Studies on the phospholipases of rat intestinal mucosa

1. Subcellular distribution and characteristics of different phospholipases of rat intestinal mucosa were studied. 2. The presence of free fatty acid was necessary for the maximal hydrolysis of lecithin (phosphatidylcholine), but there was no accumulation of lysolecithin (1 or 2-acylglycerophosphorylcholine);lysolecithin accumulated when the reaction was carried out in the presence of sodium deoxycholate and at or above pH8.0. 3. The fatty acid-activated phospholipase B as well as lysolecithinase showed optimum activity at pH6.5, whereas for the phospholipase A it was about pH8.6. 4. The bulk of the phospholipase A was present in the microsomal fraction, whereas the phospholipase B and lysolecithinase activities were distributed between the microsomal and soluble fractions of the mucosal homogenate. 5. Phospholipase A was equally distributed between the brush border and brush-border-free particulate fraction, with the brush border having highest specific activity, whereas the other two activities were distributed between the brush-border-free particulate and soluble fractions. 6. Various treatments showed marked differences between the phospholipase A and phospholipase B activities, but not between phospholipase B and lysolecithinase activities. 7. By using (beta[1-(14)C]-oleoyl) lecithin it was shown that the mucosal phospholipase A was specific for the beta-ester linkage of the lecithin molecule.     

Fat metabolism in higher plants. Properties of the palmityl acyl carrier protein: stearyl acyl carrier protein elongation system in maturing safflower seed extracts

Palmityl acyl carrier protein is elongated specifically to stearyl acyl carrier protein by a system which required palmityl acyl carrier protein, malonyl CoA, and NADPH. Extracts from maturing safflower seeds, avocado mesocarp, and stroma from spinach chloroplasts contain the elongation system. The system differs from the de novo fatty acid synthetase system in that (1) it is inactivated at 37 °C whereas the de novo system remains fully active, (2) the pH optimum of the elongation system is 7.8–8.6 whereas the de novo system has a narrow pH optimum at 7.0, (3) NADPH is specifically required whereas the de novo system requires both NADPH and NADH, and (4) the elongation system is relatively insensitive to cerulenin whereas the de novo system is highly sensitive. Acetyl CoA does not serve as a C₂ donor. Stearyl acyl carrier protein, lauryl CoA, myristyl CoA, and palmityl CoA are inactive.     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.     

Screening of filamentous fungi for lipase production: Hypocrea pseudokoningii a new producer with a high biotechnological potential

Abstract

Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40°C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0–8.0 at room temperature. The intracellular lipase was thermostable up to 60°C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.     

Phospholipase A2 activity of rat stomach

Phospholipase A activity in rat stomach wall and in gastric content was studied using [1-14C]dioleoylphosphatidylcholine as substrate. The optimum activity of the stomach wall was found to take place at pH 7.0. During optimal phospholipase action about 40% of the [1-14C]oleic acid released was due to an active intracellular lysophospholipase. The gastric phospholipase required 5 mM Ca2+ for full activity and is inhibited by EDTA. It specifically hydrolyzed the sn-2 position of the phospholipid molecule. The enzyme was heat labile and inactivated by acidification at pH 3.0. The gastric content enzyme had a lower specific activity and an optimum pH of 8.0. It was heat stable and was not inactivated by acidification. These results indicate that gastric content phospholipase A is of pancreatic origin, via a duodenal reflux. By ligating the stomach we were able to further confirm that the gastric wall phospholipase was different from that of the gastric content. It originated from the stomach mucosa. Subcellular fractionation suggests that the gastric phospholipase A2 is essentially bound to the plasma membrane. About 6% of the activity was found to be soluble. Biopsies of human gastric mucosa displayed a phospholipase A activity which had similar properties to that of rat gastric enzyme. The physiological function of this enzyme is discussed in terms of prostaglandin synthesis via the release of arachidonic acid.     

Production and characterization of extracellular lipase from Calvatia gigantea

Summary A number of factors affecting production of extracellular lipase by the edible fungus Calvatia gigantea were investigated. Consecutive optimization of carbon and nitrogen sources, initial pH of culture medium and growth temperature resulted in an increase in lipase activity of 87%. Under optimum conditions, activities as high as 22.4 units ml^–1 of culture medium were obtained, competing favourably with most activities reported for other lipase hyperproducing microorganisms. The enzyme was optimally active at pH 7.0 and 30°C and had, at optimum pH, half-lives of 75.7 and 22.9 min at 45 and 55°C. Both high activity and kinetic characteristics of the enzyme make this process worthy of further investigation.Correspondence to: B. J. Macris     

Phospholipid removal during degradation of rat plasma very low density lipoprotein in vitro.

