Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design

The thermoalkalophilic lipase from Bacillus thermocatenulatusBTL2 exhibits a low phospholipase activity (lecithin/tributyrinratio 0.03). A single round of random mutagenesis of the BTL2gene followed by screening of 6000 transformants on egg-yolkplates identified three variants with 10–12-fold increasedphospholipase activities, corresponding to lecithin/tributyrinratios of 0.16–0.36. All variants were specific for thesn-1 acyl ester bond of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.Mutations occurred predominantly in the N-terminal part of BTL2with regions surrounding the predicted helix     

Positional specificity of adipose tissue lipoprotein lipase

The positional specificity of preparations of lipoprotein lipase derived from rat epididymal adipose tissue was investigated. The enzyme preparations were a crude extract of acetone powder of the whole tissue, partially purified lipoprotein lipase fractions a and b separated by gel chromatography from such an extract, and lipoprotein lipase activity eluted from adipose tissue into a medium by incubation with heparin in vitro. The enzyme preparations were incubated with triglyceride substrate labeled with³H in the glycerol moiety and with¹⁴C in the fatty acid esterified to the 2 position of the glycerol. The reaction products were separated by thin layer chromatography. All preparations preferentially hydrolyzed the 1(3) ester bonds of the tri- and diglycerides, indicating that, like lipoprotein lipase from other sources, the adipose tissue enzyme has a specificity for the 1(3) position.     

Sugar ester-modified lipase for the esterification of fatty acids and long-chain alcohols

The esterification reaction of a long-chain fatty acid and a fatty alcohol with a surfactant-modified lipase in a microaqueousn-hexane system was studied. Various lipases from different sources were first modified with a surfactant of the sugar ester type to improve their dispersibility in apolar organic solvents. This enzyme modification technique converted inactive crude lipases to highly active biocatalysts for the esterification of long-chain fatty acids and fatty alcohols in a microaqueous n-hexane system. The hydrophilic-lipophilic balance value and chainlength of the fatty acid residue of the fatty acid sugar ester, used for modifying the lipases, significantly influenced the amount of precipitated lipase that was dissolved in the aqueous solution, the protein content of the lipase-surfactant complex and its esterification activity.     

Esterification kinetics of long-chain fatty acids and fatty alcohols with a surfactant-coated lipase in n-hexane

The esterification reaction kinetics of long-chain fatty acids and fatty alcohols catalyzed with a surfactant-coated lipase in a microaqueous n-hexane system were studied. The biocatalytic complex, surfactant-lipase adduct, showed 40 times the activity after a reaction time of 5 h compared to the unmodified lipase in the same reaction system. Various factors that may affect the activity of the modified lipase were studied, such as the influence of substrate fatty acid chainlength, water content, and temperature. By varying the concentration of each of the two substrates while keeping that of the other substrate constant, it was found that the esterification reaction follows Michaelis-Menten kinetics. The surfactant-enzyme complex kinetic parameters were determined with respect to both substrates. It was suggested that the kinetics of the lipase-catalyzed esterification reaction model follow a Ping-Pong Bi Bi mechanism with no substrate or product inhibition.     

Changes in lipid content,fatty acid composition and lipoprotein lipase activity in dry goat omental adipose tissue according to tissue site

Water and lipid contents, fatty acid distribution, and lipoprotein lipase activity were determined in 11 samples taken from theOmentum Majus of five dry Alpine goats. Samples were chosen to standardize sampling sites using geometric guide marks representative of different adipose tissue sites. Sample location explained between 20% and 30% of the total variance in water and lipid contents and in lipoprotein lipase activity, and from 5.5% to 45.4% of the total variance in fatty acid distribution. Increased sample thickness was associated with an increase in lipid content and in saturated fatty acid percentages. Samples taken in proximity of the omentum tissue attached to the rumen and abomasum had the highest content. We furthermore found that the levels of 18∶2n−6, 18∶1n−7, and of branched chain fatty acids were high close to a pila of the rumen which also corresponded to high lipoprotein lipase activity. Concomitant high levels of 16∶1n−7, 17∶1n−8, and 18∶1n−9 may reflect high levels of Δ9 desaturase activity.     

