodology (RSM) analysis showed that the optimum values of the most significant factors are MgSO4 (0.85 g/L), inoculum size (65.9 ml/L) and volume of medium (51.57 ml). Under the optimal conditions, lipase production by Pseudomonas sp. ZBC1 was enhanced by approximately 2.30 fold (from 8.50 to 19.50 U/ml). Keywords : Pseudomonas , lipase, thermophilic, response surface methodology African Journal of Biotechnology Vol. 12(9), pp. 949-957

Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression

A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta.

A neutral lipase applicable in biodiesel production from a newly isolated Streptomyces sp. CS326

In an attempt to isolate a biocatalyst able to catalyze biodiesel production from microbial source, Streptomyces sp. CS326 was screened from hundreds of soil isolates collected from various parts of Korea. In 16S rRNA sequence analysis, the strain showed high degree of similarity with Streptomyces xanthocidicus (99.79%); therefore, it is classified as Streptomyces sp. CS326. An extracellular lipase produced by the strain (LP326) was purified using a single step gel permeation chromatography on Sepharose CL-6B. Molecular weight of LP326 was estimated to be 17,000 Da by SDS-PAGE. The activity was optimum at 40 °C and pH 7.0 and was stable at pH 5.0-8.0 and below 50 °C. It preferred p-nitrophenyl palmitate (C16), a long chain substrate; and K (m) and V (max) for the substrate were determined to be 0.24 mM and 4.6 mM/min mg, respectively. First 10 N-terminal amino acid sequences were APDLVALQSE, which are different from so far reported lipases. LP326 catalyzed biodiesel production using methanol and various oils; therefore, the enzyme can be applicable in the field of biofuel.

Comparative studies of vertebrate lipoprotein lipase: A key enzyme of very low density lipoprotein metabolism

Lipoprotein lipase (LIPL or LPL; E.C.3.1.1.34) serves a dual function as a triglyceride lipase of circulating chylomicrons and very-low-density lipoproteins (VLDL) and facilitates receptor-mediated lipoprotein uptake into heart, muscle and adipose tissue. Comparative LPL amino acid sequences and protein structures and LPL gene locations were examined using data from several vertebrate genome projects. Mammalian LPL genes usually contained 9 coding exons on the positive strand. Vertebrate LPL sequences shared 58–99% identity as compared with 33–49% sequence identities with other vascular triglyceride lipases, hepatic lipase (HL) and endothelial lipase (EL). Two human LPL N-glycosylation sites were conserved among seven predicted sites for the vertebrate LPL sequences examined. Sequence alignments, key amino acid residues and conserved predicted secondary and tertiary structures were also studied. A CpG island was identified within the 5′-untranslated region of the human LPL gene which may contribute to the higher than average (×4.5 times) level of expression reported. Phylogenetic analyses examined the relationships and potential evolutionary origins of vertebrate lipase genes, LPL, LIPG (encoding EL) and LIPC (encoding HL) which suggested that these have been derived from gene duplication events of an ancestral neutral lipase gene, prior to the appearance of fish during vertebrate evolution. Comparative divergence rates for these vertebrate sequences indicated that LPL is evolving more slowly (2–3 times) than for LIPC and LIPG genes and proteins.

Digestion of lipids and carbohydrates from microalgae (Chaetoceros muelleri Lemmermann and Isochrysis aff. galbana clone T-ISO) in juvenile scallops (Pecten maximus L.)

The digestion of laminaran and neutral lipids from the microalgae Chaetoceros muelleri or mixtures of C. muelleri and Isochrysis galbana clone T-ISO in 4-mm scallops Pecten maximus were studied. The microalgae were cultivated at fixed low and high growth rate (5% and 60% of μmax). The cultivation method clearly affected the lipid and carbohydrate contents, as well as their enzyme activity. Chaetoceros muelleri and T-ISO grown at low growth rate exhibited a considerably higher content of lipids and carbohydrates than those algae grown at high growth rate. The specific activity of laminaranase and neutral lipase was the highest in C. muelleri grown at the low growth rate. Growth rate of scallops was best when they were fed a mono-species diet of C. muelleri grown at low growth rate. The growth rate of the scallops was positively correlated to the dietary carbohydrate content as well as to the specific activity of neutral lipase and laminaranase in the diet. The capacity to digest carbohydrates (laminaran) might be limiting for the young scallops, and the content of carbohydrates and enzymatic activities of laminaran in the diet should be considered when formulating an optimal diet, both when the diet consist of live microalgae and formulated diets.