The hydrolysis of glycerophospholipids in very low density lipoprotein by enzyme(s) released into circulation after the injection of heparin to rats was studied. [32P]Lysolecithin was formed rapidly from [32P]lecithin when very low density lipoprotein, labeled biosynthetically with 32P, was incubated with postheparin plasma. The [32P]lysolecithin was associated with the plasma protein fraction of density greater than 1.21 g/ml, whereas [32P]lecithin exchanged between very low and high density lipoproteins. Inhibition of the plasma lecithin: cholesterol acyl transferase activity did not change the excess [32P]lysolecithin formation in postheparin plasma, and only a negligible amount of radioactivity was associated with blood cells when the incubation was repeated in whole blood. Analysis of the results has demonstrated that phospholipids are removed from VLDL by two pathways: hydrolysis of glycerophospholipids by the heparin-releasable phospholipase activity (greater than50%) and transfer to high density lipoproteins (less than50%). The tissue origin of the postheparin phospholipase was studied in plasma obtained from intact rats and supradiaphragmatic rats using specific inhibitors of the extrahepatic lipase system (protamine sulfate and 0.5 M NaCl). The phospholipase activity could be ascribed to both the hepatic and extrahepatic lipase systems. It is concluded that hydrolysis of glycerophospholipids is the major mechanism responsible for the removal of phospholipids from very low density lipoprotein during the degradation of the lipoprotein. It is suggested that phospholipid hydrolysis occurs concomitantly with triglyceride hydrolysis, predominantly in extrahepatic tissues.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

BIOSYNTHESIS OF PHOSPHATIDIC ACID IN RAT BRAIN VIA ACYL DIHYDROXYACETONE PHOSPHATE

Abstract— The enzymes for the biosynthesis of phosphatidic acid from acyl dihydroxyacetone phosphate were shown to be present in rat brain. These enzymes were mainly localized in the microsomal fraction of 12–14 day old rat brains. The brain microsomal acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42), exhibited a broad pH optimum between pH 5 and 9 with maximum activity at pH 5.4. K _m for DHAP at pH 5.4 was 0.1 m m and V _max was 0.86nmol/min/mg of microsomal protein. The corresponding microsomal enzyme for the glycerophosphate pathway (acyl CoA: sn -glycerol-3-phosphate acyl transferase EC 2.3.1.15) was shown to have a different pH optimum (pH 7.6). On the basis of the differences in pH optima, differential effects of sodium cholate in the enzymes and a common substrate competition study, these acyl transferases were postulated to be two different microsomal enzymes.
Acyl DHAP:NADPH oxidoreductase (EC 1.1.1.101) in brain microsomes was found to be quite specific for NADPH as cofactor, being able to utilize NADH only at very high concentrations. This enzyme exhibited a K _m of 8.6 μ m with NADPH and V _mx of 0.81 nmol/min/mg protein. The presence of these two enzymes and the known presence of l-acyl- sn -glycerol-3-phosphate: acyl CoA acyl transferase in brain (F leming & H ajra , 1977) demonstrated the biosynthesis of phosphatidic acid in brain via acyl dihydroxyacetone phosphate. Phosphatidic acid was shown to form when dihydroxyacetone phosphate, acyl CoA, NADPH and other cofactors were incubated together with brain microsomes. Further properties of the enzymes and the probable importance of the presence of this pathway in brain were discussed.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

Lipid monolayers: action of phospholipase A of Crotalus atrox and Naja naja venoms on phosphatidyl choline and phosphatidal choline

The activity of phospholipase A on phosphatidyl choline and phosphatidal choline spread as monolayers on phosphate buffers containing snake venom (Crotalus atrox or Naja naja) was studied by measuring the fall of surface potential as a function of time, pH, film pressure, temperature, and concentrations of phosphate and venom. At 25 degrees C, pH 7.0, and 0.2 micrograms of venom per ml, optimal activity was observed with both venoms on both substrates at 12 dynes/cm film pressure on 0.04 m phosphate. Under these conditions, the pH optimum for C. atrox was broad (6.6-7.4) and that for N. naja was sharp (8.0) for the action on phosphatidyl choline, whereas both venoms had a sharp optimum at pH 8.0 in their action on phosphatidal choline. The optimal temperature with phosphatidyl choline was 27.5 degrees C for N. naja and 40 degrees C for C. atrox. In line with studies of phospholipase A activity in bulk phase in ether, phosphatidal choline was attacked much more slowly than phosphatidyl choline by C. atrox. Under conditions where both venoms had equal activity on phosphatidyl choline, C. atrox was only half as active as N. naja on phosphatidal choline. The studies suggest that the linkage of the hydrophobic chains in glycerophosphatides may affect their interaction with proteins.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