酶法合成长链不饱和脂肪酸酯

以固定化南极洲假丝酵母脂肪酶为催化剂合成长链不饱和脂肪酸酯。重点讨论了带水工艺、带水溶剂种类、脂肪酸碳链长度和反应温度等因素对反应的影响。结果表明,固定化南极洲假丝酵母脂肪酶对长链脂肪酸、脂肪醇的酯化反应有很好的催化效果,随脂肪酸碳链长度增加,脂肪酶的催化活力提高;有机溶剂和抽真空都可以带出反应中产生的水,溶剂带水有更好效果;相同反应时间内,反应温度60℃比50℃时有更高脂肪酸转化率;脂肪酶在50℃条件下使用30 h,酶活力与未使用前几乎完全相同。以低沸点石油醚(40~60℃)为带水溶剂,酶用量0.5%,反应温度50~60℃,反应时间6 h,可以得到酸值低于1的不饱和长碳链脂肪酸(C16~C22)油醇酯。     

Estimate of fatty acid turnover in porcine adipose tissue

Fatty acid turnover in the domestic pig was estimated by measuring the half-life of linolenic acid depletion in adipose tissue depots which had been made abnormally high in linolenic acid by feeding large quantities of linseed oil. The measured half-life of linolenate in 8- to 12-month-old pigs was 300 days. The apparent half-life of linolenate in muscle lipids was less than that of subcutaneous backfat.     

Site-specific differences in the fatty acid composition of human adipose tissue

The fatty acid composition of triacylglycerols from fifteen distinct adipose depots taken from each of seven adult male human subjects was compared. Oleic, palmitic, linoleic, stearic, myristic, palmitoleic and vaccenic acids accounted for more than 90% of the triacylglycerol fatty acids in all sites from all subjects; a number of other fatty acids were also identified and quantified. There were large differences in theaverage fatty acid composition between individual subjects. There were no site-specific differences in the proportions of myristic (3.8–4.7% of triacylglycerol fatty acids), palmitic (23–29%), linoleic (6.7–9.8%) or vaccenic (4.1–4.7%) acids or in the proportions of any of the less abundant fatty acids. There were some significant site-specific differences in the proportions of palmitoleic, oleic and stearic acids. The calf depot contained more palmitoleic acid (6.41±1.09%) than the trapezius (3.12±0.55%), perirenal (3.59±0.50%) and mesenteric (3.70±0.43%) depots, more oleic acid (42.13±1.27%) than the trapezius (36.03±2.18%), perirenal (36.50±1.56%) and breast (37.13±1.55%) depots and less stearic acid (5.18±0.89%) than the trapezius (8.57±0.97%), perirenal (8.49±0.75%), mesenteric (7.87±0.42%), breast (8.02±0.75%) and clavicular (8.34±0.78%) depots. The buttock depot contained less stearic acid (6.06±0.65%) than the perirenal, mesenteric and clavicular depots, while the anterior thigh depot contained less stearic acid (6.07±0.70%) than the perirenal depot. These findings indicate that, while most human adipose depots differ little in fatty acid composition, some sites, in particular the calf, perirenal, trapezius and mesenteric depots, have site-specific properties.     

Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis

When whale oil triglycerides were subjected to pancreatic lipase hydrolysis, eicosapentaenoic and docosahexaenoic acids were found mainly in the di- and triglyceride products, suggesting that they are in the 1,3-positions but resistant to the action of the lipase. Their presence in the 1,3-positions was confirmed. Their resistance to pancreatic lipase hydrolysis was demonstrated by analysis of the products of the enzyme action on: (a) a concentrate of highly unsaturated whale oil triglycerides; (b) the latter after randomization; and (c) synthetic 1,2-di-octadecenoyl-3-eicosapentaenoyl glycerol. Docosapentaenoic acid was also shown to be present in the 1,3-position of whale oil triglycerides but was not lipase resistant. It is postulated that the presence of a double bond near the carboxyl group exercises an inhibitory effect, or that the location of the double bonds in the resistant acids places their terminal methyl groups close to the carboxyl, producing a steric hindrance effect.     

Some aspects of fatty acid metabolism in brown adipose tissue

The action of catecholamines on white and brown adipose tissue is compared. The available evidence indicates that lipolysis is initiated in both tissues by hormonal action. There are, however, some differences in the behavior of the lipolytic process in the two tissues in their response to theophylline, nicotinic acid and insulin which remain unexplained. It would appear that the release of free fatty acids triggers the stimulation of O₂ consumption in both tissues. In brown adipose tissue no evidence has been obtained to indicate that the increased O₂ consumption is geared to the production of ATP for the purpose of reesterification of the released free fatty acids, as is the case in white adipose tissue. One of nine papers to be published from the Symposium “Brown Adipose Tissue,” presented at the AOCS-AACC Joint Meeting, Washington, D.C., March 1968.     

Circum-annual changes in triglyceride fatty acids of bat brown adipose tissue

A circum-annual study of the fatty acids of brown adipose tissue triglycerides ofEptesicus fuscus has demonstrated a rhythmic pattern of change. This is seen as a reciprocal shift of the levels of oleic and linoleic acids. Oleic acid levels are lower during the summer months and higher in the winter months. Levels of palmitic and linoleic acids reach maximal values in midsummer and fall significantly during the winter. Homogenates of brown adipose tissue produce more¹⁴CO₂ from 1-¹⁴C-palmitic acid than from 1-¹⁴C-oleic acid when incubated at temperatures below 20C. The formation of¹⁴CO₂ from either substrate was maximal in the neighborhood of 30C, and the temperature effect was enhanced by stimulation with DL-carnitine. It is proposed that the rhythmic change in brown adipose tissue triglyceride composition is a reflection of the different rates of fatty acid oxidation and the absence of normal food intake for extended periods of time.     

Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis

Mammalian hormone-sensitive lipase (HSL) has given its nameto a family of primarily prokaryotic proteins which are structurallyrelated to type B carboxylesterases. In many of these     

Effects of ethanol intake on lipoprotein lipase activity in adipose tissue of fasting subjects

Ethanol (ca. 1 g/kg body weight) was given alone or together with glucose or lipid (mixed triglycerides) perorally to young, fasting subjects. The changes with time (0–6 hr) of lipoprotein lipase activity (LLA) in adipose tissue, plasma glycerol, triglyceride, insulin, blood glucose, and alcohol concentrations were followed. A maximal mean blood alcohol concentration of 0.09% (w/v) was obtained 1 hr after ingestion with no apparent intoxicating effects. Ethanol intake prevented the previously observed [Nilsson-Ehle, P., S. Carlström and P. Belfrage,Scand. J. Clin. Lab. Invest. 35∶373 (1975)] glucose-induced rapid elevation of adipose tissue LLA but had small effects on this enzymatic activity when given alone or together with lipid. Confirming results by others, ethanol intake decreased plasma glycerol concentration and increased plasma triglycerides, especially after intake of lipid. It is suggested that ethanol intake interferes with the normal carbohydrate-induced elevation of adipose tissue LLA after a mixed meal, thereby decreasing the removal capacity for circulating dietary lipid and causing enhanced and prolonged alimentary hyperlipemia.     

The effect of dietary fish oil on muscle and adipose tissue lipoprotein lipase

The activity of lipoprotein lipase (LPL) in the adipose tissue and skeletal muscle of rats fed glucose- or fructose-based diets containing fish oil, corn oil or tallow was examined. In addition, heart LPL activity was measured in rats fed a glucose-based diet containing either corn oil or fish oil. Adipose tissue LPL activity was unaffected by dietary fat. In both heart and skeletal muscle, LPL activity was higher in rats fed the fish oil diet. These results suggest that increased removal of triglyceride by muscle may contribute to the blood triglyceride lowering effect of dietary fish oil.     

Transesterification of oil by fatty acid-modified lipase

Fatty acid was covalently attached to lipase (EC 3.1.1.3.) fromPhycomyces nites, yielding a modified lipase of higher specific activity in hydrolytic and synthetic reactions in organic solvents. Attached long-chain fatty acids solubilized the lipase in organic solvent and, therefore, promotion of dispersibility in organic solvent resulted in much higher reactivity. The initial rate of transesterification by modified lipase was almost 40 times that of native lipase in organic solvent. The specificities and selectivity of the modified lipase depended on the kind of attached fatty acid.     