A comparison of glucoside formation by liver preparations from the rabbit and the mouse

Liver homogenates from mice and from rabbits transfer glucose from UDP-[6-(3)H]glucose, at pH7.0, to oestradiol-17alpha, oestradiol-17beta, oestradiol-17alpha 3-glucuronide, p-nitrophenol and diethylstilboestrol. In the rabbit the phenolic steroids were better substrates than p-nitrophenol for the glucosyltransferase, whereas the reverse was true in the mouse. At pH8.0, rabbit liver, but not mouse liver, transferred glucose to oestradiol-17alpha 3-glucuronide in better yield than that at pH7.0. Evidence is presented for the presence of two glucosyltransferases in rabbit liver. One of these has a pH optimum at about 8.0, and is highly specific for oestradiol-17alpha 3-glucuronide, whereas the other, which has a pH optimum at about 7.0, is similar in this respect to the transferase in mouse liver.     

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Metabolism of sulfoquinovosyl diglyceride in Chlorella pyrenoidosa by sulfoquinovosyl monoglyceride: fatty acyl CoA acyltransferase and sulfoquinovosyl glyceride: fatty acyl ester hydrolase pathways

Cell-free preparations of Chlorella pyrenoidosa catalyze the transfer of the fatty acyl moiety of fatty acyl CoA derivatives to sulfoquinovosyl monoglyceride to form sulfoquinovosyl diglyceride. This reaction is stimulated by Triton X-100 concentrations of up to 0.6 mg/ml and has a pH optimum of 7.7. Similar Chlorella preparations catalyze the stepwise removal of both fatty acyl groups from sulfoquinovosyl diglyceride to form sulfoquinovosyl monoglyceride and then sulfoquinovosyl glycerol. This reaction is inhibited by both calcium and magnesium. The nonionic surfactant Triton X-100 inhibits the enzymatic deacylation at concentrations of less than 0.5 mg/ml but stimulates it at higher concentrations. The pH optimum for the deacylation of sulfoquinovosyl glycerides is 8.2, with little activity observed below pH 8. The enzymatic activities for both the transacylation and deacylation reactions are associated with a 30,000 g particulate fraction of Chlorella. Sulfoquinovosyl glycerol was found not to be an acceptor of the fatty acyl moiety of fatty acyl CoA derivatives. Methods are described for the preparation of sulfoquinovosyl monoglyceride, sulfoquinovose, and 3-sulfo-1,2-propanediol.     

High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM

The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0 mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg⁻¹), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65 °C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50 °C for more than 150 min.     

一株耐热脂肪酶产生菌的筛选及酶学性质研究

从云南省富油地采取了60份土样中,利用透明圈法筛选出一株耐热脂肪酶产生菌。对其酶学性质和发酵条件进行了研究,酶学性质表明,该酶最适作用温度为50℃,最适pH6.0,在pH3.0-8.0范围内稳定,在60℃保温60 min酶活还保留70%;70℃保温60 min残余50%;具有良好的热稳定性;不同金属离子有不同的作用,Ca+,K+对酶有激活作用,Fe3+、Pb2+、Mn2、Cu2+、Al3+、Zn2+对酶活有抑制作用。EDTA对酶影响不大。产酶最佳条件为:MgSO4.7H2O 0.05 g,K2HPO40.1 g,CaCO30.25 g,可溶性淀粉2.5 g,大豆粉2.5 g,装液量50 mL。这株细菌通过培养基优化酶活达到20.3 U/mL。     

A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene

An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. Keywords: Bacter     

Studies on sterol-ester hydrolase from Fusarium oxysporum. I. Partial purification and properties.

1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium. 2. The cholesterol esterase from Fusarium oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography. 3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60 degrees C for 30 min, at pH 7.0). 4. The optimum pH of the enzyme was found to be around 7.0. 5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable substrate. 6. Cholesterol esters in serum were also hydrolyzed by this enzyme.