Changes in hydrolysis specificities of lipase from Rhizomucor miehei to polyunsaturated fatty acyl ethyl esters in different aggregation states

Hydrolysis specificities of lipase from Rhizomucor miehei were compared for various fatty acyl ethyl esters. Activity yields of immobilized lipases, measured with 1 mM substrate, were more than 100%. Differences in hydrolysis rate and affinity for the substrates between lipase preparations were also typically higher during hydrolysis of substrates at 100 mM than at 1 mM, indicating better mass transfer effects for 1-mM substrates. The native lipase showed higher affinity for polyunsaturated fatty acid substrates at 1 mM than at 100 mM. Hydrolysis rates for 1-mM substrates were observed with immobilized lipases, fixed on anion exchange resin with glutaraldehyde and on cation exchange carrier with carbodiimide, and suggested some modification of the basic amino acid related to the lid of R. miehei lipase. Activation with these bifunctional reagents was not observed for 100-mM substrates, indicating that interfacial activation always occurred because of aggregation of 100-mM substrates. These results show that lipase from R. miehei recognizes molecular aggregation of lipids, and that various changes occur in the hydrolysis specificities for fatty acids.     

Uptake and release of fatty acids by rat adipose tissue: Last in—First out?

Fatty acid labeled chyle was administered iv to ad lib fed rats. At intervals from 1 hr to 50 days later rats were killed and pieces of their parametrial adipose tissue were incubated in vitro with norepinephrine. The specific radioactivity of the mobilized free fatty acids was compared to that of tissue glycerides and that of tissue free fatty acids. The results indicate that fatty acids taken up by the adipose tissue do not mix immediately with the bulk of tissue lipids and that the mobilized free fatty acids do not pass through the tissue free fatty acid pool.     

The variation of triglyceride structure with fatty acid composition in pig adipose tissue

Forty-five triglyceride samples with a wide range of fatty acid compositions were selected from a large number of pig adipose tissue samples (inner and outer back fats and perenephric fat) available from nutritional experiments. These samples were subjected to stereospecific analysis to determine the changes occurring in the positional distribution of the component fatty acids. The oleic acid content of the triglycerides was taken as the standard of comparison and as this increased, the proportions of the other unsaturated fatty acids also increased in a linear manner and the concentrations of the saturated components decreased proportionately. In position 1, the palmitic acid concentration remained constant while the stearic acid concentration decreased linearly and the concentrations of the unsaturated fatty acids increased. In position 2 the stearic acid concentration remained almost constant while the palmitic acid concentration decreased linearly in response to increases in the concentrations of the unsaturated acids. The least change occurred in position 3 where there were slight decreases in the concentrations of saturated acids as the concentrations of unsaturated acids increased. The precise quantitative relationships depended on the tissue examined. Constant proportions of the available myristic and palmitoleic acids were found in all three positions and constant proportions of the available stearic and oleic acids were found in position 1. These results are discussed in relation to possible pathways of triglyceride biosynthesis in pig adipose tissues.     

Identification of methyl-branched fatty acids from the triacylglycerols of subcutaneous adipose tissue of lambs

A concentrate of branched chain fatty acids (as methyl esters) was prepared from the triacylglycerols of subcutaneous adipose tissue lipids of lambs receiving a carbohydrate-rich (cereal) diet. This was accomplished by procedures which allowed the removal of unsaturated components by peroxidation and straight chain saturated components by urea-adduct formation. The concentrate was analyzed by high resolution gas chromatography in combination with mass spectrometry and was shown to consist of a complex mixture of saturated methyl-substituted fatty acids. Methyl substitution occurred on even-numbered carbon atoms (relative to the carboxyl group) and the chain lengths of the acids ranged from 10 to 18 carbon atoms. Acids with one methyl substituent in the fatty acyl chain were most abundant; di-, tri- and tetramethyl-substituted acids were also present. The biosynthesis of these methyl-substituted acids is discussed briefly.